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Macrophages: the overlooked target for pulmonary fibrosis and COPDBoorsma, Carian Eline

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Macrophages: the overlooked target for pulmonary fibrosis and COPD

Carian Eline Boorsma

Noordelijke Cara Stichting

The work described in this thesis was performed at the Department of Pharmacokinetics, Tox-icology and Targeting at the University of Groningen, The Netherlands. The research project was financially supported by a grand from the Dutch Northern Cara Foundation (Noordelijke Cara Stichting) and the Pender Foundation (Pender Fonds). Research communication was made possible by financial support from the Dutch Respiratory Society (Nederlandse Respi-ratory Society) and the lung fund (het Longfonds).

Printing this thesis was financially supported by the University of Groningen and Greiner bio-one.

Macrophages: the overlooked target for pulmonary fibrosis and COPD© copyright 2016 Carian Eline BoorsmaAll rights reservedNo part of this thesis may be reproduced or transmitted in any form or by any means without prior written permission from the author.

isbn: 978-90-367-9328-5isbn electronic version: 978-90-367-9327-8

Cover: Graphical display of the lungs with three windows showing the inside structure of the lungs

Cover design: Bert HoltkampLayout: Bert HoltkampIllustrations: made possible by lipi, curve, Andrejs Kirma, Katrin Leo Pako, Wes Breazell, David Padrosa and Nut Chanut from the Noun Project.Illustrations design: Nina BoorsmaPrinting: Ridderprint BV – www.ridderprint.nl

Macrophages: the overlooked target for pulmonary fibrosis

and COPD

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

vrijdag 2 december 2016 om 14.30 uur

door

Carian Eline Boorsma

geboren op 18 maart 1987 te Groningen

Promotores Prof. dr. B.N. Melgert Prof. dr. W. Timens Prof. dr. K. Poelstra Beoordelingscommissie Prof. dr. R. Gosens Prof. dr. M.C. Harmsen Prof. dr. J.C. Grutters

Paranimfen Fransien van Dijk Nina Eliisa Boorsma

Er gebeurt meer achter de schermen dan je ziet op het toneel.

Inhoud

Chapter 1 General introduction 11

Chapter 2 The RANK/RANKL/OPG axis has a role in regulating tissue repair processes in lung 43

Chapter 3A Potent Tartrate Resistant Acid Phosphatase Inhibitor to Study the Function of TRAP in Alveolar Macrophages 77

Chapter 4Current smoking is associated with fewer CD16+ monocytes in peripheral blood of male healthy controls and patients with COPD 105

Chapter 5Characterization of Interstitial Macrophages Around Airways of Current and Ex-smoking COPD Patients and Controls 123

Chapter 6General discussion 141

Chapter 7APPENDIX | PATENT 155

Chapter 8Dankwoord 198Curriculum Vitae 203List of publications 204

Chapter 1

General introduction

Parts are published in:“Macrophage heterogeneity in respiratory diseases. Carian E. Boorsma1,2, Christina Draijer1,2 and Barbro N. Melgert1,2. Mediators of Inflammation 2013”

1 Department of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute for Pharmacy, University of Groningen, Groningen, The Netherlands.2 GRIAC Research Institute, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands

12 Macrophages: the overlooked target for pulmonary fibrosis and COPD

Introduction

Macrophages are among the most abundant immune cells in the respiratory tract and these resident immune cells can be broadly divided into two populations depending on their local-ization: alveolar macrophages (AM) on the air-side that line the surface of alveoli and inter-stitial macrophages (IM) that reside in the lung tissue itself 1. The role of these macrophages in the lungs in steady state or diseased conditions has increasingly been the subject of var-ious studies, but a lot remains unknown regarding their phenotype, function and contribu-tion to lung diseases such as pulmonary fibrosis and chronic obstructive pulmonary disease (COPD). Therefore, in this thesis, we studied different macrophage phenotypes in COPD and tried to alter the (undesired) function of pulmonary macrophages (irrespective of their local-ization) in pulmonary fibrosis and COPD.

Resident macrophagesRecent studies have shown that alveolar macrophages are derived from embryonic progeni-tors (yolk sac macrophages and fetal monocytes) and self-maintain during steady-state con-ditions, i.e. they have the ability to divide and replenish the pool of resident AMs during life without contribution of adult monocytes derived from hematopoietic stem cells 2-4. AMs are situated in the inner lining of the lungs in order to respond quickly upon inhaled foreign particles or microbes that may potentially be pathogenic. They specialize in phagocytosing these foreign particles from the airway lumen and generate oxygen radicals and inflammatory cytokines as part of their host defense function 5.

Besides alveolar macrophages, interstitial macrophages populate the lungs within the lung tissue itself instead of the airway lumen. The origin of these IMs has not been defined yet. IMs have a higher turnover rate as compared to AMs, which may indicate that these cells are replenished from blood monocytes, but this is much debated 6,7. In addition, there is some evidence that IMs function as a pool for new AMs and may therefore also be derived from yolk sac macrophages and fetal monocytes 8.

Both AMs and IMs can have homeostatic properties 9. In steady state it was shown that both types of macrophages express MHC class II, even though the expression was higher on IMs than on AMs. Furthermore, they were found to produce high levels of IL-10 to sup-press inflammation following an allergic event or activation by inflammatory cytokines 10-12. In addition, AMs are able to regulate the response to an inert antigen by controlling antigen presenting function of dendritic cells and suppress T cell activation 13. Even though AMs and IMs may have different origins they are both able of adopting different phenotypes depending on the signals they receive from their surroundings, which will be explained in more detail later on in this chapter.

MonocytesIn cases of tissue damage, steady state conditions change and tissue resident macrophages may be supplemented with macrophages derived from incoming monocytes. In mice, two populations of monocytes have been identified based on the expression of the surface mol-


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ecule Ly6C (lymphocyte antigen 6C). Monocytes with high expression of Ly6C are gener-ally called classical or inflammatory monocytes and these patrol the extravascular tissues in homeostatic conditions 14. During this patrolling function they remain monocytic and do not commit to being macrophages. During inflammation, however, they respond by rapidly infiltrating into affected tissues and they can readily transform into macrophages with lim-ited potential for migration 14. Monocytes with low expression of Ly6C are called nonclassical monocytes and patrol the blood vessels to monitor endothelial cell homeostasis 15,16. They develop from the Ly6C-hi subset and this can also take place in injured or inflamed tissue with subsequent conversion from inflammatory monocyte to wound-healing macrophages that can proliferate locally 3,17-20.

In humans, similar monocyte subsets are found based on the expression of CD14 and CD16 21. Classical monocytes express high levels of CD14 and no CD16, while nonclassical monocytes express low levels of CD14 and high levels of CD16. Both in humans and mice, an interme-diate third subset is suggested to exist characterized in humans by high levels of CD14 and intermediate levels of CD16. The functions of this subset are not well understood, although they have been found to preferentially accumulate in inflamed human livers and have been postulated to play a role in fibrogenesis 22.

Macrophage activation statesIn addition to their role in host defense mechanisms, macrophages in the lung have also been associated with many other processes, including tissue repair processes and destruction of tissue, fibrosis and downregulation of inflammation. In order to combine these diverse and sometimes contradictory activities, macrophages exert the ability to adopt the most effec-tive phenotype based on signals from surrounding tissue. This results in a myriad of pheno-types among macrophages, irrespective of their origin, and the most investigated types are described below.

To distinguish the various macrophage phenotypes, a nomenclature was proposed similar to the Th1/Th2 dichotomy based on results from in vitro studies, with M1 macrophages being known as classically activated macrophages induced by interferon gamma (IFNγ) and tumor necrosis factor alpha (TNF-α) and M2 being known as alternatively activated macrophages induced by interleukin (IL)-4 and IL-13 23,24. Recent new insights led to changes within the macrophage field concerning the nomenclature of the macrophage phenotypes 25. It was already apparent that in vivo macrophage phenotypes appear as a continuum rather than discrete entities 26-28. In the new nomenclature, the macrophage phenotype is defined on the origin of the macrophage, the substance that induces the specific macrophage phenotype and/or on the markers it expresses. This in contrast to the previously used nomenclature that focused mainly on the expression of a single or a few markers or on the function of the mac-rophage, i.e. inflammatory macrophage (M1) versus the tissue repairing macrophage (M2). In this thesis, macrophage subsets will mostly be referred to according to the expression of specific markers or, if necessary, the old and new nomenclature will both be mentioned as some cited articles used the old nomenclature. The exact markers will be discussed in more detail later in this section.


IRF5+ M1 macrophagesThis macrophage phenotype is activated by pro-inflammatory cytokines and microbial products like IFNγ, TNF-α and/or lipopolysaccharide (LPS) under the influence of the tran-scription factor interferon-regulatory factor 5 (IRF5) 29. These macrophages are essential in host defense against intracellular pathogens by generating reactive oxygen species (ROS) and nitric oxide (NO) through upregulated expression of inducible nitric oxide synthase (iNOS) and amplifying Th1 immune responses by producing pro-inflammatory cytokines like IL-12, IL-1β and TNF-α (see also figure 1) 28. In addition, they show enhanced phagocytosis of micro-organisms, antigen presentation capabilities and enhanced production and secretion of matrix metalloproteinases (MMPs) such as MMP7 and MMP9 30-33. The secretion of MMPs enables macrophage migration during inflammatory responses, but excessive or unregulated production results in tissue damage 26,33.

Figure 1. Schematic representation of three macrophage phenotypes and their characteristics. Abbreviations: IFNγ: interferon gamma; TNF-α: tumor necrosis factor alfa; LPS: lipopolysac-charide; MHC class II: major histocompatibility complex class II; IL: interleukin; NO: nitric oxide; IRF5: interferon regulatory factor 5; Fe: iron; TGM2: transglutaminase 2; YM1: chiti-nase-3–like protein-3; FIZZ1/Relmα: resistin-like molecule-α; Arg-1: arginase-1; TGFβ: trans-forming growth factor beta; TLR: toll-like receptor; PGE2: prostaglandin E2; PPARγ: peroxi-some proliferator-activated receptor gamma.


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M2 macrophages: CD206+ M2 or IL-10+ M2-like macrophages Alternatively activated or M2 macrophages were named to indicate their activation status was distinctly different from the classically activated macrophages. First discovered to be induced by IL-4 and IL-13 this phenotype was soon found to have more siblings, closely resembling each other but distinctly different in function 26,34-36. A variety of different names have been suggested according to the expression of various markers 26,36. They have suggested alterna-tively activated or M2 macrophages for the phenotype induced by IL-4/IL-13 and regulatory macrophages or M2-like cells for the phenotype characterized by high IL-10 production that are induced by a variety of stimuli (see also figure 1). In this thesis, we will mainly distinguish between these two subsets and refer to them as CD206+ when mentioning the “M2-ma-rophages” and the “M2-like-macrophages” as IL-10+ macrophages.

1: CD206+ M2 macrophagesMacrophages induced by IL-4/IL-13 under the influence of the transcription factor IRF4 37, have a role in protection against helminthes and are considered wound-healing macro-phages because of their association with physiological and pathological tissue remodeling 26,38. They are characterized by upregulated expression of mannose receptors (CD206) and transglutaminase 2 (TGM2) in man and mice and by upregulated expression of arginase-1, chitinase-3–like protein-3 (Chi3l3, also known as Ym1), and resistin-like molecule-α (Relmα, also known as FIZZ1) in mice only (see also figure 1) 24,35,38-40. They have poor antigen present-ing capabilities and exhibit increased release of iron and increased clearance of apoptotic cells (efferocytosis) and extracellular matrix components 41-43.

2: IL-10+ M2-like macrophagesOne of the CD206+ macrophage subtypes produces high levels of IL-10 (see figure 1). These macrophages are induced by a number of stimuli that need to be combined with a Toll-like receptor (TLR) signal. The initial signals include glucocorticosteroids, prostaglandin E2 (PGE2), antibody immune complexes, transforming growth factor beta (TGFβ) and IL-10 itself 26. Transcriptional control of this phenotype is unclear but may involve peroxisome prolifer-ator-activated receptor gamma (PPARγ) and the cAMP-responsive element-binding protein (CREB)-CCAAT/enhancer-binding protein-β (C/EBPβ)-axis 44. As a result of their high IL-10 production, these macrophages have strong anti-inflammatory activity. This can be bene-ficial during later stages of immune responses to limit inflammation, but may also permit tumor progression when associated with tumors 26,28. They may also be the macrophages that produce TGFβ in addition to IL-10, but this has not been rigorously shown due to the overlap in markers among the CD206+ macrophages 45-48. Only IL-10 production would be a reliable marker but is used seldom to identify these macrophages 26. Nevertheless, we will use IL-10 to identify this macrophage phenotype.

Lung diseasesThe distinct phenotypes and functions of macrophages and the switching between them in lung tissue appear to have developed to maximize defense against external threats without impeding gas exchange. It therefore does not come as a surprise that changes in the number of macrophages and changes in macrophage phenotypes have been associated with many


pulmonary diseases. Thus, depending on the function, macrophages could be beneficial or harmful. In this thesis we studied what macrophage functions/phenotypes are associated with disease processes and how we could change macrophage function/phenotype in order to have beneficial effects on lung diseases. Using the natural functions of macrophages may be an interesting novel opportunity to induce resolution of lung diseases.

The lung consists of conducting airways and an alveolar compartment. The latter is respon-sible for gas exchange between air and blood over a thin layer of ECM and epithelial cells (the interstitium). Increased deposition of ECM in the interstitium, as observed in patients with pulmonary fibrosis, or loss of interstitial tissue, as observed in patients with COPD, has an enormous effect on the gas exchange homeostasis and therefore lung function. The reg-ulation of this interstitial ECM in the lung is poorly understood, but macrophages may play an important role in maintaining integrity of this thin layer. In addition, airway interstitial macrophages may also be involved in regulating processes leading to airway remodeling, another important characteristic of COPD. Below we describe what is known regarding the role of macrophages in the light of pulmonary fibrosis and COPD and which gaps in knowl-edge still remain.

Pulmonary fibrosisPulmonary fibrosis is a disease that encompasses a collection of restrictive pulmonary disor-ders characterized by progressive and irreversible destruction of lung architecture by exces-sive deposition of ECM in parenchymal areas of the lung 49,50. While ECM formation usually functions as an essential process of tissue repair after lung injury, continuous lung damage may result in abnormal wound healing and progress to fibrosis 51. Fibrosis of the intersti-tium ultimately leads to organ malfunction because of the disturbed architecture of the lung, leading to impaired gas exchange, and eventually death from respiratory failure 49. In some cases, fibrotic lesions remain localized to a limited area of the lung because the initial trigger is removed, for example after tuberculosis or a fungal infection, while in others such as in sarcoidosis and idiopathic pulmonary fibrosis (IPF) the fibrotic process continues to progress throughout the lungs in a diffuse manner 52.

Idiopathic pulmonary fibrosis (IPF) is the most common and most dangerous of the fibrotic interstitial lung diseases. The chronic and slowly progressing character of the disease together with an unknown etiology, makes it a difficult disease to diagnose and treat. The incidence of IPF appears to be increasing and is currently estimated at 7-16 cases per 100.000 persons 53. Patients diagnosed with IPF have a poor life expectancy with a median survival of 2-5 years 54. Currently there are no effective therapies available for these patients, as no therapy has yet been proven to cure or even halt the progression of fibrosis 52.

Pathogenesis of pulmonary fibrosisTo describe the pathogenesis of pulmonary fibrosis, tissue repair after injury can be divided into four different stages: the clotting phase for emergency wound healing, then the inflam-matory phase to fight the inciting agent, followed by formation of scar tissue in the fibrotic phase for more permanent repair and eventually reduction of scar tissue and restoration


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of tissue homeostasis in the resolution phase. During fibrosis some or all of these stages of wound healing are dysregulated as will be discussed below and the same framework will be used to discuss the role of macrophages in fibrosis later on.

Pulmonary fibrosis is thought to be the result of repetitive injury to the epithelial cell layer lining the alveoli. This damage initiates a blood coagulation cascade to prevent severe blood loss and maintain some sort of homeostasis. This includes platelet accumulation and produc-tion of fibrin by epithelial cells, which is essential for fibrin-containing clot formation 55. To restore the function of damaged tissue, plasminogen activator (PA) eventually breaks down this fibrin matrix again. In pulmonary fibrosis, changes in both the coagulation cascade itself and the resolution of the wound-healing clot are known to affect the disease. Impaired fibrin degradation for instance has been shown to worsen epithelial cell survival, that can be caused by either the absence of PA or by increased production of PA inhibitors PAI-1 or PAI-2 56-58.

As a result of the cell damage, an inflammatory reaction is triggered. Unfortunately, it has been difficult to investigate the contribution of this phase to the fibrosis because patients usually present to the clinic with end-stage disease. Nevertheless, the inflammatory response has been extensively studied in LPS-induced inflammation in humans (reviewed by Rossol et al. 59). They concluded that epithelial cell damage induces the release of several cytokines and chemokines that trigger an influx of neutrophils, closely followed by monocytes to fight the inciting agent 60. Epithelial cells also release growth factors like TGFβ, TNF-α and epidermal growth factor alpha (EGFα) to stimulate tissue healing through the activation of fibroblasts, which are main producers of collagen and other ECM proteins 61,62.

Control of the inflammatory event, however, is crucial for a proper wound healing process 62. Apoptosis and subsequent removal of inflammatory macrophages, initiated by T cells, has proven to be essential in this step 63. Dysregulation of the inflammatory phase with a prominent role for M1 macrophages has long been thought to be important to the process of fibrosis. However, the fact that anti-inflammatory drugs such as corticosteroids have no therapeutic merit in patients with pulmonary fibrosis has made this assumption unlikely 64. Now the new prevailing hypothesis is that pulmonary fibrosis probably develops when the fibrotic phase itself and/or resolution phase become dysregulated 65.

To progress from the inflammatory phase to the next phase of tissue repair, inflammation needs to be dampened. The release of IL-10 and TGFβ by epithelial cells and macrophages dampen inflammation and promotes ECM production by myofibroblasts 66. Under the influ-ence of TGFβ and PDGF, produced by damaged epithelial cells, platelets, and macrophages, fibroblasts differentiate into myofibroblasts, proliferate and produce ECM proteins 67-69. Fur-thermore, they start producing their own TGFβ to maintain the tissue healing process 70. In pulmonary fibrosis this phase is probably dysregulated as increased numbers of myofibro-blasts and increased production of ECM are found in fibrotic lungs. Increased numbers of CD206+ macrophages are associated with this phase and these macrophages are therefore suggested to play an important role in the development of fibrosis 62.


Eventually repair of the epithelial cell barrier and removal of excess ECM are essential to recover normal lung function. To overcome the loss of alveolar epithelial type I cells (AEC I), alveolar epithelial type II cells (AEC II) become hyperplastic and provisionally restore the epi-thelial cell layer along ECM produced by myofibroblasts 61. Normally these type II cells would revert back to AEC I and homeostasis is restored. However, when injury is repetitive this does not seem to occur; ECM is produced continuously and AEC II continue to proliferate without differentiating to AEC I. In a proper wound healing response, the excess of ECM products is removed to gain full function of the lungs again. Macrophages play an important role in degrading and taking up ECM components. In order to do so, they produce cathepsins, MMPs and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs). A balance between the activities of proteolytic enzymes and their inhibitors determines the ECM-balance within the tissue 71. Although levels of both, MMPs, TIMPs and cathepsins are all elevated in patients and mouse models of pulmonary fibrosis, their balance is clearly disrupted as the net result is an excess of ECM in fibrotic lungs 72-74.

Macrophages and pulmonary fibrosisMacrophages play an important role in the pathogenesis of lung fibrosis, but their role is com-plex 69. They are involved in many of the dysregulated tissue healing responses in fibrosis and adopt numerous phenotypes depending on the stage and condition of the tissue. This com-plicates studies into their role in fibrosis tremendously. In the next part we will discuss what is known about the contribution of each macrophage phenotype to what stage of fibrosis.

IRF5+ M1 macrophages in pulmonary fibrosisTo our best knowledge, we have found no studies reporting on the presence of IRF5+ macro-phages in pulmonary fibrosis except for one study by Nagai et al. showing that folate receptor beta (FRβ)-positive macrophages were higher in patients with IPF as compared to controls 75. These macrophages have previously been shown to produce TNF-α and oxygen radicals and could therefore be IRF5+ macrophages 76.

Several lines of evidence suggest that IRF5+ macrophages may play a role in both the inflam-matory phase as well as resolution phase of pulmonary fibrosis. In response to epithelial cell damage, monocytes are recruited to the site of inflammation and may differentiate into IRF5+ macrophages under the influence of pro-inflammatory cytokines. Once activated, IRF5+ macrophages themselves produce TNF-α, IL-1β, and oxygen radicals to kill and phagocytose microbes to fight an infection or remove an exogenous agent 77. Many studies indicate that these pro-inflammatory cytokines and oxygen radicals are associated with fibrosis develop-ment 78-86. In the study by Nagai et al. ablation of FRβ-expressing macrophages during the inflammatory phase of bleomycin-induced fibrosis, abrogated fibrosis development 75. How-ever, the importance of the contribution of inflammation to established fibrosis has been challenged because anti-inflammatory drugs such as corticosteroids have no therapeutic effects in patients with pulmonary fibrosis 64. This view was confirmed by a study from Gib-bons et al. They studied newly recruited inflammatory macrophages in a mouse model of bleomycin-induced lung fibrosis and showed that depletion of tissue-resident macrophages


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and/or circulating inflammatory monocytes during the inflammatory phase did not affect the onset or degree of fibrosis that developed after this inflammatory phase 87.

During the resolution phase, macrophages are involved in the process of ECM degradation and the uptake of matrix components 71,87,88. Depletion of macrophages during this recovery phase impaired the resolution of fibrosis by slowing down the degradation of ECM 87. It is unclear what type of macrophage is responsible for degradation of ECM, but a case can be made for IRF5+ macrophages as these have been shown to produce several cathepsins and MMPs including MMP7 and MMP9. Levels of MMP9 have been found increased in lungs of IPF patients and this may reflect a failing attempt of the lungs to remove excess ECM caused by a simultaneous increase of the inhibitor TIMP-1 89-91. In addition two other studies pointed out that IRF5+ macs may even be beneficial for fibrosis since they showed that the inflam-matory cytokine TNF-α, mainly produced by the IRF5+ phenotype, promoted alveolar epi-thelial cell recovery and therefore also contributed to resolution 92. Furthermore, Redente et al. showed that TNF-α-knockout mice exhibited delayed resolution of bleomycin-induced pulmonary fibrosis, but they also showed that pulmonary delivery of TNF-α reduced the fibrotic burden in these mice 93. Furthermore, macrophages are also important in the sub-sequent removal of ECM components through endocytosis-mediated mechanisms. Again it is unclear if this is restricted to one particular phenotype but the receptors involved would suggest more of a CD206+ M2 phenotype and this will therefore be discussed in the next part.

In summary, IRF5+ (M1) macrophages are important in the inflammatory phase but their presence does not appear to affect the subsequent fibrotic phase. During resolution of scar tissue, macrophages are indispensable for degradation of ECM. This may be related to an IRF5+ phenotype and it may therefore be beneficial to stimulate recruitment of these mac-rophages to reverse fibrosis.

CD206+ M2 macrophages in pulmonary fibrosisThere is a great deal of evidence that Th2 responses are important in the development of fibrosis and it appears that IL-13 is the predominant cytokine of profibrotic responses 94-101. Levels of IL-13 are higher in patients with pulmonary fibrosis as compared to controls and macrophages isolated from these fibrotic lungs produce more IL-13 than macrophages from control lungs 102. It therefore comes as no surprise that CD206+ macrophages are associated with pulmonary fibrosis, although we could not find publications directly showing numbers of CD206+ macrophages are increased in lung tissue of patients with pulmonary fibrosis. We did find one study showing higher numbers of CD206+ macrophages and higher levels of other CD206 related markers in BALF of IPF patients as compared to controls and two studies showing higher numbers of insulin-like growth factor-I (IGF-I)-positive and PDGF+ interstitial macrophages in lung tissue of IPF patients as compared to controls 103-105. Both these markers are important profibrotic mediators and a recent study by Chen et al. showed that expression of IGF-I colocalized with arginase-1 and not with IL-10 expression in macro-phages suggesting genuine CD206+ macrophages express IGF-I and not the IL-10+ M2-like subset 106. This was a study in mice. It therefore remains to be investigated whether this is also true in humans.


Other markers found on or produced by CD206+ macrophages have also been found increased in pulmonary fibrosis. Levels of galectin-3, a carbohydrate-binding lectin that is necessary for alternative activation, were higher in BALF of IPF patients as compared to con-trol patients 107,108. Furthermore, macrophages from IPF patients produced more of the human marker CCL18, related to CD206+ M2 macrophages, than control macrophages and this cor-related negatively with pulmonary function test parameters 109. IPF patients were also found to have higher serum and pulmonary levels of chitinase-like protein YKL-40 as compared to controls, although it is still unclear whether chitinases are true markers of alternative activa-tion in human macrophages 110. This is also the case for arginase-1, which is a marker of the CD206+ M2 macrophages in mice, but its specificity is debated in humans 38. Nevertheless, lung tissue from IPF patients had higher expression of arginase-1 in macrophages than nor-mal lung tissue 111. Lastly, circulating monocytes from systemic sclerosis patients with pulmo-nary fibrosis showed enhanced profibrotic phenotype by increased expression of CD163, a marker of alternative activation in humans 112.

Experimental models of pulmonary fibrosis have revealed more about the role of CD206+ macrophages in fibrosis in the lung. Depletion of macrophages during the fibrotic phase of lung fibrosis reduced the deposition of ECM in this organ 87. To confirm a role for CD206+ macrophages, levels of Ym1 and arginase-1 were measured before and after macrophage depletion. Both markers showed decreased expression in the lungs after removal of macro-phages. The inflammatory macrophage marker iNOS did not show a reduction in expression, indicating that the YM1+/Arg1+ macrophages are predominantly responsible for the develop-ment of fibrosis. Venosa et al. depleted macrophages that originate from the spleen by per-forming a splenectomy before an intratracheal nitrogen mustard exposure to induce fibrosis 113. The spleen functions as a reservoir of inflammatory monocytes that migrate into injured tissue and they found a decrease in the mature subset of M2 macrophages, accompanied by a larger M1 population and more tissue damage after splenectomy. Especially levels of IL-10, a key cytokine to dampen inflammatory reactions, were much lower in the splenectomy groups as compared with the sham group. Unfortunately, no further distinction was made between M2 and M2-like macrophages, but the reduction in IL-10 production suggests a reduction in the number of M2-like macrophages. Furthermore, the M2 marker MMP12 was shown to be essential in the development of fibrosis induced by excessive activation of Fas and in a model of IL-13 dependent fibrosis 114,115.

There is some evidence regarding the mechanisms behind CD206+ macrophages and their contribution to the development of fibrosis. The aforementioned production of IGF-I and PDGF contribute to proliferation of fibroblasts and stimulate their transformation towards ECM-producing myofibroblasts 67. Furthermore, FIZZ1 (also known as resistin-like molecule alpha (RELMα) expressed by alternative activated macrophages 116) was found to increase ECM production in fibroblasts 117. The same group found that FIZZ1 is induced in bleomy-cin-induced lung fibrosis and significantly less fibrosis was seen in FIZZ1 knock-out mice 117,118. Moreover, FIZZ1, expressed by skin macrophages, aids an effective wound healing pro-cess following injury when stimulated with IL-4. Here, FIZZ1 activation stimulated collagen fiber cross-linking that promotes tissue repair 119. However, a paper by Pesce et al. showed that FIZZ1 actually ameliorated fibrosis development by negatively regulating Th2-dependent


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responses 120. These contradictory findings highlight other new findings that also suggest that FIZZ1+ macrophages could be antifibrotic. A mechanistic study in a model of Schistosoma-in-duced liver fibrosis with specific deletion of IL-4Rα on myeloid cells, preventing alternative activation of macrophages, showed that IL-4-induced macrophages are not required for fibro-sis development 121. In addition, related studies with mice lacking arginase-1 in macrophages showed that the arginase-1-expressing macrophages were even required for suppression and resolution of fibrosis 120. This correlates well with findings that uptake of ECM components appears to be mediated by CD206+ macrophages in general. Uptake of these components is mediated by different mannose receptors and by glycoprotein milk fat globule epidermal growth factor 8 (Mfge8) 42,122. Mannose receptors are of course known alternative activation markers but for Mfge8 this is unclear, even though both the mannose receptor 2 and Mfge8 were shown to attenuate fibrosis in different models 122,123.

Besides the conventional cytokines that promote the development of the M2 macrophages, like IL-13 and IL-4, several studies have looked at additional regulatory mechanisms behind M2 polarization. The protein IL-1R-associated kinase-M (IRAK-M), upregulated by surfac-tant proprotein A (SP-A), was first identified as an inhibitor of TLR-mediated inflammation in human macrophages 124. Macrophages with upregulated IRAK-M expression produced less TNF-α and IL-6 in response to LPS exposure. Then recently, Ballinger et al. showed that besides suppression of M1 cytokines, IRAK-M seems positively influence the differentiation of macrophages towards an M2 phenotype 125. IRAK-M expression in macrophages was higher following bleomycin exposure and was accompanied by an increased IL-13 production, while macrophages from IRAK-M-/- mice showed lower IL-13 production. In humans, IRAK-M expression was higher in IPF patients than in controls and correlated positively with arginase expression, suggesting a link between IRAK-M and fibrosis. In addition, inhibitory factors of M2 activation have also been revealed. Disrupted activity of tyrosine phosphatase ShP2 pro-moted alternative activation 126. ShP2 is able to suppress the JAK1/STAT signaling pathway that is activated after IL-4 stimulation, thereby suggesting that the phosphatase activity of Sph2 is able to inhibit alternative activation.

To summarize, CD206+ M2 macrophages are firmly associated with fibrosis development but new evidence suggests they may actually contribute to resolution of fibrosis. Their presence during fibrosis may be explained as a failing attempt to clear the excess ECM. The conflicting roles described in literature may be the result of difficulties separating the effects of CD206+ M2 and IL-10+ M2-like macrophages simply because these two subsets are difficult to dis-tinguish. IL-10+ M2-like macrophages may be a more likely candidate for the promotion of fibrosis as will be discussed below.

IL-10+ M2-like macrophages in pulmonary fibrosisThe signature marker of these macrophages is IL-10, which is the canonical anti-inflammatory cytokine with profibrotic actions. Elevated levels of IL-10 and enhanced production of IL-10 by alveolar macrophages have been reported in several fibrotic diseases, including IPF and in systemic sclerosis patients with interstitial lung disease 112,127-130. Its anti-inflammatory actions in lung are illustrated by a study from Armstrong et al., showing that IL-10 inhibited TNF-α production by alveolar macrophages after LPS stimulation 131. Interestingly, induction of the


IL-10 production by a low dose of LPS protected lung tissue against a subsequent lethal dose of LPS 132. In addition, several studies in mice using the model of bleomycin-induced fibrosis suggest that IL-10 attenuates bleomycin-induced inflammation and can thereby attenuate fibrosis development 80,133,134. However, overexpression of IL-10 in lungs of mice was found to be profibrotic 135. Sun et al. found that inducible IL-10 overexpression in Clara cells induced fibrosis by fibrocyte recruitment and activation of IL-13+IL10+ macrophages expressing Arg1. The increased levels of IL-10 found in lungs of IPF patients may therefore contribute to the fibrotic process.

MMP28 has also been shown to regulate M2 polarization and dampen the M1 polarization. MMP28-/- mice showed high levels of M1 markers at baseline and less M2 markers following bleomycin induced experimental fibrosis 136. Moreover, MMP28 influenced the IL-10 produc-tion; MMP28-/- mice showed lower basal levels of IL-10 compared to wild type 136. The mac-rophage receptor with collagenous structure (MARCO) also seems to play a role in the alter-native polarization of macrophages in fibrosis 130. Following instillation of chrysotile asbestos, MARCO-/- mice expressed significantly lower levels of M2 markers: Ym1 and active TGFβ and had less fibrosis. In contrast, inflammatory mediators related to the M1 phenotype, TNF-α and IL-1β, were also increased in these knockout mice. Furthermore, patients with asbestosis, characterized by chronic inflammation and scarring of lung tissue, showed a predominant M2 phenotype with high levels of MARCO 130. The high levels of IL-10 and TGFβ that are asso-ciated with MARCO suggest that these macrophages are likely to be IL-10 M2-like macro-phages. The high levels of the profibrotic, anti-inflammatory cytokine TGFβ that often coexist with high IL-10 levels would also fit with the role of dampening inflammation and promoting tissue repair by this macrophages subset 127,130,137. Whether TGFβ production is restricted to the IL-10-producing macrophage subtype remains to be investigated.

Figure 2. Schematic representation of the presence of IRF5+, CD206+, and IL-10+ macrophages in lung tissue during homeostatic conditions and after injury to the lung. Normally after lung injury a process of tissue repair is initiated with four distinct phases leading to homeostatic con-ditions again. In lung fibrosis this normal tissue repair response is suppressed or dysregulated leading to deposition of excess extracellular matrix and little resolution of scar tissue.


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In summary, IL-10+ macrophages are likely candidates for the promotion of fibrosis. They may be recruited or induced by damage to the epithelium to dampen inflammation and start up tissue repair processes. In the event of on-going damage they are continually induced or recruited and may contribute to fibrosis by the overexpression of IL-10. Since corticosteroids are also capable of inducing IL-10+ macrophages, this would explain why these drugs are not effective against fibrosis and may even be disadvantageous 138. This was also illustrated by our finding that when corticosteroids are specifically delivered to liver macrophages in a model of liver fibrosis, fibrosis actually becomes worse 139.

Overall, current data on the role of macrophages in the development of pulmonary fibro-sis show that macrophages are important cells in the pathogenesis of this disease (see also figure 2). IRF5+ M1 macrophages are important in the inflammatory phase and may also be important for resolution of the disease, although this hypothesis needs testing. CD206+ and CD206+IL-10+ macrophages are highly associated with fibrogenesis. However, new data sug-gest that the CD206+ macrophages may actually protect against development of fibrosis while the CD206+IL-10+ macrophages contribute to fibrosis. Therefore, key to understanding how these two phenotypes contribute to pulmonary fibrosis are studies differentiating between these two subpopulations. Eventually, macrophages may prove important in the resolution of fibrosis due to their expression of various proteolytic enzymes.

COPDCOPD is one of the most common respiratory diseases and affects around 320.000 people in the Netherlands (Annual Report 2011 Dutch Lung Fund). It is projected to be the fourth leading cause of death worldwide by 2030 and places a huge economic burden on society 140. COPD is caused by lung inflammation due to inhalation of noxious gasses and particles: in the Western World most commonly from cigarette smoking and in developing countries from indoor biomass cooking and heating 141. COPD is characterized by airflow limitation that is not fully reversible, which is caused by a combination of obstructive bronchiolitis (also known as chronic bronchitis) and destruction of alveoli resulting in airspace enlargement (also known as emphysema) 142. The relative contributions of chronic bronchitis and emphysema to the COPD phenotype can vary from person to person.

Pathogenesis of COPDExposure to smoke and particles leads to an exaggerated chronic inflammation in lungs of people susceptible to the development of COPD. Excess mucus production and progressive narrowing of the respiratory bronchioles characterize chronic bronchitis. The mucosa, sub-mucosa, and glandular tissue become infiltrated with inflammatory cells and the walls of the respiratory bronchioles become thickened because of edema and fibrosis 143. Chronic mucus hypersecretion is induced by goblet cell hyperplasia and hypertrophy of submucosal glands 144, which further contributes to occlusion of small airways. This progressive narrowing leads to obliteration or even complete disappearance of respiratory bronchioles. Not much is known about the role of macrophages in this part of the disease, but pigmented macrophages were found to cluster around small airways and these were associated with peribronchiolar fibrosis 145.


Within the peripheral compartment, alveolar destruction that characterizes emphysematous COPD is the result the infiltration of inflammatory cells with a prominent role for macro-phages. Both neutrophils and macrophages are being recruited to the lung because smoke/particle exposure injures epithelial cells that subsequently release cytokines and chemokines to recruit inflammatory cells 146,147. Neutrophils and macrophages have been postulated to be the main effector cells contributing to the excess tissue damage seen in emphysema because of their ability to produce proteolytic MMPs like neutrophil elastase and macrophage elas-tase (MMP12) 147,148. Increased numbers of macrophages and neutrophils have been found in airways and lung parenchyma of patients with COPD 149-151. However, only the number of parenchymal alveolar macrophages was directly proportional to the severity of lung destruc-tion in emphysematous lung tissue from COPD patients 152. Animal studies confirmed the dominant role for macrophages, because deletion of neutrophils in smoke-exposed rats did not prevent cigarette smoke smoke-induced emphysema, whereas deletion of macrophages did 153. In addition, mice deficient in MMP12 (mainly produced by macrophages), were com-pletely protected from cigarette-smoke-induced emphysema even though they could still produce neutrophil elastase 154. Similarly, inhibiting MMP12 reduced smoke-induced airway inflammation in mice 155.

Macrophages and COPDThe role of the different macrophage phenotypes in COPD is the topic of quite a few studies recently and the subject of much debate as the results have been somewhat counterintui-tive. Based on studies in mice and results from patient studies, IRF5-directed polarization is expected to play an important role in the pathogenesis of COPD. However, the results of other studies have questioned this view and this is nicely illustrated by studies from Shaykhiev et al. and Hodge et al. 156,157. The first ones recently studied the transcriptome of alveolar macro-phages from healthy smokers and nonsmokers and compared them to alveolar macrophages from COPD smokers 156. Their results showed a mixed phenotype for alveolar macrophages after smoking with downregulation of genes associated with IRF5+ M1 macrophages and partial upregulation of CD206+ M2 genes, which was progressively worse in COPD. Hodge et al. showed a mixed phenotype in alveolar macrophages of smoking COPD patients with some M1 (MHC II expression) and M2 (efferocytosis) markers going down and some going up (pro-inflammatory cytokine production and DC-SIGN expression) 157. In the next part we will touch upon this debate as we discuss the separate phenotypes in the pathogenesis of COPD.

IRF5+ M1 macrophages in COPDSeveral lines of evidence support a role for IRF5+ macrophages, but also a role for dysregu-lated IRF5+ macrophages in the development of COPD, suggesting an undesired function or an actual genetic defect. First of all, exposure to compounds in smoke appears to induce inflammatory/M1 polarization of macrophages. Smoking is the most important risk factor for COPD and cigarette smoke contains many thousands of compounds, including LPS that can activate macrophages in the lung 158. Indeed, increased expression of iNOS in alveolar macrophages was found in COPD patients 159-161. Upregulation of iNOS increases ROS and NO production and can then cause oxidative stress, an important contributor to the pathogenesis


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of COPD 162. Smoking itself can also cause oxidative stress and thereby add to the stress caused by the increase in iNOS activity in M1 macrophages 163-165.

Furthermore, many studies have shown that smoke exposure enhances the release of the M1 pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α 166-171. Inflammatory/M1-derived cytokines also play a role in the pathogenesis of COPD. IL-1β, IL-6, IL-8, and TNF-α have all been found elevated in COPD and in experimental settings have been found to contribute to the development of persistent airway inflammation, emphysema and mucus production 166,172-187. TNF-α was found to drive most of the emphysema development in the alveolar com-partment in mice after smoking because mice lacking receptors for TNF-α only developed mild emphysema 188. This increase in TNF-α production was shown to correlate with the influx of inflammatory monocytes in mice in a mouse model for acute lung injury, together with high MMP12 levels in the lung 189. MMP12 has been indicated as an M2 marker, but the dif-ferent phenotypes were not distinguished in this study 190. Though, both TNF-α and MMP12 are known to contribute to emphysema. Mice overexpressing TNF-α in lung tissue develop chronic inflammation and emphysema 183,191,192. However, in humans antibodies against TNF-α seem to be ineffective in COPD, questioning the relevance of this cytokine for human COPD 193. In addition to TNF-α, IL-1β, also an inflammatory/IRF5+ macrophage cytokine, was found to play a role 166,187. Overexpression of IL-1β in lung caused lung inflammation, emphysema, mucus metaplasia, and airway fibrosis in mice 184. Taken together these data suggest cytokines produced by IRF5+ M1 macrophages at least play a role in the pathogenesis of COPD.

Another important inflammatory/M1-related cytokine with a role in COPD is IFNγ. It is pro-duced by CD8+ T cells that infiltrate the lungs in COPD and can stimulate IRF5-directed polarization. Inducible overexpression of IFNγ in lungs of mice caused emphysema with alterations in the balance of MMPs and antiproteases 152,194-196. However, in human alveolar macrophages from smokers a reduction in IFNγ receptor expression and reduced IFNγ signal-ing were found, suggesting IRF5-directed polarization may be impaired after smoking. This of course is in line with the above-cited finding by Shaykhiev et al. that inflammatory genes are downregulated in alveolar macrophages of healthy smokers and smoking COPD patients as compared to nonsmokers 156.

IRF5+ M1 macrophages have also been found to produce MMP9, presumably to enable mac-rophage migration during inflammatory responses 26,33. In addition, MMP9 is associated with the breakdown of extracellular matrix in the parenchyma in COPD as macrophages from patients with COPD have a significantly higher production of MMP9 as compared to control macrophages 197. This was confirmed by Foronjy et al., showing that overexpression of human MMP9 in mouse macrophages induced emphysema and loss of alveolar elastin pointing at a role for these macrophages in COPD development 198.

Finally, an important property of IRF5+ macrophages that appears to be dysregulated is the phagocytosis of microorganisms. IRF5+ macrophages are geared towards killing and disposal of microbial threats and phagocytosis of micro-organisms is part of that function 30,31. COPD is often exacerbated by infections and there is accumulating evidence that reduced macro-phage phagocytosis in COPD may be responsible for the persistence of micro-organisms in


the lungs 199-202. The cause of the impaired phagocytosis may be a result of reduced expression of essential proteins or a general genetic defect. Presence of micro-organisms even correlated positively with disease severity but seems to be pathogen specific 203. This dysfunction of phagocytosis is not restricted to micro-organisms but also appears to be present for CD206+ macrophage-related phagocytic functions such as efferocytosis and mannose receptor-medi-ated uptake 204,205. This overall inhibition of phagocytosis irrespective of macrophage pheno-type was further confirmed by the later study of Hodge et al. that has already been mentioned before 157.

Taken together, the available data suggest that a dysregulated IRF5+ macrophage, either in function or a genetic defect, response plays a role in COPD rather than an increased num-ber of these macrophages. Some aspects of the IRF5+ macrophage activation signature are upregulated in COPD (ROS generation, pro-inflammatory cytokines, production of MMP9), but some aspects and functions are downregulated (phagocytosis, IFNγ responsiveness).

CD206+ M2 macrophages in COPDOverexpression of prototypical IRF5+ macrophage-inducer IFNγ may be able to induce emphysema, but so does overexpression of prototypical M2-macrophage inducer IL-13. Zheng et al. showed that overexpression of IL-13 in mouse lung tissue caused a pathology mirroring human COPD with macrophage- and lymphocyte-rich inflammation, emphysema, and mucus metaplasia 206. Unfortunately, macrophages were not further characterized in this study, so it is not known how IL-13 overexpression affected macrophage polarization. Fur-ther evidence for a role for CD206+ IL13-induced macrophages in emphysema came from a study by Kim et al. who showed that viral infections could induce an IL-13-producing pheno-type through interactions with natural killer T cells leading to chronic airway inflammation 207. They also showed higher numbers of IL-13-positive macrophages in lung tissue of COPD patients.

In mice, CD206+ macrophages produce large amounts of chitinases like Ym1 and Ym2 116. Whether their human counterparts are also induced by alternative activation is unclear, but another member of this family, stabilin-1 interacting chitinase-like protein (SI-CLP), has been found upregulated following IL-4 and dexamethasone stimulation 208. Many members of the chitinase family associate with COPD. Chitotriosidase levels, for instance, were increased in bronchoalveolar lavage of smokers with COPD and they also had more chitotriosidase-posi-tive cells in bronchial biopsies and an elevated proportion of alveolar macrophages express-ing chitotriosidase as compared to smokers without COPD or never-smokers 209. Furthermore, macrophage chitinase-1 was selectively increased in a subset of patients with severe COPD and serum concentrations of YKL-40 were significantly higher in smokers with COPD as com-pared to nonsmokers or smokers without COPD and correlated negatively with lung func-tion 210-213. Interestingly, YKL-40 also stimulated the production of pro-inflammatory cytokines and MMP9 by macrophages from COPD patients similar to the effects of TNF-α, suggesting YKL-40 itself actually induces a slightly different macrophage phenotype as compared to the CD206+ M2 phenotype 213.


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Data from studies investigating MMPs indicate a possible role for CD206+ macrophages. As mentioned above MMP12 plays an important role in mouse emphysema and MMP12 was found specifically induced in IL-4-stimulated macrophages 154,155,214. Furthermore, Woodruff et al. showed increased CD206+ polarization of alveolar macrophages in smokers using MMP12 as a marker for alternative activation and many others showed that smoke induces MMP12 in macrophages 190,215-221. Interestingly, MMP12 production by macrophages was also found to be necessary to terminate both neutrophil and macrophage influx at the end of an inflammatory response and may therefore be an instrument of CD206+ macrophages to dampen inflam-mation to be able to start remodeling of damaged tissue 222. How that ties in with the potential pro-emphysematous role of M2 macrophages remains an open question.

Summarizing, there is some evidence for a role of CD206+ activation in COPD and this evi-dence points at a role contributing to the development of COPD. The data by Hodge et al. suggest that, similar to dysfunctional IRF5+ M1 activation, CD206+ M2 activation is also dys-regulated with reduced efferocytosis but increased expression of M2 marker DC-SIGN 157.

Figure 3. Schematic representation of the presence of M1, M2, and M2-like macrophages in lung tissue during homeostatic conditions, during healthy smoking, and in COPD. Please note the dysfunctional state of macrophages during COPD.


IL-10+ M2-like macrophages in COPDNo attempts have been made to distinguish the roles of CD206+ and CD206+IL-10+ macro-phages in COPD. Two studies reported on IL-10 in the context of COPD. A study by Hackett et al. showed diminished IL-10 production in lung tissue of COPD patients after LPS stimulation as compared to response in lung tissue of patients with normal lung function 223. Takanashi et al. demonstrated that the level of IL-10 and the number of IL-10-positive macrophages in sputum from COPD patients and healthy smokers was decreased as compared to healthy non-smokers, although Li et al. found no difference in IL-10 levels in sputum from COPD patients as compared to controls 224,225. This would suggest that IL-10+ macrophages are impaired in smok-ing and COPD and therefore cannot suppress the ongoing inflammation induced by smoke.

Combining the data available for IRF5+, CD206+ and IL10+ macrophages (see also figure 3), it appears that COPD is a disease of dysfunctional macrophages rather than a disease of one particular polarization state. Macrophages in COPD are promoting ongoing inflammation and tissue damage but are unable to effectively dampen inflammation because they have lost the ability to phagocytose microorganisms and apoptotic bodies and produce anti-in-flammatory cytokines like IL-10. Currently, the most common treatment of COPD patients is by glucocorticosteroids, and this most common (chronic) treatment causes contradicting shifts in macrophage function 226. Glucocorticosteroids do inhibit inflammation and may improve lung function, but also increase pneumonia susceptibility 227. Thus, more insight into the role of macrophages in COPD could give direction to the search for more optimal treatment options.

Scope of the thesisAs discussed before, pulmonary fibrosis and COPD are diseases accompanied by changes in tissue structure, macrophage number and macrophage function in the lung. Macrophages are important in maintaining tissue ECM homeostasis in the lung and their dysfunction is related to the (over)production of ECM, contributing to pulmonary fibrosis and remodeling of airways in COPD, and with tissue destruction, contributing to emphysema. There is still debate regarding which macrophage phenotypes are involved in each of these processes. Our aim was therefore to identify the different macrophage subsets present in the diseased state and try to influence macrophage function in order to restore tissue homeostasis. Using an experimental model for pulmonary fibrosis, we aimed to stimulate the differentiation of pro-teolytic macrophages in order increase ECM degradation processes in the lung as described in chapter 2. In contrast, in chapter 3 we aimed to dampen the proteolytic activity of mac-rophages with the aid of a specific inhibitor of tartrate resistant acid phosphatase to improve our understanding of the role of macrophages in COPD. Furthermore, in chapter 4, we char-acterized blood monocytes in patients with COPD and compared them with healthy indi-viduals to assess whether we can detect disease-related changes in these macrophage-pre-cursors cells. In addition to the monocyte phenotypes, we also studied the presence of the three macrophage phenotypes described above in the central airways of COPD patients in chapter 5. Chapter 6 gives a general discussion on the findings in this thesis and we discuss future perspectives regarding the role of macrophages in lung diseases and their potential as targets for drug development.


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225. Takanashi, S. et al. Interleukin-10 level in sputum is reduced in bronchial asthma, COPD and in smokers. Eur Respir J 14, 309–314 (1999).

226. van de Garde, M. D. B. et al. Chronic exposure to glucocorticoids shapes gene expres-sion and modulates innate and adaptive activation pathways in macrophages with dis-tinct changes in leukocyte attraction. J Immunol 192, 1196–1208 (2014).

227. Yang, I. A., Clarke, M. S., Sim, E. H. A. & Fong, K. M. Inhaled corticosteroids for stable chronic obstructive pulmonary disease. Cochrane Database Syst Rev 7, CD002991 (2012).

Chapter 2

The RANK/RANKL/OPG axis has a role in regulating tissue repair processes in lung

Carian E Boorsma1,7, Kurnia S.S. Putri2, Christina Draijer1,7, Peter Heukels3, Bernt van den Blink3, Robbert H Cool4, Corry-Anke Brandsma5,7, George Nossent6, David M. Brass8, Peter Olinga2, Wim Timens5,7, Barbro N. Melgert1,7

1University of Groningen, Department of Pharmacokinetics, Toxicology and Targeting, Gron-ingen Research Institute for Pharmacy, Groningen, The Netherlands. 2University of Gron-ingen, Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute for Pharmacy, Groningen, The Netherlands. 3Erasmus MC, Department of Pul-monary Medicine, Rotterdam, The Netherlands. 4University of Groningen, Department of Pharmaceutical Gene Modulation and Department of Pharmaceutical Biology, Groningen Research Institute for Pharmacy, Groningen, The Netherlands. 5University of Groningen, Uni-versity Medical Center Groningen, Department of Pathology, Groningen, The Netherlands. 6University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases, Groningen, The Netherlands. 7University of Groningen, University Medical Center Groningen, GRIAC Research Institute, Groningen, The Netherlands. 8Department of Medi-cine, Pulmonary Division, Duke University Medical Center, Durham, NC, USA.


ABSTRACTOsteoprotegerin (OPG) is associated with fibrotic processes, but its role in pulmonary fibrosis (PF) is unknown. OPG is a decoy receptor for Receptor-Activator-of-NF-kB Ligand (RANKL). RANKL can bind to RANK on macrophages, inducing degradation of extracellular matrix (ECM) in bone and OPG prevents this. Pulmonary macrophages also express RANK. We hypothesized that RANKL similarly induces ECM degradation in lung, while high levels of OPG dampen ECM degradation by macrophages and contribute to fibrosis development.

OPG levels were higher in fibrotic human and mouse lung tissue and correlated with disease severity in mice. Expression was found in fibroblasts and isolated fibroblasts of PF patients had higher OPG production as compared to control fibroblasts. TGFβ stimulated OPG production in fibroblasts and in precision-cut-lung slices. RANKL-treatment of mice with fibrosis did not result in resolution but greatly induced OPG-production and epithelial cell numbers in lung.

In conclusion, the induction of OPG and the increase in epithelial cell numbers in lung following RANKL-treatment of fibrosis suggest that the RANK/RANKL/OPG-axis is not only active in bone, but also has a role in regulating tissue repair processes in lung. As OPG produc-tion is closely linked to fibrosis it is interesting new avenue to explore for drug and biomarker development.

The rank/rankl/opg axis has a role in regulating tissue repair processes in lung

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Introduction

Pulmonary fibrosis is characterized by relentless scarring of the lung with extracellular matrix (ECM) deposition and progressive destruction of lung architecture that leads to impaired lung function with high mortality rates1-9. Fibroblasts and myofibroblasts are abundantly present in fibrotic lung tissue and are considered the main producers of excess ECM. Mac-rophages are also abundantly present in fibrotic lung tissue and can contribute to fibrosis by producing profibrotic cytokines1,10-12. However, macrophages have also been shown to be antifibrotic by virtue of their ability to degrade ECM with matrix metalloproteinases (MMPs), cathepsins and their ability to internalize degraded collagens4,7,13-17. Gibbons et al. showed that resolution of experimental lung fibrosis in mice was attenuated when lung macrophages were depleted during the resolution phase of the disease10,18,19. However, it has not yet been elucidated whether this is a distinct population of macrophages and how their behavior is regulated.

OPG is a decoy receptor for receptor activator of nuclear factor kb ligand (RANKL), which is best known for its role in the regulation of bone ECM20-28. RANKL can stimulate macrophages in bone via its receptor RANK towards osteoclast differentiation and these can degrade bone ECM. As the decoy receptor for RANKL, OPG blocks the interaction of RANKL with RANK thereby inhibiting osteoclast differentiation and bone ECM degradation. Increased osteopro-tegerin (OPG) expression has recently been associated with fibrotic processes in liver2,5,21,23,29, heart30-32 and in the vasculature19,33,34. Increased OPG expression has also been observed in a mouse model of chronic lipopolysaccharide-induced airway remodelling1 and in silica-3 and bleomycin-induced lung fibrosis1. OPG levels increase early after bleomycin treatment and wane in time accompanied by a decrease in collagen deposition, suggesting that OPG may be important in active fibrotic processes. However, how OPG plays a role in fibrosis and whether it is involved in human pulmonary fibrosis remains to be determined.

As pulmonary macrophages were shown to express RANK as well (www.proteinatlas.com), we hypothesized that RANK activation could initiate a matrix resolving phenotype in the lung in the presence of RANKL, which may be hampered by high levels of OPG in fibrotic condi-tions. We therefore characterized the expression of RANK, RANKL and OPG in lung tissue samples of patients with pulmonary fibrosis and of mice with experimental silica-induced lung fibrosis. To address the role of OPG in lung fibrosis, mice with silica-induced fibrosis were treated therapeutically with soluble RANKL (sRANKL) to neutralize OPG and we exam-ined the progression of fibrosis in lung tissue.

Materials and methods

More detailed information for each part is available in the online supplementary “Material and Methods” section.


Human tissueThe study protocol was consistent with the Research Code of the University Medical Center Groningen, Dutch national ethical and professional guidelines, and the Medical Ethical Com-mittee of Rotterdam. Patient characteristics are displayed in table 1.

Table 1. Characteristics of patients whose lung tissue was used for determining RANK, RANKL and OPG levels (medians with range are presented).

Controls Pulmonary Fibrosis

Cancer (n=15) COPD (n=9) (n=11)

Sex, male/female 1/5 9/0 10/1

Age, years 63 (57-81) 58 (50-62) 56 (37-64)

Smoking status 4 non-smokers/2 ex-smokers

1 non-smokers/8 ex-smokers

4 non-smokers/6 ex-smokers/1 unknown

FEV1 % predicted 103 (85-125) 32 (10-72) 49 (45-89)

Diagnosis 3 lung cancer/3 lung metastases

1 GOLD stage 3/8 GOLD stage 4

11 IPF/UIP

Definition of abbreviations: FEV1 = forced exhaled volume; COPD = chronic obstructive pulmonary disease; IPF = idiopathic pulmonary fibrosis; UIP = usual interstitial pneumonia.

Isolation and culture of primary fibroblasts Primary lung fibroblasts were isolated from lung explants using a technique described pre-viously35,36. Patient characteristics are summarized in table 2. Cells used in the experiments were maximally passaged three times. Plated cells were grown to confluence and transferred to low-serum medium (0.5% FCS). After 72h, supernatants were collected for analysis.

Animal experimentsMale C57BL/6 mice were obtained from Harlan (Zeist, The Netherlands). All animal experi-ments were approved by the Institutional Animal Care and Use Committee, (DEC6064) and (DEC6416AA). Animal experiments were performed in the animal facility of the University of Groningen according to strict governmental and international guidelines on animal exper-imentation.

Silica-induced lung fibrosis in miceFibrosis was induced using a single intratracheal dose of Min-U-Sil 5 crystalline silica (0.2 g/kg in 50 ml 0.9% saline, a kind gift from Dr. Andy Ghio, US EPA, Chapel Hill, NC) following isoflurane anesthesia. Control animals received an equivalent volume of 0.9% saline. Mice were sacrificed after 28 days and serum and lung tissue were collected (n=6-8).


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Table 2. Characteristics of patients whose lung tissue was used for the isolation of primary lung fibroblasts (medians with range are presented).




Age, years 65 (45-74) 55 (53-57) 64 (58-66)

Smoking status 1 non-smoker/2 ex-smoker

2 ex-smoker 2 non-smoker/ 4 ex-smoker

FEV1 % predicted 98 (90-130) 22.5 (20-25) 62.5 (52-75)

Diagnosis Lung cancer COPD 2 IPF/UIP, 1 ILD/EAA, 1 NSIP/dust-exposed, 1 FNSIP/UIP

Definition of abbreviations: FEV1 = forced exhaled volume; COPD = chronic obstructive pulmonary disease; IPF = idiopathic pulmonary fibrosis; UIP = usual interstitial pneumonia; ILD = interstitial lung disease; EAA = extrinsic allergic alveolitis; NSIP = non-specific interstitial pneumonia; FNSIP = fibrosing NSIP.

In mice treated with sRANKL, silica was administered on day zero and sRANKL treatment began after 28 days and was continued for an additional 2 weeks. sRANKL was adminis-tered intranasally in 40 ml saline or 40 ml saline (vehicle) three times per week (n=12). On day 43, mice were sacrificed and lungs were collected for flow cytometry, western blot analysis, ELISA, or histology. See figure 1 for an overview of the experimental setup.

Figure 1. Experimental setup of the animal experiments: pulmonary fibrosis was induced intra-tracheal (i.t.) instillation of silica on day 0. After 28 days, mice were either sacrificed to assess pulmonary fibrosis development or treated intranasally (i.n.) with sRANKL or vehicle for two additional weeks.


Mouse lung precision-cut lung slicesLungs of six male C57BL/6 mice (20-30gr, age 6-8 weeks) were used to make precision-cut lung slices. This method was previously described by us37. Lung slices were incubated in trip-licate for 48h with TGFβ (5 ng/ml) or 48h with TGFβ followed by 24 h of either vehicle or RANKL (200ng/ml) stimulation. Three slices from each condition were pooled for further analyses and tissue slice supernatants were collected separately.

Mouse cell cultures NIH 3T3 fibroblasts and RAW264.7 macrophages were purchased at the American Type Cul-ture Collection. Self-propagating mouse alveolar-like macrophages (MPI cells) were a kind gift from Dr. G. Fejer, Plymouth University, Devon, UK.

Before TGFβ stimulation of 3T3 fibroblasts, cells were incubated in 0.5% FCS medium. After 24h, cells were stimulated with TGFβ (5 ng/ml, Peprotech, Rocky Hill, NJ) for 48h after which the supernatant was collected.

To induce a proteolytic phenotype, RAW264.7 macrophages (until max. passage 12) or MPI macrophages were stimulated with 200 ng/ml soluble RANKL (sRANKL, produced at our own facility). For each experiment, the data presented are representative of at least three independent replicates.

Protein isolation from lung tissueProtein was isolated from frozen human or mouse lung tissue (30-40 mg) in buffer (0.25 M Tris/HCl buffer, 2.5% Igepal, 0.5% SDS, Protease Inhibitor Cocktail (Boehringer Mannheim, Germany), pH 7.5). Overall protein concentration was determined using an RC DCTM Protein Assay based on the Lowry method (cat#5000111, Bio-Rad, Hercules, CA).

Western blot analysisOne hundred micrograms of tissue lysate per sample was used for western blot analysis. The following primary antibodies were used: mouse RANK (1:200, Acris, Herford, Germany), human RANK (1:100, R&D Systems, Minneapolis, MN), RANKL (human and mouse, 1:100, Acris) and β-actin (1:20,000 Sigma-Aldrich). RANK and RANKL expression levels were nor-malized to β-actin expression.

ELISAHuman and mouse OPG levels in lung tissue and culture supernatants were assessed using ELISA (cat#DY805 (human), cat#DY459 (mouse), R&D Systems) according to the instructions provided by the manufacturer. One hundred micrograms of tissue lysate total protein in a total of 100 μl was analyzed per sample. Cell culture media was analyzed undiluted while lung slice culture medium was diluted 1:5 before analysis.


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ImmunohistochemistryImmunohistochemical analysis of collagen I, RANK, and OPG was performed on 3 mm par-affin sections of mouse lung tissue using anti-mouse collagen I (Southern Biotech, Birming-ham, AL), anti-human RANK (R&D Systems), and anti-mouse OPG (Antibodies-online, Atlanta, GA). The amount of collagen deposition in the lung was determined using ImageS-cope software (Aperio, Burlingame, USA).

Cathepsin K activityCathepsin K activity of RAW264.7 macrophages was measured using a Cathepsin K Activity Kit (Cat#KA0769, Abnova, Taipei, Taiwan) following instructions provided by the manufacturer. Cathepsin K activity was normalized against the activity observed in unstimulated cells.

Quantitative Real-time PCRTotal mRNA was isolated from cells or tissues using a Maxwell® LEV simply RNA Cells/Tissue kit (cat# AS1280, Promega, Madison, WI) according to the instructions provided by the man-ufacturer. The following primers were used: collagen IαI, fibronectin, MMP9, OPG, β-actin, YWHAZ and 18s (Sigma-Aldrich, Zwijndrecht, The Netherlands). mRNA expression was nor-malized to β-actin for cell culture samples, to YWHAZ for tissue samples, and to 18S for pre-cision-cut lung slices and expressed as fold-change of healthy controls or control conditions.

Production and purification of sRANKL proteinRecombinant sRANKL was produced by R. Cool following standard procedures at our facility.

Flow cytometryMouse-left lungs were used for the isolation of a single cell suspension for flow cytome-try. Total cell numbers were determined using a FACS Array cell counter (BD Biosciences) and used to calculate total numbers of specific cell types. Single lung cell suspensions were stained for T cells, epithelial cells, neutrophils, interstitial and alveolar macrophage, and RANKL expression by flow cytometry. See supplemental Table 1 and 2 for antibody details. Samples were analyzed using an LSRII flow cytometer (BD Biosciences). Data was analyzed using FlowJo software (Tree Start, Ashland, USA). See supplemental data figure 3 and 4 for the gating strategy.

Statistical analysisAll groups were considered non-normally distributed due to small samples sizes. Statistical differences between two groups were assessed using Mann-Whitney U tests. Multiple-group comparison was performed with Kruskal Wallis. When significant, a Mann-Whitney-U test with a Bonferroni adjustment for multiple comparisons was used as a post-test. Correlations were assessed by calculating the Spearman correlation coefficient (R). Significance was con-sidered when p<0.05. The data were analyzed using GraphPad Prism 6.


Results

OPG levels are higher in fibrotic lung tissueWe observed higher OPG levels in human lung tissue with end stage fibrotic lung disease than in control lung tissue (figure 2A). OPG levels in the lungs of silica-treated mice were also sig-nificantly higher when compared with control lungs (figure 2B). Fibrosis in our mouse model was confirmed by significantly higher collagen expression and deposition in lungs of mice exposed to silica (supplemental data figures 1A and 1B). Staining for OPG in mouse lung tissue showed that OPG expression localized in the smooth muscle layers around vessels and airways in control lung tissue (figures 2C-D). In mouse lungs exposed to silica, OPG expression was also observed within areas of active fibrosis development, which was difficult to pinpoint to a specific cell type (figures 2E-F, positive staining is indicated by the arrows). To assess whether OPG could be produced by fibroblasts in the fibrotic areas, human primary lung fibroblasts were tested for their ability to produce OPG. We found they produced copious amounts of OPG and lung fibroblasts from fibrotic lungs produced significantly more OPG than lung fibro-blasts isolated from lung tissue of patients with lung carcinoma or COPD (figure 2G). We also assessed whether bronchial or alveolar epithelial cells and macrophages could produce OPG and found that cultures of these cells did not produce any OPG (data not shown).

RANKL is expressed by epithelial cells and T cells in lung tissueAs OPG is the decoy receptor of RANKL, we assessed RANKL expression patterns in human and mouse lung tissue. We found clear expression of RANKL in both human and mouse lung tissue (figure 3). Overall RANKL protein levels were similar in fibrotic lung tissue compared with control lung tissue in both humans (figure 3A) and mice (figure 3B).

˘ Figure 2. ELISA analysis of OPG levels on lung tissue lysates showed that OPG protein levels were higher in fibrotic conditions in both human (A) and mouse lung tissue (B). OPG levels were measured in control human lung tissue obtained from 15 patients undergoing surgical resection for carcinoma or COPD and pulmonary fibrosis tissue obtained from 11 patients undergoing lung transplantation. Mouse lung tissue was collected from mice 28 days after receiving silica (experimental fibrosis, n=7) or saline (healthy control, n=8). (C-D). Immu-nohistochemical staining for OPG in paraffin-embedded mouse lung tissue revealed expres-sion in smooth muscle cells underneath endothelial cells and bronchial epithelial cells (E-F). Under fibrotic condition, OPG expression was also observed in fibrotic areas as indicated by the arrows, but it was unclear which cells were responsible for this staining. (G). Cell cultures of human primary fibroblasts isolated from lung tissue of patients with pulmonary fibrosis (n=6) or patients undergoing surgical resection for carcinoma or COPD (control patients, n=5) revealed that fibroblasts from fibrotic lung tissue produced significantly higher levels of OPG in culture supernatant than fibroblasts from control lung tissue. Groups were compared using a Mann-Whitney U test and p<0.05 was considered significant.


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To assess which cells in mouse lung tissue express RANKL, we analyzed RANKL expression in by flow cytometry. We found clear RANKL+ expression on CD3+ T cells and EpCAM+ epi-thelial cells (figure 3C and D) and no significant expression on macrophages. Induction of fibrosis by silica resulted in higher number of RANKL+ T cells (figure 3C) and no change in the number of RANKL+ epithelial cells (figure 3D).

Figure 3. (A) Western blot analysis of RANKL shows expression of this protein in human lung tissue. RANKL levels were similar in lung tissue lysates from control patients undergoing sur-gical resection for carcinoma or COPD (n=15) and pulmonary fibrosis patients (n=11). (B) Western blot analysis of RANKL shows expression of this protein in murine lung tissue. Mouse lung tissue of healthy controls and mice with silica-induced pulmonary fibrosis showed similar RANKL protein expression levels. (C-D) Flow cytometric analysis of cells from murine lung suspensions showed RANKL expression on T cells (C) and epithelial cells (D). More T cells positive for RANKL were found in fibrotic as compared to healthy conditions, while the number of RANKL+ epithelial cells was similar between fibrotic and control conditions. Groups were compared using a Mann-Whitney U test and p<0.05 was considered significant.


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RANK expression on lung macrophages in decreased in fibrotic conditionsRANK, the membrane-bound receptor of RANKL, was also expressed in both human and mouse lung tissue and levels were similar between fibrotic and control lung tissue in both humans and mice (figures 4A and 4B). When staining for RANK we found expression by bron-chial epithelial cells and interstitial as well as alveolar macrophages (figure 4C-F).

OPG levels correlated with collagen I levels and are induced by TGF-_To assess its relationship with fibrosis, we studied whether OPG levels in murine lung tissue correlated with markers of fibrosis. Indeed, OPG levels in the lung correlated with the colla-gen-I content of the lung (figure 5A) and serum OPG levels reflected the OPG levels in the lung (figure 5B). To confirm OPG is produced by lung tissue under the control of a profibrotic cyto-kine, we incubated murine precision-cut mouse lung slices with TGFβ, the hallmark cytokine of fibrosis. We found that precision-cut mouse lung slices produced more OPG following TGFβ stimulation than unstimulated slices (figure 5C). Moreover, 3T3 murine fibroblasts produced detectable levels of OPG at baseline and stimulation with TGFβ resulted in even higher OPG levels in culture supernatant (figure 5D).

RANKL induces ECM-degrading enzymes in macrophages with a hematopoietic originWe tested whether RANKL is able to induce a proteolytic phenotype in either macrophages with an embryonic origin (alveolar macrophages) or with a hematopoietic origin (infiltrating mono-cytes) 20. We found that 48 hours of RANKL stimulation induced significant expression of MMP9 mRNA (figure 6A) and activity of cathepsin K (figure 6B) in monocyte-derived RAW264.7 mac-rophages as compared to unstimulated cultures. No polynuclear cells were seen, which would be an indication of osteoclast formation. In contrast, we found that RANKL did not induce MMP9 mRNA expression in the embryonic alveolar-like MPI macrophages (figure 6C).

RANKL treatment of mice with silica-induced pulmonary fibrosis does not reverse fibrosis, but does result in more OPG and epithelial cellsBy treating mice with silica-induced pulmonary fibrosis with recombinant sRANKL, we aimed to overcome the high levels of OPG and induce the development of ECM-degrading macrophages. Induction of fibrosis by silica resulted in higher collagen I deposition and more neutrophils, more macrophages (both interstitial and alveolar) and, as expected, more pro-duction of OPG as compared to nonexposed healthy mice (figures 7A-K). Exposure to silica did not change the number of infiltrating T cells (figure 7B) or the number of epithelial cells (figure 7D), but did lead to a higher number of RANKL+ T cells in lung tissue (figure 7C) as compared to nonexposed healthy mice. Treatment with 5 or 10 mg sRANKL had no effect on collagen I deposition and the number of (RANKL+) T cells, neutrophils, and (alveolar and interstitial) macrophages as compared to silica-exposed nontreated animals. Collagen IαI, MMP9 and fibronectin mRNA expressions also increased after silica exposure and remained unchanged following sRANKL treatment (Supplemental figures 2A, 2B and 2C).


Figure 4. Western blot analysis of RANK shows expression of this protein in human and murine lung tissue (A-B). No differences were found in RANK expression between control and fibrotic lung tissue in humans (A) and mice (B). Lung tissue lysates were collected from 15 control patients undergoing surgical resection for carcinoma or COPD and 11 samples were collected from pulmonary fibrosis patients undergoing lung transplantation. Mice were either silica-ex-


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posed to induce pulmonary fibrosis (n=4) or vehicle-exposed (n=3) for 28 days. (C-D) Immu-nohistochemical staining for RANK in paraffin-embedded control mouse lung tissue revealed expression by alveolar and interstitial macrophages and bronchial epithelial cells. (E-F) Immu-nohistochemical staining of fibrotic murine lung tissue showed a similar expression pattern of macrophages and bronchial epithelial cells as for control lung tissue.

Figure 5. (A) OPG levels in lysates of lung tissue from mice exposed to silica correlated with collagen I levels in that same lung tissue. (B) OPG serum levels correlated with OPG levels in murine fibrotic lung tissue. A Spearman correlation coefficient was calculated for each cor-relation and p<0.05 was considered significant. (C) OPG production was higher in murine precision-cut lung slices following 48 hours of TGFβ stimulation compared tot vehicle-treated controls (n=7 independent experiments). (D) OPG levels in culture supernatant of murine 3T3 fibroblasts were higher after 24 hours of TGFβ stimulation than in supernatant from vehi-cle-stimulated fibroblasts (n=7 independent experiments). Groups were compared using a Mann-Whitney U test and p<0.05 was considered significant.


Figure 6. In RAW264.7 monocytic macrophages, MMP9 mRNA (A) and cathepsin K activ-ity (B) were higher following RANKL stimulation (24h and 72h respectively) as compared to unstimulated controls. (C) sRANKL stimulation of MPI alveolar-like macrophages did not result in higher MMP9 expression as compared to unstimulated controls. Groups were com-pared using a Mann-Whitney U test and p<0.05 was considered significant.


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sRANKL treatment did result in a strong induction of OPG expression (figure 7J) and this also resulted in in higher OPG levels in lung tissue as compared to silica-exposed nontreated animals (figure 7K). In addition, sRANKL treatment resulted in a dose-dependent signifi-cant increase in the number of epithelial cells in lung tissue as compared to silica-exposed nontreated animals and this was paralleled by a similar pattern for the number of RANKL+ epithelial cells (figures 7D and E).To investigate whether sRANKL could directly induce the expression of OPG in lung tissue, we incubated precision-cut murine lung slices with TGFβ for 72 hours and added sRANKL during the last 24 hours to mimic our in vivo experiment. sRANKL treatment for 24 hours did not lead to higher expression of OPG mRNA in lung slices as compared to nontreated slices (figure 7L).

Discussion

Our study shows that OPG protein levels are higher in lung tissue of both humans and mice with (silica-induced) pulmonary fibrosis and that in addition to OPG, its ligand RANKL and the membrane-bound receptor RANK are all present in lung tissue. In mice with silica-in-duced fibrosis, these lung tissue levels of OPG correlate positively with the amount of collagen in the lung. The findings that sRANKL treatment increases epithelial cell numbers and OPG expression in lung tissue suggests that the RANK/RANKL/OPG axis is not only active in bone, but also has a role in regulating tissue repair processes in the lung.

OPG has previously been found to associate with fibrosis-related diseases like vascular fibro-sis38, cystic fibrosis39 and liver fibrosis2,5. In addition, high OPG levels were found broncho-alveolar lavage fluid of mice with silica- and bleomycin-induced fibrosis1,3 and in a model of endotoxin-induced airway remodeling1. We now show that OPG expression is also high in human fibrotic lung tissue and that it is produced by fibroblasts isolated from this lung tissue. The higher OPG production resulted from production in lung tissue itself, as OPG mRNA expression in lung tissue was also higher in experimental fibrosis. Some have suggested that OPG levels are higher to counteract increased RANKL levels that may be elevated due to the actions of IL-6 and TNF-α13. However, we showed that TGFβ, the hallmark cytokine of fibrosis, is capable of increasing OPG production. Therefore our findings strongly support the concept that OPG is associated with the fibrotic process itself and is produced locally in the lung.

In bone, OPG prevents the development of osteoclasts by binding RANKL to reduce bone ECM degradation. We hypothesized a similar role for OPG in the lungs. However, when treat-ing with sRANKL to overcome the excess OPG in fibrosis, we found no differences in collagen I deposition in lung tissue, our outcome measure for pulmonary fibrosis. In contrast, sRANKL treatment even further induced OPG levels, potentially still preventing the induction of ECM degradation. Possibly, the sRANKL administered binds to free OPG, triggering a pulmonary or extrapulmonary feedback mechanism to compensate for the lower levels of free OPG. To explain this phenomenon we studied whether RANKL could directly induce OPG in preci-sion-cut lung slices and found it did not change OPG mRNA expression. It therefore seems likely that RANKL somehow indirectly induces the expression of OPG in lung tissue. Clearly,


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our findings have provided interesting leads to further investigate the regulation of OPG in the lung.

Interestingly, the treatment with sRANKL clearly was able to affect cells within lung tissue as we found a strong dose-dependent increase in the number of Epcam+ cells, which is indica-tive of an increase in the number of epithelial cells or an upregulation of the Epcam protein by these cells. Immunohistochemical staining for RANK showed expression of this receptor of RANKL on bronchial epithelial cells and these cells could therefore potentially be respon-sive to the effects of RANKL. To the best of our knowledge no reports on RANK expression on lung epithelial cells have been published nor on the effects of stimulating this receptor with RANKL in lung tissue. However, both mammary epithelial cells as well as a subset of thymic epithelial cells were found to expand after exposure to RANKL which may suggest RANKL is a more general epithelial growth factor 40,41. Further studies investigating proliferation effects of RANKL on bronchial and alveolar epithelial cells are needed to address this question. In combination with the clear pulmonary regulation of OPG during fibrosis, these data do sug-gest a RANK/RANKL/OPG axis is operating in lung tissue during tissue repair.

RANKL could theoretically still play a role in the resolution of fibrosis by stimulating the expression of ECM-degrading enzymes in macrophages, However, its usefulness seems lim-ited because of its ability to upregulate OPG production. In vitro stimulation of embryonic alveolar-like macrophages with sRANKL did not induce MMP9 expression, but it did induce a proteolytic phenotype in monocyte-derived RAW264.7 macrophages as shown by our data and those of others previously18,20,22,24-28. MMP9 and cathepsin K can both contribute to the ECM degradation process1,4,6-9 and these monocyte-derived macrophages may be therapeu-tically more relevant as Gibbons et al. previously showed that infiltrating monocyte-derived macrophages contributed largely to the development of fibrosis, whereas resident alveolar macrophages did not3,10-12. The development of ligands that induce this proteolytic state in monocte-derived macrophages without inducing OPG production may be a novel way of inducing ECM degradation and treating advanced fibrotic disease42.

¯ Figure 7. Mice were treated with sRANKL (3x per week for two weeks, 5 or 10 mg per mouse/administration) four weeks after inducing pulmonary fibrosis with silica. Collagen I depo-sition (A), numbers of CD3+ T cells (B), RANKL+CD3+ T cells (C), Epcam+ epithelial cells (D), RANKL+Epcam+ epithelial cells (E), neutrophils (F), total macrophages (G), alveolar macrophages (H), interstitial macrophages (I), and levels of OPD mRNA expression (J) and protein expression (K) were assessed. Silica-exposure led to higher collagen I deposition in lung tissue, more RANKL+CD3+ T cells, neutrophils, total, interstitial and alveolar macrophages, and higher expression of OPG mRNA and protein in lung tissue as compared to nonexposed animals. sRANKL treatment did not change collagen I deposition but did dose-dependently increase the number of (RANKL+) epithelial cells and also resulted in higher OPG mRNA and protein levels in lung tissue as compared to mice with untreated fibrosis. Groups were compared using a Kruskall Wallis test for multiple testing and p<0.05 was considered significant. (L) OPG mRNA expression did not change in TGFβ + sRANKL-treated murine precision-cut lung slices compared to TGF + vehicle-treated controls (n=6 independent experiments).


Based on our findings that serum OPG levels correlated significantly with OPG levels in murine lung tissue, OPG may be considered as a biomarker of disease severity or treatment effects. OPG serum levels in patients with liver cirrhosis have been included into a panel of serum biomarkers to improved diagnostic accuracy of a noninvasive test to assess liver fibrosis severity1,4,7,14-17. OPG may be similarly useful in pulmonary fibrosis based on our in vivo data, even though McGrath et al. found no differences between controls and IPF patients OPG serum levels and no association with lung function parameters10,19,35,36. Further studies could aim at investigating whether OPG correlates with non-invasive diagnostic parameters for (early) detection of the disease or with disease progression.

A limitation of our findings is the fact that our mouse model of silica-induced pulmonary fibrosis only represents one cause of pulmonary fibrosis; while in humans the causes are diverse and usually not known. The patients from whom the lung tissue samples were obtained, however, represented many types of pulmonary fibrosis (idiopathic pulmonary fibrosis, fibrotic non-specific interstitial pneumonia and progressed chronic extrinsic aller-gic alveolitis). All samples showed high levels of OPG, while we found low levels of OPG in COPD and in control lung tissue obtained from patients undergoing surgical resection for carcinoma with normal lung function. This suggests that the induction of OPG production is specific for fibrotic lung diseases, though other lung diseases should be considered to confirm this assumption.

RANKL is just one of the ligands for OPG and we focused on in relation to fibrosis since RANKL possesses the highest affinity for OPG out of all other possible ligands21,23,37. OPG is expressed in various organs and can also function as a decoy receptor of TNF-related apop-tosis-inducing ligand (TRAIL), heparin and glycosaminoglycans2,5,20,21,23,29. Involvement of one of these ligands may also explain possible actions of OPG in the lung or generally in fibrosis30-32,38. For example, by binding TRAIL, OPG may be able to prevent TRAIL-induced apoptosis of lung myofibroblasts and thereby allowing myofibroblasts to continue produc-ing ECM19,33,34,39. To investigate all ligands together instead of investigating them separately, a lung-specific conditional OPG knockout mouse could be considered in combination with models of experimental pulmonary fibrosis to determine the impact of OPG on the develop-ment and onset of fibrosis.

In conclusion, OPG expression is higher in fibrotic lung tissue as compared to control lung tissue and the profibrotic mediator TGFβ induces its production by fibroblasts. The induction of OPG and the increase in epithelial cell numbers in lung tissue following sRANKL treatment of pulmonary fibrosis suggest that the RANK/RANKL/OPG axis is not only active in bone, but also has a role in regulating tissue repair processes in the lung. Even though the exact role of OPG in fibrosis remains undetermined, the fact that OPG production is closely linked to fibrosis makes it an interesting new avenue to explore for drug and biomarker development.


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Acknowledgements

The Pender Foundation is gratefully acknowledged for their support of the exchange of fibrotic lung tissue between the Groningen and Rotterdam centers (BM and BB). A Young Investigator Award of the Netherlands Respiratory Society for our research on OPG in pul-monary fibrosis is gratefully acknowledged (CB). Barbro N Melgert is an active member of COST action BM1201. We would like to thank the following Bachelor students of Pharmacy and Master students of Pharmacy for their practical contributions to the manuscript: Burak Güney, Axel Haak, Annemarie Broesder, Thirsa Tuin and Marcelina Stel.

COMPETING INTERESTSDr. Timens reports personal fees from Pfizer, personal fees from GSK, personal fees from Chiesi, personal fees from Roche Diagnostics/Ventana, grants from Dutch Asthma Fund, per-sonal fees from Biotest, personal fees from Merck Sharp Dohme, personal fees from Novartis, personal fees from Lilly Oncology, outside the submitted work.

Dr. van den Blink reports grants and personal fees from Boehringer Ingelheim, grants and personal fees from Intermune-Roche, grants from Promedior, outside the submitted work.


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Supplementary file

Manuscript: Exploring the Role of Osteoprotegerin in Pulmonary FibrosisCarian E Boorsma1, Kurnia S.S. Putri, Christina Draijer, Peter Heukels, Bernt van den Blink, Robbert H Cool, Corry-Anke Brandsma, George Nossent, David M. Brass, Peter Olinga, Wim Timens, Barbro N Melgert


Human tissueHuman fibrotic lung tissue was collected with informed consent from patients with end-stage pulmonary fibrosis undergoing lung transplantation at either the University Medical Center Groningen (UMCG) or at the Erasmus Medical Center Rotterdam. In Groningen, the study protocol was consistent with the Research Code of the University Medical Center Gronin-gen (http://www.umcg.nl/EN/Research/Researchers/General/ResearchCode/Paginas/default.aspx (accessed 18 Feb2016)), and Dutch national ethical and professional guidelines (http://www.federa.org (accessed 18 Feb2016)). In Rotterdam, the Medical Ethical Committee approved all protocols followed in that center.

Control lung tissue was obtained at the UMCG from patients undergoing surgical resection for carcinoma or chronic obstructive pulmonary disease (COPD). In the cases of tumor resec-tions, histologically normal lung tissue was taken as far distally as possible from the tumor and assessed visually for abnormalities with standard haematoxylin and eosin staining.

For the analyses of protein levels of RANK, RANKL and OPG, samples of frozen lung tissue were used of patients as characterized in table 1.

Primary lung fibroblasts were isolated from lung tissue obtained from 6 patients with pulmo-nary fibrosis, 2 patients with COPD and 3 controls. These patient characteristics are summa-rized in Table 2. Technical details on the isolation of the lung fibroblasts are described in “Iso-lation and culture of primary fibroblasts”. Additionally, central and peripheral lung tissue was fixed in formalin and embedded in paraffin or frozen and stored at -80°C until further use.

Isolation and culture of primary human fibroblasts Lung fibroblasts were isolated from lung explants using a technique described previ-ously[18,19]. In short, fresh parenchymal lung tissue, excluding visible vessels and airways, was cut into 1-2 mm pieces that were then cultured for 4-5 weeks in 12-well plates (Greiner bio-one, Alphen aan de Rijn, The Netherlands) in the presence of complete Ham’s F12 medium [10% fetal calf serum (FCS, Invitrogen, The Netherlands), Penicillin/Streptavidin, fungizone, glutamine] at 37°C in an atmosphere of 5% CO

2. At 25% confluency, the cells were

transferred to T25 flasks. After 1 week, the cells were plated in 12-wells plates (50,000 cells/well) for experiments or cryopreserved in FCS with 10% DMSO under slow cooling conditions


and eventually stored at -150°C. Cells used in the experiments described were maximally passaged three times. Plated cells were grown to confluence and transferred to low-serum medium (0.5% FCS). After 72h, supernatants were collected for ELISA analysis of OPG levels and stored at -80°C for later analysis.

Animal experimentsMale C57BL/6 mice were obtained from Harlan (Zeist, The Netherlands). Animals were main-tained with permanent access to food and water in a temperature-controlled environment with a 12h dark/light cycle regimen. All animal experiments were approved by the Institutional Animal Care and Use Committee. Mice were used in experiments with silica-induced pulmo-nary fibrosis (DEC6064) and for the preparation of precision cut lung slices (DEC6416AA). Animal experiments were performed in the animal facility of the University of Groningen according to strict governmental and international guidelines on animal experimentation.

Silica-induced lung fibrosis in miceFibrosis was induced using Min-U-Sil 5 crystalline silica (a kind gift from Dr. Andy Ghio, US EPA, Chapel Hill, NC). Animals were anesthetized using isoflurane before they received a sin-gle administration of crystalline silica (0.2 g/kg) in 50 ml 0.9% saline by intratracheal installa-tion, while control animals received an equivalent volume of 0.9% saline. Mice were sacrificed after 28 days and serum and lung tissue were collected (n=6-8).

In mice treated with sRANKL, silica was administrated on day zero and sRANKL treatment began after 28 days and was continued for an additional 2 weeks. We calculated that the administered sRANKL dose should exceed the calculated OPG content (0.8 mg OPG/lung) in fibrotic lungs to be able to see a biological effect of sRANKL. We therefore used doses of 5 μg (n=12) and 10 μg RANKL (n=12) per mouse three times per week. sRANKL was administered intranasally in 40 ml saline. Control mice with pulmonary fibrosis received 40 ml saline (vehi-cle) three times per week (n=12). On day 43, mice were sacrificed and lungs were collected for flow cytometry, western blot analysis, ELISA, or histology. See figure 1 for an overview of the experimental setup.

Mouse lung precision-cut lung slicesAn ex vivo model of early lung fibrosis development was used to investigate the effect of TGFβ stimulation on OPG production by lung tissue. Lungs of male C57BL/6 mice (20-30gr, age 6-8 weeks) of in total six mice were used to make precision-cut lung slices. After sacrifice by exsanguination via the aorta abdominalis under isoflurane anaesthesia, mouse lungs were filled with 1.5% low-melting temperature agarose in 0.9% NaCl (Sigma-Aldrich) and trans-ferred directly into ice-cold University of Wisconsin organ preservation solution (DuPont Critical Care, Waukegab, IL). Lung slices of 5-mm in diameter and 10-15 cell layers thickness, were prepared with a Krumdieck tissue slicer (Alabama Research and Development, AL) using ice-cold Krebs-Henseleit Buffer [25 mM D-glucose (Merck, Darmstadt, Germany), 25 mM NaHCO3 (Merck), 10 mM HEPES (MP Biomedicals, Aurora, OH), saturated with carbo-gen (95% O

2/5% CO

2) and adjusted to pH 7.4] as we have previously described[20].


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Lung slices were incubated in 12-well plates (Greiner bio-one) in DMEM + Glutamax medium [4.5g/L D-glucose and pyruvate (Gibco) supplemented with a non-essential amino acid mix-ture (1:100), 100U/ml penicillin, 100g/ml streptomycin, 45 µg/ml gentamycin and 10% FCS]. After a 1h pre-incubation at 37°C in a 95% O

2/5% CO

2 atmosphere with continuous shaking

at 90 rpm, the slices were transferred into fresh medium and incubated in triplicate for 48h with TGFβ (5 ng/ml) or 48h with TGFβ followed by either vehicle or RANKL (200ng/ml) stim-ulation for 24 hours. Three slices from each condition were pooled for further analyses and tissue slice supernatants were collected separately. All samples were stored at -80°C for fur-ther analyses.

Supplemental table 1. Characteristics of patients whose lung tissue was used for determining RANK, RANKL and OPG levels (medians with range are presented).




Age, years 63 (57-81) 58 (50-62) 56 (37-64)

Smoking status 4 non-smokers/2 ex-smokers

1 non-smokers/8 ex-smokers

4 non-smokers/6 ex-smokers/1 unknown

FEV1 % predicted 103 (85-125) 32 (10-72) 49 (45-89)

Diagnosis 3 lung cancer/3 lung metastases

1 GOLD stage 3/8 GOLD stage 4

11 IPF/UIP

Definition of abbreviations: PF = pulmonary fibrosis; FEV1 = forced exhaled volume; COPD = chronic obstructive pulmonary disease; IPF = idiopathic pulmonary fibrosis; UIP = usual interstitial pneumonia.

Supplemental table 2. Characteristics of patients whose lung tissue was used for the isolation of primary lung fibroblasts (medians with range are presented).




Age, years 65 (45-74) 55 (53-57) 64 (58-66)

Smoking status 1 non-smoker/2 ex-smoker

2 ex-smoker 2 non-smoker/ 4 ex-smoker

FEV1 % predicted 98 (90-130) 22.5 (20-25) 62.5 (52-75)

Diagnosis Lung cancer COPD 2 IPF/UIP, 1 ILD/EAA, 1 NSIP/dust-exposed, 1 FNSIP/UIP

Definition of abbreviations: PF = pulmonary fibrosis; FEV1 = forced exhaled volume; COPD = chronic obstructive pulmonary disease; IPF = idiopathic pulmonary fibrosis; UIP = usual interstitial pneumonia; ILD = interstitial lung disease; EAA = extrinsic allergic alveolitis; NSIP = non-specific interstitial pneu-monia; FNSIP = fibrosing NSIP.


Mouse cell cultures NIH 3T3 fibroblasts (American Type Culture Collection) were cultured in Dulbecco’s modi-fied Eagle’s medium (Invitrogen, The Netherlands) [10% FCS, penicillin/streptomycin] at 37°C in an atmosphere of 5% CO

2.

RAW 264.7 macrophages (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, The Netherlands) [10% FCS, L-Glutamine, Gentamy-cin] and self-propagating mouse alveolar-like macrophages (MPI cells, a kind gift from Dr. G. Fejer, Plymouth University, Devon, UK) were cultured in RPMI 1640 medium (Gibco, Bleis-wijk, The Netherlands) [10% FCS, 20 ng/ml GM-CSF][17]. Macrophages were cultured at 37°C in an atmosphere of 5% CO

2.

3T3 cells were stimulated with TGFβ to transform them to myofibroblasts. Before TGFβ stimu-lation, plated cells were incubated in 0.5% FCS medium. After 24h, cells were stimulated with TGFβ (5 ng/ml, Peprotech, Rocky Hill, NJ) for 48h. Afterwards, the supernatant was collected and stored at -80°C until further use.

To induce a proteolytic phenotype, RAW macrophages (until max. passage 12) or MPI mac-rophages were stimulated with 200 ng/ml soluble RANKL (sRANKL, produced at our own facility. Technical details are described in “Production and purification of sRANKL protein”). Cells were lysed after 24h for the isolation of mRNA or after 72 hours for protein analysis. Samples were stored at -80°C for later analysis. For each experiment, the data presented are representative of at least three independent replicates.

Protein isolation from lung tissueProtein was isolated from frozen human or mouse lung tissue (30-40 mg) in buffer (0.25 M Tris/HCl buffer, 2.5% Igepal, 0.5% SDS, Protease Inhibitor Cocktail (Boehringer Mannheim, Germany), pH 7.5) by incubating for 1 hour at 4°C, homogenizing and centrifuging at 13,200 rpm for 30 min. Supernatants were collected and the overall protein concentration of the tissue lysates was determined using an RC DCTM Protein Assay based on the Lowry method (cat#5000111, Bio-Rad, Hercules, CA). Samples were stored at -80°C until further analyses by Western blot or ELISA analysis.

Western blot analysisWestern blot analysis was used to compare levels of mouse and human RANK and RANKL in control and fibrotic lung tissue. One hundred micrograms of total protein per sample was loaded into the well of a 10% SDS-PAGE gel and transferred to a polyvinylidene-fluoride membrane. Membranes were blocked with 5% nonfat milk in Tris-Buffered saline with 0.05% Tween 20 (TBST) for 2 hours, following incubation with the first antibody in blocking buf-fer overnight at 4°C. The following primary antibodies were used: rabbit-anti-mouse RANK (1:200, Acris, Herford, Germany), goat-anti-human RANK (1:100, R&D Systems, Minneapolis, MN), rabbit-anti-RANKL (human and mouse, 1:100, Acris) and mouse-anti β-actin (1:20,000 Sigma-Aldrich) as a loading control. After washing 30 min with TBST, the membrane was incubated with an appropriate secondary antibody in blocking buffer for 2 hours at room


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temperature. Subsequently, membranes were washed with TBST and with TBS before the bands were visualized with enhanced chemiluminescence (Perkin-Elmer Life Sciences, Bos-ton, MA). Band intensity was measured and quantified using GeneSnap (SynGene, Synoptics, Cambridge, UK). RANK and RANKL expression levels were normalized to β-actin expression.

ELISAHuman and mouse OPG levels in lung tissue and culture supernatants were assessed using ELISA (cat#DY805 (human), cat#DY459 (mouse), R&D Systems) according to the instructions provided by the manufacturer. One hundred micrograms of tissue lysate total protein in a total of 100 μl was analyzed per sample. Cell culture media was analyzed undiluted while lung slice culture medium was diluted 1:5 before analysis.

ImmunohistochemistryImmunohistochemical analysis of collagen I, RANK, and OPG was performed on 3 mm paraf-fin sections of mouse lung tissue. Tissue sections were deparaffinized in xylene, rehydrated in alcohol, rinsed in milliQ water and placed in PBS. When applicable, sections were subjected to antigen retrieval (0.1 N Tris-HCl pH 9.0 buffer, over night at 80°C). Prior to the incubation with the OPG antibody, sections were blocked with 1% BSA in 5% nonfat milk (Sigma-Aldrich). Primary antibodies were then incubated for 1h at room temperature in the presence of 5% normal mouse serum. Antibodies used were goat-anti-mouse collagen I (1:75, Southern Bio-tech, Birmingham, AL), rabbit-anti-mouse OPG (1:400, Antibodies-online, Atlanta, GA), and goat-anti-human RANK (1:100, R&D Systems). Primary antibody incubation was followed by incubation with species-specific PO-labeled secondary and optional tertiary polyclonal antisera. PO-labeled antibodies were visualized using ImmPACT NovaRED kit (Vector, Burl-ingame, USA) with a hematoxylin counterstain when appropriate.

The amount of collagen deposition in the lung was determined using ImageScope software (Aperio, Burlingame, USA). After selecting the stained area of the lung sections excluding edges of the tissue, large airways and vessels, a threshold was set to identify positive staining of the tissue (represented by collagen). The percentage of stained-tissue surface per total-tis-sue surface analyzed was then calculated for each section.

Cathepsin K activityCathepsin K activity of RAW264.7 macrophages was measured using a Cathepsin K Activity Kit (Cat#KA0769, Abnova, Taipei, Taiwan) following instructions provided by the manufacturer. Cathepsin K activity was normalized against the activity observed in unstimulated cells.

Quantitative Real-time PCRTotal mRNA was isolated from cells or tissues using a Maxwell® LEV simply RNA Cells/Tis-sue kit (cat# AS1280, Promega, Madison, WI) according to the instructions provided by the manufacturer. Final mRNA concentrations were determined using a Nanodrop ND-100 spec-


trophotometer (Nanodrop Technologies, Wilmington, DE). All primers were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). The following primers were used: collagen IαI forward: 5’-TGACTGGAAGAGCGGAGAGT-‘3; collagen IαI reverse: 3’-ATCCATCGGTCAT-GCTCTCT-‘5; fibronectin forward: 5’-gcgactctgactggccttac-‘3; fibronectin reverse: 5’-ccgtg-taagggtcaaagcat-‘3; MMP9 forward: 5’-GTCCAGACCAAGGGTACAGC-‘3; MMP9 reverse: 3’-GCCTTGGGTCAGGCTTAGAG-‘5; OPG forward: 5’-ACAGTTTGCCTGGGACCAAA-‘3; OPG reverse: 3’-CTGTGGTGAGGTTCGAGTGG-‘5; β-actin forward: 5’-ATCGTGCGTGACAT-CAAAGA-‘3; β-actin reverse: 3’-ATGCCACAGGATTCCATACC-‘5; YWHAZ forward: 5’-ATCGT-GCGTGACATCAAAGA-‘3; YWHAZ reverse: 3’-ATGCCACAGGATTCCATACC-‘5; 18s forward: 5’-CTTAGAGGGACAAGTGGCG-‘3; 18s reverse: 5’-ACGCTGAGCCAGTCAGTGTA-‘3. Tran-scription levels of these genes were measured by using 20 ng cDNA per sample in a quantita-tive real-time PCR (SensiMix™ SYBR kit (Bioline, Taunton, MA)) and an ABI7900HT sequence detection system (Applied Biosystems, Foster City, CA). For each sample, the threshold cycles (Ct values) were calculated using SDS 2.3 software (Applied Biosystems), and mRNA expres-sion was normalized to β-actin for cell culture mRNA samples, YWHAZ for tissue mRNA samples, and 18S for precision-cut murine lung slices. Results are expressed as fold-change of healthy controls or control conditions.

Production and purification of sRANKL proteinPlasmid pET15b-mRANKL, encoding the extracellular domain of murine RANKL (sRANKL; amino acids 160-316), was transformed into Escherichia coli strain BL21(DE3). A 10 mL over-night culture was used to inoculate 1L 2xYT [16g Bacto Tryptone, 10g Bacto Yeast Extract, 5g NaCl, pH 7.0] medium containing 0.1mg/L ampicillin. Cells were grown at 370C until the absorbance at 600 nm was 0.5 after which sRANKL production was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside and incubation continued for another 16 hours at 200C.

After harvesting and resuspending wet cells at 1 g/3 ml in buffer A [50 mM MES, pH 5.8, 2 mM dithiothreitol, 10 % glycerol], cells were carefully sonicated on ice. Cell debris was removed by centrifugation for 60 min at 38,000xg and 4°C. Supernatants were loaded on an 8 mL cation exchange column (Source 30S, GE Healthcare, USA). sRANKL eluted early from a linear 0-500 mM NaCl gradient in buffer A. Pooled fractions at 40C were brought to pH 7.5 by adding drops of 1 M NaOH and to 1.3 M (NH

4)

2SO

4 by slowly adding pulverised salt crystals. The protein was

loaded on a 5 mL HiTrap phenyl sepharose column (GE Healthcare), pre-equilibrated with buffer B [20 mM sodium phosphate buffer, 2 mM dithiothreitol, 10% glycerol, pH 7.5] and eluted early from a linear 1.3-0 M (NH

4)

2SO

4 gradient in buffer B. Pooled fractions were run

on a HiTrap Superdex75 column (GE Healtcare) in 20 mM sodium phosphate buffer NaPi, [NaH2PO4, Na2HPO4, 10 % glycerol, pH 7.5]. Protein purity was checked by SDS-PAGE and sRANKL concentrations were measured with a Coomassie Bradford protein assay kit (Pierce, USA) using BSA as reference and with a RANKL ELISA kit (cat#DY462, R&D Systems). Values from both assays were always in the same range and the concentration obtained from the ELISA analysis was used for further experiments. Purified RANKL is routinely tested for endo-toxin contamination comparing heat-inactivated RANKL with native RANKL using RAW264.7 macrophages and no measurable contamination was found.


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Flow cytometryMouse left lungs were minced and incubated in RPMI containing 10% FCS, 10 μg/ml DNase I (grade II from bovine pancreas, Roche Applied Science, Almere, The Netherlands), and 0.7 mg/ml collagenase A (Sigma-Aldrich) in a shaking water bath (37°C) for 45 min. Digested lung tissue was mashed through a 70 μm nylon strainer (BD Biosciences, Breda, The Netherlands) resulting in a single cell suspension. To remove contaminating erythrocytes, the cells were incubated with Pharmlyse (BD Biosciences) for 2 min at room temperature. Total cell num-bers were determined using a FACS Array cell counter (BD Biosciences) and used to calculate total numbers of specific cell types.

Single lung cell suspensions were stained for T cells, neutrophils, epithelial cells and mac-rophage subsets by flow cytometry (see supplemental Table 1 and 2 for antibody details). A viability dye was included (Fixable Viability Dye eFluor® 506, eBioscience, San Diego, CA) to exclude dead cells. T cells were identified using anti-CD3-APC/Cy7 and epithelial cells were identified using anti-EpCAM-Alexafluor 647. Interstitial macrophages were identified as CD68+CD64negCD11c-low-expressing cells and alveolar macrophages were identified as CD68+CD64varCD11C-high-expressing cells using anti-CD68-PerCp/Cy5.5, anti-CD64-PE/Cy7 and anti-CD11-Brilliant Violet 785. Neutrophils were identified as CD68negGR1+ cells. In addition, the expression of RANKL on T cells, epithelial cells, and macrophages was deter-mined by using anti-RANKL-FITC. See supplemental data figure 3 and 4 for the gating strat-egy. Approximately 106 cells were incubated with cell surface marker antibodies in the presence of 1% normal mouse serum (30 min, on ice, protected from light). The cells were then washed with PBS containing 2% FCS and 5 mM EDTA and fixed and permeabilized using a fixation and permeabilization buffer (30 min, eBioscience) to stain for intracellular markers. After washing the cells with permeabilization buffer, all samples were resuspended in FACS lysing solution (Biosciences) and stored at 4°C until analysis. Samples were analyzed using an LSRII flow cytometer (BD Biosciences). The data were analyzed using FlowJo software (Tree Start, Ashland, USA). For each antibody, a Fluorescent Minus One (FMO) control was included for proper gating during data analyses.

Statistical analysisAll groups were considered non-normally distributed due to small samples sizes. Therefore, statistical differences between two groups were assessed using nonparametric Mann-Whit-ney U tests. For the comparison of multiple groups, we used Kruskal Wallis. When significant, a Mann-Whitney-U test with a Bonferroni adjustment for multiple comparisons was used as a post-test to calculate statistical differences between specific groups. Correlations between OPG levels in lung and serum, and the correlation with collagen content in the lungs were assessed by calculating a nonparametric Spearman correlation coefficient (R). Significance was considered *= p<0.05. The data were analyzed using GraphPad Prism 6.


Supplemental table 3. Overview of flow cytometry antibodies used to stain for T cells and epithelial cells.

Antigen Fluorochrome Cataloge # Company

αCD3 APC/Cy7 100222 Biolegend

αRANKL 488 510002 Biolegend

αEpCAM A647 118211 Biolegend

Live/Dead e506 65-0866 eBioscience

Supplemental table 4. Overview of flow cytometry antibodies used to stain for macrophages.

Antigen Fluorochrome Cataloge # Company

αCD11c BV785 563735 eBioscience

αGR1 A700 108421 Biolegend

Live/Dead e506 65-0866 eBioscience

αCD68 PerCp/Cy5.5 137009 Biolegend

αCD64 PE/Cy7 139314 Biolegend

Supplemental data figure 1. A. Collagen I mRNA expression was higher in lungs of mice exposed to silica (fibrosis, n=7) as compared to healthy controls (n=8). Levels were corrected for YHWAZ as housekeeping gene. B. Representative pictures of an immunohistochemical staining for collagen I deposition, showing that collagen I deposition was higher in the lungs of mice exposed to silica as compared to saline-exposed controls. Collagen I expression is indicated by the red colour.


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¯ Supplemental data figure 2. mRNA expressions of collagen IαI (A), fibronectin (B) and MMP9 (C) were higher in fibrotic mouse lungs following silica exposure for 28 days (n=6) and vehicle treatment for two weeks as compared to healthy controls (n=3), but unchanged in fibrotic lungs of mice that received sRANKL treatment for two weeks (5μg/mouse, n=6 and 10ug/mouse, n=6) as compared to vehicle treat-ment. Groups were compared using a Kru-skall Wallis test for multiple testing and p<0.05 was considered significant.


Supplemental data figure 3. Gating strategy for T cells and epithelial cells.

Supplemental data figure 4. Gating strategy for macrophages and neutrophils.

Chapter 3

A Potent Tartrate Resistant Acid Phosphatase Inhibitor to Study the Function of TRAP in Alveolar Macrophages

Carian E Boorsma1,6, Kurnia SS Putri2, Andreia de Almeida1, Christina Draijer1,6, Thais Mauad3, Corry-Anke Brandsma4,6, Maarten van den Berge5,6, Yohan Bossé7, Don Sin8,9, Ke Hao10,11,12, Peter Olinga2, Wim Timens4,6, Angela Casini1,3, Barbro N Melgert1,6*

1University of Groningen, Department of Pharmacokinetics, Toxicology and Targeting, Gron-ingen Research Institute for Pharmacy, Groningen, The Netherlands. 2University of Gron-ingen, Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute for Pharmacy, Groningen, The Netherlands. 3School of Chemistry, Cardiff Univer-sity, Park Place, Cardiff CF10 3AT, United Kingdom. 4São Paulo University, Department of Pathology, São Paulo, Brazil. 5University of Groningen, University Medical Center Groningen, Department of Pathology, Groningen, The Netherlands. 6University of Groningen, Univer-sity Medical Center Groningen, Department of Pulmonology, Groningen, The Netherlands. 7University of Groningen, University Medical Center Groningen, GRIAC Research Institute, Groningen, The Netherlands. 8Institut universitaire de cardiologie et de pneumologie de Québec, Department of Molecular Medicine, Laval University, Québec, Canada. 9UBC James Hogg Research Center, Providence Heart + Lung Institute, St. Paul’s Hospital. 10Respiratory Division, Department of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada. 11Merck Research Laboratories, Boston, Massachusetts, United States of America. 12Merck, Rahway, New Jersey, United States of America, 3 Genetics, Rosetta Inphar-matics. 13Merck, Seattle, Washington, United States of America.

Manuscript submitted

Of note: Results from this manuscript have contributed to and are included in the patent: “Gold(III) compounds as Tartrate Resistant Acid Phosphatase inhibitors, and therapeutic uses thereof.”, as presented in appendix I.


AbstractThe enzyme tartrate resistant acid phosphatase (TRAP) is expressed in alveolar macrophages in high quantities but its function is unclear and stable inhibitors are lacking We studied changes in TRAP expression and activity in patients with chronic obstructive pulmonary disease (COPD), (fatal) asthma and found higher TRAP activity and expression in patients with COPD or fatal asthma compared to controls. In mouse models, more TRAP activity was detected in the lungs of mice following cigarette smoke or house dust mite exposure. Stimuli related to asthma or COPD (Receptor activator of NF-kb ligand (RANKL), M-CSF, IL-4, ATP, Xanthine/Xanthine Oxidase) were tested for their capacity to induce TRAP in macrophages in vitro and in lung slices. RANKL and Xanthine/Xanthine Oxidase induced TRAP mRNA expression in mouse macrophages, but only RANKL induced TRAP activity in mouse lung slices. Several gold(III) coordination compounds were tested for their ability to inhibit TRAP activity and function. The gold(III) compound AubipyOMe strongly inhibited human recombinant TRAP activity (IC50 0.48 mM), and TRAP activity in mouse cell and human tissue extracts. Function-ally, AubipyOMe inhibited mouse macrophage migration. In conclusion, this TRAP inhibitor could provide a useful tool to study the function of TRAP in lung.

A potent tartrate resistant acid phosphatase inhibitor to study the function of trap in alveolar m

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Introduction

Tartrate resistant acid phosphatase (TRAP) is a metalloenzyme and member of the purple acid phosphatases, containing a binuclear iron (Fe2+) center that facilitates the hydrolysis of phosphate esters and the generation of reactive oxygen species (ROS) 1-4. It is highly expressed in osteoclasts and alveolar macrophages and lower expression can be found in activated mac-rophages and dendritic cells 1,2,4-9. TRAP exists in two isoforms: the 5a isoform is a monomer expressed in lysosomes, while the 5b isoform is a dimer derived from 5a by proteolytical cleavage and is the more active form 5,10-15. Alveolar macrophages have especially high expres-sion of TRAP5a while osteoclasts express high levels of TRAP5b. The function of TRAP5b in bone has been studied in relation to bone remodeling, in which TRAP activity was found to mediate osteoclast migration. Osteoclasts are attached to bone matrix through an osteopon-tin - integrin alphav-beta3 (α

vβ

3) bond. Migration of osteoclasts can only occur when this

bond is disconnected by TRAP-dependent dephosphorylation of osteopontin 10,13,14,16. The role of TRAP5a in alveolar macrophages has not been clarified yet and specific, stable inhibitors to study its function lack.

Little is known about the regulation of TRAP in alveolar macrophages, in addition to its unknown function. One study investigated the expression of TRAP in lung tissue and another specifically measured TRAP expression in alveolar macrophages and both found higher expression in smokers 3,17-19. As TRAP may be involved in defending the lung against airborne dangers by its ability to generate oxygen radicals, we investigated whether its expression and/or activity is altered in patients with fatal asthma or chronic obstructive pulmonary disease (COPD) 1,2,4,14. In both diseases we found either higher expression or higher activity of TRAP as compared to controls and we therefore continued to investigate its regulation by asthma and/or COPD-specific cytokines and mediators.

Exploring the function of TRAP activity has been hampered due to lack of (specific) stable inhibitors. Hayman et al demonstrated inhibitory effects of gold chloride on TRAP activ-ity 1-4,17-19. However, gold chloride is a reactive compound with a highly oxidative character and unspecific protein binding, whose activity may interfere with many different cellular pathways. In recent years, gold-based compounds of different families have been shown to possess ideal enzyme/protein inhibition properties, which allow them to be designed and exploited as chemical probes to study protein functions in biological systems and to possibly be developed as therapeutic agents 5-9,20,21. Thus, we decided to screen a series of gold coordi-nation complexes with N-donor ligands, in which the gold(III) ions are known to be highly stabilized by the ancillary organic ligands. The presence of the ligand also allows designing the compounds to be selective for specific enzyme inhibition. Among the newly tested gold complexes, the compound [Au(4,4’-dimethoxy-2,2’-bipyridine)Cl

2][PF

6] (AubipyOMe, fig-

ure 1) was found to be the most potent inhibitor of TRAP activity. Therefore, we used this compound to investigate functional aspects of TRAP activity in macrophages, such as mac-rophage migration.


Figure 1. Chemical structure of the gold(III) compound [Au(4,4’-dimethoxy-2,2’-bipyridine)Cl

2][PF

6] (AubipyOMe).

Our starting hypothesis was that TRAP activity in alveolar macrophages is involved in regu-lation of their migration, similar to osteoclasts in bone. Osteopontin expression in lung has been shown on the luminal side of epithelial cells 11,15,22. As alveolar macrophages are present in the lumen of airways and alveoli and express α

vβ

3 integrins, we hypothesized they may also

need TRAP to migrate 10,12-14,23. We used this migration hypothesis to test the functionality of our new gold-based TRAP inhibitor, AubipyOMe.

Materials and Methods

Human tissue

COPDGene expression data of TRAP was obtained from a large gene expression study comparing lung tissue from 311 patients with COPD and 270 non-COPD controls that were part of the Lung eQTL consortium. Details of this population can be found in supplemental table 1. Lung tissue samples were derived from patients with or without COPD undergoing lung tumor resection surgery or lung transplantation for severe COPD. In case of tumor resections, mac-roscopically nontumorogenic lung tissue was taken far distant from the tumor and histology of all samples was checked for abnormalities using standard haematoxylin and eosin staining. Lung samples were obtained in accordance with local ethical guidelines. A detailed descrip-tion of the whole genome mRNA profiling has been previously published by Brandsma et al. and Hao et al. 16-18,24.

AsthmaPost mortem lung tissues from subjects with fatal asthma or subjects who died from non-pulmonary causes (controls) were retrieved from the Department of Pathology of São Paulo University (São Paulo, Brazil). Patient characteristics can be found in supplemental table 2. A detailed clinical and demographic description of this population has been previously published by Mauad et al. 25. For this study we investigated the presence of TRAP activity in


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paraffin-embedded peripheral lung tissue samples of 10 asthma patients and 8 controls as described below.

Animal experimentsDuring the experiments, all animals were held under specific pathogen-free conditions in groups of 4-6 mice per cage in a temperature-controlled room with a 12h dark/light cycle and permanent access to food and water. The Institutional Animal Care and Use Committee approved these experiments according to strict governmental and international guidelines on animal experimentation (DEC2857, DEC5318, and DEC6416AA-001).

Smoke-induced lung inflammationTo model COPD, we exposed five male A/JOlaHsd mice (Harlan, Horst The Netherlands, 8-10 weeks old) nose-only to mainstream cigarette smoke for 9 months in an experimental set-up as described before by us 26. In short, mice were exposed daily to mainstream smoke from four 2R1 Reference Cigarettes (University of Kentucky, KY). This protocol was continued for 5 days/week for 9 months. Six control mice were sham-exposed to room air under similar conditions, following the same duration of exposure as the smoke-exposed group. Mice were sacrificed after 9 months and lungs were collected, formalin-fixed and embedded in paraffin for histological analysis and TRAP-activity.

Allergic lung inflammationTo model asthma, we exposed male and female BALB/c mice (Harlan, Horst The Netherlands, 8-10 weeks old) intranasally to whole body house dust mite (HDM) extract (Dermatophagoi-des pteronyssinus, Greer laboratories, Lenoir, USA) in 40 ml phosphate-buffered saline (PBS) according to a protocol we have described before 27. In short, mice (n=8) were exposed to HDM extract under isoflurane anesthesia: one time to a high dose of HDM (100 mg) in the first week and 5 times to a low dose (10 mg) in the second week. Control animals (n=8) were exposed to 40 ml PBS according to this same schedule. Mice were sacrificed on day 24, three days after the last HDM exposure and the right lung was inflated with 0.5 ml 50% Tissue-Tek® O.C.T.™ compound (Sakura, Finetek Europe B.V., Zoeterwoude, The Netherlands) in PBS and formalin-fixed for histological analyses and TRAP activity. Other parameters of allergic lung inflammation of these animals are described in detail by Draijer et al. 1,2,4,27.

Enzyme histochemistry for TRAP activityPresence of active TRAP was assessed using a standard enzyme histochemical method. In short, deparaffinated lung sections (3mm) were rehydrated and incubated overnight in a buffer containing 0.1 M Tris at pH 7.4 supplemented with 30 mM calcium acetate, 23mM zinc acetate and 37mM zinc chloride, followed by pre-incubation in a 0.2 M acetate buffer at pH 5.0. TRAP-active cells were visualized by incubating sections for 0.5-4 hours at 37°C in a reaction solution containing 0.2 M acetate buffer with 0.5 mg/ml Naphtol AS-MX phosphate (Sigma-Aldrich, St. Louis, USA) and 1.1 mg/ml fast red TR salt (Sigma-Aldrich) at pH 5.0. After hematoxylin counterstaining, sections were imbedded in VectaMount Permanent mounting medium (Vector Laboratories, Burlingame, CA). The number of positive cells was counted


manually with the aid of ImageScope software (Leica Biosystems, Son, The Netherlands) in human and murine lung tissue sections, on average, a surface of 9 mm2 was measured of each section), and corrected for the surface area of the corresponding lung tissue (mm2).

Cell culture of macrophagesSelf-propagating murine alveolar-like macrophages (MPI macrophages, a kind gift from dr. G. Frejer, Plymouth University, Devon, UK) were cultured in RPMI 1640 medium (Gibco, Bleis-wijk, The Netherlands) supplemented with 10% fetal bovine serum (FCS), gentamycin (10 mg/ml), and GM-CSF (20 ng/ml) at 37°C under 5% CO

2 and humidified conditions 1,2,4,28. MPI

macrophages were plated at a density of 50,000 cells/well. The next day, cells were stimulated for 16h with a superoxide-generating system (0.2 mM Xanthine + 10mU/ml Xanthine oxidase (Sigma-Aldrich, St. Louis, USA)) to mimic oxidative stress, for 24h with the damage-asso-ciated molecular pattern ATP (1, 10 or 100 mg/ml), for 24h with RANKL (200 ng/ml, kindly provided by dr. R.H. Cool, University of Groningen, The Netherlands), for 24h with IL-4 (10 ng/ml), or for 24h with M-CSF (10 ng/ml). Cells were harvested for mRNA isolation purposes.

RAW 264.7 macrophages (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, The Netherlands) supplemented with 10% FCS, 2mM L-glutamine, and Gentamycin (10 mg/ml) and cultured at 37°C, 5% CO

2, and

humidified con-

ditions. RAW macrophages were used in transwell and cell-tracking experiments, as further explained in “Inhibition of macrophage migration by AubipyOMe”.

Quantitative Real-Time PCRFor TRAP mRNA expression, RNA was isolated using the Maxwell® LEV simply RNA Cells/Tissue kit (Promega, Madison, WI). Final mRNA concentrations after isolation were deter-mined using a Nanodrop ND-100 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Subsequently, cDNA was prepared using 10 ml mRNA (20 mg/ml) with 5x RT-buffer, 10 mM dNTP’s, 5 units Rnasin, random primers and 40 units M-MLV Rev transcriptase. The Eppendorf Thermocycler (Eppendorf, Hamburg, Germany) was used for the amplification of the cDNA. Transcription levels of TRAP were measured in 20 ng cDNA per sample in a quantitative real-time PCR (SensiMix™ SYBR kit, Bioline, Taunton, MA) and the ABI7900HT sequence detection system (Applied Biosystems, Foster City, CA). Primers used for RT-PCR were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands): TRAP forward: 5’-GCT-GTCCTGGCTCAAAAAGC-3’; TRAP reverse: 5’-CACACCGTTCTCGTCCTGAA-3’; GAPDH forward: 5’-ACAGTCCATGCCATCACTGC-3’; GAPDH reverse: 5’-GATCCACGACGGACA-CATTG-3’. For each sample, the threshold cycles (Ct values) were calculated with the SDS 2.3 software program (Applied Biosystems) and mRNA expression was normalized against GAPDH. The experiment was repeated at least four times.

Stimulation of precision cut lung slicesLungs of male C57BL/6 mice (20-30 gr) of in total six mice were used to make precision-cut lung slices. Mice were sacrificed under isoflurane anaesthesia by exsanguination via the aorta


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abdominalis. Lungs were filled with low-melting temperature agarose (1,5% in 0.9% NaCl) (Sigma-Aldrich) and transferred directly into ice-cold University of Wisconsin organ preser-vation solution. Lung slices, diameter 5-mm and weight ±5 mg, were prepared with a Krum-dieck tissue slicer (Alabama Research and Development, AL) using ice-cold Krebs-Hense-leit Buffer [25 mM D-glucose (Merck, Darmstadt, Germany), 25 mM NaHCO3 (Merck), 10 mM HEPES (MP Biomedicals, Aurora, OH), saturated with carbogen (95% O

2/5% CO

2) and

adjusted to pH 7.4] as described by us before for liver slices 5,29.

After slicing, murine lung slices were transferred to 12-well plates (Greiner bio-one, Alphen aan de Rijn, The Netherlands) with pre-warmed DMEM + Glutamax medium (1.3 ml) [4.5g/L D-glucose and pyruvate (Gibco) supplemented with non-essential amino acid mixture (1:100), 100 U/ml penicillin, 100 mg/ml streptomycin, 45 µg/ml gentamycin, and 10% FCS]. Following 1h pre-incubation at 37⁰C in 95% O

2/5% CO

2 conditions and continuous shaking (90

rpm), slices were transferred into fresh medium and incubated in triplicate with the following stimulants: vehicle, RANKL (200 ng/ml), ATP (10 µg/ml), or Xanthine (0.2 mM) + Xanthine oxidase (10 mU/ml). After 24 hours of incubation, the three slices of each condition were pooled and directly snap frozen in liquid nitrogen and stored at -80⁰C. For the TRAP activity analysis, 400 ml acetate buffer was added to each sample. Slices were then homogenized using small glass pearls and a Mini-Beadbeater-24 (45s) (Biospec products, Bartlesville, UK), centrifuged (10 min, 13.2 rpm, 4⁰C), and used immediately for the TRAP activity assay.

TRAP activity assay on lysates of MPI macrophages and precision-cut lung slicesTRAP activity levels were determined with a TRAP activity assay. Lung slice homogenates or MPI-macrophage lysates were incubated at 37°C for an hour at a 1:1 ratio with an L-pa-ra-Nitrophenylphosphate (PNPP) solution [100 mM PNPP, 200 mM sodium citrate, 200 mM sodium chloride, 80 mM sodium tartrate (L+), pH 4.5]. To stop the reaction, 1M NaOH was added and absorption at 410 nm, with 490 nm as a reference value, was measured using a spectrophotometer. Each sample was applied in duplicate and stimulus outcome was calcu-lated relative to the nonstimulated control absorption level. Finally, the average TRAP-activity was calculated over six individual cell experiments or in lung tissue slices of six mice.

Identification of TRAP inhibitors Initially, a small library of gold compounds was tested for TRAP inhibition using a TRAP activ-ity assay (R&D, Minneapolis, USA) with recombinant human TRAP. The gold(III) compounds [Au(terpy)Cl]Cl

2 (terpy = terpyridine, Auterpy) 11,30, [Au

2(μ-O)

2(bipy)

2](PF

6)

2 (bipy = 2,2’-bipyr-

idine, Auoxo) 10,13,14,31 and [Au(bipyOMe)Cl2][PF

6] (bipyOMe = 4,4’-dimethoxy-2,2’-bipyridine,

AubipyOMe) 17-19,32 were synthesized according to literature procedures and their purity was confirmed by elemental analysis and showed to be >98 %. The anti-rheumatic gold(I) com-pound sodium aurothiomalate (Authiomalate) was purchased from Sigma-Aldrich. NaAuCl

4

was used as a reference compound.

Inhibitor dilutions were prepared in acetate buffer from freshly prepared stock solutions (10 mM in DMSO). Recombinant TRAP (1.25ng/ml) was incubated at 37°C for 30 minutes with


PNPP solution (1:1 ratio) and increasing concentrations of NaAuCl4 (range 0-40 μM) or gold

compounds (range 0-5.1 μM). To stop the reaction, 1M NaOH was added and absorption at 410 nm, with 490 nm as a reference value, was measured using a spectrophotometer.

Testing of inhibitors on cell and tissue lysatesMouse alveolar macrophage lysates were obtained by resuspending 500.000 MPI macro-phages in 1 ml acetate buffer. The cell suspension was sonicated, spun down (10 min, 13,200 rpm), and the supernatant was collected for TRAP activity assays. Human lung lysates from COPD patients were used to test the inhibitor on human TRAP present in lung tissue. Lysates from in total 18 COPD patients were pooled (see supplemental table 3 for patients charac-teristics). To obtain the tissue lysates, 10-20 mg frozen lung tissue was collected and 50 μl acetate buffer was added. Then, the tissue was homogenized manually using a small plunger. After spinning the samples down (13.200 rpm, 10 min, 4°C), all supernatants were pooled and stored at -80°C until use.

AubipyOMe was tested in the range 0-40 μM with MPI lysates or pooled lung tissue lysates from COPD patients using a TRAP activity assay. MPI lysates were diluted 1:1 and COPD tissue lysates 1:20 with acetate buffer and incubated with increasing concentrations inhibitor, that were prepared from freshly made stock solutions (10 mM in DMSO) and diluted with acetate buffer. Lysates were incubated at 37°C for 30 min in the presence of PNPP solution (1:1 ratio). Adding 1M NaOH stopped the reaction and absorption was measured at 410 nm with 490 nm as a reference value.

The inhibitory effects of gold compounds were calculated as the ratio of absorbance between the treated and untreated wells. The IC50 value was calculated as the concentration of inhib-itor causing a 50% reduction in TRAP activity. The average IC50 of three independent exper-iments was calculated.

MTT assayThe effect of AubipyOMe and NaAuCl

4 on cell growth was assessed with a classical MTT assay.

RAW macrophages were seeded in 96-well plates (Greiner bio-one) at a concentration of 10,000 cells/well and grown for 24h in complete medium. Compound dilutions were pre-pared in complete medium from a freshly prepared stock solution (10 mM in DMSO) and added to the wells (200 μl) to obtain a final concentration (0 to 100 μM). Cells were incubated for 72h after which 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the cells at a final concentration of 0.50 mg/ml and incubated for 3h. After 3h, the MTT-solution was removed and the violet formazan crystals dissolved in DMSO. The opti-cal density of each well was quantified in quadruplicate at 540 nm, using a multi-well plate reader, and the percentage of surviving cells was calculated from the ratio of absorbance between treated and untreated cells. The IC50 value over four individual experiments was cal-culated as the actual concentration of inhibitor reducing the proliferation of the cells by 50%.


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Inhibition of macrophage migration by AubipyOMe

Transwell experiment Migration of RAW macrophages was assessed using a transwell-culturing system with inserts (Sigma-Aldrich, St. Louis, USA) coated with osteopontin (R&D, Minneapolis, UK). Bovine collagen-coated inserts (Advanced Biometrix, Carlsbad, USA) were used as a matrix control. RAW macrophages, pre-stimulated with 200 ng/ml RANKL for 72h to induce TRAP activity, were seeded on 8 mm pore inserts coated with osteopontin (10 mg/ml, 250,000 cells/well) or collagen (10 μg/ml, 250,000 cells/well). Cells were cultured for 16h in the presence or absence of AubipyOMe (128 nM) in quadruplicate. The next day, total cell numbers in the lower com-partment, including dead cells, were counted. The number of cells migrated to the lower well was calculated relative to cells not stimulated with RANKL and an average of six individual experiments was calculated.

Confocal imaging of macrophage migrationBriefly, RAW macrophages were plated on osteopontin-coated (10 mg/ml, R&D) Lab-tek chamber slides (Nunc, Hatfield, USA) at a density of 7500 cells/well. Half of the wells were stimulated with RANKL (200 ng/ml) for 48 hours to induce TRAP activity, followed by CFSE labeling (5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester, CFSE, Invitrogen, Life Technologies Europe BV, Bleiswijk, The Netherlands) to visualize cells for live cell imaging using a confocal microscope (Solamere Nipkow Confocal Live Cell Imaging system, Solamere Technology Group, Salt lake city, USA). A solution of 4mM CFSE in PBS (400 ml) was added to the cells for 20 min. After washing the cells with medium, new medium was applied (200 ml) with or without AubipyOMe (128 nM). Cell movement was tracked overnight in the pres-ence of 1% zymosan solution (Sigma-Aldrich, St. Louise, USA) to stimulate cell movement. Environmental conditions were kept at 37°C and 96% O

2/4% CO

2. Pictures were taken every

10 min and were transformed into movies each representing one well with Image J 1,2,4,33 and Imaris x64 (Bitplane, Zurich, Switzerland) software.

Statistical analysisAll data were nonparametrically distributed. Statistical differences between two groups were calculated using a Mann-Whitney U test. When comparing multiple conditions, a nonpara-metric Kruskal-Wallis with a Dunn’s multiple comparisons test was performed. When study-ing correlations, a Spearman coefficient was calculated to test for significance. P<0.05 was considered significant.

Results

TRAP expression and TRAP activity in COPD and (fatal) asthma To assess whether TRAP mRNA expression is changed in COPD versus control lung tissue we did a single gene look-up for TRAP in a genome wide gene expression dataset comparing 311 COPD patients and 270 non-COPD controls 16,34. Amongst the upregulated genes was TRAP,


which was significantly higher in COPD patients compared to control patients (p<0.0008, figure 2A). To investigate the effect of current smoking on TRAP expression, we additionally compared control individuals currently smoking with individuals that had stopped smoking for at least 5 years in the same dataset. This comparison showed higher expression of TRAP in the individuals that are currently smoking versus the ex-smokers (figure 2B). A similar analysis among the COPD patients showed no differences between current and ex-smokers.

In addition, we examined whether TRAP mRNA expression correlated with lung function in COPD patients (as defined by FEV

1) and found a negative correlation, meaning higher TRAP

expression was linked with lower FEV1 values (p<0.04, figure 2C). This significant correlation

Figure 2. High TRAP expression is associated with COPD. (A) TRAP mRNA expression was significantly higher in patients with COPD (n=311) as compared to their respective controls (n=270). (B) TRAP mRNA expression was significantly higher in control individuals that were current smokers (n=73) in contrast to control exsmokers after at least 5 years of smoking cessa-tion (n=106). (C) TRAP mRNA expression correlated negatively with lung function (FEV

1). (D)

TRAP mRNA expression is only significantly higher in patients with severe COPD (GOLD stage IV) as compared to controls. Differences were tested using a Mann-Whitney U test, * P<0.05, ** P<0.01, *** P<0.001. The correlation between TRAP mRNA expression and FEV

1 was calculated

using a Spearman correlation coefficient, * P<0.05.


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is mainly caused by the high expression of TRAP in lung tissue of patients with severe COPD: patients with the most severe disease, i.e. highest GOLD stage and therefore lowest FEV1 value, had significantly higher expression of TRAP in lung tissue as compared to non-COPD controls, while the patients with less severe COPD had similar TRAP expression as compared to controls (figure 2D).

Figure 3. High TRAP activity is associated with fatal asthma. (A) Parenchymal lung tissue of patients with fatal asthma (n=10) contained higher numbers of TRAP-positive macrophages as compared to controls dying of nonpulmonary causes (n=8). (B) Representative pictures of lung tissue sections of a control individual stained for TRAP activity. (C) Representative pictures of lung tissue sections of a fatal asthma patient stained for TRAP activity. Cells positive for TRAP (purple) are alveolar macrophages as indicated by the arrows. Differences were tested using a Mann-Whitney U test, * P<0.05.


To investigate the activity levels of TRAP in fatal asthma, we compared lung sections of patients who had died during an asthma attack with lung sections from controls who had died of nonpulmonary causes. Staining for TRAP activity showed that the number of cells positive for TRAP activity was higher in lung tissue from patients with fatal asthma as com-pared to control subjects (figure 3A-C). Only alveolar macrophages were found to be TRAP activity-positive (figure 3B, indicated by the arrows), though not all alveolar macrophages seemed positive for TRAP activity.

Figure 4. High TRAP activity is associated with exposure to smoke and house dust mite. (A) Parenchymal lung tissue of mice that were exposed to cigarette smoke for 9 months (n=5) contained a higher number of TRAP+ alveolar macrophages than mice exposed to room air (n=6) as determined by the number of positive cells/mm2. (B-C) Representative pictures of lung tissue sections of a control mouse and a smoke-exposed mouse stained for TRAP activity. Alveo-lar macrophages stain positive for TRAP (purple) as indicated by the arrows. (D) Mice exposed to house dust mite (n=8) showed higher numbers of TRAP+ alveolar macrophages in paren-chymal lung tissue compared with control mice (n=8) as determined by the number of positive cells/mm2. (D-F) Representative pictures of lung tissue sections of a control mouse and a house dust mite-exposed mouse stained for TRAP activity. Alveolar macrophages stain positive for TRAP (purple) as indicated by the arrows. Differences were tested using a Mann-Whitney U test, ** P<0.01, *** P<0.001.


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In lungs of mice that were exposed to cigarette smoke for 9 months we found that the number of TRAP-active cells was significantly higher as compared to air-exposed mice (figure 4A). Sections of the lung show that alveolar macrophages stain positive for TRAP activity (figure 4B-C, indicated by the arrows). In a model of house dust mite (HDM)–induced asthma, we also found significantly higher numbers of TRAP-positive cells in the parenchyma of mice exposed to HDM as compared to vehicle-exposed control mice (figure 4D). Again, alveolar macrophages were the only cells that stained positive for TRAP activity (figure 4E-F, indicated by the arrows).

TRAP expression is upregulated by RANKL and oxidative stressIn order to study what causes the higher activity and/or expression of TRAP in alveolar mac-rophages, we exposed MPI alveolar-like macrophages and murine precision-cut lung slices to various stimuli related to COPD and asthma, namely IL-4, M-CSF and RANKL, the dam-age-associated molecular pattern ATP, and oxidative stress mimicked by the xanthine/xan-thine oxidase (X/XO) system. TRAP mRNA expression in MPI alveolar-like macrophages was significantly higher after stimulation with RANKL and the X/XO system (figure 5A). TRAP expression was significantly lower after M-CSF incubation. There was no significant effect observed of ATP or IL-4 stimulation. To subsequently study TRAP activity we used lung slices incubated with RANKL, ATP and the X/XO system and measured TRAP activity in the slices. RANKL treatment resulted in significantly higher TRAP activity, but ATP and X/XO treatment did not (figure 5B).

The gold compound AubipyOMe inhibits TRAP activityTo be able to study TRAP function, we evaluated a group of gold-containing compounds as possible TRAP activity inhibitors. Thus, a series of gold coordination compounds (including mono- and di-nuclear Au(III) compounds with N-donor ligands, as well as the anti-rheu-matic agent sodium aurothiomalate (Myochrysine®, see supplemental figure 1 for the struc-tures of the compounds tested) were tested for their ability to inhibit recombinant human TRAP activity.

The initial screening revealed that the compound AubipyOMe possessed the best TRAP activity inhibitory capacities compared to the previously described TRAP activity inhibitor NaAuCl

4 3,35. Both AubipyOMe and NaAuCl

4 inhibited the activity of recombinant TRAP in a

dose-dependent manner (figure 6A) with IC50 values in the nM range (see table 1 for a com-plete overview of the IC50 values). We therefore continued testing AubipyOMe and NaAuCl

4,

the latter used as


Table 1. Effect of gold compounds on TRAP activity from different samples.

IC50 (mM)

CompoundsIC50 (mM)

Recombinant TRAP MPI macrophages cell lysates

COPD patients cell lysates

NaAuCl4 0.280 ±0.095 0.470 ±0.477 4.12 ±2.65

AubipyOMe 0.476 ±0.666 2.951 ±2.095 13.98 ±5.61

Authiomalate No inhibition - -

Auterpy* 1.643 ±1.437 - -

Auoxo1 No inhibition - -

*: Auterpy was found to be toxic to macrophages.

reference compound, in more relevant biological settings. Indeed, TRAP activity in cell lysates of MPI macrophages was also significantly inhibited in the presence of AubipyOMe or NaAuCl

4 with IC50 values of 2.95±2.1 mM and 0.47±0.48 mM respectively (figure 6B). Impor-

tantly, the inhibitory effects of AubipyOMe and NaAuCl4 were also tested on TRAP activity in

pooled lung lysates from COPD patients. AubipyOMe significantly inhibited TRAP activity in these lysates with an IC50 value of 14.0±5.6 mM, while NaAuCl

4 inhibited the activity with an

IC50 value of 4.1± 2.7 mM (figure 6C). In addition, AubipyOMe only had cytotoxic effects on RAW macrophages in very high concentrations (IC50 ca. 30 mM, figure 6D) and AuCl

4 did not

display significant toxicity (IC50>200 mM).

Figure 5. RANKL and oxidative stress are inducers of TRAP. (A) TRAP mRNA expression in MPI macrophages was higher after stimulation with xanthine/xanthine oxidase and after RANKL (200ng/ml) stimulation. M-CSF (10ng/ml) significantly downregulated TRAP mRNA expression. ATP (10mg/ml) and IL-4 (10ng/ml) had no effect. (B) TRAP activity was higher in precision-cut lung slices incubated with RANKL (200ng/ml) and no effect on TRAP activity was seen following stimulation with ATP (10mg/ml) or oxidative stress (10mU/ml). Stimulus out-come was calculated relative to the vehicle control. Results shown represent at least four inde-pendent experiments and differences were tested by a two-way Kruskal-Wallis and a Dunn’s post test, *P<0.05.


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Macrophage migration depends on TRAP activity and is inhibited by AubipyOMeOsteoclast migration is suggested to be TRAP-dependent through the ability of TRAP to dephosphorylate osteopontin 14,36. To investigate if this is also the case for macrophages we used RAW 264.7 macrophages because we could modulate TRAP activity from low to high by pretreating with RANKL in these cells, which we could not do in the same extent with MPI macrophages. We induced TRAP activity in macrophages with RANKL and incubated cells with or without AubipyOMe to investigate effects of TRAP activity and the inhibition of TRAP

Figure 6. AubipyOMe is a potent TRAP inhibitor. (A) TRAP activity of commercially pur-chased recombinant human TRAP was inhibited by NaAuCl

4 (IC50: 0.28±0.09 mM) and by

AubipyOMe (IC50: 0.48±0.67 mM). (B) TRAP activity in lysates of murine MPI macrophages was inhibited by NaAuCl

4 (IC50: 0.47±0.48 mM) and by AubipyOMe (IC50: 3.0±2.1 mM). (C)

TRAP activity in lysates from COPD lung tissue was inhibited by NaAuCl4 (IC50: 4.1±2.7 mM)

and by AubipyOMe (IC50: 14.0±5.6 mM). The inhibitory effect was calculated as the ratio of absorbance between treated and untreated cells. The IC50 value was calculated as the con-centration of inhibitor causing a 50% reduction in TRAP activity. Data represent at least three independent experiments. (D) MTT assay performed on RAW 264.7 macrophages resulted in an IC50 value of 29.3±4.6 mM for AubipyOMe. NaAuCl4 inhibited cell growth with an IC50 value of >200 mM. The percentage of cell viability was calculated from the ratio of absorbance between treated and untreated cells. The IC50 value was calculated as the concentration inhib-itor reducing the proliferation of the cells by 50%. Data is presented as a mean ±SEM of four independent experiments.


activity on macrophage cell migration in a transwell setup. Cells prestimulated with RANKL migrated significantly more through an osteopontin-coated membrane into the lower com-partment as compared to unstimulated cells (figure 7A). RANKL stimulation in combination with the inhibitor AubipyOMe led to significantly less migration of macrophages as compared to just RANKL stimulation. Incubation with the inhibitor alone did not affect macrophage migration. This effect was osteopontin-specific because RANKL stimulation did not lead to enhanced migration through a collagen-coated membrane and this was also not influenced by the inhibition of TRAP activity by AubipyOMe (figure 7B).

Figure 7. TRAP is involved in macrophage migration. (A) In a transwell set-up, RAW 264.7 macrophages were allowed to migrate over an osteopontin-coated membrane with or with-out RANKL stimulation (200ng/ml). RANKL-stimulated macrophages migrated significantly more through an osteopontin-coated membrane as compared to vehicle-stimulated macro-phages. (B) RAW 264.7 macrophages were allowed to migrate through a collagen-coated mem-brane with or without RANKL stimulation (200ng/ml). No differences were observed between RANKL stimulated and unstimulated conditions. The number of cells that had migrated from the insert to the bottom well was calculated relative to the number of unstimulated cells that had migrated. Each experiment was performed in quadruplicate and performed 6 times. (C) Live cell tracking of macrophages in an osteopontin-coated well revealed that macrophage migratory behavior was higher in the presence of RANKL (200ng/ml) as compared to the vehi-cle-treated control and AubipyOMe inhibited this migration (movies are provided in the online supplemental data, Movies 1-4). Differences were tested with a Kruskal-Wallis test and a Dunn’s post test, *P<0.05.


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Furthermore, we investigated whether RANKL stimulation of RAW macrophages led to increased macrophage migration using a live cell imaging. Cells that were plated on wells coated with osteopontin showed more migratory behavior following RANKL stimulation (figure 7C, movie 1 versus 2 in supplemental data) than unstimulated cells. This increased migratory behavior was not observed in the presence of AubipyOMe (figure 7C, movies 3 and 4, supplemental data).

Discussion

We observed high TRAP expression in the lungs of COPD patients and high TRAP activity in the lungs of fatal asthma patients and mouse models of COPD and asthma. This increase may be triggered by RANKL and/or oxidative stress, though the functional consequences of this higher expression and activity remain to be determined. To aid the elucidation of the role of TRAP in the lungs, we developed a new type of TRAP-inhibitor: AubipyOMe. This gold(III) bearing compound is a potent inhibitor of the phosphatase function of TRAP and using this inhibitor we first studied whether AubipyOMe was able to inhibit macrophage migration, as this is one of the known functions of TRAP in osteoclasts and this may also apply to (alveo-lar) macrophages. We indeed showed that higher TRAP activity may be related to enhanced migration of (alveolar) macrophages and that our inhibitor was able to limit migration.

To the best of our knowledge, TRAP expression levels have never been studied before in rela-tion to COPD. Only two studies looked at the effect of smoking, the most important risk fac-tor of COPD, on TRAP mRNA and/or protein expression: one reported higher TRAP mRNA expression in the lungs of smokers and the other showed higher TRAP protein levels in alve-olar macrophages obtained from smokers 17-19,37,38. Our results confirm that current smoking results in higher TRAP mRNA expression and we additionally show that TRAP activity is higher in the lungs of mice exposed to cigarette smoke. Moreover, TRAP mRNA expression is also higher in lung tissue of COPD patients compared with controls, independent of smoking status. TRAP mRNA expression is higher with the highest disease severity, therefore, a dis-ease-specific factor may be causing the increase in TRAP expression on top of smoking-re-lated induction of TRAP.

TRAP has not been studied in the context of asthma as well. This chronic lung disease has a different pathogenesis from COPD but was also characterized by higher numbers of TRAP-ac-tive macrophages in lung tissue in both fatal asthma and a murine model of asthma as com-pared to controls. This higher number of TRAP-active macrophages most likely results from an increase in TRAP activity, as all alveolar macrophages express TRAP protein, but not all of it is found to be active (data not shown). Furthermore, Draijer et al. showed that the actual number of alveolar macrophages remained unchanged after induction of asthma 27, confirm-ing this conclusion. Hence, we investigated some overlapping cytokines and conditions of both obstructive lung diseases to identify the cause of the high TRAP expression and TRAP activity in these lung diseases.


When considering the regulation of TRAP in lung diseases, in vitro experiments showed that the most likely candidate to induce TRAP expression/activity is RANKL. RANKL is a well-known inducer of TRAP expression in osteoclasts (multinuclear bone macrophages) and was included as a positive control. However, higher levels of RANKL have also shown to be present in patients with COPD, especially those suffering from osteoporosis, a known comorbidity of emphysematous COPD 20,21,39. Therefore, the high levels of circulating RANKL may explain the high TRAP expression and activity in lung tissue of COPD patients. The fact that especially GOLD stage IV patients, transplanted for severe COPD, have the highest mRNA expression of TRAP is in line with this observation. The high RANKL levels may explain the high TRAP expression and activity in COPD; it does not explain the high TRAP activity found with fatal asthma patients. To our knowledge, no reports have been published about the levels of RANKL in (fatal) asthma patients, therefore it is unclear whether and how RANKL could play a role in (fatal) asthma.

Our results furthermore showed that TRAP mRNA expression is higher in macrophages cul-tured under high-oxidative-stress conditions. Indeed, oxidative stress is a known inducer of osteoclast formation accompanied by increased TRAP5b expression in these cells 22,31. Patients with COPD have been shown to have high levels of oxidative and nitrosative stress in their lungs 34,40 and in asthma basal oxidative stress levels are elevated as a result of chronic inflam-mation 35,41,42. Local anti-oxidants apparently possess insufficient capacity to counteract this oxidative stress 36,41,43, which in turn may affect macrophage TRAP mRNA expression 1,37,38,43,44. Interestingly, the higher TRAP expression following oxidative stress in macrophages in vitro did not result in higher TRAP activity in lung slices that were cultured under similar condi-tions. The reason for this disconnect is unclear but may relate to the slicing and subsequent incubation of lung tissue, which may have exhausted reducing agents like superoxide dis-mutase in the tissue that are necessary for optimal TRAP activity 4,41,42.

TRAP itself may also contribute to oxidative stress levels in the lung through its oxygen radical producing potential. Alveolar macrophages are an important first-line defense against patho-gens and their TRAP expression was shown to contribute to ROS formation and bacterial killing 41,43,45,46. In addition, evidence already supports a role for TRAP in immune-regulatory processes in the lung 1,43,44,47. The monomeric, intracellular TRAP5a isoform can generate cel-lular oxidative stress through oxidation of one of the iron atoms of TRAP 43,48,49. As both COPD and asthma are characterized by high oxidative stress conditions, high TRAP expression/activity may even contribute to these levels.

To further aid the study of the function of (elevated) TRAP activity in alveolar macrophages, we developed a new potent TRAP inhibitor, namely the gold(III) compound AubipyOMe, that effectively inhibits the activity of human and murine TRAP. In addition to its contribu-tion to intracellular ROS production, TRAP can act as a phosphatase and dephosphorylate osteopontin 41,43,50,51. Our inhibitor has been tested as a phosphatase inhibitor only, and we therefore investigated a possible function of the phosphatase activity. For osteoclasts it has been shown that the extracellular TRAP5b isoform activity contributes to osteoclast migration by dephosphorylation of osteopontin thereby reducing (α

v3-integrin-mediated) cell adhesion

of osteoclasts 14,52-58. We now show that TRAP activity in macrophages has a similar function,


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because upregulation of TRAP by RANKL stimulation resulted in more macrophage migra-tion. In addition, subsequent inhibition of the phosphatase function of TRAP by AubipyOMe inhibited macrophage migration.

Functional consequences of enhanced macrophage migration in lung tissue in diseases like COPD and asthma remain to be investigated. Work of the group of Väänänen et al. showed that TRAP is involved in matrix degrading processes by assisting in trafficking of collagens in vesicles through the cell and that TRAP colocalized with phagocytosed material within alveolar macrophages 4,41,59-62. Therefore, alveolar macrophage TRAP may be involved in the tissue remodeling processes that play a role in both asthma and COPD. Another possible role for TRAP in COPD and asthma could be the regulation of interferon alpha (IFNα) production. Both COPD and asthma are characterized by exacerbations, often induced by viral infections 16-18,45,46. IFNα is important in defenses against viruses and Briggs et al. recently showed that TRAP inhibits IFNα production by regulating intracellular levels of phosphorylated osteo-pontin in dendritic cells 24,47. Expression of nonfunctional TRAP led to higher levels of IFN and therefore high expression of TRAP (like in COPD and asthma) may result in a lower IFNα production. Indeed, lower levels of IFN have been detected in patients with asthma and COPD 25,48,49. The gold-based TRAP inhibitor AubipyOMe could be used to investigate the role of TRAP in IFNα production by alveolar macrophages. An unexpected outcome was the clear downregulation of TRAP expression in alveolar-like macrophages after M-CSF stimula-tion. In the bone field, many publications have shown that M-CSF is necessary for osteoclast formation as determined by the formation of TRAP-expressing/TRAP-active multinucleated cells. This discrepancy in M-CSF responsiveness may be caused by the fact that we used alve-olar-like macrophages, which are derived from fetal monocytes/yolk sac macrophages and self-maintain in lung tissue during life, while osteoclasts are derived from hematopoietic stem cells and replenished from bone marrow 27,52,54,56,57. Since alveolar macrophages are particu-larly dependent on GM-CSF for their development, this could explain the discrepancy in the response to M-CSF 59,61,62.

There are still some unknown aspects regarding the inhibitor we developed. For example, its selectivity should be studied in more detail to elucidate whether AubipyOMe also inhibits other phosphatases or enzymes. In addition, it would be of interest to determine the affinity of AubipyOMe towards the two TRAP isoforms, namely the TRAP5a monomer and the more active TRAP5b dimer, as their functions differ 41. Therefore, structural modifications to the inhibitor could be considered to improve its selectivity and affinity. Moreover, we have shown AubipyOMe inhibits the phosphatase function of TRAP, but it is unknown whether this inhib-itor can affect ROS production.

In conclusion, TRAP expression and activity are high in COPD and fatal asthma and in rele-vant mouse models. One of the roles of TRAP may be to facilitate macrophage migration and ROS production, but the consequences of such effects on the pathogenesis of COPD and (fatal) asthma are still unclear. The development of our potent gold-based TRAP inhibitor now allows more detailed studies into the function of TRAP in the lung and diseases of the lung characterized by higher TRAP activity such as asthma and COPD.


Acknowledgements

AC and BM are both members of COST action CM1106. BM and CAB are both members of COST action BM1201. Both COST actions are gratefully acknowledged for financial support and fruitful discussions. Thais Mauad is funded by CNPq (Brazilian National Research Coun-cil) and gratefully acknowledges this financial support. All master students that contributed to this paper are acknowledged for their hard work: Tirza Timmerman, Bram Maillie. And fellow PhD students/research technicians: Fransien van Dijk and Christina Draijer were of great help performing the experiments. YB holds a Canada Research Chair in Genomics of Heart and Lung Diseases.


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Supplementary file captions

Supplemental table 1. Patient characteristic of patients with and without fatal asthma (median + range is displayed).

Control (n=8) Fatal asthma (n=10)

Sex, male/female 5/3 5/5

Age, years 49.5 (37-58) 35 (20-66)

Smoking history

Nonsmokers 8 5

Smokers 0 5

Exsmokers 0 0

Corticosteroid use

Oral 0 2

Inhaled 0 0

Cause of death

Asthma 0 10

Cardiovascular disease 7

Acute pancreatitis 1

Supplemental table 2. Patient characteristic of COPD patients from whom lung tissue was used for the preparation of lung tissue lysates (median + range is displayed).

COPD (n=18)

Sex, male/female 12/6

Age, years 66 (35-80)

Smoking status 18 ex-smoker

FEV1 % pred. 62 (36-77)

GOLD stage GOLD II = 15GOLD III = 3

Supplemental able 3. Patient characteristics of control and COPD patients used to assess TRAP mRNA expression (median + range is displayed).

Control (n=270) COPD (n=311)


Age, years 61 (37-80) 63 (33-84)

Smoking status 197 ex-smoking73 current smoking

222 ex-smoking89 current smoking

FEV1 % pred. 98% (80-136) 57 (11-83)

FEV1/FVC 76 (70-91) 53 (16-70)

GOLD stage - GOLD I = 1, GOLD II = 216GOLD III = 27, GOLD IV = 56 Undetermined = 11


Supplemental figure 1. Chemical structures of the gold-containing compounds tested for their potential TRAP inhibitory capacities.

Movies 1-4Live cell tracking of macrophages on an osteopontin-coated well revealed that macro-phage migration was higher in the presence of RANKL (200ng/ml) as compared to the vehi-cle-treated control and AubipyOMe inhibited this mobility. Live cell tracking of macrophages was conducted in the presence of a vehicle control (Movie 1), RANKL (200ng/ml) (Movie 2), the inhibitor AubipyOMe (320ng/ml) (Movie 3) and RANKL (200ng/ml) in combination with the inhibitor AubipyOMe (320ng/ml) (Movie 4).

Chapter 4

Current smoking is associated with fewer CD16+ monocytes in peripheral blood of male healthy controls and patients with COPD

Barbro N. Melgert1,4, Carian E. Boorsma1,4, Dirkje S. Postma2,4, Wouter H. van Geffen2,4, Huib A.M. Kerstjens2,4, Machteld N. Hylkema3,4, Wim Timens3,4 and Corry-Anke Brandsma3,4

1University of Groningen, Groningen Research Institute of Pharmacy, Department of Phar-macokinetics, Toxicology and Targeting, The Netherlands. 2 University of Groningen, Univer-sity Medical Center Groningen, Department of Pulmonary Diseases and Tuberculosis, The Netherlands. 3 University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, The Netherlands. 4 University of Groningen, University Medical Center Groningen, GRIAC Research Institute, The Netherlands.

Manuscript in preparation.


Abstract Macrophages play an important role in the pathogenesis of smoking-related lung disorders like chronic obstructive pulmonary disease (COPD) and can develop from monocytes entering the lungs. Human monocytes comprise CD14+ classical and CD16+ intermediate and non-classical subsets and have different capacities to replenish tissue macrophages. It is currently unknown if circulating monocyte subsets are altered by smoking and/or having COPD and we therefore compared percentages of monocytes of the different subsets and expression of oxidant-sensing TLR2/4 receptors in current and ex-smoking COPD patients (n=20) and healthy controls (n=19).

The presence of total, classical, nonclassical and intermediate monocytes and their TLR2/4 expression was assessed by flow cytometry in blood of 20 male smoking and ex-smoking COPD patients (age range 58-80) and 29 healthy male smoking, never and ex-smoking indi-viduals (age range 46-84).

We found higher percentages of inflammation-associated classical and intermediate mono-cytes in COPD patients than in healthy individuals, but this was not seen anymore after correcting for age. Higher age was associated with more classical and intermediate mono-cytes. Current smoking resulted in lower percentages of CD16+ intermediate and nonclassical monocytes with more TLR2/4 expression compared to ex-smoking. CD16+ intermediate and nonclassical monocytes had higher expression of TLR2/4 as compared to CD14+ classical monocytes.

The disease COPD was not associated with changes in monocyte subsets or their expression of oxidant sensors TLR2/4, whereas current smoking was. The lower percentages of CD16+ monocytes with higher TLR2/4 expression in peripheral blood of current smokers as compared to ex-smoking individuals may suggest migration of this subset, with heightened sensitivity to cigarette smoke components, into lung tissue.

Current sm

oking is associated with few

er cd16+ m

onocytes in peripheral blood of male healthy controls and patients w

ith copd

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Introduction

COPD is a chronic lung disease characterized by infiltration of inflammatory cells into the lungs and is mainly caused by long-term smoking. Accumulation of macrophages is par-ticularly found in lungs of smokers and COPD patients and their numbers are proportional to disease severity in COPD patients 1-7. This increase in macrophages can be the result of infiltrating monocytes and/or local proliferation of tissue-resident macrophages, but the exact contributions have not been determined yet. There is some evidence that suggests that at least part of the increase may be explained by infiltrating monocytes differentiating into macrophages 8.

Human monocytes consist of three subsets according to the Nomenclature Committee of the International Union of Immunological Societies and appear to have different functions 9,10. These monocytes are classified by expression of CD14 (lipopolysaccharide receptor) and CD16 (Fcγ receptor III) 10. CD14hiCD16-negative monocytes are commonly referred to as clas-sical monocytes, CD14hiCD16-positive monocytes as intermediate monocytes, and CD14-pos-itiveCD16hi monocytes as nonclassical monocytes. Classical monocytes can migrate to sites of injury and contribute to inflammation by producing cytokines like IL-6, IL-8 and reactive oxygen species (ROS) 11. Intermediate monocytes are also associated with inflammation and are the main producers of TNFα and IL-1β 11,12. Nonclassical monocytes patrol resting vascu-lature and remove debris and may have a more anti-inflammatory profile 13. They have also been thought to replenish some tissue resident macrophages and dendritic cells 11,14.

Both smoking and COPD are associated with an increase in total monocytes in the circu-lation, but it is unclear if this is associated with a specific increase in any of the predefined monocyte subsets 15-19. Since pro-inflammatory cytokines and ROS, produced by classical and intermediate monocytes, are known to contribute to the pathogenesis of smoke-induced lung damage and COPD, we postulated that these subsets would be increased in COPD and/or by current smoking 20. In addition, these subsets may be characterized by changed expressions of receptors responsible for inflammatory responses by monocytes, such as certain types of Toll-like Receptors (TLR). Especially TLR2 and TLR4 were found to be important for the responses of monocytes and macrophages to cigarette smoke 21-23. Data by Paul-Clark et al. showed that TLR2 is essential for initial sensing of oxidants in smoke-induced inflammation and that TLR4 is critically involved in a later phase of this type of inflammation 24.

In order to investigate whether current smoking and/or having COPD are associated with changes in types of monocytes and their expression of oxidant-sensing receptors TLR2/4, we quantified the percentages of total monocytes and the three subsets in current smoking and ex-smoking male individuals with COPD and compared these numbers with never-smok-ing, ex-smoking and current smoking healthy control individuals. We also investigated the expression of TLR2/4 on the three monocyte subsets in relation to smoking cigarettes and/or having COPD.



Patient populationCOPD patients and healthy individuals participated in this study that was also used to study peripheral B lymphocytes 25. Inclusion criteria for COPD patients were; clinical diagnosis of COPD, post bronchodilator FEV1 < 80% predicted, post bronchodilator FEV1/FVC < 70%, and no exacerbation in the six weeks preceding the study. Inclusion criteria for healthy individ-uals were; no symptoms of pulmonary disease, FEV1 > 90% predicted, and FEV1/FVC > 70%. All participants met the following criteria: age > 40 years, negative skin prick tests for the most common aeroallergens, no use of (inhaled or systemic) corticosteroids in the 6 weeks preced-ing the study and no major comorbidities. To avoid the known effects of sex on monocytes, only males were included in the study 26-28. Smokers and ex-smokers had a smoking history of at least 10 packyears and ex-smokers had quit smoking for a least one year. The Medical Ethics Committee of the University Medical Center Groningen approved the study and all participants gave their written informed consent.

Cell isolation All participants donated 20 ml of peripheral blood and peripheral blood mononuclear cells (PBMC) were isolated using ficoll-paque plus (GE Healthcare, UK) density gradient centrif-ugation. Total isolated cells were counted using a Sysmex pocH-100i cell counter (Sysmex, Roche, Germany) and were used for flow cytometry as described below.

Flow cytometric analysisPercentages of total classical, nonclassical and intermediate monocytes were assessed in PBMC using flow cytometry by staining with pacific blue-labeled anti-human-CD14 (1:12, Biolegend, San Diego, USA) and PerCP/Cy5.5-labeled anti-human-CD16 (1:4, BD Biosciences, NL). Expressions of TLR2 and TLR 4 were assessed using antibodies x (1:4, eBioscience, San Diego, CA) and y (1:4, eBioscience) in the same cocktail. Briefly, PBMC were transferred to a 96-well plate at a density of 1x106 cells. Samples were incubated protected from light for 30min at 4°C with the antibody cocktail for the identification of CD14, CD16, TLR2, and TLR4. Following washing with 2% bovine serum albumin (Serva, Heidelberg, Germany) in phos-phate-buffered saline, cells were resuspended in FACS lysing solution (BD Biosciences) and kept in the dark on ice until flow cytometric analysis. Fluorescent cell staining was measured on an LSR-II flow cytometer (BD) and data were analyzed using FlowJo software (Treestar, Ashland, USA). Our gating strategy is depicted in figure 1. Counts of monocytes and the sub-sets were expressed as percentage PBMC.

Statistical analysisThe normal distribution of the residuals of the other data was analyzed with a D’Agostino & Pearson normality test and when needed data were log-transformed to normalize distribu-tions. When this did not normalize the distribution a nonparametric test was chosen instead.

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Clinical characteristics of COPD patients versus controls (never, current and ex-smoking) and of current versus ex-smoking individuals (both COPD and control) were compared using a Mann-Whitney U test. A value of p<0.05 was considered significant.

Linear regression analysis was used to determine whether percentages of total monocytes and the three subsets were different by disease and smoking status and packyears level. Since COPD patients were slightly older than healthy individuals and age has been shown to affect monocyte subsets, we also added age as an independent factor to our regression model and evaluated relationships between monocyte subsets and age with Pearson correlation. P<0.05 was considered significant. Posthoc testing between ex- and current smokers was done using a Student’s t test and in the case of the TLR4 expression using a Mann-Whitney U test.

TLR2/4 expression between the different monocyte subsets was compared using a nonpara-metric Kruskal Wallis test. A post-hoc Dunn’s test was used to compare expression levels on the three subsets. P<0.05 was considered significant.

Data was analyzed using GraphPad Prism (Graphpad Software, La Jolla, CA).

Figure 1 Gating strategy for monocyte subsets based on their expression pattern of CD14 and CD16.


Results

Patient characteristics The characteristics of the twenty COPD patients (current and ex-smokers) and twenty-nine healthy volunteers (never, current and ex-smokers) that were included in the study are shown in table 1. Healthy individuals were significantly younger than the COPD patients which was mainly caused by the young age of the healthy smokers. Additionally, COPD patients had more packyears of smoking when compared to healthy current and ex-smokers. One healthy person was included as “never smoker” who had a smoking history of 2.5 packyears and had stopped smoking for 40 years, the other never smokers had no smoking history at all. When comparing current and ex-/never smoking individuals (COPD and control combined) no significant differences in age, FEV1 % predicted after bronchodilator, FEV1/FVC % predicted after bronchodilator, and packyears of smoking were found.

Table 1: Characteristics of COPD patients and healthy individuals. COPD patients did not receive corticosteroid treatment six weeks before and during the study.

Healthy individuals COPD patients

Never smokers Ex-smokers Current smokers

Ex-smokers Current smokers

Subjects (n) 10 10 9 10 10

Age (years) 58 (7) 61 (9) 53 (4)* 67 (7)† 66 (4)†

Packyears 0 (1) 21 (6) 25 (11) 37 (18)† 34 (14)†

FEV1 post BD (%pred)

111 (12) 116 (16) 106 (9) 61 (15)† 45 (15)†‡

FEV1/FVC post BD (%)

79 (4) 78 (6) 76 (4) 44 (11)† 38 (10)†

Mean (standard deviation) is depicted. Mann Whitney U tests were used to test differences between the groups. FEV1 = Forced expiratory volume in 1 second. FVC = Forced vital capac-ity. BD = Bronchodilator.* Healthy current smokers versus healthy ex-smokers p = 0.01 and versus healthy never smokers p = 0.045† COPD patients versus healthy individuals; packyears p = 0.006, age p = 0.000‡ COPD smokers versus COPD ex-smokers p = 0.03

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Figure 2 COPD patients (n=20, closed symbols) had significantly higher percentages of total monocytes (p=0.02) (A), classical monocytes (p=0.03) (B) and intermediate monocytes (p=0.008) (C) than healthy controls (n=29, open symbols). * p<0.05 as tested with a Students’ t test. No differences between COPD patients and healthy controls were seen for nonclassical monocytes (D).

Monocyte subsets: COPD versus controlCOPD patients had significantly higher percentages of total monocytes as compared to con-trols (figure 2A), most likely caused by the higher percentages of classical and intermediate monocytes in COPD patients than controls (figures 2B and C). Percentages of nonclassical monocytes were similar between the two groups (figure 2D). Our control subjects were sig-nificantly younger than the COPD patients and we therefore added age to our regression model. We found that percentages of total, classical and intermediate monocytes were influ-enced by age. In fact, the effect of age was larger than the effect of COPD. Age was significantly associated with total (p=0.02) and classical (p=0.01) monocytes in controls and associations with COPD for these populations were no longer significant when correcting for age. To untangle the effects of COPD and age, we assessed correlations between age and monocyte subsets separately for COPD patients and healthy individuals. Although power was relatively low for these analyses, we found that total, classical and intermediate monocytes correlated positively with age in healthy individuals, even within the narrow age range of 46-84 years (figures 3A-C), but they did not correlate with age in the group of COPD patients (age range: 58-80). Percentages of nonclassical monocytes did not correlate with age in both healthy controls as well as in COPD patients (figure 3D). When we subsequently excluded all young control individuals (age younger than 55) to match the ages of control individuals and COPD patients the correlation between age and monocyte subsets disappeared in the controls (data


not shown). Importantly, no significant differences for the monocyte subsets were found anymore between COPD patients and controls when we corrected for age (data not shown).

Figure 3 In healthy controls, percentages of total monocytes (A), classical monocytes (B), and intermediate monocytes (C) correlated significantly with age (open symbols with the dotted line), while such correlation was not found in COPD patients (closed symbols with the solid line). Tested with Pearson, p<0.05. No significant correlations were found for age and nonclas-sical monocytes (D) in healthy controls and COPD patients.

Monocyte subsets: current smoking versus never/ex-smokingWe also assessed whether current or never/ex-smoking influenced monocyte subsets. As current and never/ex-smoking individuals had a similar distribution with regard to age, dif-ferences between these individuals for the monocyte subsets were not influenced by age like they were for the COPD-control comparison. Our regression model, which included never, ex- and current smoking individuals, showed that only intermediate monocytes were lower in current smoking individuals as compared to never/ex-smoking (p=0.073). This comparison could have been influenced by a difference in FEV1 as current smokers had a significantly

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lower FEV1 % predicted than the never/ex-smokers. We therefore also compared ex-smokers with the current smokers, as these were completely similar with respect to age, lung function parameters and packyears. We found trends for lower percentages of total and intermediate monocytes (p=0.088 and p=0.075 respectively) and significantly lower percentages of non-classical monocytes (p=0.028) in current smokers as compared to ex-smoking individuals (figures 4A-D). Percentages of classical monocytes were similar between current smoking and ex-smoking individuals.

Figure 4 Current smoking resulted in trends towards lower percentages of total monocytes (A) and intermediate monocytes (C) when compared with ex-smoking individuals. A signif-icant lower percentage of nonclassical monocytes was found in current smokers compared with ex-smokers (D). Current smoking had no effect on the percentages of classical monocytes (A). Significance was tested with a two-way ANOVA on the ex- and current smokers with a Holm-Skidak’s post test, P <0.05 (*) was considered significant.

Expression of TLR2 and TLR4 on monocyte subsets: COPD versus controlWe further investigated what the expression levels of TLR2 and TLR4 were on the different monocyte subsets, irrespective of COPD or smoking status. We found that the CD16+ subsets, intermediate and nonclassical monocytes, had significantly higher expression of TLR2 and TLR4 than classical monocytes (figure 5A and B). When assessing all controls and COPD


patients, nonclassical monocytes had higher expression of TLR2 than intermediate mono-cytes (figure 5C). No differences in expression levels of TLR4 were found between interme-diate and nonclassical monocytes (figure 5D). We then continued to examine the effects of COPD and smoking status and their interaction using our linear regression model. No effects of having COPD and no interaction between COPD and smoking status were found with respect to TLR2/4 expression. We also did not find an effect of age on the expression of these two receptors either.

Figure 5 TLR 2 and 4 expressions were higher on CD16+ monocytes than on classical CD16-neg-ative monocytes (A-B). Nonclassical monocytes had significantly more TLR2 expression as compared with intermediate monocytes (C) but not TLR4 (D). Significance was tested with a nonparametric Kruskal Wallis test. A post-hoc Dunn’s test was used to compare expression levels on the three subsets. P<0.05 was considered significant.

Expression of TLR2 and TLR4 on monocyte subsets: current smoking versus never/ex-smokingWe then solely compared expression levels of TLR2 and TLR4 between current and never/ex-smokers. We found that TLR2 expression on nonclassical monocytes was significantly higher for current smokers as compared to never/ex-smokers (figure 6C, p=0.038). Further-more, a trend for higher TLR4 expression on intermediate monocytes of current smokers as compared to never/ex-smoking individuals (figure 6E, p=0.087). When taking the never smokers out of the equation by comparing ex- and current smoking individuals directly we found that current smoking was associated with significantly higher expression levels of TLR4 on intermediate and nonclassical monocytes and a trend for higher expression of TLR2 on nonclassical monocytes.

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Figure 6 No differences in TLR2 expression were found on any of the other subsets (A and B). TLR2 expression on nonclassical monocytes was higher in current smoking individuals as compared with never/ex-smoking individuals (C). TLR4 expression showed a trend towards a higher expression on intermediate monocytes (E), but no changes were found on any of the other subsets (D and F). TLR2/4 expression between groups was compared using a nonpara-metric Kruskal Wallis test. Posthoc testing between ex- and current smokers was done using a Student’s t test and in the case of the TLR4 expression using a Mann-Whitney U test. P<0.05 was considered significant.


Discussion

Our study is, as far as we know, the first to investigate the effects of COPD and smoking on the different monocyte subsets in peripheral blood. Our data have shown that percentages of classical and intermediate monocytes were higher in our COPD patients as compared to the healthy individuals but our data also showed that this difference was completely deter-mined by a difference in age between the two groups. Our COPD patients were older than the control individuals and we found that the older the individual is, the higher the percentages of classical and intermediate monocytes are (and therefore also total monocytes). Therefore, having COPD does not appear to affect the percentages of the different monocyte subsets. However, we did find that current smoking has an effect: both types of CD16+ monocytes were lower in current smokers than in ex-smokers and had higher expression of TLR2 (nonclassical monocytes) and TLR4 (intermediate and nonclassical monocytes).

COPD is characterized by lung inflammation with higher levels of pro-inflammatory cyto-kines and higher production of ROS, we therefore hypothesized that the subsets of monocytes being able to produce these mediators, i.e. classical and intermediate monocytes, would be higher in COPD. When correcting for age this is not what we found. It may be possible that the contribution of monocytes to these phenomena in COPD is minor and that tissue resident cells, like macrophages, are producing most of these pro-inflammatory mediators.

The strong effect of age we found on the percentages of monocytes in all individuals (healthy and COPD combined) and in all healthy individuals separately is in line with recent stud-ies showing associations of innate immune activation and shifts in monocyte subsets with aging in healthy individuals 28,29. The lack of this relationship in only COPD patients may be the result of the relatively small age range available for these COPD patients or may indicate these patients have already reached a plateau of innate immune activation. Younger COPD patients are not available to us in our institute, therefore, inclusion of more COPD patients could increase power and may help us to untangle the effects of age and COPD.

Interestingly, we did find an effect of current smoking on the presence of the different mono-cyte subsets. Lower percentages of the CD16+ subsets intermediate and nonclassical mono-cytes were associated with current smoking. Of the studies investigating the effects of smok-ing on monocytes, most, but not all, showed that more total monocytes were associated with current smoking 15-19,30,31. As intermediate and nonclassical monocytes only make a minor contribution to the total population, we found a trend towards lower percentages of total monocytes as well, which is opposite of most previous findings. We have no clear explanation for this difference other than the fact that in most of these studies monocytes were deter-mined using a hematology analyzer or differential staining and not flow cytometry, which may explain some of the difference. In addition, most of these studies report significantly higher absolute counts but no differences in monocytes when expressed as percentage of white blood cells. Our data are expressed as percentage PBMC, but when we calculate abso-lute numbers we still do not find a significant higher number of total monocytes in smokers as compared to never smokers.

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What could be the functional consequence of having less CD16+ monocytes in the circula-tion when smoking? It is conceivable that these subsets are specifically recruited from the circulation to lung tissue for specific tasks to deal with the stress of cigarette smoke exposure. CD16+ intermediate and nonclassical monocytes express high levels of the chemokine recep-tor CX3CR1 (fractalkine receptor) and this receptor has been shown to play a critical role in attracting mononuclear cells like monocytes to the lung when exposed to cigarette smoke 32,33. In addition, expression of the ligand for this receptor, CX3CL1, was shown to be higher in lung tissue of smoking humans and mice explaining why CX3CR1+CD16+ monocytes can be specifically recruited to lung tissue 34.

Current smoking also had an effect on the expression of TLR receptor 2 and 4 and interest-ingly specifically on the subsets of monocytes of which percentages were affected by smok-ing. CD16+ intermediate and nonclassical monocytes had higher expression of TLR2 and 4 than classical monocytes and we found even higher expression of these in current smokers as compared to ex-smokers. The differential expression of TLR2 and 4 between the different monocyte subsets has been shown before for the combined CD16+ monocytes versus clas-sical monocytes. Iwahashi et al. showed higher expression of TLR2 on CD16+ monocytes and Skinner et al. showed higher expression of TLR4, but not TLR2, on CD16+ monocytes compared to classical monocytes 35,36. We now show that CD16+ nonclassical monocytes have the highest expression of TLR2 of the three subsets and that both CD16+ subsets have higher expression of TLR2 and 4 than classical monocytes. Why TLR4 expression on intermediate monocytes and TLR2/4 expression on nonclassical monocytes are even higher in smokers compared to ex-smokers is an open question. Iwahashi et al. showed that TLR2 expression on CD16+ monocytes was induced by IL-10 and M-CSF and reduced by TGFβ. Smoking was found associated with higher levels of M-CSF in bronchoalveolar lavage and serum and this could potentially also have systemic effects, possibly explaning the higher expression of TLR2 on CD16+ monocytes 37,38. We could not find any studies investigating regulation of TLR4 expression in CD16+ monocytes specifically, but TLR4 expression on monocytes was found inducible by IFNγ 39. Smoking was found associated with higher levels of IFNγ and could potentially have increased TLR4 expression on CD16+ monocytes 40.

The fact that TLR2 and 4 expressions increase on CD16+ monocytes may have some interest-ing functional consequences. TLR2 was shown to be essential for sensing oxidants (like found during smoking) and TLR4 played a critical role in the inflammation induced by oxidants 23. Having monocytes with increased expression of these TLRs may make them more sensitive to the effects of smoking. Combining this with the possibly increased migration of these CD16+ monocytes into lung tissue, may suggest the lung is trying to respond to the stress of cigarette smoking.

In conclusion, the disease COPD was not associated with changes in monocyte subsets or their expression of oxidant sensors TLR2/4. Instead we found a very strong effect of age on the presence of classical and intermediate monocytes, with higher percentages of these mono-cytes being present in older subjects. Current smoking did affect the percentages of the dif-ferent monocyte subsets. The lower percentages of CD16+ monocytes with higher TLR2/4 expression in peripheral blood of current smokers as compared to ex-smoking individuals may suggest migration of this subset, with heightened sensitivity to cigarette smoke compo-nents, into lung tissue.


References



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19. Rumora, L. et al. Levels changes of blood leukocytes and intracellular signalling path-ways in COPD patients with respect to smoking attitude. Clin Biochem 41, 387–394 (2008).


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21. Hansel, T. T. & Barnes, P. J. New drugs for exacerbations of chronic obstructive pulmo-nary disease. The Lancet 374, 744–755 (2009).

22. Karimi, K. et al. Toll-like receptor-4 mediates cigarette smoke-induced cytokine pro-duction by human macrophages. Respir Res 7, 66 (2006).

23. Paul-Clark, M. J. et al. Toll-like receptor 2 is essential for the sensing of oxidants during inflammation. Am J Respir Crit Care Med 179, 299–306 (2009).

24. Noble, P. W. & Homer, R. J. Back to the future: historical perspective on the pathogenesis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol 33, 113–120 (2005).

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32. Rius, C. et al. Critical role of fractalkine (CX3CL1) in cigarette smoke-induced mononu-clear cell adhesion to the arterial endothelium. Thorax 68, 177–186 (2013).

33. Xiong, Z., Leme, A. S., Ray, P., Shapiro, S. D. & Lee, J. S. CX3CR1+ lung mononuclear phagocytes spatially confined to the interstitium produce TNF-alpha and IL-6 and pro-mote cigarette smoke-induced emphysema. J Immunol 186, 3206–3214 (2011).

34. McComb, J. G. et al. CX3CL1 Up-Regulation Is Associated with Recruitment of CX3CR1+ Mononuclear Phagocytes and T Lymphocytes in the Lungs during Cigarette Smoke-In-duced Emphysema. Am J Pathol 173, 949–961 (2008).

35. Iwahashi, M. et al. Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis. Arthritis Rheum 50, 1457–1467 (2004).

36. Skinner, N. A., MacIsaac, C. M., Hamilton, J. A. & Visvanathan, K. Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens. Clin Exp Immunol 141, 270–278 (2005).

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39. Rehli, M. et al. PU.1 and interferon consensus sequence-binding protein regulate the myeloid expression of the human Toll-like receptor 4 gene. J Biol Chem 275, 9773–9781 (2000).

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Chapter 5

Characterization of Interstitial Macrophages Around Airways of Current and Ex-smoking COPD Patients and Controls

Carian E Boorsma1,3, Christina Draijer1,3, Corry-Anke Brandsma2,3, Wim Timens2,3, Barbro N Melgert1,3

1University of Groningen, Department of Pharmacokinetics, Toxicology and Targeting, Gron-ingen Research Institute for Pharmacy, Groningen, The Netherlands. 2University of Gronin-gen, University Medical Center Groningen, Department of Pathology, Groningen, The Neth-erlands. 3University of Groningen, University Medical Center Groningen, GRIAC Research Institute, Groningen, The Netherlands.

Manuscript in preparation.


AbstractMacrophages significantly contribute to the development of chronic obstructive pulmonary disease (COPD). Little is known about their relationship with airway remodeling and which polarization types of macrophages are present in airways of COPD patients. Therefore, we studied numbers of airway interstitial macrophages, their polarization states M1, M2, or M2-like and basement membrane thickness in central airways of COPD patients and non-COPD controls.

Large airway sections from ex- and current smoking individuals with COPD (n=27) or with-out COPD (n=29), that underwent resection surgery for suspected cancer, were stained for macrophages with CD68 in combination with a macrophage polarization marker: IRF5+ for M1 macrophages, CD206+ M2 macrophages and IL-10+ for M2-like macrophages. Effects of disease and smoking status were assessed and macrophage numbers were correlated with lung function (FEV1 %pred.) and basement membrane thickness.

Numbers of total macrophages and the three subsets as well as basement membrane thick-ness were similar between COPD patients and controls. Lower FEV1 was associated with hav-ing more IRF5+ M1 macrophages and a thicker basement membrane in COPD patients. Base-ment membrane thickness correlated negatively with M2 macrophages in ex-smoking COPD patients. Current smoking was associated with lower numbers of total and M1 macrophages compared to ex-smoking.

In conclusion, having COPD was not associated with changes in interstitial airway macro-phage phenotypes or greater basement membrane thickness, whereas current smoking did lead to lower numbers of interstitial M1 macrophages. The negative associations of lung func-tion with M1 macrophages and basement membrane thickness in COPD patients stress the importance and the possible contributions of the different macrophage subsets to obstructive airway disease.

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Introduction

A number of reports have shown that macrophages in the lung play an important role in main-taining tissue homeostasis but that they can also significantly contribute to the development of chronic obstructive pulmonary disease (COPD) 1-4. COPD is characterized by pulmonary inflammation, often caused by smoking, leading to a varying combination of chronic bron-chitis with fibrosis around airways and emphysema with loss of parenchymal lung tissue 1-7.

Macrophages can polarize into various phenotypes depending on tissue-derived or exog-enous signals and these phenotypes present themselves as a spectrum rather than distinct phenotypes 8,9. Within this spectrum three main phenotypes are recognized: 1. A proinflam-matory type (also known as M1), induced by IFNγ, TNFα, and microbial products, and char-acterized by expression of interferon regulatory factor 5 (IRF5) 10,11; 2. A tissue repair-associ-ated type (also known as M2), induced by IL4 and/or IL13, and characterized by expression of uptake receptors like CD206 and Mfge8 12,13; 3. An anti-inflammatory type (also known as M2-like or M2c), induced by IL10, corticosteroids and PGE2, and characterized by production of high levels of IL10 and expression of CD163 8,14. For the sake of simplicity, the names M1/M2/M2-like are adopted for these three main subsets throughout this report.

Although a role of macrophages in the development of COPD is generally accepted, little is known about the presence of the different phenotypes in lung tissue of COPD patients and their association with different aspects of the disease. To the best of our knowledge no quan-tification of macrophage phenotypes in COPD compared to controls have been published for either alveolar or interstitial macrophages. Alveolar macrophages have been studied most in COPD with respect to changes on a genetic, cytokine-producing and cellular level. Shaykhiev et al. demonstrated that bronchoalveolar lavage macrophages of COPD patients show sub-stantial suppression of M1-related genes and partial induction of genes associated with M2 polarization as compared to healthy individuals 15. This partial suppression/induction of the M1/M2 repertoire was also shown by Hodge et al. in studies showing a mixed phenotype in alveolar macrophages of smoking COPD patients with some M1 (MHC II expression) and M2 (efferocytosis) markers going down and some going up (proinflammatory cytokine produc-tion and DC-SIGN expression) compared to healthy individuals 2-5,16,17.

None of the studies to date, however, have investigated polarization changes in interstitial macrophages around airways. This is of interest because COPD is characterized by chronic bronchitis with remodeling of the airways. Especially M2 macrophages are associated with fibrotic processes and we therefore hypothesized that the macrophages around the airways in COPD patients would show preferential polarization towards M2. To investigate this hypothe-sis and to gather information about changes in interstitial macrophage polarization, we quan-tified the three main polarization states of macrophages in the central airways of subjects undergoing lung resection for suspected cancer with or without mild to moderate COPD (GOLD stage II and III). Macrophage phenotypes were identified based on their expression of IRF5, CD206, or IL10 in combination with the general macrophage marker CD68 and we correlated their presence with various clinical parameters and basement membrane thick-ness as a marker of airway remodeling.


Materials and Methods

SubjectsThe study protocol was consistent with the Research Code of the University Medical Center Groningen (UMCG) and Dutch national ethical and professional guidelines 6,7,18. Lung tis-sue included in this study was obtained from individuals with COPD (n=27) and non-COPD control subjects (n=29) undergoing lung surgery for suspected cancer. These subjects were part of a larger cohort and the material was previously collected by the UMCG (for more detailed information on this study see Hao 2012 8,9,19). The group of COPD patients included patients with GOLD stage II and III and both ex- and current smokers were included. COPD was defined as FEV

1/FVC ratio <70%. The control group consisted of subjects with normal

lung function (FEV1/FVC >70%), no history of lung disease and included ex-smokers (min 5 years of smoking cessation) and current smokers. Subjects with other lung diseases such as asthma, cystic fibrosis, or interstitial lung diseases were excluded. Patient characteristics are reported in table 1.

Table 1. Characteristics of patients whose central airway lung tissue was used to determine the number of macrophages in airway walls.

Control (n=26) COPD (n=27)


Age, years 60 (45-76) 64 (35-82)

Smoking status Ex-smokersCurrent smokers

1214

1512

FEV1 % predicted 98 (84-114) 66 (45-78)

FEV1/FVC 76 (71-90) 55 (40-69)

Diagnosis Suspected lung cancer COPD + suspected lung cancer

GOLD stage Stage II: 25Stage III: 2

Corticosteroid use 1/26 2/27

Patients underwent surgical resection for suspicion of carcinoma. Noncancerous normal lung tissue was obtained as far distant from the tumor as possible and lung tissue of each patient was stained with a standard haematoxylin and eosin staining and checked for abnormalities by a lung pathologist (WT). Collected tissue was fixed with 4% formalin and paraffin imbed-ded. Lung tissue sections of 2.5 mm thickness were used for all analyses.

HistologyWe assessed the total number of macrophages and the three main subsets in the submu-cosa of a transversally cut large airway (average internal diameter of 5 mm ±SD 2.0 mm). The total number of macrophages in each section was identified by a single CD68 staining




127 5

(anti-CD68, DAKO, Heverlee, Belgium) with a haematoxylin nuclear counterstain. Numbers of M1 macrophages were determined by a double staining of CD68 and IRF5 (anti-IRF5, ProteinTech Europe, Manchester, UK), M2 macrophages by combining CD68 (anti-CD68, Abnova, Heidelberg, Germany) with CD206 (anti-CD206, Serotec, Puchheim, Germany), and M2-like macrophages by a combination of CD68 and IL10 (anti-IL10, Hycult Biotech, Uden, The Netherlands) using standard immunohistochemical procedures. The double stainings were not counterstained with haematoxylin to facilitate identification of double-positive cells.

To stain for IRF5+ or CD206+ macrophages, sections were deparaffinized and antigen retrieval was performed by overnight incubation in Tris-HCL buffer pH 9.0 at 80°C; thereafter sections were incubated with rabbit anti-IRF5 followed by mouse anti-CD68 or mouse anti-CD206 fol-lowed by rabbit anti-CD68. Next, sections were incubated with the two secondary antibodies together: horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody and alkaline phosphatase (AP)-conjugated goat-anti-rabbit antibody.

To stain for IL10+ macrophages, antigen retrieval was performed by heating the sections in citrate buffer at pH 6.0 for 10 minutes at sub-boiling temperatures. Sections were pretreated with 1% bovine serum albumin (Sigma Aldrich, St Louis, MO) and 5% milk powder in PBS for 30 minutes and incubated with rabbit anti-IL-10 overnight. The next day, sections were incu-bated with mouse anti-CD68 followed by the two secondary antibodies together: HRP-con-jugated goat-anti-rabbit antibody and alkaline AP-conjugated goat-anti-mouse antibody.

First the AP-conjugated antibodies were visualized using 5-bromo-4-chloro-3-indolyl-phos-phate/ nitro blue tetrazolium (BCIP/NBT) as chromogen (Vector, Burlingame, CA). Next, the HRP-conjugated antibodies were visualized with ImmPACT NovaRED (Vector, Burlingame, CA) as chromogen. Figure 1 displays representative pictures of each double staining (arrows indicate double positive cells).

Stainings were quantified by morphometric analysis using ImageScope analysis software (Aperio, Vista, CA). Sample slides were blinded for an observer who manually counted CD68-single positive cells or double positive cells in a central airways section per length of intact basement membrane, extending 100 μm into the intact submucosa, excluding vessel and smooth muscle. Data are expressed as the number of positive cells per 0.1 mm2. Along the same length, we measured the basement membrane thickness at 4 different, randomly picking points per mm airway in the CD68+hematoxylin stained sections.

Statistics Data were non-normally distributed; therefore, the data were log-transformed to obtain a Gaussian distribution. Comparisons between two groups were then conducted using a two-sided, unpaired Students T-test. The effects of COPD and smoke exposure were determined with a two-way ANOVA. When significant, a Sidak’s multiple comparisons test was used to test whether differences between groups were significant. When log-transformation did not result in a Gaussian distribution (basement membrane thickness) groups were compared using a Kruskal-Wallis and a Dunn’s post-test to correct for multiple testing (P<0.05).


Correlations were calculated for normally distributed data or log-transformed data using a Pearson test. A Pearson Rho was calculated for the interactions between macrophage sub-types and lung function (FEV

1 and FEV

1/FVC), age and pack years. Lung function correlations

were solely determined for COPD patients, as lung function is more variable in COPD and is a marker of disease severity. A nonparametric Spearman coefficient was calculated for the correlations between basement membrane thickness (no Gaussian distribution) and FEV

1 or

macrophage subtypes. P-values <0.05 were considered significant. Data was analyzed using Graphpad Prism 6 (Graphpad Software, La Jolla, CA).

Results

Macrophages and the three subsets were identified by double staining central airway sec-tions of patients with and without COPD. Figure 1 shows representative pictures of each of the double staining: (A-B) CD68+IR5+, (C-D) CD68+CD206+ and (E-F) CD68+IL-10+ cells (double positive cells are indicated by the arrows). We then related the numbers of double positive macrophages to having COPD or not, smoking status, age, packyears and basement membrane thickness.

No differences in macrophage numbers or polarization states in central airways between COPD patients and controlsFirst we investigated whether patients with COPD had different numbers of total macrophages compared to controls and found no significant differences between the groups (figure 2A). Furthermore, no differences were found with respect to the three macrophage polarization states (figure 2B-D). Correlations of FEV

1 with macrophage polarization states (only calculated

for COPD patients as FEV1 is variable among these patients) showed a trend towards a negative

correlation between FEV1 and numbers of M1 macrophages (p=0.10, data not shown).

When we subsequently correlated the number of macrophages and FEV1 in patients with

COPD separately for ex- and current smokers, we found a significant correlation between FEV

1 and the number of IRF5+ M1 macrophages in ex-smoking COPD patients (figure 3B,

Pearsons coefficient: -0.56, P<0.04), but no correlation was found in current smoking COPD patients. No correlations were found for any of the other populations (figure 3A, C and D). In addition, we found no correlations between the number of macrophages and FEV

1/FVC,

packyears and age (data not shown).

Lower numbers of total and IRF5+ macrophages in central airways of current smokersSmoking is a major risk factor for COPD. We therefore assessed the effect of smoking status on the number of macrophages around the central airways. Figure 4A shows that overall, current smokers had lower numbers of macrophages around central airways as compared to ex-smokers. Among the different macrophage phenotypes, the number of IRF5+ M1 mac-rophages was significantly lower in current smokers compared with exsmokers (figure 4B). Specifically, current -smoking COPD patients showed a significantly lower number of IRF5+ M1 macrophages than ex-smoking COPD patients (figure 4B). A similar effect was observed




129 5

as a trend among the smoking control individuals as compared to exsmokers (p=0.06). No differences were found between current and ex-smokers regarding CD206+ M2 and IL10+ M2-like macrophages (figure 4C-D).

Basement membrane thickness was unchanged in COPDThickening of the basement membrane in the central airways is an indication of remodeling of the airways and this is associated with COPD. Thus, we assessed basement membrane thickness and correlated these findings with the number of macrophages in airways of COPD patients. No correlations were found between any of the macrophage subtypes and basement membrane thickness when assessed separately for all current smokers or ex-smokers (COPD and controls combined, data not shown). Among the COPD patients, the number of CD206+ M2 macrophages showed a significant negative correlation with basement membrane thick-

Figure 1. Representative pictures of large airway sections double stained for CD68 and IRF5 (A-B), CD68 and CD206 (C-D), CD68 and IL-10 (E-F). Double –positive cells are indicated by the arrows.


ness, exclusively in ex-smokers (figure 5C), whereas other macrophage phenotypes did not significantly associate with basement membrane thickness (figure 5A, B and D). In addition, basement membrane thickness did not correlate with total number of macrophages or any of the macrophage subsets in the group of control subjects (data not shown).

COPD patients did not show altered thickening of the basement membrane as compared to controls, nor was there a difference with respect to current or ex-smoking (figure 5E). More-over, we assessed basement membrane thickness and correlated these findings with lung function (FEV

1 %pred.) and showed a trend towards a negative correlation with FEV

1 for COPD

patients (P<0.06, figure 5F).

Figure 2. No differences were detected in the number of macrophages present 100 μm into intact submucosa of large airways of COPD patients compared with controls,. Macrophages were characterized as followed: CD68+ was used as a general macrophage marker (A), M1 macrophages were detected with CD68 and IRF5 (B), M2 macrophage by CD68 and CD206 (C), and M2-like macrophages with CD68 and IL10 (D). Data are presented with a mean and statistical differences were tested with a student-T test and P<0.05 was considered significant.




131 5

Discussion

In this study we aimed to investigate whether having COPD or not would be associated with changes in macrophage polarization states around the airways, in particular with having more M2-polarized macrophages as these have been shown to be associated with remodel-ing processes. Surprisingly, no differences were found in the number of total macrophages or the three main polarization states M1, M2, and M2-like between COPD patients and control subjects. We did find that current smokers have significantly less total macrophages around the airways as compared to ex-smokers and this seemed to be caused by a significant selective decrease in the number of IRF5+ M1 macrophages in current smokers with COPD. We found no evidence for more remodeling of the airways in COPD patients as compared to controls, because basement membrane thickness was similar between the two groups. However, a thicker basement membrane correlated negatively with the number of CD206+ M2 macro-phages in the group of COPD patients.

Figure 3. Total numbers of macrophages showed no correlation with FEV1 %pred. in COPD

patients (A). FEV1 did negatively correlate with the number of CD68+IRF5+ M1 macrophages in

ex-smoking COPD patients (Pearson coefficient -0.56, P<0.04) (B). Numbers of CD68+CD206+ M2 macrophages (C) or CD68+IL10+ M2-like macrophages (D) did not correlate with FEV

1. For

each correlation a Pearsons correlation coefficient was calculated and P<0.05 was considered significant.


Current literature has not provided a straightforward view on the presence of different macro-phage subsets in COPD 3,4,10,11,15,20-22. Macrophages are known to be involved in the pathogenesis of COPD, but the presence of each subset separately has hardly been studied. Most studies investigating macrophage polarization have focused on alveolar macrophages and the pic-ture that arises from these studies is that COPD may be characterized by increased numbers of macrophages with dysfunctional M1 and M2 characteristics 4,12,13,20,23-27.

Information on polarization changes of interstitial macrophages around the airways is even less available. Airway inflammation plays a key role in the development of COPD and mac-rophages are important inflammatory cells involved in addition to neutrophils and CD8+ T cells 2,8,14,28-30. However, our current results did not show a higher number of macrophages or any changes in their polarization state in the central airways of COPD patients as compared to control subjects. For the total number of macrophages, this is in line with other studies using lung tissue from lung resection surgeries for suspected cancer 15,31-34. However, higher num-

Figure 4. Smoking status affected numbers of macrophages in the airway wall. Current smokers showed lower numbers of CD68+ total macrophages (p<0.04) (A) and CD68+IRF5+ M1 macro-phages (P=0.001) (B) compared with ex-smokers. In COPD patients, current smoking resulted in lower numbers of CD68+IRF5+ M1 macrophages (P=0.017) (B). No differences were detected for CD68+CD206+ M2 macrophages (C) or CD68+IL-10+ M2-like macrophages (D). Data are presented with a mean and statistical differences were tested with a two-way ANOVA with a Sidak’s multiple comparison post-test. P<0.05 was considered significant.




133 5

bers of CD68+ macrophages have been found in airway wall biopsies from COPD patients as compared to healthy controls 28,35-37. The differences between these studies are firstly that these three latter studies used airway wall biopsies while we and Saetta et al., Grashoff et al., Turato et al. and Battaglia et al. used large airways in specimens of dissected lung tissue. Secondly, these biopsy studies had genuine healthy control groups as opposed to our con-trol group that consists of macroscopically unaffected tissue patients undergoing lung tissue

Figure 5. Among CD68+CD206+ M2 macrophages, the number of macrophages correlated negatively with basement membrane thickness in ex-smoking COPD patients (Spearman coef-ficient -0.63, P=0.018) (C). No correlations were detected between basement membrane thick-ness and CD68+ total macrophages (A), CD68+IRF5+ M1 macrophages (B) or CD68+IL10+ M1 macrophages (D). Statistical differences were tested using a Kruskal-Wallis with a Dunn’s post-test to correct for multiple testing. For each correlation a Spearman correlation coefficient was calculated and P<0.05 was considered significant. Basement membrane thickness was not different between controls and COPD patients (E). We observed a trend (P=0.06) towards a negative correlation between FEV

1 and basement membrane thickness (F).


resection surgery for suspection of a tumor. Comparing the number of total macrophages per 0.1 mm2 of tissue between the two types of studies, we find comparable numbers of total macrophages for COPD patients, suggesting biopsy results are comparable to results from dissected airways. However, our non-COPD controls have more total macrophages as com-pared to healthy controls. This suggests changes in total macrophage numbers due to the presence of a tumor or differences in the clinical phenotype of the control subjects. As we do not have access to dissected lung tissue of healthy controls, this hypothesis is difficult to test. Nevertheless, our COPD patients also underwent lung resection surgery for suspected tumors and we found that having COPD did not additionally affect interstitial macrophages around the airways or their phenotype. A cautionary remark to be made about the data is the fact that both M1 and M2 macrophages were characterized based on phenotypical markers that say little about their function. Disturbed functions of both subsets of macrophages have been suggested before in COPD and this may play a role in the pathogenesis of COPD 3,4,20,23,26,38-42.

Interestingly, we did see that current smoking was associated with lower numbers of total macrophages and IRF5+ M1 macrophages. There are several possible explanations for this finding. Cigarette smoke components may directly or indirectly kill macrophages in the inter-stitium, affect antigen exposure and therefore staining quality or influence chemokine release by bronchial epithelial cells, which may have drawn macrophages out from the interstitium into the lumen of the airway. The latter may seem the more logical explanation as many studies have found higher numbers of alveolar macrophages in (healthy) smokers in BAL/sputum 43-45. Moreover, smoking has been found to induce epithelial expression of chemo-attractants for macrophages such as monocyte chemoattractant protein-1 (MCP-1 or CCL2) and IL-8 (CXCL8) 46-49. Why this appears to affect IRF5+ M1 macrophages more than the other two M2 subsets may be explained by a differential expression of both chemokine receptors by the different macrophage subsets. A recent publication by Xuan et al. showed that M1 mac-rophages have higher expressions of CXCR1 and CXCR2 (the receptors for CXCL8) than M2 macrophages and are indeed specifically attracted by CXCL8 as opposed to M2 macrophages. CXCL8 is well known for the fact that it is produced by bronchial epithelial cells after smoke exposure, particularly with respect to its role in attracting neutrophils 49. It may therefore also promote M1 migration out of the interstitial-tissue compartment in current smokers. This may also explain the trend we observed in the control group that also pointed towards less M1 macrophages in current smokers.

The lower number of IRF5+ M1 macrophages in specifically current smoking COPD patients as compared to ex-smoking COPD patients, however, is more difficult to explain using the IL-8 hypothesis. Many studies have shown either unchanged or even higher IL-8 levels in sputum of ex-smoking as compared to current smoking COPD patients 50-54. Therefore it is likely that other mechanisms or chemokines may also play a role in explaining the lower numbers of M1 macrophages in smoking COPD patients. As we do not see a concomitant increase in either M2 or M2-like macrophages, we think it is unlikely that cigarette smoke induces repolarization into a M2 subset as has been suggested by the studies of Yuan et al. 55.

We did not find any evidence for our main hypothesis that airway remodeling, as defined as a thicker basement membrane, was associated with the presence of more CD206+ M2




135 5

macrophages. On the contrary, our data suggest that lower numbers of CD206+ M2 macro-phages were associated with a thicker basement membrane in ex-smoking COPD patients. In addition, a thicker basement membrane was associated with a trend towards a lower FEV

1

in all COPD patients. A possible explanation could be the finding that M2 macrophages also appear to be involved in inhibiting fibrotic processes and can be important in clearing away excess extracellular matrix through mannose receptors and Mfge8 56-60. These differences in function within the M2 subset are not detectable by staining for just two phenotypic markers (CD206 and CD68). A more functional marker or read-out could help to get direction in this discussion. Why the association would only be present in ex-smoking COPD patients cannot be explained at present.

We found no differences in basement membrane thickness between patients with COPD and controls. Results regarding basement membrane thickness in COPD patients compared to controls have been inconsistent going from thicker in COPD, to no differences and even thin-ner in COPD as compared to control 36,61-64. Of note, our patient populations may differ from these earlier studies due to the presence of cancer as we did find a trend in COPD patients towards lower FEV

1 being associated with a thicker basement membrane. Therefore, within

COPD patients, basement membrane thickness does appear to influence lung function. Fur-thermore, the number of IRF5+ M1 macrophages showed a trend towards a negative correla-tion with FEV

1 in all COPD patients (which was significant in ex-smokers only). This is in line

with data we obtained from a cohort of asthma patients in which IRF5+ M1 macrophages also negatively correlated with FEV

1 (Draijer et al. manuscript submitted). The reason for the

stronger effect in ex-smokers is not clear to us at present.

In conclusion, having COPD was not associated with changes in numbers of interstitial airway macrophages, their polarization states, or basement membrane thickness as com-pared to controls. However, lower FEV

1 was associated with a thicker basement membrane

and having more IRF5+ M1 macrophages. Furthermore, a thicker basement membrane was associated with having less CD206+ M2 macrophages in ex-smoking COPD patients. These associations stress the importance and the possible different contributions of macrophage subsets to COPD.


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Chapter 6

General discussion


General Discussion

Macrophages are important immune cells in the lungs constituting a first line of defense towards inhaled airborne threats. Their main function in host protection is presented through their ability to produce reactive oxygen species and inflammatory cytokines 1-4. Besides this function, macrophages can adopt many more phenotypes in order to fulfill other roles 5-13. Depending on their environment, macrophages respond to signals they receive and adopt one of these phenotypes in order to execute the desired role. In the past, two main mac-rophage subtypes were distinguished matching the Th1/Th2 dichotomy: the predominantly inflammatory subtype M1 and the alternatively activated subtype M2. However, it was later shown that these phenotypes appear in a spectrum rather than as distinct phenotypes 10,14. Therefore, recently a group of expert macrophage researchers recommended to describe the different macrophage phenotypes according to the markers they express or the stimulus that was used to induce them 15-17. In this thesis, we have used the following markers to identify three different macrophage subtypes: IRF5 for pro-inflammatory M1 macrophages, CD206 for repair-associated M2 macrophages and IL-10 for anti-inflammatory M2-like macrophages.

An important function of macrophages is to participate in the maintenance of tissue extra-cellular matrix (ECM) homeostasis 1,4,18,19. Especially in the lungs, external influences, such as smoke or dust exposure, can affect the macrophage environment and therefore alter mac-rophage function. As a result, this can cause an imbalance in the ECM production versus tissue breakdown. The contribution of different macrophage subsets and their individual functions has hardly been studied in this context. Therefore, the aim of this thesis was to investigate which macrophage subtypes are present in diseased lung tissue and how manip-ulation of macrophages could help restore the balance in tissue ECM homeostasis. To do so, we have studied the presence and role of macrophages in the context of two pulmonary dis-eases: pulmonary fibrosis and chronic obstructive pulmonary disease (COPD). In pulmonary fibrosis, macrophages have been shown to contribute to stimulation of extracellular matrix production, whereas in COPD, macrophages were shown to play a role in tissue destruction or, alternatively, in insufficient tissue repair contributing to the progression of emphysema 5,11,20,21. These opposing roles macrophages can have in tissue homeostasis is the central theme of this thesis.

Proteolytic macrophages in lung diseasesIn chapters 2 and 3 of this thesis we focused on proteolytic macrophages. Actions of these macrophages affect lung architecture considerably and thereby impair lung function, with either insufficient proteolytic activity or too much proteolytic activity. In fibrosis, macro-phages can contribute to overproduction of ECM and abnormal remodeling. This causes impaired gas exchange due to extensive parenchymal fibrosis with often an extensive dis-tortion of parenchymal architecture. In COPD, airways show fibrotic changes in their walls whereas in the parenchyma the opposite is seen; Destruction of alveoli results in emphysema and eventually leads to airway obstruction and loss of diffusion capacity. Here macrophages may also play a role. In the parenchyma, macrophages may adapt a more proteolytic phe-

General discussion

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notype in the alveoli, resulting in the destruction of alveoli or in an abnormal regulation of normal tissue repair. Considering the different diseases, we studied the possibility of either promoting the differentiation of macrophages towards a proteolytic phenotype in fibrosis or inhibition of proteolytic macrophages in emphysema.

Proteolytic macrophages in fibrosisMacrophages are known to stimulate the production of ECM proteins by myofibroblasts through their ability to produce Transforming Growth Factor beta (TGFβ), Platelet Derived Growth Factor (PDGF) and Resistin-like molecule alfa1 (Relmα/FIZZ1), which are essential cytokines in tissue repair processes 2,4,10,15,22-27. However, these tissue repair processes can esca-late towards fibrosis when mechanisms that restore tissue function lack or fail 1,6,8,10,12,13,28-30. Under healthy conditions, feedback mechanisms correct for superfluous tissue-repair pro-cesses, stimulate the reduction of scar tissue and restore tissue function. Macrophages con-tribute to this reduction of scarred tissue with proteolytic capacities through their production of proteolytic enzymes such as MMPs and cathepsins 10,15-17,31-37. Gibbons et al. emphasized this important task by showing that macrophage depletion during the recovery phase slowed down the resolution of fibrosis induced by a single administration of bleomycin 1,3,4,38-42. In fibrotic diseases, however, these feedback mechanisms fail to balance tissue-repair processes, resulting in excessive and uncontrolled ECM production. Attempts by the proteolytic macro-phage subpopulation to restore a balance in ECM homeostasis appear insufficient.

We hypothesized that the impaired actions of macrophages in the tissue repair process result from dampened macrophage differentiation into a proteolytic phenotype. We proposed a communication mechanism between fibroblasts and macrophages via the RANK/RANKL/OPG-axis, known from activity in in bone-matrix homeostasis, that controls the proteolytic activity of osteoclasts 5,7,9,11,43-45. RANKL, receptor activator of Nf-kb ligand, is able to induce a proteolytic phenotype in osteoclasts, for which we proposed a similar role in lung mac-rophages 14,46,47,. We confirmed that RANKL stimulation induces cathepsin K activity and MMP9 expression in macrophages (chapter 3, and 15,17,48,49. RANKL binds to its receptor RANK, located on macrophages/osteoclasts to induce a proteolytic phenotype, while osteoprote-gerin (OPG) functions as a decoy receptor of RANKL, preventing the interaction between RANKL and RANK. Interestingly, Brass et al. had already showed that OPG levels are higher in silica- and bleomycin-induced mouse models of pulmonary fibrosis 18,19,50-52. This led to our hypothesis that the elevated OPG levels in pulmonary fibrosis may influence fibrotic pro-cesses by repression of the development of proteolytically active macrophages.

We showed that RANK, RANKL and OPG are all expressed in the lungs and in accordance with the findings of Brass et al., we found elevated OPG protein levels in both human and (experimental) mouse pulmonary fibrosis compared to their respective controls. Together with a decreased RANK expression in macrophages, this pointed towards profibrotic con-ditions in the lung. The elevated OPG levels resulted from local OPG production since OPG mRNA expression in lung tissue was also higher than in control lungs. One of the inducers of OPG appeared to be TGFβ, the hallmark cytokine of fibrosis, suggesting a significant link between OPG and fibrosis. Thus, reducing the high levels of OPG might improve conditions for proteolytic macrophages in the lungs and lead to positive changes regarding the resolu-


tion of fibrosis. We continued with treating mice with established pulmonary fibrosis with soluble recombinant RANKL. Surprisingly, the balance between OPG and RANKL seemed very tight in the lungs, because we found a significant increase in OPG levels after RANKL treatment and no reduction in collagen deposition. This tight balance shows that OPG and RANKL are involved in the lungs, but their exact roles remain unknown. It can be speculated that apart from the role of OPG, other, secondary, signals are neces-sary to activate the (proteolytic) macrophages. So, another approach would be to stimulate macrophages directly towards a proteolytic macrophages. Direct stimulation of antifibrotic macrophages has been shown to be a promising clinical approach. Pentraxin 2, also known as serum amyloid P, inhibits de development of profibrotic macrophages and supports the development of an antifibrotic phenotype 20,50,53. A recent phase I clinical trial showed PRM-151, a human recombinant form of pentraxin 2, was well tolerated in patients with idiopathic pulmonary fibrosis (IPF) in a range of doses. In addition, a stable or slightly improved lung function and lung volume were observed as compared to placebo treatment 22,23,54,55. Definite efficacy of PRM-151 in IPF patients will be tested in future clinical trials.

OPG may also act profibrotically through other mechanisms. For example, besides RANKL, TNF-related apoptosis-inducing ligand (TRAIL) is also an OPG ligand, with cytotoxic prop-erties 28,46. By binding TRAIL, OPG may be able to inhibit this cytotoxic function and thereby prevent TRAIL-induced apoptosis of lung fibroblasts. Fibroblasts within the fibroblast foci are able to respond to TRAIL as they express death receptors (DR) 4 and 5 35,56-62. However, Akram et al. also conclude that these fibroblasts exert very low p53 and p21 expression, two pro-apoptotic markers, suggesting an apoptotic-resistant fibroblast phenotype. This apop-totic-resistant phenotype may be enforced through the binding of TRAIL to OPG. The high levels of OPG in lungs of patients with pulmonary fibrosis could then create a condition for myofibroblasts to continue producing ECM 31-34,63,64. TRAIL and other ligands may be consid-ered when investigating the role of OPG in pulmonary fibrosis.

Furthermore, timing with respect to the increased expression of OPG is also unknown. What is the first trigger for (over)production of OPG in the lungs? Is it a primary response or is it a reaction to compensate for other mechanisms? One of the difficulties with respect to answering these questions is the fact that these are initially slowly progressing diseases in which patients report to the clinic in a relative late stage of the disease. Up until now it has been proven difficult to identify these patients in an early stage of the disease. Depending on the timing of OPG production and its specificity for fibrotic lung diseases, this protein may be further studied as a possible (early) biomarker in fibrosis.

Proteolytic macrophages in COPDIn contrast to pulmonary fibrosis, one component of COPD, emphysema, is characterized by excessive loss of alveolar lung tissue resulting in reduced lung diffusion capacity. The effects of proteolytic macrophages in emphysema have been shown in many studies 38-42,65,66. They contribute to the degradation of parenchymal lung tissue through their MMP and cathep-sin activity 9,43-45,67-73. A marker associated with proteolytic activity and highly expressed by alveolar macrophages is tartrate resistant acid phosphatase (TRAP). In bone, proteolytic macrophages are characterized by TRAP expression and are induced by RANKL. In these

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osteoclasts, TRAP is involved in motility of the osteoclast and associated with bone matrix degradation 46,47. We found that TRAP activity is higher in lungs of patients with COPD com-pared with controls (chapter 3). Moreover, cigarette-smoke exposure, the most important risk factor for COPD, was also associated with higher numbers of TRAP active macrophages in mice and more TRAP expression in humans. Up until now, the reason for the upregulation of TRAP in COPD is unknown and also its function in the lung was not investigated before in detail. Taken together, this provided a strong trigger to investigate the regulation and possible role of TRAP in the lungs.

By staining for TRAP activity, it was apparent that TRAP was only expressed by alveolar mac-rophages and not by interstitial macrophages 48,49. This may be explained by the fact that interstitial macrophages are likely to have a different origin than alveolar macrophages 50-52. Resident alveolar macrophages already originate early in development from fetal monocytes and self-maintain during life 50,53. The origin of interstitial macrophages has not been studied in detail yet, but a difference in origin may explain the lack of TRAP expression in interstitial macrophages. Furthermore, we discovered in vitro that oxidative stress, an important trait of COPD, is able to induce TRAP expression in macrophages 54,55. This may explain the higher expression of TRAP in lung tissue of COPD patients as compared to controls. In addition, other factors or a combination of factors may be responsible for influencing TRAP expression in alveolar macrophages.

Importantly, we developed a gold-based TRAP inhibitor (AubipyOMe) to study TRAP func-tion in macrophages and we successfully showed its ability to inhibit human and mouse TRAP. To study the function of TRAP in macrophages and show the inhibitory effect of AubipyOMe, we focused on the role of TRAP in the regulation of macrophage motility. The best-proven function of TRAP in osteoclasts is being involved in osteoclasts motility 46. We therefore first investigated if TRAP is also involved in macrophage motility. We showed that induction of TRAP expression in macrophages indeed led to higher macrophage motility and that we could inhibit this higher motility with AubipyOMe. This result contributes to our notion that AubipyOMe could be an effective tool to study the function of TRAP in alveolar macrophages in more detail.

Monocytes and macrophages in COPDAlthough COPD is an inflammatory disease of the lungs, we hypothesized that macrophage progenitors in blood, the monocytes, may also be affected in patients with COPD. This may be the case even before they infiltrate the lungs and differentiate into macrophages, as the high inflammatory load in the lungs of COPD patients has also been shown to have systemic influ-ences 56-62. Monocytes exist in three subsets: two predominantly inflammatory subsets called classical monocytes (CD14hiCD16-negative) and intermediate monocytes (CD14hiCD16-pos-itive) 63,64. The third subset, the nonclassical CD14-positiveCD16hi monocytes, have a more anti-inflammatory phenotype. We found higher percentages of these inflammatory mono-cyte subsets in blood from COPD patients as compared to controls, but this was completely explained by our COPD patients being older than our controls (chapter 4). Age has been linked to changes in monocyte subsets towards more inflammatory activation 65,66. Since


COPD can also be characterized as a disease of accelerated aging, our results may point towards monocytes in COPD having aged prematurely compared to healthy individuals, but this hypothesis needs further testing.

Smoking is an important risk factor for COPD and, therefore, we assessed the influence of smoking on monocyte subsets separately and furthermore, studied their expression patterns of Toll-like receptors (TLR). Percentages of CD16+ monocytes were lower in current smok-ers than ex-smokers and we found a trend towards a lower percentage of total monocytes. Previous studies showed higher numbers of monocytes in smokers, however, these studies only considered the total number of monocytes and did not investigate the subsets 67-73. We observed a significantly lower number of nonclassical monocytes in current smokers. This suggests a loss of anti-inflammatory potential due to smoking or, alternatively, recruitment into the inflamed tissue to respond to the effects of smoke exposure and therefore not being detectable in blood anymore. Moreover, current smoking was associated with higher expres-sion of TLR2 and TLR4 on both types of CD16+ monocyte subsets. This may have functional consequences as TLR2 has shown to be involved in oxidant sensing and TLR4 plays a critical role in the inflammation induced by oxidants 2,74.

In addition to changes in circulating monocyte subsets, macrophages in the lungs of COPD patients are also likely to change due to the disease and/or cigarette smoke exposure. Remark-ably, following characterization of interstitial macrophages around large airways by their expression of CD68 or their expression of the following markers: IRF5 (M1 macrophages), CD206 (M2 macrophages) and IL-10 (M2-like macrophages), we found no differences in numbers of any of these airway-based macrophage subsets in COPD patients when compar-ing them with controls (chapter 5). Current smoking did affect macrophages numbers and we found lower numbers of total and IRF5+ macrophages in current smoking individuals, which is different from previous findings in alveolar macrophages. These previous studies showed higher expression of markers usually associated with IRF5+ macrophages in macrophages in sputum of patients with COPD, as well as in serum of these patients 6,8,10,12,13,54,75,76. This differ-ence may be explained by migration of interstitial macrophages towards the air compartment to fight the effects of smoking from the direct-exposure side. Recent data from Draijer et al. in a mouse model of asthma has shown that interstitial macrophages may be able to replenish the alveolar macrophages when needed to respond to house dust mite (manuscript in prepa-ration). A similar mechanism in response to smoke may play a role here as well. Furthermore, we found that the number of IRF5+ macrophages correlated negatively with FEV

1 in ex-smok-

ing COPD patients, indicating that these macrophages could influence the airways. Our data indicate this is likely caused by an overactive or impaired function of IRF5+ macrophages rather than an increase or decrease number of macrophages, as we found no differences in numbers between COPD patients and controls. This is in line with previous findings by others 2,10,77-85. Characterization of macrophages with functional markers would highly contribute to our knowledge regarding the induced or impaired function of macrophages in COPD.

In chapter 5, we hypothesized that the number of CD206+ macrophages is associated with basement membrane thickness as a feature of airway remodeling. CD206+ (M2) macrophages are associated with tissue repair/fibrosis mechanisms and may contribute to airway remodel-

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ing 16,75,86,87. Results from previous studies looking at basement membrane thickness in COPD patients were variable and we too found no differences in basement membrane thickness between COPD patients and control individuals 1,4,88-92. In addition, no positive association with CD206+ macrophages was found, suggesting these macrophages have no role in airway remodeling. A similar finding was also reported in asthma by Draijer et al., further confirming this finding (manuscript submitted). However, basement membrane thickness did show a trend towards a negative correlation with FEV

1, suggesting that a thicker basement membrane

is associated with worsening of the disease, but possibly other processes are responsible the production of the excess ECM.

Future perspectivesThis thesis shows the results of investigations of the importance of macrophage subtypes and their marked differences regarding function in lung diseases and especially their contribution to maintaining lung tissue homeostasis. Future research should keep in mind the different macrophage phenotypes, as their functions differ greatly. It is apparent that it is important to study how altering macrophage functions could effectuate the best possible outcome for a disease. In pulmonary fibrosis, the possible contribution of macrophages to the reduction of scar tissue and thereby to the resolution of fibrosis has generally been overlooked, while we strongly believe that macrophages are essential in this process. In that respect, the first results of the pentraxin-2 trials in pulmonary fibrosis are promising because they show that stimulating antifibrotic behavior in macrophages may indeed be a successful new avenue to contribute to treatment of fibrosis 5,11,20-23.

The plasticity that macrophages exert should be used to our advantage, not only in fibrosis but also in emphysema as a component of COPD. Here, macrophages contribute to lung tissue destruction, as been acknowledged many times 2,4,10,15,24-27,38-42. Therefore, future experi-ments should focus on investigating how to inhibit or change these proteolytic macrophages. Assuming that TRAP has a function in proteolytic macrophages, our TRAP inhibitor Aubi-pyOMe may be a useful tool to explore the role of TRAP in lung tissue destruction. TRAP is clearly involved in macrophage motility, but also other functions are suggested for TRAP in the lungs. For example intracellular TRAP was shown to be involved in collagen degrada-tion and to add to oxidative stress load in the lungs 1,6,8,10,12,13,29,30,36,37,93-95. It was also shown to be important in regulating IFNα production 15-17,96. Thus, future experiment should focus on studying these functions and reveal whether inhibition of these processes may benefit disease outcome.

Finally, as previously mentioned, including functional macrophage markers when studying macrophage subsets is crucial. In this thesis, we have identified macrophages according to certain markers they express. However, this does not give us any information regarding the actual function of the macrophage or their contribution to lung disease. Investigating func-tional macrophage markers and functional tests will help us understand the functional con-tribution of each macrophage subset and, hopefully, provide us with promising targets for future drug development.


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Chapter 7

APPENDIX | PATENT

Principle investigators:

B.N. Melgert1A. Casini1,2

1University of Groningen, Department of Pharmacokinetics, Toxicology and Targeting, Gro-ningenResearch Institute for Pharmacy, Groningen, The Netherlands. 2School of Chemistry, Cardiff University, Park Place, Cardiff CF10 3AT, United Kingdom.

Filed September 2015


Gold(III) compounds as Tartrate Resistant Acid Phosphatase inhibitors, and therapeutic uses thereof.

The invention relates to the fields of organometallic chemistry, enzymology, and medicine. In particular, it relates to gold (III) compounds as Tartrate Resistant Acid Phosphatase (TRAP) inhibitors, and to uses thereof, among others in the treatment of respiratory/pulmonary dis-eases like chronic obstructive pulmonary disease (COPD) and asthma.

COPD is a common but relatively unknown disease in which airways are chronically inflamed (chronic bronchitis) and lung tissue is destroyed (emphysema). Patients are characterized by varying contributions of both phenotypes. It affects around 10% of the population worldwide and is caused by cigarette smoking, air pollution and/or indoor biomass cooking. COPD is the fourth cause of mortality and morbidity in the world and is projected to be the third by 2030. Currently no effective therapy exists and transplantation is the only option for end-stage disease.

The pathogenesis of emphysema is incompletely understood but macrophages have been shown to be important in the process (6). Exposure to cigarette smoke and air pollution induces inflammation in the lungs with infiltration of macrophages and neutrophils and, after prolonged exposure, development of emphysema (7). Macrophages are considered to be the more relevant effector cells because deletion studies in mice have shown that these were indispensable to developing emphysema, while neutrophils were not (8) . Macrophages can produce a host of proteolytic enzymes like matrix metalloproteins (MMPs) and cathepsins that have the ability to degrade extracellular matrix (ECM) and therefore lead to lung tissue destruction. In addition, alveolar macrophages are one of the few macrophage subsets in the body that highly express the enzyme TRAP, which in bone is involved in ECM turnover and may have a similar function in the lung (1).

Asthma is a chronic inflammation of the airways after becoming allergic to airborne allergens and affects 5-20% of the people in the Western world. Treatment consists of anti-inflamma-tory drugs and bronchodilators. However, a substantial group of patients with severe asthma are not treated effectively with the currently available drugs and are particularly susceptible to acute worsening of their disease.

An important clinical problem for patients with lung diseases like COPD or severe asthma is their susceptibility to exacerbations. These exacerbations are an acute worsening of symp-toms mostly caused by viral and/or bacterial infections. Exacerbations are the major cause of morbidity and mortality in COPD and asthma. Dysfunction of alveolar macrophages has been presumed to be an important cause of impaired responses to infections (9).

A study by Capelli et al. (10) reported that the enzyme TRAP was found to be upregulated in alveolar macrophages of smokers as compared to nonsmokers. Vuillenemot et al. (11) reported upregulation of TRAP gene ACP5 in an animal model of COPD.

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157 7

The present inventors hypothesized that TRAP expressed by macrophages in the lung may be involved in the pathogeneses of asthma and COPD. Indeed, in both diseases a higher expression of TRAP was found in lung tissue of patients as compared to controls (Figure 1A-B), suggesting TRAP may be involved in alveolar macrophage dysfunction. In addition, increased TRAP expression was found in mouse models of asthma and COPD (Figure 1C and D).

ACP5 encodes the enzyme tartrate-resistant acid phosphatase (TRAP; EC3.1.3.2), which belongs to the family acid phosphatases and distinguishes itself by its resistance to inhi-bition by tartrate. It is a metalloenzyme and contains two iron atoms at its active site. It is primarily found in lysosomes, and similar organelles, of osteoclasts, alveolar macrophages, and activated macrophages and dendritic cells. Its function is unknown but two main func-tions have been proposed based on the two enzymatic activities the protein harbours. Its only known substrate is the heavily phosphorylated protein osteopontin (OPN). OPN can be present extracellularly as part of lung ECM and intracellularly as a signalling molecule. One of TRAP’s proposed functions is the dephosphorylation of extracellular OPN to inhibit adhesion of macrophages to ECM and promote migration of macrophages (12,13). TRAP has also been shown to promote the metastasizing behaviour of tumors, confirming the idea it may be involved in cell migration (14).

In addition, TRAP may be involved in impaired antimicrobial responses by dephosphory-lating intracellular OPN. For instance, viral stimulation of TLR9 induces phosphorylation of OPN and subsequently leads to increased IFNα production to combat the virus. In patients lacking TRAP expression, IFNα levels have been found to be increased (15). Conversely, in patients with COPD and asthma lower levels of IFNα have been found, suggesting overpro-duction of TRAP, and explaining their impaired responses to viral infections (16,17).Thus, from a therapeutic point of view, ACP5/TRAP is an interesting molecular target. Unfor-tunately, the only known inhibitors of TRAP are the toxic metal compounds molybdate, vana-date, lead acetate, mercuric acetate and gold(III) chloride. The in arthritis therapeutically active gold(I) compounds Aurothioglucose and Aurothiomalate inhibit TRAP only at high concentration (mM range) (20). Gold chloride has been shown to be able to inhibit TRAP (Hayman et al., Cell Biochem Funct 2004, 22L 275-280), but is too unstable to be used ther-apeutically since it is prone to undergo reduction to colloidal gold, which may also result in toxic effects. No other substances that are capable of inhibiting TRAP and suitable for thera-peutic or diagnostic applications have been reported so far.Recognizing the potential of ACP5/TRAP as therapeutic target, the present inventors there-fore set out to search for novel inhibitors that can be used in vivo to inhibit TRAP. In partic-ular, they aimed at providing non-toxic inhibitory compounds showing an IC50 value in the nanomolar range.

This goal was met by the surprising finding that various gold(III) coordination compounds bearing N-donor ligands are potent TRAP inhibitors in vitro with IC50s in the nM range. Moreover, these compounds are not cytotoxic to macrophages in the concentration range showing an inhibitory effect. Interestingly, TRAP was shown to be involved in macrophage migration and this migration could be inhibited using the novel gold-based inhibitors. Lastly,


it was demonstrated that inhibition of TRAP modulates the responses of macrophages to microbial compounds.

Accordingly, in one embodiment the invention provides a gold(III) coordination compound bearing N-donor ligands, the compound having the general formula A, B, C, D or E

wherein dotted lines can be absent or present; each of R

1 through R

11 is independently

selected from the group consisting of H; aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; amines (e.g. NH

2, aliphatic amines –R-NH

2); halogens

(e.g. chloride, iodide); moieties with hydroxyl functional groups (e.g. –OH or–Y-OH); ether containing moieties of general formula –Y-O-Y’; carbonyl containing moieties (-Y-C(O)OH); or of amide bonds (-Y-C(O)N-Y’-); sulfonamidic groups; nitrile/nitro groups, and peptide moi-eties, wherein Y and Y’ are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and het-erocycloaliphatic groups comprising up to four C-atoms; L and L’ are independently selected from the group consisting of halogen, hydroxyl, acetate, phosphane, and thiol-bearing groups (e.g. thio-sugars, cysteine and methionine groups); and Z is a cyclic moiety selected from the group consisting of homocyclic and heterocyclic aromatic/aliphatic moieties, preferably 6,6-, 5,6- or 6,5-fused bi-homocyclic and bi-heterocyclic aromatic/aliphatic moieties, wherein the heterocyclic moieties may include nitrogen, oxygen and/or sulfur atoms, or a pharmaceuti-cally acceptable salt or solvate thereof, for use in a method of treating and/or alleviating the symptoms of a disease associated with increased tartrate-resistant acid phosphatase (TRAP) activity.

Some gold (III)-compounds for use in the present invention are known in the art. For exam-ple, Messori et al. (J Med Chem 2000, 43, 3541-3548) discloses gold complexes wherein the gold (III) center is coordinated to a polydentate ligand with nitrogen donors, such as the

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159 7

compound [Au(terpy)Cl]Cl2 that belongs to the general formula B shown herein above. Dis-

cussed is the use of the gold complexes as cytotoxic and antitumor drug. Casini et al. (Dalton Trans., 2010, 39, 2239-2245) disclose the synthesis, characterization and biological properties of square planar gold(III) compounds with modified bipyridine and bipyridylamine ligands according to the general formula A. The complexes showed cytotoxicity in vitro towards the A2780 human ovarian carcinoma cell line and a cisplatin resistant variant thereof.

WO2013/005170 relates to metal-based modulators that selectively bind to cell transmem-brane proteins aquaglyceroporins AQPs, leading to its inhibition. The selective inhibition of AQP channels was accomplished among others by compounds tetracoordinated to gold (III) complexes according to the general formula A, B, C or E as shown herein above. Also dis-closed is the use thereof in manufacturing pharmaceuticals, cosmetics and chemical reagents for diagnostic, treatment, prophylaxis and prevention of clinical conditions directly or indi-rectly related to aquaglyceroporins AQPs functions.

Hayman et al. (2014, Cell. Biochem. Funct. 22: 275-280) investigated whether gold (III) com-pounds mediate their effect on osteoclastic bone resorption by modification of TRAP activity. It was found that gold chloride was a powerful TRAP inhibitor whereas aurothioglucose or aurothiomalate had no effect.

Thus, the art fails to teach or suggest the use of the gold (III) compounds as claimed herein for the inhibition of TRAP or for the treatment of a disease associated with TRAP activity, like respiratory or pulmonary diseases such as COPD and asthma.

In a gold(III) compound for use according to the invention, each of R1 through R

11 on the

N-donor ligands is independently selected from the group consisting of H; aliphatic, het-eroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; amines (e.g. NH


2); halogens (e.g. chloride, iodide); moieties with hydroxyl

functional groups (e.g. –OH or –Y-OH); ether containing moieties of general formula –Y-O-Y’; carbonyl containing moieties (-Y-CO-OH); or of amide bonds (-Y-CO-N-Y’-); sulfonamidic groups; nitrile/nitro groups, and peptide moieties, wherein Y and Y’ are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, het-eroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms.

In a preferred embodiment, each of R1 through R

11 (also referred to herein as ‘’R substituents’’

or ‘’R groups’’) is independently selected from the group consisting group consisting of H; aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-het-eroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms. For example, each of R

1 through R

11 is independently selected from H, C

1-C

4 alkyl and C

1-C

4

alkoxy.

In another embodiment, at least one of the R substituents is selected from the group consist-ing of amines (e.g. NH


2); halogens (e.g. chloride, iodide); moieties


with hydroxyl functional groups (e.g.-OH; –Y-OH); ether containing moieties of general for-mula –Y-O-Y’; carbonyl containing moieties (-Y-CO-OH); or of amide bonds (-Y-CO-N-Y’-); sulfonamidic groups; fluorophores; nitrile/nitro groups, and peptide moieties, wherein Y and Y’ are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms. In some embodiments, one or more of R

1 though R

11 is

F, -CH3, -CH

2CH

3, -OH, -OCH

3, or -OCH

2CH

3. In some embodiments, at least one of R

1 through

R11 is -CH

3 or –OCH

3.

In one embodiment, at least one of R1 through R

11 is other than H, so that the (hetero)aromatic

rings contain one or more substituents. The number of R substituents other than H can vary. In one embodiment, the compound contains up to 6, up to 5, or up to 4 R-groups other than H. For example, suitable compounds for use according to the invention are those having one, two, three or four R substituents selected from aliphatic, heteroaliphatic, aromatic, heteroar-omatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocy-cloaliphatic groups comprising up to four C-atoms; amines; halogens; moieties with hydroxyl functional groups; ether containing moieties of general formula –Y-O-Y’; carbonyl containing moieties (-Y-CO-OH); or of amide bonds (-Y-CO-N-Y’-); sulfonamidic groups; nitrile/nitro groups, and peptide moieties, while the remaining R groups are H. In another embodiment, all of the R groups are H, i.e. the gold(III) coordination compound bears unsubstituted N-do-nor ligands.

L and L’ are independently selected from the group consisting of halogen, hydroxyl, acetate, phosphane, and thiol-bearing groups e.g. thio-sugars, cysteine and methionine groups. In a preferred embodiment of the invention, L and L’ are halogen, preferably chloride or iodide.Compounds according to any one of formula A, B, C, D or E can be screened for their efficacy using methods known in the art. For example, US 6,451,548 discloses an assay employing (recombinant) TRAP that has been activated with a cysteine protease and a phosphotyro-sine or phosphoserine containing test compound. Disclosed herein below is a method using recombinant TRAP or a cell lysate as source of TRAP, and para-nitrophenylphosphate (PNPP) as enzyme substrate.

In one aspect, the invention provides a compound for use as TRAP inhibitor according to formula A, being an [Au(substituted-2,2’-bipyridine)LL’]n+ with n = 1 or 2 or an [Au(2-phen-ylpyridine)LL’] n+ with n = 0 or 1. In a preferred aspect, the invention provides the use of a compound of formula A based on a bipyridine i.e. wherein X is N.

In an inhibitor according to formula A, all substituents R1 through R

8 can be H. In one aspect,

at least one, two, three or four of R1 through R

8 is other than H. The substituents R

1 through R

8

can be symmetric or asymmetric. Symmetrically substituted formula A compounds include those having substituents other than H at positions R

1 and R

8; R

2 and R

7; R

3 and R

6; R

4 and

R5; R

1, R

2, R

7 and R

8; R

3, R

4, R

5 and R

6, and so forth. In one embodiment, the substitutions are

(only) at positions 3,3’; 4,4’; 5,5’ or 6,6’ of the bipyridine or phenylpyridine ring. Preferably, the substituents other than H are (solely) at positions 4, 4’. For example, provided is a TRAP

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161 7

inhibitor according to formula A wherein X is N, and wherein the substitutions are at posi-tions 4, 4 of the bipyridine ring.

In another embodiment, the formula A compound is asymmetrically substituted. Exemplary asymmetric compounds include those having a single substituent other than H, and those having substituents other than H at positions R

1 and R

7; R

2 and R

8; R

3 and R

5; R

4 and R

8; R

1,

R2, R

6 and R

7; R

3, R

4, R

7 and R

8, and so forth. In a specific aspect, a compound of formula A is

symmetric having two aliphatic or heteroaliphatic substituent at positions R3 and R

6, such as

those wherein R3 and R

6 are methoxy or ethoxy.

In one aspect, each of substituents R1 through R

8 of a formula A inhibitor compound is inde-

pendently selected from the group consisting of H, C1-C

4 alkyl and C

1-C

4 alkoxy. An exemplary

Formula A compound is [Au(bpOMe)Cl2][PF

6] (bpOMe = 4,4’-dimethoxy-2,2’-bipyridine]

(Aubipy-O-Me). Useful variants of Aubipy-O-Me include compounds having different substit-uents at the same positions, and compounds having one or more C

1-C

4 alkoxy substituent(s)

at different positions of the bipyiridine ring.

In a further embodiment, the inhibitor is a substituted-2,2’,2”- terpyridine compound of for-mula B, wherein preferably up to four of substituents R

1 through R

11 are other than H. In one

aspect, it is an [Au(substituted-2,2’,2”- terpyridine)L]n+ with n = 2 or 3. An exemplary formula B compound is [Au(2,2’:6’,2’’-terpyridine)Cl]Cl (Auterpy).

In a still further embodiment, the compound for use according to the invention is a polypyr-idyl compound of formula C. The polypyridyl moiety depicted by Z is a cyclic moiety selected from the group consisting of homocyclic and (hetero)cyclic aromatic and aliphatic moieties. The heterocyclic moiety may include nitrogen, oxygen and/or sulfur atoms. In one embodi-ment, Z is a fused ring system comprising one, two or three (hetero)cyclic moieties. Prefera-bly, Z is a 6,6-, 5,6- or 6,5-fused bi-homocyclic or bi-heterocyclic aromatic/aliphatic moiety. Preferably, Z is a heterocyclic aromatic moiety, more preferably a heterocyclic aromatic moi-ety comprising two nitrogen atoms. In one aspect, the compound is an [Au(polypyridyl)LL’]n+ with n = 1 or 2.

The polypyridyl scaffold can for example be based on dipyrido [3, 2-f:2’, 3’-h] quinoxaline (DPQ), dipyrido [3, 2-a:2’, 3’-c] phenazine (DPPZ), or dipyrido [3,2-a:2’,3’-c] (6,7,8, 9-tetrahy-dro) phenazine (DPQC).

NN

NN

NN

NN

NN

NN

DPQ DPPZ DPQC


Accordingly, exemplary inhibitory compounds according to formula C include those wherein the polypyridyl moiety is dipyrido[3,2-f:2’,3’-h]quinoxaline, dipyrido[3,2-a:2’,3’-c]phenazine, or dipyrido[3,2-a:2’,3’-c](6,7,8,9-tetrahydro)phenazine.

In a further embodiment, the inhibitory compound has a structure according to formula D. The dotted lines in the general formula D can be present or absent. For example, the com-pound may bear bidentate or tridentate nitrogen donor ligands selected from 2-(2-pyridyl)imidazole, 2-phenylimidazole, 2,6-bis(benzimidazol-2-yl)pyridine, and 1-methyl-2-[2-pyr-idyl]-1H-benzo[d]imidazole). Very good results were observed with compounds wherein up to four of R

1 through R

9 are other than H. Preferably, at least R

5 is other than H, for example R

5

is C1-C

4 alkyl or C

1-C

4 alkoxy. In a specific embodiment, only R

5 is other than H, for example

R5 is C

1-C

4 alkyl or C

1-C

4 alkoxy. For example, the inhibitory compound is [Au(1-methyl-2-[2-

pyridyl]-1H-benzo[d]imidazole)Cl2]Cl (AuPbImMe). A compound according to formula D has

not been disclosed or suggested in the art. Hence, the invention also provides a compound according to formula D and its use as medicament. Preferably, the compound is [Au(1-meth-yl-2-[2-pyridyl]-1H-benzo[d]imidazole)Cl2]Cl (AuPbImMe) or functional equivalent thereof. Also provided is a pharmaceutical composition comprising a compound according to for-mula D, and a pharmaceutical carrier, diluent or excipient.

Still further aspects of the invention relate to inhibitory compounds according to formula E, being based on a phenantroline scaffold. Preferably, the compound is an [Au(substitut-ed-1,10-phenantroline)L2]n+ with n= 1, 2 or 3. Good results can be observed with compounds wherein up to four of R

1 through R

8 are other than H.

An inhibitor compound for use in the present invention can be synthesized using methods known in the art. See for example Casini et al., Dalton Trans. 2010, 39, 2239– 2245 or Hollis et al., J. Am. Chem. Soc. 1983, 105, 4293 –4299. Compounds of formula D can be synthesized adapting the conditions from Casini et al., Dalton Trans. 2010, 39, 2239– 2245, and also from Serratrice et al., Inorg. Chem. 2012, 51, 3161−3171.

A gold (III) compound disclosed herein is suitably used as in vitro or in vivo inhibitor of tar-trate-resistant acid phosphatase (TRAP). In addition, it can be modified to serve as (chemical) probe for detecting TRAP activity. For example, a probe can be a fluorescent derivative of a gold(III) compound of the invention. The probe finds its use for example in assessing the role of TRAP activity in a certain physiological process or cellular pathway. For example, the role of TRAP in the interplay between macrophages and cancer stem cells can be determined. In one aspect, the probe compound contains in at least one of the R positions a fluorophore such as an anthracenyl or coumarin moiety. Compounds containing 1,10-phenantroline are also fluorescent per se.

Specifically, one of the R groups is an anthracenyl or coumarin moiety.

Appendix | patent

163 7

R = anthracenyl moeity

O

O

O

R = coumarin moeity

In a further aspect, the gold (III) inhibitory compound is used, e.g. for experimental, scientific and/or drug-screening purposes, as in vitro inhibitor of TRAP.

In a preferred embodiment, an inhibitor compound finds it use in a method of treating and/or alleviating the symptoms of a respiratory disease associated with increased tartrate-resistant acid phosphatase (TRAP) activity. Respiratory disease is a medical term that encompasses pathological conditions affecting the organs and tissues that make gas exchange possible in higher organisms, and includes conditions of the upper respiratory tract, trachea, bron-chi, bronchioles, alveoli, pleura and pleural cavity, and the nerves and muscles of breath-ing. Respiratory diseases range from mild and self-limiting, such as the common cold, to life-threatening entities like bacterial pneumonia, pulmonary embolism, acute asthma and lung cancer. The study of respiratory disease is known as pulmonology.

In a specific embodiment of the present invention, an inhibitor compound finds it use in a method of treating and/or alleviating the symptoms of a pulmonary disease associated with increased expression of TRAP, like asthma, COPD, smoking-induced inflammation (cigarette smoke-induced pulmonary inflammation or (pulmonary) sarcoidosis.

In a preferred embodiment, the disease is a pulmonary disease such as chronic obstructive pulmonary disease (COPD) or asthma. Without being bound by theory, upregulation of ACP5 / TRAP in pulmonary diseases like COPD assists in degradation of collagen by macrophages and, therefore, aids parenchymal lung tissue destruction and assists in stimulation of mac-rophage migration through the lung. This enhanced migration could magnify the areas in which lung tissue can be destroyed. In addition, we propose that increased expression of ACP5/TRAP impairs antimicrobial responses in both asthma and COPD by yet unknown mechanisms, causing enhanced disease and an increased susceptibility to exacerbations. The inventors hypothesize that inhibition of TRAP in emphysema will slow down lung tissue destruction, and in COPD and asthma it will improve responses to infections.

Accordingly, also provided is a pharmaceutical composition formulated for respiratory or pulmonary administration and/or inhalation, comprising one or more TRAP inhibitory com-


pounds as disclosed herein. The inhalable formulation is a solution, suspension, emulsion, colloidal dispersion, or dry powder, wherein the formulation is suitable for administration to the lungs of a mammal.

In a specific aspect, the TRAP inhibitor compound is formulated for use or contained in a medical device called a metered dose inhaler (MDI) or a dry powder inhaler (DPI). A metered dose inhaler is a handheld device that delivers a specific amount of medication in aerosol form. The MDI consists of a pressurized canister inside a plastic case, with a mouthpiece attached. Its portability makes it easy to use anywhere, anytime. MDIs use a chemical pro-pellant to push medication out of the inhaler. In a preferred embodiment, the compound is in the form of a dry powder and suitable for use in a dry powder inhaler, which is also a handheld device. A DPI delivers medication to the lungs as you inhale through it. It doesn’t contain propellants or other ingredients.

To give the most effective dry powder aerosol, therefore, the particles should be large while in the inhaler, but small when in the respiratory tract. In an attempt to achieve that situa-tion, one type of dry powder for use in dry powder inhalers may include carrier particles to which the fine active particles comprising the TRAP inhibitor adhere whilst in the inhaler device, but which are dispersed from the surfaces of the carrier particles on inhalation into the respiratory tract to give a fine suspension. In a specific aspect of the invention, the TRAP inhibitory compound is administered by the multidose breath-actuated dry powder inhaler (DPI) known under the tradename Novolizer.

A further embodiment of the invention relates to a method of treating and/or alleviating in a subject the symptoms of a respiratory disease associated with increased tartrate-resis-tant acid phosphatase (TRAP) activity, comprising administering to the subject an effective amount of a TRAP inhibitor as herein disclosed. In a specific aspect, the method comprises administering at least one compound selected from the group consisting of [Au(bpOMe)Cl

2]

[PF6] (bpOMe = 4,4’-dimethoxy-2,2’-bipyridine] (Aubipy-O-Me); an [Au(substituted-2,2’,2”-

terpyridine)L]n+ with n = 2 or 3; [Au(2,2’:6’,2’’-terpyridine)Cl]Cl (Auterpy); a compound of formula C, being an [Au(polypyridyl)LL’]n+ with n = 1 or 2.; or a compound of formula C, based on dipyrido[3,2-f:2’,3’-h]quinoxaline, dipyrido[3,2-a:2’,3’-c]phenazine, or dipyrido[3,2-a:2’,3’-c](6,7,8,9-tetrahydro)phenazine.

In another embodiment, the method comprises administering a compound of formula D, bearing bidentate or tridentate nitrogen donor ligands selected from 2-(2-pyridyl)imidazole, 2-phenylimidazole, 2,6-bis(benzimidazol-2-yl)pyridine, and 1-methyl-2-[2-pyridyl]-1H-ben-zo[d]imidazole; [Au(1-methyl-2-[2-pyridyl]-1H-benzo[d]imidazole)Cl

2]Cl (AuPbImMe); or a

compound of formula E, being an [Au(substituted-1,10-phenantroline)L2]n+ with n= 1, 2 or 3.Exemplary diseases to be treated include those mentioned herein above, in particular respi-ratory diseases associated with increased expression of TRAP such as asthma, COPD, smok-ing-induced inflammation and sarcoidosis.

Also provided herein is a method of inhibiting TRAP activity in a mammal, comprising administering to the mammal an inhalable formulation described herein. In one aspect, the

Appendix | patent

165 7

mammal has at least one symptom of a TRAP-dependent or TRAP-mediated (respiratory) disease or condition.

Legend to the figures

Figure 1:A: Numbers of macrophages showing TRAP activity are higher in parenchymal lung tissue of patients with fatal asthma as compared to controls dying of nonpulmonary causes. B: TRAP gene ACP5 is a major upregulated gene in lung tissue of COPD patients compared to non-COPD controls. C: Numbers of macrophages showing TRAP activity are higher in parenchymal lung tissue of mice exposed to house dust mite to induce experimental asthma than in healthy control mice. D: Numbers of macrophages showing TRAP activity are higher in parenchymal lung tissue of mice exposed to cigarette smoke to induce experimental COPD than in control mice exposed to air.

Figure 2: Structures of exemplary compounds showing TRAP inhibitory activity.

Figure 3: Recombinant human TRAP was used to assess the TRAP inhibitory capacity of the gold (III) compounds Aubipy-O-Me, Auterpy, Auoxo1 and Aurothiomalate as compared to NaAuCl

4

(see also figure 4). TRAP can catalyze the formation of p-nitrophenyl (PNP) from the colori-metric substrate p-nitrophenyl phosphate, which can be measured photospectometrically. Increasing concentrations of the gold compounds were used to study their inhibiting poten-tial of this conversion. NaAuCl

4 and Aubipy-O-Me were the most potent inhibitors (n=3).

Figure 4: Cytotoxicity of the gold compounds of the gold (III) compounds Aubipy-O-Me, Auterpy, Auoxo1 and Aurothiomalate as compared to NaAuCl

4 was measured by a proliferation assay

using 3H-thymidin.

Figure 5: Cell lysates of murine alveolar macrophages (MPI, kind gift of G. Fejer) were used to assess the TRAP inhibitory capacity of our gold compounds Aubipy-O-Me, AuPblmMe, (dipyNH)AuCl

2 and (pyb-H)AuCl

2 as compared to NaAuCl

4. PNP formation was assessed in the pres-

ence of tartrate to eliminate the contribution of other phosphatases to the dephosphorylation of PNPP. NaAuCl

4, Aubipy-O-Me and AuPblmMe were the most potent inhibitors (n=3).

Figure 6: Pooled tissue lysate of lung tissue obtained from human COPD patients was used to assess the TRAP inhibitory capacity of the gold compound Aubipy-O-Me as compared to NaAuCl

4.


PNP formation was determined in the presence of tartrate to eliminate the contribution of other phosphatases to the dephosphorylation of PNPP.

Figure 7:A. In a transwell experiment, RAW264.7 macrophages were allowed to migrate over osteo-

pontin-coated membranes with or without RANKL stimulation to induce TRAP expres-sion and with or without 128 nM Aubipy-O-Me. RANKL-stimulated cells had a trend towards more migration over the membrane as compared to vehicle-stimulated cells, and co-incubation with Aubipy-O-Me was able to inhibit this increased migration (n=4).

B. The effect of RANKL-induced TRAP expression on migration behaviour was specific for osteopontin, as no effects of RANKL or Aubipy-O-Me were seen on macrophages cultured on collagen-coated membranes.

Figure 8:Human monocyte-derived macrophages (n=3) have higher expression of several pro-in-flammatory genes when stimulated with 100 ng/ml lipopolysaccharide (LPS) as compared to control macrophages. Inhibition of TRAP during this LPS stimulation with 300 nM or 1 μM Aubipy-O-Me lowered the expression of these pro-inflammatory genes.

Appendix | patent

167 7

Experimental section


Cell cultureThe Murine RAW 267.4 macrophage cell line was obtained from American Type Culture Col-lection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invi-trogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, Penicillin/Streptavidin (Invitrogen, Carlsbad, CA) and L-glutamine (Invitrogen, Carlsbad, CA) at 37°C under 5% CO

2

and humidified conditions.

Gold inhibitorsThe following compounds were tested for their ability to inhibit TRAP activity: [AuCl

4]-, Auth-

iomalate , AubipyOMe , Auterpy, Auoxo1, AuPbImMe, (dipyNH)AuCl2 and (pyb-H)AuCl

2,

wherein dipyNH means… and pyb means…. AubipyOMe and (dipyNH)AuCl2 were prepared

and characterized using the procedure described by Casini et al. (Dalton Trans. 2010 Mar 7;39(9):2239-45). Auoxo1 was prepared according to reported procedure by Casini et al. (J Med Chem. 2006 Sep 7;49(18):5524-31). (pyb-H)AuCl

2 was prepared following the procedure

reported by Cinellu et al. (J. Chem. Soc., Dalton Trans., 1996, 4217–4225). Auterpy was syn-thesized according to Hollis et al. (J. Am. Chem. SOC. 1983, 105, 4293-4299). [AuCl

4]- and

Authiomalate are commercially available from Sigma-Aldrich. AuPbImMe was synthesized according to conditions adapted from Casini et al. (Dalton Trans. 2010, 39, 2239– 2245).

TRAP activity assayTRAP activity was assessed by incubating either recombinant TRAP (1.25ng/ml, R&D, Min-neapolis, USA) or cell lysate (1:2 ratio) with L para-Nitrophenylphosphate (PNPP) solution [100 mM PNPP, 200 mM sodium citrate, 200 mM sodium chloride, 80 mM sodium tartrate (L+), pH 4.5]. Cell lysate was prepared by collecting cells directly from a culture flask in ace-tate buffer (pH 5.0) at a density of 500.000 cells/ml and sonication of cells. Subsequently, recombinant TRAP or cell lysate was incubated with increasing concentrations of AubipyOMe (range 0-40 mM). Samples were incubated with PNPP solution at a 1:1 ratio up to an hour at 37°C. The reaction was stopped using 1M NaOH and the absorption at 410 nm was measured using a spectrophotometer. The inhibitory effect was calculated from the ratio of absorbance between the treated and untreated cells. The IC50 value was calculated as the concentration of inhibitor caused a 50% reduction in TRAP activity. Data was presented as mean ±SEM and consisted of at least three independent experiments.

Proliferation assayCytotoxicity of the compounds was assessed using 3H-thymidin. In short, RAW macrophages were grown in 96-well plates (Corning Incorporated, NY) in a medium volume of 100ul, at a


density of 5000 cells/well and grown for 48h. Cells were exposed to various concentrations of inhibitor were prepared from a freshly made stock solution (10mM) in DMSO and diluted in medium including relevant negative controls (range 31-3934nM). After 28h hours, tritium thymidine (0.25uCi/ml) (Perkin Elmer, Waltham, USA) was added for 10 hours, after which excess of thymidine was washed away. Cells were subsequently incubated with 100ml of 10.5% TCA solution at 4°C for 30 min and lysed in 100ml of 1M NaOH. The lysate was transferred to a scintillation vial with 4 ml of scintillation fluid (Perkin Elmer). Samples were measured using the LS 6500 Multi-purpose Scintillation Counter (Perkin Elmer).

Macrophage motilityMotility of RAW macrophages was assessed by either live cell tracking using a confocal micro-scope or a Corning transwell culturing system (Sigma-Aldrich, St. Louis, USA). For the live cell tracking experiments, RAW cells were plated on osteopontin (OPN, 10mg/ml) coated Lab-tek chamber slides (Nunc, Hatfield, USA) at a density of 7500 cells/well. Cells were stimulated with RANKL (200ng/ml, kindly provided by dr. R.H. Cool, University of Groningen, The Neth-erlands.) for two days to increase TRAP expression, followed by CFSE labeling (Invitrogen, Life Technologies Europe BV, Bleiswijk, The Netherlands), over night tracking in the presence of 1% zymosan solution (Sigma-Aldrich, St. Louise, USA), and presence or absence of 128 nM AubipyOMe. Video material was analyzed using Imaris software.

In addition to live cell imaging, migration ability was assessed using a transwell culturing sys-tem with inserts (Sigma-Aldrich, St. Louis, USA) coated with osteopontin (R&D, Minneapolis, USA) or collagen (Advanced Biomatrix, Delta, Canada) to determine macrophage migration over the membrane. Briefly, RAW macrophages, pre-stimulated with or without 200ng/ml RANKL for 72h to increase TRAP expression, were seeded with 250,000 cells/well on 8 mm pore inserts coated with 10mg/ml osteopontin or collagen. Cells were cultured for 16h in the presence or absence of 128 nM AubipyOMe. The next day, total cell numbers in the lower compartment, including dead cells, were counted. The number of cells migrated was calcu-lated relative to cells not stimulated with RANKL.

Generation of monocyte-derived macrophagesMonocytes were isolated from buffy coats of healthy blood donors (NHS Blood and Trans-plant, UK) by Lymphoprep (Axis-Shield, Oslo, Norway) density gradient centrifugation fol-lowed by plastic adherence in IMDM (Lonza, Basel, Switzerland) containing 10% human pool serum, 1% penicillin/streptomycin (Invitrogen, San Diego, CA), and 0.25% ciprofloxacin (Bayer, Leverkusen, Germany). PBMCs were incubated, and nonadherent cells were removed after 2 h incubation by extensive washing with PBS. Subsequently, adherent monocytes were detached using PBS containing 10 mM EDTA at room temperature and plated for macrophage maturation at a density of 1 × 105 cells/cm2 in IMDM containing 10% human pool serum, 1% penicillin/streptomycin, and 0.25% ciprofloxacin.

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TRAP-dependent macrophage responses to lipopolysaccharide (LPS)For microarray analysis LPS -responses, MDMs were cultured for 7 days. At day 7, LPS (100 ng/ml; Salmonella minnesota R595; Alexis Biochemicals, Lausen, Switzerland), and 300 nM , or 1 mM Aubipy-O-Me were added 6 h prior to harvesting the cells (n = 3). Control cells received vehicle.

RNA extraction and analysisRNA was extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. After extraction, the sample was incubated with Turbo DNase at 37 °C for 30 minutes and subsequently re-purified using RNeasy clean-up protocol. Expression of he following genes were subsequently analyzed as compared to the expression of β-actin: Interferon-regulatory factor-1 (IRF1), Serpin Peptidase Inhibitor, Clade G (C1 Inhibitor), Member 1 (SERPING1), Apolipoprotein L3 (APOL3), interleukin-6 (IL6), tumor necrosis factor alpha (TNF), Chemo-kine (C-C motif) ligand 20 (CCL20), Interleukin-12 p40 (IL12B), ndoleamine 2,3-dioxygenase 1 (IDO1), and Chemokine (C-C Motif) Ligand 4 (CCL4).

AnimalsMale A/JOlaHsd mice and male and female BALB/c mice (8-10 weeks old) were obtained from Harlan (Horst, The Netherlands). Animals were kept in a temperature-controlled room with a 12h dark/light cycle and with permanent access to food and water. All animal exper-iments were approved by the Institutional Animal Care and Use Committee. The animal experiment was performed in the animal facility of the University of Groningen according to strict governmental and international guidelines on animal experimentation.

Smoke-induced lung inflammation in miceTo model COPD, we exposed male A/JOlaHsd mice nose-only to mainstream cigarette smoke for 9 months in an experimental setup as described before by us (Van der Strate, 2006). In short, mice were exposed daily to main-stream smoke from four 2R1 Reference Cigarettes (University of Kentucky, KY). This protocol was continued for 5 days/week for 9 months. Control mice were sham-exposed to room air under similar conditions, following the same duration of exposure as the smoke-exposed group. The method used to check the delivery of total particulate matter by the smoking equipment has previously been described by Griffith and Hancock. The smoking equipment was calibrated before every smoking session to ensure accurate and standardized smoke exposure. Carboxyhaemoglobin levels were measured to determine actual smoke exposure.

Allergic lung inflammationTo model asthma, we exposed male and female BALB/c mice intranasally to whole body house dust mite (HDM) extract (Dermatophagoides pteronyssinus, Greer laboratories, Lenoir, USA) in 40 ml phosphate-buffered saline (PBS) according to a protocol we have described before (Draijer med inflame 2013). In short, mice were exposed to HDM extract under isoflu-rane anesthesia: one time to a high dose of HDM (100 mg) in the first week, 5 times to a low dose (10 mg) in the second week and were sacrificed on day 21. Control animals (n=8) were


exposed to 40 ml PBS according to this same schedule. Mice were sacrificed on day 24, three days after the last HDM exposure and the right lung was inflated with 0.5 ml 50% Tissue-Tek® O.C.T.™ compound (Sakura, Finetek Europe B.V., Zoeterwoude, The Netherlands) in PBS and formalin-fixed for histological analyses. Other parameters of allergic lung inflammation of these animals are described in detail by Draijer et al {Draijer 2013}.

Enzyme histochemistry TRAP activity was measured via enzyme histochemistry. The staining was performed on 3mm sections of mouse lung tissue imbedded in paraffin. Deparaffinated, rehydrated sections were incubated over night in a zinc-buffer solution (0.1M Tris, pH 7.4 complemented with 30mM calcium acetate, 23mM zinc acetate and 37mM zinc chloride), following pre-incubation in a 0.2M acetate buffer (0.2M sodium acetate, 50mM L(+) tartaric acid, pH 5.0). Slides were then incubated for 2 hours at 37°C with the reaction solution (0.2M acetate buffer containing 0.5 mg/ml Naphtol AS-MX phosphate (Sigma-Aldrich, St. Louis, USA) and 1.1 mg/ml fast red TR salt (Sigma-Aldrich, St. Louis, USA). A hematoxylin couterstaining was applied and stained sections were imbedded in DEPEX mounting medium (VWR, Murarrie, Australia). The num-ber of positive cells (#/mm2) was counted manually with the aid of ImageScope software.

Patient materialCOPDGene expression data of TRAP was obtained from 311 patients with COPD and 270 non-COPD controls who were part of the Lung eQTL dataset from three academic sites. Details of this population as well as a detailed description of the whole genome mRNA profiling has been previously published by Brandsma et al. {Thorax 2015}. Data was corrected for the following potential confounders: age, gender, pack-years and smoking status. Lung tissue samples were collected from patients undergoing lung tumor resection or lung transplantation. In case of tumor resections, macroscopically normal lung tissue was taken far distant from the tumor and histology of all samples was checked for abnormalities using standard haematoxylin and eosin staining. Lung samples were obtained in accordance with local ethical guidelines.

AsthmaPost mortem lung tissues from subjects with fatal asthma or death from nonpulmonary causes (controls) were retrieved from the Department of Pathology of São Paulo University (São Paulo, Brazil). A detailed clinical and demographic description of this population has been previously published by Mauad et al. { Rev Panam Salud Publica 2008; 23: 418–423}. For this study we investigated the presence of TRAP activity in paraffin-embedded peripheral lung tissue samples of 10 asthma patients and 10 controls as described below.

EXAMPLE 1: Gold(III) compounds inhibit TRAP activityThe inhibitory capacity of four different Gold(III) coordination compounds on TRAP was assessed by using recombinant TRAP in combination with increasing concentrations of can-didate inhibitors and these were compared to the positive control NaAuCl

4 (figure 3).Two out

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171 7

of four compounds were able to reduce TRAP activity within the used concentration range, namely Aubipy-O-Me and Auterpy. Aubipy-O-Me was almost as potent as positive control NaAuCl

4 in inhibiting TRAP activity. Its IC50 value was 476 nM as compared to 280 nM for

NaAuCl4 (table 1). Notably, the lowest concentration of Aubipy-O-Me already gave a signif-icant reduction of TRAP activity compared to vehicle. The inhibitory effect of Aubipy-O-Me further increased in a dose-dependent manner and the highest concentration Aubipy-O-Me reduced TRAP activity significantly. Auterpy was less potent than Aubipy-O-Me and had an IC50 value of 1643 nM.Cytotoxicity of these gold compounds was measured by a proliferation assay using 3H-thy-midin (figure 4). None of the compounds except for Auterpy showed severe cytotoxicity in the concentration range needed for successful inhibition of TRAP activity. The IC50 value for inhibiting cell proliferation was lower than the value for inhibiting TRAP (1024 nM for prolif-eration inhibition versus 1643 nM for inhibition of TRAP), rendering Auterpy unsuitable for use of TRAP inhibition in cells (table 1 and 2).

EXAMPLE 2: Inhibition of cell-derived TRAPThe inhibitory capacity of three other different Gold(III) coordination compounds on TRAP in comparison to Aubipy-O-Me and NaAuCl4 was assessed by using cell lysates of high TRAP-expressing macrophages in combination with increasing concentrations of the can-didate inhibitors (figure 5). AuPblmMe was equally effective as Aubipy-O-Me in this system, while the other two, (pyb-H)AuCl2 and (dipyNH)AuCl2, were less potent.In this more complex cell mixture Aubipy-O-Me was less effective in inhibiting cell-derived TRAP as compared to using recombinant TRAP. The IC50 value increased 6-fold to 3 mM (table 3).

To test whether the Gold (III) compounds could also inhibit TRAP in lung tissue of COPD patients, pooled lung tissue lysates of COPD patients were incubated with increasing concen-trations of Aubipy-O-Me or NaAuCl

4 (see figure 6). Aubipy-O-Me could inhibit human TRAP

in lung tissue of COPD patients with an IC50 of 8 mM.

Tables 1-3 herein below summarize the IC50 data obtained.

Table 1. IC50 of recombinant TRAP inhibition.

recombinant

TRAP inhibition IC50 (nM)

Authiomalate No inhibition found

Auterpy 1643

Auoxo1 No inhibition found

Aubipy-O-Me 476

[AuCl4]- 280


Table 2. IC50 of the cytotoxicity measured with proliferation

Cytotoxicity

Proliferation IC50 (nM)

Authiomalate No toxicity found

Auterpy 1024

Auoxo1 No toxicity found

AubipyOMe 7494

[AuCl4]- No toxicity found

Table 3. IC50 of cell-derived TRAP inhibition.

TRAP isolated

from cells IC50 (nM)

[AuCl4]- 470

AubipyOMe 2951

AuPblMe 3559

(dipyNH)AuCl2 4093

(pyb-H)AuCl2 10396

EXAMPLE 3: Macrophage motility depends on TRAP activity and is inhibited by AubipyOMeMacrophage motility is suggested to be TRAP-dependent. Macrophages require TRAP activity to detach and enable cell movement when attached to an osteopontin surface. Therefore, macrophages were seeded on osteopontin-coated surfaces when macrophage motility was assessed using live tracking of macrophages by confocal microscopy or using a transwell-in-cubation system in the absence or presence of the TRAP inhibitor AubipyOMe.Videos of live-tracked macrophages (data not shown), indicated little movement of macro-phages incubated with the vehicle, but a significant amount of movement in the presence of RANKL, a cytokine that induces the expression of TRAP in these macrophages. Following incubation with the inhibitor, macrophages’ movement is less, suggesting that TRAP activity is necessary for macrophage movement. These results were quantified in a transwell-migra-tion assay, in which more RANKL-stimulated macrophages migrated through an osteopon-tin-coated membrane compared to vehicle-stimulated cells (figure 7A), while this migration was suppressed in the presence of Aubipy-O-Me. This effect was specific for an osteopon-tin-coating, because migration through a coating of collagen was not affected by RANKL stimulation and/or Aubipy-O-Me incubation (figure 7B).

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EXAMPLE 4: Human macrophage responses to the bacterial compound lipopolysaccharide (LPS) are altered when co-incubated with AubipyOMe.Macrophage responses to micro-organisms such as bacteria and viruses is suggested to be influenced by TRAP. For instance intracellular TRAP was shown to inhibit production of inter-feron alpha (15). To investigate if TRAP influences the responses to Gram-negative bacteria, human macrophages were stimulated with LPS in the presence or absence of 2 concentra-tions of Aubipy-O-Me. LPS clearly induced the expression of genes associated with inflam-mation such as IRF1, SERPING1, APOL3, IL6, TNF, CCL20, IL12B, IDO1, and CCL4 (figure 8). Aubipy-O-Me was found to inhibit these responses.

References1. Halleen JM, et al., Clin Chem 2001;47:597-600.2. Adams LM, et al., Cell Biol Int 2007;31:191-195.3. Honig A, et al., BMC Cancer 2006;6:199.4. Hayman AR, et al., J Histochem Cytochem 2000; 48:219-227.5. Hayman AR, et al., J Histochem Cytochem 2001;49:675-684.6. Barnes PJ. COPD 2004;1:59-70.7. Boorsma CE, et al. Mediators Inflamm 2013; 2013:1-19.8. Ofulue AF, et al., Am J Physiol 1999;277:L97-105.9. Kurai D, et al., Front Microbiol 2013;4:293.10. Vuillemenot BR, et al., Am J Respir Cell Mol Biol 2004;30:438-448.11. Capelli A, et al., Chest 1991;99:546-550.12. Andersson G, et al., J Bone Miner Res 2003;18:1912-1915.13. Ek-Rylander B, et al., Exp Cell Res 2010;316:443-451.14. Xia L, et al., Oncogene 201315. Briggs TA, et al., Nat Genet 2011;43:127-131.16. Tversky JR, et al., Clin Exp Allergy 2008;38:781-788.17. See H, et al., Paediatr Respir Rev 2008;9:243-250.18. Roberts HC, et al., Calcifi Tissue Int 2007;80:400-410.19. Halleen JM, et al., J Bone Miner Res 2003;18:1908-1911.20. Hayman AR, et al., Cell Biochem Funct 2004;22:275-280.21. Harada K, et al., PLoS ONE 2013;8:e78612.


Claims

1. A gold(III) coordination compound bearing N-donor ligands, the compound having the general formula A, B, C, D or E

wherein each of R

1 through R

11 is independently selected from the group consisting of H;

aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphat-ic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; amines (e.g. NH

2, aliphatic amines); halogens (e.g. chloride, iodide); moieties

with hydroxyl functional groups (e.g. –OH or –Y-OH); ether containing moieties of general formula –Y-O-Y’; carbonyl containing moieties (-Y-CO-OH); or of amide bonds (-Y-CO-N-Y’-); sulfonamidic groups; fluorophores; nitrile/nitro groups, and peptide moieties, wherein Y and Y’ are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; L and L’ are independently selected from the group consisting of halogen, hydroxyl, acetate, phosphane, and thi-ol-bearing groups (e.g. thio-sugars, cysteine and methionine groups); and Z is a cyclic moiety selected from the group consisting of homocyclic and heterocyclic aromatic/ali-phatic moieties, wherein the heterocyclic moieties may include nitrogen, oxygen and/or sulfur atoms; dotted lines can be absent or present; or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treating and/or alleviating the symptoms of a respiratory disease associated with increased tartrate-resistant acid phosphatase (TRAP) activity.

2. Compound for use according to claim 1, wherein L and L’ are halogen, preferably chloride or iodide.

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175 7

3. Compound for use according to any one of the preceding claims, wherein each of R1

through R11 is independently selected from the group consisting of H, C

1-C

4 alkyl and C

1-C

4

alkoxy. 4. Compound for use according to any one of the preceding claims according to formula A,

being an [Au(substituted-2,2’-bipyridine)LL’]n+ with n = 1 or 2 or an [Au(2-phenylpyri-dine)LL’]n+ with n = 0 or 1.

5. Compound for use according to any one of the preceding claims according to formula A, wherein the substitutions are (only) at positions 6,6’; 3,3’; 4,4’; or 5,5’ of the bipyridine or phenylpyridine ring.

6. Compound for use according to claim 4 or 5, wherein X is N, preferably with the substi-tutions of the bipyridine ring at positions 4, 4’.

7. Compound for use according to any one of claims 4-6, wherein each of R1 through R

7 is

independently selected from the group consisting of H, C1-C

4 alkyl and C

1-C

4 alkoxy.

8. Compound for use according to claim 7, being [Au(bpOMe)Cl2][PF

6] (bpOMe = 4,4’-dime-

thoxy-2,2’-bipyridine] (Aubipy-O-Me). 9. Compound for use according to any one of claims 1-3 of formula B, preferably wherein

up to four of R1 through R

11 are other than H.

10. Compound for use according to claim 9, being an [Au(substituted-2,2’,2”- terpyridine)L]n+ with n = 2 or 3;

11. Compound for use according to claim 9, being [Au(2,2’:6’,2’’-terpyridine)Cl]Cl (Auterpy).12. Compound for use according to any one of claims 1-3 of formula C, being an [Au(poly-

pyridyl)LL’]n+ with n = 1 or 2.13. Compound for use according to any one of claims 1-3 of formula C, based on dipyr-

ido[3,2-f:2’,3’-h]quinoxaline, dipyrido[3,2-a:2’,3’-c]phenazine, or dipyrido[3,2-a:2’,3’-c](6,7,8,9-tetrahydro)phenazine.

14. Compound for use according to any one of claims 1-3 of formula D, bearing bidentate or tridentate nitrogen donor ligands selected from 2-(2-pyridyl)imidazole, 2-phenylimidaz-ole, 2,6-bis(benzimidazol-2-yl)pyridine, and 1-methyl-2-[2-pyridyl]-1H-benzo[d]imidaz-ole).

15. Compound for use according to any one of claims 1-3 of formula D, wherein up to four of R

1 through R

9 are other than H.

16. Compound for use according to claim 15, being [Au(1-methyl-2-[2-pyridyl]-1H-benzo[d]imidazole)Cl

2]Cl (AuPbImMe).

17. Compound for use according to any one of claims 1-3 of formula E, being an [Au(substi-tuted-1,10-phenantroline)L2]n+ with n= 1, 2 or 3

18. Compound for use according to any one of the preceding claims, wherein said disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), smoking-induced inflammation and sarcoidosis.

19. Compound for use according to claim 18, wherein said disease is COPD or asthma. 20. Compound as recited in any one of claims 1 to 17, for use as in vitro inhibitor of tartrate-re-

sistant phosphatase (TRAP).21. A pharmaceutical composition formulated for pulmonary administration / inhalation,

comprising one or more compounds as recited in any one of claims 1-17.22. Compound according to formula D as defined in any of claims 1-3, 14 and 15.23. Compound according to claim 22, for use as medicament.


24. Pharmaceutical composition comprising a compound according to claim 22, and a phar-maceutical carrier, diluent or excipient.

25. A medical device comprising a TRAP-inhibitory compound as recited in any one of claims 1 to 17.

26. Medical device according to claim 25, being a metered dose inhaler (MDI) or a dry pow-der inhaler (DPI).

27. Medical device according to claim 25 or 26, wherein said TRAP-inhibitory compound is present in the form of a dry powder.

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Title: Gold(III) compounds as Tartrate Resistant Acid Phosphatase inhibitors, and thera-peutic uses thereof.

Abstract The invention relates to gold (III) compounds as Tartrate Resistant Acid Phosphatase (TRAP) inhibitors, and to uses thereof, like in the treatment of pulmonary diseases like COPD and asthma. The inhibitory compounds have the general formula A, B, C, D or E


Fig.

1

0.15

0.10

0.05

0.00

-0.0

5

-0.1

0

10 8 6 4 2 0

300

200

100 0

600

400

200 0

TRAP activity in parenchymallung tissue (semiquantitative)

1/12

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Fig. 2

CIAU-

CI

CI CI Na+ NAU

N

CICI

M3O OM3

(PF6)

O

NaO

AuS O

ONaxH2O

NaAuCI4 Aubipy-O-Me

Auterpy Auoxo-1

Aurothiomalate

2/12


Fig.

3

3/12

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Fig.

4

4/12


Fig.

5

5/12

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Fig.

6

6/12


Fig

. 7C

Fig

. 7A

Fig

. 7B

7/12

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Fig. 8

8/12


Fig. 8, contd.

9/12

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Fig. 8, contd.

10/12


Fig. 8, contd.

11/12

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Fig. 8, contd.

12/12

Chapter 8

Nederlandse samenvatting

Dankwoord

Curriculum Vitae

List of publications


Nederlandse samenvatting

MacrofagenMacrofagen zijn immuuncellen die in de longen in grote getalen aanwezig zijn om een eerste verweer te vormen tegen pathogenen en kleine deeltjes in de lucht. Deze longmacrofagen zijn in twee populaties onderverdeeld: alveolaire macrofagen en interstitiële macrofagen. Alveo-laire macrofagen zitten aan de luchtkant van de longen in de alveoli (longblaasjes), waar ze snel kunnen reageren op (potentiële) ziekteverwekkers. De interstitiële macrofagen zitten in de wanden van de alveoli en de grote luchtwegen vlak onder het luchtwegepitheel en houden de gesteldheid van het longweefsel in de gaten. Beide macrofaag populaties spelen een rol bij het initiëren van een ontstekingsreactie.

Ondanks het feit dat macrofagen al lange tijd geleden ontdekt zijn, blijven de exacte functies van dit celtype in de longen onduidelijk. Dit heeft ook vooral te maken met de diversiteit aan functies die macrofagen kunnen uitvoeren. Zowel interstitiële als alveolaire macrofagen kun-nen een ontstekingsreactie veroorzaken in het geval van een infectie. Wanneer de infectie is bestreden, zorgen macrofagen ook weer voor longweefselherstel. Macrofagen zijn in staat om van de ene functie te switchen naar een andere functie, afhan-kelijk van wat er nodig is om het longweefsel gezond te houden. De specifieke functie wordt bepaald door de signalen die zij ontvangen van de omringende cellen. Om de verschillende subtypen macrofagen te kunnen identificeren, worden ze in dit proefschrift ingedeeld in drie categorieën. Ze worden gekarakteriseerd aan de hand van merkers, dat wil zeggen verschil-lende eiwitten, die ze tot expressie brengen. De drie subtypen die wij onderscheiden zijn: 3. M1 macrofagen die ook wel de pro-inflammatoire macrofagen genoemd worden en wor-

den gekenmerkt door de merker IRF5. Dit subtype ontwikkelt zich na blootstelling aan bepaalde cytokinen zoals interferon gamma (IFNγ) en producten van micro-organismen zoals lipopolysaccharide (LPS). Naast het feit dat M1 macrofagen infecties kunnen bestrij-den, zijn deze ook in staat hulp in te roepen van het adaptieve immuunsysteem door het aantrekken en activeren van T cellen.

4. M2 macrofagen, ook wel alternatief geactiveerde macrofagen genoemd, beschermen tegen infecties door grotere organismen zoals wormen en worden gekenmerkt door expressie van de merker CD206. Dit subtype ontwikkelt zich na blootstelling aan inter-leukine 4 en/of 13 en kunnen stervende cellen verwijderen tijdens een ontstekingsreactie.

5. M2-like macrofagen kenmerken zich door een ontstekingsremmende functie via produc-tie van het ontstekingsremmende cytokine interleukine 10 (IL-10). Naast het feit dat IL-10 ontstekingsreacties kan remmen, kan dit subtype ook het wondhelingsproces stimuleren door productie van de groeifactor transforming growth factor beta (TGFβ).

De verschillende subtype macrofagen en hun merkers zijn schematisch weergegeven in figuur 1.


193 8

Figuur 1. Schematische weergave van de drie macrofaag subtypen en de merkers waardoor ze van elkaar kunnen worden onderscheiden. Afkortingen: IFNγ: interferon gamma; TNF-α: tumor necrosis factor alfa; LPS: lipopolysaccharide; MHC class II: major histocompatibility complex class II; IL: interleukin; NO: nitric oxide; IRF5: interferon regulatory factor 5; Fe: iron; TGM2: transglutaminase 2; YM1: chitinase-3–like protein-3; FIZZ1/Relmα: resistin-like molecule-α; Arg-1: arginase-1; TGFβ: transforming growth factor beta; TLR: toll-like receptor; PGE2: prostaglandin E2.

LongziektenMacrofagen blijken essentieel in het handhaven van de gezondheid van de longen. Ze beschermen tegen infecties en helpen bij het herstelproces naderhand. Het is echter geble-ken dat macrofagen ook kunnen bijdragen aan ziekteprocessen. In bepaalde longziekten zijn er bijvoorbeeld te veel macrofagen aanwezig en verdwijnen ze niet meer. In andere longziek-ten is juist de functie van de macrofagen verstoord. Er is nog veel onbekend over het exacte type macrofaag dat een rol speelt bij longziekten zoals longfibrose en chronisch obstructieve longziekten (COPD). Welk type macrofaag is er aanwezig en waar zitten ze precies? Waar komen deze macrofagen vandaan? Welke functie hebben macrofagen die in de longen zit-ten? En kan deze functie ook worden beïnvloed om op deze manier longziekten te genezen?


Longfibrose en macrofagenLongfibrose is een ziekte waarbij overmatig bindweefsel gevormd wordt in de longen. Dit is een ongecontroleerd en voortdurend proces. Het bindweefsel verdikt de wanden van de longblaasjes, waardoor de uitwisseling van koolstofdioxide en zuurstof wordt bemoeilijkt. Dit veroorzaakt benauwdheid bij patiënten met longfibrose. Het is een progressieve, chro-nische ziekte die wordt veroorzaakt door een langdurige irritatie van de longen (15-20 jaar) door het inademen van kleine stofdeeltjes zoals metaal, hout of andere stofdeeltjes. Tot op heden zijn er nog maar twee geneesmiddelen beschikbaar: pirfenidon en nintedanib, maar die kunnen de ziekte slechts een beetje vertragen. In de meeste gevallen is de enige oplos-sing een longtransplantatie. De behoefte aan meer kennis over het ontstaan maar vooral ook het genezen van de ziekte is dus groot, waarbij de nadruk zou moeten liggen op het herstelproces. Patiënten met longfibrose komen vaak pas in een heel laat stadium naar het ziekenhuis, waardoor het remmen van de aanmaak van bindweefsel minder effectief lijkt te zijn. Een andere manier van behandelen zou zijn de afbraak van overmatig bindweefsel te stimuleren. Macrofagen kunnen een belangrijke rol spelen bij dit afbraakproces. Wanneer ze de juiste signalen ontvangen, kunnen ze diverse enzymen tot expressie brengen die hen de capaciteit geven om bindweefsel af te breken. Echter, bij longfibrose lijken macrofagen deze functie niet te hebben.

In hoofdstuk 2 hebben we daarom onderzocht waarom macrofagen niet aangezet worden tot het afbreken van overmatig bindweefsel. Hierbij hebben we ons gericht op de communicatie tussen bindweefsel-produceren cellen, fibroblasten, en macrofagen en hebben we ontdekt dat er waarschijnlijk een verstoorde communicatie is tussen deze celtypen. Dit communica-tiemechanisme hebben we afgekeken van hoe in botten de productie van bindweefsel wordt gereguleerd en hebben we vervolgens onderzocht in de longen. Onze hypothese was dat fibroblasten het eiwit receptor activator of Nfkb ligand (RANKL) produceren dat macrofagen aan kan zetten tot afbraak van bindweefsel door een interactie aan te gaan met de receptor activator of Nfkb (RANK) op macrofagen. Echter, RANKL kan worden weggevangen door osteoprotegerin (OPG). Dit eiwit functioneert als een ongebonden, vrije receptor van RANKL en komt zeer hoog tot expressie in de longen van patiënten met longfibrose. Dit kan in theorie de communicatie tussen RANKL en RANK sterk verstoren, met als gevolg dat macrofagen niet worden geactiveerd tot afbraak van bindweefsel.

Uit onze resultaten bleek dat RANKL niet door fibroblasten, maar door T cellen en epitheel-cellen wordt geproduceerd en dat OPG wel door fibroblasten wordt gemaakt, met name na fibrotische stimuli. Verder hebben wij in een muismodel van longfibrose de muizen extra RANKL toegediend om zo de grote hoeveelheid OPG in de longen te kunnen compenseren. We ontdekten dat de hoeveelheid OPG als reactie hierop alleen maar toenam. Dit geeft aan dat dit communicatieproces een rol speelt in de longen en wij hebben voor het eerst aange-toond dat er een delicate balans bestaat voor de hoeveelheid OPG in de longen. Aangezien we met de behandeling van RANKL nog niet in staat waren om de bindweefsel-afbrekende macrofagen te activeren, zullen we moeten zoeken naar andere mogelijkheden om OPG te blokkeren of om via een andere weg macrofagen te stimuleren om het overmatige bindweef-sel af te breken.


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COPD en macrofagenNaast de rol van macrofagen in longfibrose, hebben we ook macrofagen in COPD bestudeerd. COPD is een ziekte die gekarakteriseerd wordt door een chronische ontsteking in de longen, in de meeste gevallen als gevolg van roken. Patiënten kunnen in verschillende mate last heb-ben van chronische bronchitis (ontsteking van de grote luchtwegen) en emfyseem (afbraak van longweefsel). Hierdoor daalt de longfunctie en zijn patiënten vaak benauwd hetgeen verergerd als de ziekte vordert. Veelal hebben patiënten extra zuurstof nodig om het verlies aan longfunctie op te vangen.

In eerder onderzoek naar macrofagen in COPD is gevonden dat macrofagen bijdragen aan de afbraak van longweefsel. Alveolaire macrofagen brengen als een van de weinige macrofagen in hoge mate het enzym tartrate resistant acid phosphatase (TRAP) tot expressie. Dit enzym staat centraal in hoofdstuk 3 en we weten eigenlijk niet wat dit enzym doet in de longen. In botweefsel leidt verhoogde activiteit van dit enzym tot verhoogde weefselafbraak. We laten zien in hoofdstuk 3 dat de activiteit van dit enzym hoger is bij patiënten met COPD, rokers en ook in patiënten met fataal astma vergeleken met controles. De oorzaak van deze hoge acti-viteit in de longen is onbekend. Factoren die TRAP-activiteit kunnen verhogen in macrofagen zijn het al eerdergenoemde RANKL en oxidatieve stress. Het is bekend dat de oxidatieve stress hoog is in de longen van rokers, COPD-en astmapatiënten. Dit zou dus een oorzaak kunnen zijn van de verhoogde TRAP-activiteit.

Naast weefselafbraak is verhoogde TRAP-activiteit in botweefsel in verband gebracht met een grotere beweeglijkheid van bot-afbrekende cellen. Onderzoek naar de exacte functie van TRAP in de longen is lastig omdat goede remmers van dit enzym ontbreken. Er waren aanwijzingen dat het edelmetaal goud TRAP zou kunnen remmen en daarom hebben wij diverse organische verbindingen met een goud-atoom (Au) getest op hun potentie om TRAP-activiteit te remmen. Uit deze studies bleek AubipyOMe (zie figuur 2 voor de chemische ver-binding) de meest sterke remmer te zijn van TRAP. Vervolgens hebben we met deze remmer kunnen aantonen dat TRAP ook betrokken is bij de beweeglijkheid van macrofagen. Dit geeft aan dat we een eerste grote stap gezet hebben naar het ontwikkelen van een verbinding die als TRAP remmer ingezet kan worden om zo de functie van TRAP in de longen verder te kun-nen onderzoeken. Uiteraard is er nog meer onderzoek nodig naar bijvoorbeeld de specificiteit van de remmer voor TRAP en affiniteit voor TRAP.

Figuur 2. Chemische samenstelling van de TRAP remmer AubipyOMe.


MonocytenAlveolair macrofagen komen waarschijnlijk al voor de geboorte naar de longen en leven hier heel lang door zich continu te delen. In het geval van een infectie of een longziekte zoals hier-boven beschreven, kunnen deze langlevende lokale alveolaire macrofagen aangevuld worden met macrofagen die zich ontwikkelen uit monocyten, een type witte bloedcellen. Monocyten bevinden zich in het bloed en kunnen vanuit het bloed naar de longen reizen om te helpen bij de bestrijding van een infectie of te helpen bij het herstellen van beschadigd weefsel. Mono-cyten kunnen onderverdeeld worden in 3 subtypen aan de hand van expressie van de eiwitten CD14 en CD16. Monocyten genaamd “klassieke monocyten” en “intermediaire monocyten” zijn veelal betrokken bij ontstekingen, terwijl het derde subtype, de “niet-klassieke monocy-ten” meer een ontstekingsremmende functie lijken te hebben

Van de ontstekingsziekte COPD is bekend dat er ook effecten van de ziekte kunnen optre-den buiten de longen, de zogenaamde systemische effecten. In hoofdstuk 4 hebben wij ons daarom gericht op de aanwezigheid van deze drie subtype monocyten in het bloed van COPD patiënten om te kijken of de aanwezigheid van de verschillende subtypen wordt beïnvloed door de ontsteking in de longen. Daarbij verwachtten we te zien dat er meer klassieke en intermediaire monocyten in het bloed aanwezig zouden zijn dan bij controle individuen, omdat deze twee subtypen sterk geassocieerd zijn met ontstekingen. Daarnaast hebben we gekeken de expressie van twee specifieke eiwitten, Toll like receptoren (TLR2 en 4), die betrokken zijn bij het detecteren van rookschade door monocyten.

Uit onze analyses van bloed van COPD patiënten en gezonde rokers bleek dat COPD pati-enten meer klassieke en intermediaire monocyten hebben dan mensen die deze ziekte niet hebben. Deze toename bleek echter geassocieerd te zijn met leeftijd, dat wil zeggen dat het feit dat onze COPD patiënten gemiddeld ouder waren dan de controle mensen verklaarde dat zij meer klassieke en intermediaire monocyten hadden dan controle mensen en niet zozeer omdat ze COPD hadden. Dit effect van leeftijd op het aantal klassieke en intermediaire monocyten was ook al in studies van anderen aangetoond, maar verrassend was dat het ook zichtbaar was bij onze relatief kleine verschillen in leeftijd.

Naast het effect van het hebben van de ziekte COPD hebben we ook gekeken naar het effect van roken. Roken was geassocieerd met het hebben van minder ontstekingsremmende niet-klassieke monocyten en met verhoogde expressie van de rookschade-detectoren op de ont-stekingsbevorderende monocyten subtypen. Tezamen suggereren deze data dat roken het ontstekingsbevorderende gedrag van monocyten bevorderd.

Macrofaag subtypen in COPDEerder onderzoek heeft laten zien dat macrofagen bijdragen aan het ziekteproces van COPD. Echter, het is niet eerder onderzocht welke subtypen macrofagen er precies aanwezig zijn in de grote luchtwegen van COPD patiënten en in welke verhouding ze voorkomen. Daarom hebben wij aan de hand van de merkers, die al eerder beschreven zijn, deze verschillende subtypen in hoofdstuk 5 in kaart gebracht. Hiervoor hebben we het totaal aantal macrofagen en 3 verschillende subtypen M1, M2 en M2-like geteld in een grote luchtweg van COPD pati-enten en de aantallen vergeleken met die in een grote luchtweg van controle mensen, waarbij


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we rekening hebben gehouden met wel of niet roken, longfunctie en de dikte van de lucht-weg. De dikte van de luchtweg was met name interessant omdat M2 macrofagen in verband zijn gebracht met het stimuleren van de aanmaak van bindweefsel, hetgeen tot verdikking van de luchtweg kan leiden. In COPD kunnen de ontstekingsprocessen in de luchtwegen leiden tot meer bindweefsel, hetgeen weer kan leiden tot een slechtere longfunctie.

In grote luchtwegen van COPD patiënten bleken vergelijkbare aantallen totale en verschil-lende subtypen macrofagen te zitten als bij controle mensen. Wel was de longfunctie slechter van niet meer rokende COPD patiënten naarmate er meer M1 macrofagen in hun luchtwe-gen aanwezig waren. Opvallend genoeg had wel of niet roken een groter effect op het aantal macrofagen; rokers hadden significant minder macrofagen in hun luchtwegen en dan met name minder M1 macrofagen. Daarnaast vonden we dat dikkere luchtwegen gepaard gin-gen met minder M2 macrofagen. Dit was niet wat wij verwacht hadden op basis van eerder gepubliceerde onderzoeken en zou kunnen betekenen dat er binnen het subtype M2 nog verschillende functies mogelijk zijn.

Dit onderzoek is nog maar het begin van het bestuderen van macrofagen subtypes in COPD. Naast het karakteriseren van macrofagen op basis van merker eiwitten, is het van belang ook naar de functies van de subtypen bij COPD te kijken. Een belangrijk aspect hierbij is dat er meer functionele merkers voor de verschillende subtypen beschikbaar komen. Wan-neer functionele merkers gecombineerd kunnen worden met de huidige merkers voor de subtypen kan er meer gezegd worden over de bijdrage die een bepaald subtype heeft aan bijvoorbeeld een ziekteproces.

AanbevelingenIn welke mate macrofagen aanwezig zijn in de longen en wat hun bijdrage is aan ziektepro-cessen begint langzamerhand duidelijker te worden. Een belangrijk aspect is dat de macro-fagen een ziekte kunnen verergeren, maar wanneer het juiste subtype en dus de juiste functie wordt gestimuleerd, kunnen diezelfde macrofagen mogelijk bijdragen aan het herstel van de ziekte. Macrofagen hebben van nature deze plasticiteit en daar zouden we gebruik van moeten maken om longziekten proberen te genezen. Om dit soort onderzoek mogelijk te kunnen maken is het van belang eerst functionele merkers van de verschillende subtypen macrofagen in kaart te brengen.


Dankwoord

Zoals één van mijn stellingen al aangeeft: het doen van promotieonderzoek heeft mijn ver-wachtingen aan alle kanten overtroffen. Beide kanten op. Er waren ontzetten leuke, grappige en nieuwe ervaringen, maar ook minder leuke en zware momenten. Het blijkt dat je van die laatsten juist weer veel van kan leren. Uiteraard heb ik veel steun gehad en heel veel nieuwe mensen leren kennen, waarvan ik een aantal hier specifiek wil noemen en bedanken.

Allereerst wil ik uiteraard mijn promotor Barbro ontzettend bedanken. Onderhand zijn we al 6 jaar nauw met elkaar verbonden, omdat ik voor de start van mijn PhD in januari 2011 bij jou begon met een kort masterproject. Het besef hoeveel ik aan je heb gehad en hoe fijn het was om jou als begeleidster te hebben, kwam vooral naar voren naar aanleiding van je Long-fonds aanvraag. Daarin werd jou gevraagd om verbeterpunten van jezelf noemen. Je hebt toen deze vraag ook bij mij neergelegd: wat vind ik jouw verbeterpunten? Waarop mijn ant-woord was dat ik me geen betere begeleider had kunnen wensen. Je was geduldig; zelfs wan-neer mijn FACS analyse dramatisch uit de hand dreigde te lopen, hielp je me er doorheen. Aan het begin van mijn onderzoek heb je samen met me op het lab gestaan om me alles te leren: van flow analyses tot aan dierproeven, en wanneer de tijd rijp was, liet je me los. Maar altijd met een vinger aan de pols. Ik heb grote bewondering voor je doorzettingsvermogen, je nieuwsgierigheid naar nieuwe ontwikkelingen binnen het onderzoek en je vermogen om in te spelen op de persoonlijke behoeften van iedere student die je hebt begeleid. Heel erg bedankt! En last but not least: mijn introductie in de apparaten van Apple, ik wil en kan niet meer zonder ;).

Mijn promotor, Klaas. Ik heb al eens tegen je gezegd: wij spraken elkaar vaker met een bier-tje in de hand, dan op je kantoor. Niet dat daardoor de gesprekken minder leerzaam waren, in tegendeel. Ik heb genoten van onze gesprekken over groepsprocessen, leidinggeven en samenwerken. Allemaal aspecten die naar voren komen bij jouw nieuwe baan als vice-decaan en bij het doen van een PhD. Ik hoop dat we die gesprekken in de toekomst ook nog vaak zullen hebben. Dankjewel Klaas voor het verrijken van mijn promotieonderzoek door onze gesprekken.

En mijn promotor, Wim, jou wil ik ook ontzettend bedanken voor de tijd die je genomen hebt om mijn onderzoek in goede banen te leiden. Dankjewel voor de grote wetenschappelijke bijdrage die je hebt gedaan in de vorm van werkbesprekingen (zowel de wekelijkse meeting van de afdeling Pathologie als onze maandelijkse besprekingen) en voor de feedback op mijn manuscripten en mijn proefschrift.

Ook wil ik alle leden van de leescommissie, prof. dr. J. Grutters, prof. dr. M. Harmsen en prof. dr. R. Gosens, hartelijk bedanken voor de tijd en energie die jullie hebben geïnvesteerd om mijn proefschrift te beoordelen. Macro, erg jammer dat je niet bij de verdediging aanwe-zig kunt zijn, maar ik ben je dankbaar voor de feedback die je me gegeven hebt en de leuke email wisseling die daaruit voortvloeide.

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Uiteraard heb ik me tijdens het doen van mijn onderzoek nooit eenzaam gevoeld, mede dankzij het warme bad dat de afdeling FTT heet. Allereerst wil ik de vaste staf die alles moge-lijk maakt op de afdeling bedanken: Geny, Klaas, Barbro, Hans, Anna, Leonie, Inge, Daan en Jan. Bedankt voor het stellen van kritische vragen tijdens de werkbesprekingen en het stimuleren van samenwerkingen binnen en buiten de afdelingen. Geny, wat een geweldige bijdrage heb jij geleverd aan de wetenschap en ik ben blij dat de slices onderdeel uitmaken van mijn proefschrift. Hopelijk blijf je nog met de wetenschap verbonden, ondanks het aan-staande afscheid.

Chrisje (aka Christina aka princess), wat was het leuk om tegelijk met jou te kunnen begin-nen aan het avontuur van een PhD en ineens zijn we beide klaar. Ik ben ontzettend trots op je wat je bereikt hebt in deze periode en dat je de overstap hebt gemaakt naar een nieuwe baan in de VS. Hieruit bleek dat er tijdens die 4 jaar toch ruimte was voor onderzoek doen, want wat hebben wij een boel uren besteed aan kletsen over katten, sport, feestjes, familie, man-nen en nog veel meer! Dat jullie destijds voor het “Barbie en Ken” Oud&Nieuw feestje nog opzoek waren naar Kennen zal ik sowieso nooit vergeten. En je hebt er een geweldige man aan over gehouden! Ook zal ik nooit vergeten dat jij het voor elkaar kreeg om een Mac vast te laten lopen ook niet, hihi. Dankjewel voor de leuke tijd en je hulp tijdens de experimenten, humane longen en in het CPD in onze groene pakjes met hakjes. Ik vond het super leuk om deze periode met jou te delen! En inderdaad, niets gaat even.

During a PhD lifetime of four years (almost five…), many PhD students passed by and I would like to acknowledge every one of them who has shared time, laughter, hard work and/or a room with me during my PhD. Thank you for being a part of my PhD: Marlies, Inge, Karin, Mackenzie, Andreia, Amirah, Sara, Viktoriia, Xiaoyu, Adhy, Natalia, Benny, Ming, Suresh and Nia. Mijn kamergenootje, Marlies, wij hebben aan 1 woord genoeg, maar kunnen uren lullen. Wat miste ik jou als sparringpartner, toen je met je gezin naar Aruba vertrok. Maar gelukkig zijn er vliegtuigstoelen genoeg die die kant op gaan, dus deze afstand heeft ook niet in de weg gezeten. Nu weer terug in NL wens ik je al het beste voor jou, je gezin en familie! My dearest Andreia, I will always remember the first time you and Tiago visited Groningen and you asked the group if someone dances salsa, while we were having dinner in Het Pan-nenkoekenschip. That same night we went to Hemingway to dance the night away. Ever since, we have built a great friendship! I greatly admire your skills, both as a scientist and an artist and you are able to combine these in a beautiful way. And thank you for letting me and Sie-gard be a part of two of your most important days: your wedding and your PhD defense. Stay amazing and see you in Cardiff! And of course, Nia, thank you sooooo much for the time you spent doing experiments on slicing, helping out with human lungs and working so hard. You are the kindest person I know. I wish you all the best with finishing your PhD and a happy married life! Adhy, thank you for being a part of our group. It takes great courage to travel so far to an unknown country for a new job. You have grown along the way, with maybe the big-gest change when your son Zidney was born. I wish you all the best with the rest of your PhD and blessings to your wife, son and family.


For the newest generation of PhD students Valentina, Roberta, Keni, Daphne, Laura, Anienke and Gwenda. I want to wish you all the best with the rest of your PhD! En Anienke, sorry dat je in mijn voetsporen treedt ;), maar je vindt je eigen weg wel. En je mag me ALTJD stalken voor meer data/toegang tot mijn drive/uitleg/tips en tricks. Heel veel succes en ple-zier!

Zonder de steun van alle analisten op het lab, zou het lab ineenstorten. Daarom heel veel dank aan al deze mensen die ons onderzoek mogelijk maken. Eduard, steun en toeverlaat in het CDP en op het lab. Helaas verbonden ook onze knieproblemen ons, maar samen kneusje zijn is altijd beter dan alleen! Je stond je altijd klaar voor borrels, filmpjes maken, nagels lak-ken, mooie prikborden maken en mojito’s drinken! Keep it up! Catharina, een onuitputtelijke bron van ervaring op het gebied van celkweek en kleuren. Dankjewel voor het delen van je kennis! En uiteraard ook voor de gezelligheid bij de borrels, bijdrages aan de filmpjes en de mojito avonden! Jan, wie hadden er anders op het lab moeten passen tijdens de zomer dan wij twee! Geef dat roer maar aan ons over en het komt goed. Dankjewel voor je positieve ener-gie en je oneindige hulp. Marina, wij kennen elkaar natuurlijk al sinds mijn bachelor project. Ik mocht altijd bij je aankloppen voor een vraag, het was nooit te veel. Ook geweldig hoe je samen met Eduard altijd klaar stond om te helpen als er een humane long kwam! Ontzettend bedankt! Marjolijn, Alie en Marieke de Ruijter, praktisch hebben wij wat minder met elkaar te maken gehad, maar ook met jullie hulp hielden jullie alles draaiende, als de PhD studenten er een potje van dreigden te maken.

Gillian, ik heb er haast geen woorden voor om te beschrijven hoe ontzettend leuk, grappig, lief, behulpzaam en nog veel meer jij bent. Ik mis het nu al dat we de telefoon op kunnen nemen met een zwoele “Hellooo…”. Wat heb ik dubbel gelegen van het lachen om je verhalen en je vunzige dubbelzinnigheid, of was dat alleen mijn eigen dirty mind? Hoe dan ook: a joy forever!

En iedereen die op de afdeling was tijdens mijn PhD, dankjewel voor een super leuke tijd! Magdalena, Miriam, Jai, Ruchi, Hans, Frits, Marianne, Sylvia, Ventakesh, Martin, Adriana, Na, Paul, Marike van Beuge, Sanna!, Marieke Elferink, Femke Hoogstra-Berends, Bert, Christa, Henk, Amit, Kaisa en Irma. My dear Finnish girls: Sanna ja Kaisa, kiitoksia kovasti kivasta ajasta! Oli aivan mahtavaa puhua Suomea töissä! Ihanaa että olemme vielä yhteydessa seka Hollanissa että Suomessa. En Irma, dankjewel voor je aanstekelijke lach en het introdu-ceren van de hoogste frequenties om “doei” te zeggen aan het einde van de dag. Ik vraag me nog steeds af of iedereen de hoogste tonen kon horen. Al het beste voor je lieve gezinnetje!

Mijn paranimfen: mijn schat Fransien en mijn lieve zusje Nina. Lieve Frannie, je kwam als scheikunde student binnen in de farmaceutische wereld van FTT, hebt mij uit de brand geholpen als analist (“Ja baas”) en werd mijn roomie toen je aan je PhD begon. Eerst achter Chrisje, die het niet kon laten om achterstevoren op haar stoel op jouw beeldscherm te kijken, en later naast mij. Ik vraag me af of het überhaupt een moment stil is geweest op onze kamer sinds die tijd. Sieg had het idee dat het net iets TE gezellig was bij ons op de kamer :-P. Maar wat was het fantastisch! Het was ook zo bijzonder om deel uit te maken van je vrijgezellen-

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feest, waar duidelijk werd hoe ontzettend veel liefde jij hebt om te delen, maar ook krijgt van alle lieve mensen die je om je heen hebt. Heel veel succes met je promotieonderzoek (hoewel dat niet nodig is, want jij bent de beste!) en alle geluk toegewenst aan jou en lieve Jakkie. Dan mijn liefste zusje Nina, wat vind ik het geweldig dat je creatief hebt bij kunnen dragen aan mijn boekje. Het is altijd zo fijn om even te kunnen praten, relativeren en jouw raad te krijgen. Ik blijf soms een olifant in een porseleinkast en jij kan de juiste nuances aanbrengen. Dankje lieve Niensel!

Ook wil ik alle mensen bij de pathologie bedanken voor hun input tijdens de werkbespre-kingen: Machteld, Corry-Anke, Patricia, Wim, Marjan. Ik was erg blij met jullie input als “long groep”, aangezien wij ons meer in het “lever” veld begaven. Ook alle dank naar de leden van GRIAC. Het was ontzettend leerzaam om over de uiteenlopende onderzoeken te horen,vooral de klinische kant van de longziekten. Dit hielp ook mee om mijn eigen onder-zoek in een breder perspectief te kunnen zien!

We had support from different sides to be able to do this research. David Brass, you have set the base for the largest and most precious chapter of my thesis. It was a blast to meet you for the first time in Big Sky, Montana and we could not stop joking about our height difference! Thank you for your input and fruitful discussions. Ook mijn dank aan Robbert Cool. Jij hebt ons zo ontzettend geholpen met de productie van RANKL en je bijdrage aan en feedback op het OPG artikel. Daarnaast wil ik ook onze samenwerking met Rotterdam bedanken: Bernt van den Blink en Peter Heukels. Bernt, heel veel succes met je nieuwe baan en Peter, ik vrees dat die database nog steeds niet staat, haha. Alle co-auteurs die boven de manuscripten staan in dit proefschrift: bedankt voor je bijdrage en je feedback op de manuscripten.

Tijdens mijn PhD hebben een aantal Master studenten een onderzoeksproject gedaan bij ons, die ik ook wil bedanken voor hun bijdrage aan mijn onderzoek: Tirza, Burak, Marce-lina, Bram, maar ook Fiona, die na haar project mij nog als analist heeft geholpen. Daarnaast zijn er meerdere bachelor studenten van de opleiding Farmacie bij ons geweest. Ook jullie: bedankt voor je bijdrage! Als ik er zo op terug kijk, is de lijst van mensen die ik heb leren kennen tijdens mijn PhD en die mij hierbij hebben ondersteund bijna oneindig. Ook zijn de leuke, hilarische en ont-roerende momenten te veel om op te noemen. Iedereen echt ontzettend bedankt voor een fantastische tijd! En dat de echo van mijn lach maar lang mag ronddwalen over de gangen van PTT.

Het PTT nest heb ik onderhand al bijna een jaar geleden verlaten om een nieuwe uitdaging aan te gaan bij IQ Products als Technical Sales Specialist. In het kort: ik mag veel praten, ik reis veel en dat allemaal met leuke mensen om mij heen. Lieve collega’s van IQ, ik had van tevoren niet gedacht dat de sfeer bij PTT geëvenaard kon worden, maar daar zijn jullie zeker in geslaagd. Ik heb het tot nu toe heel erg naar mijn zin gehad bij jullie en kijk uit naar de komende tijd!


Dan last but not least, mijn lieve familie en vrienden. Jullie hebben me zo ontzettend gesteund en in mij geloofd. Zonder die steun was ik lang niet zo ver gekomen. Äiti, olit aina valmis kuuntelemaan minua ja aina sulla oli vyvää neuvoa, niin kuiin äidit kuuluukin antaa, hihi. Suuuukkkkoooo! Jaap, wij zijn twee handen op één buik. Je was altijd geïnteresseerd in mijn onderzoek en wilde alles weten. Dankjewel voor je luisterend oor, daddy! Mirk, ons contact is wisselend geweest, maar ik ben trots op wat je hebt bereikt en dat je de stap hebt durven nemen om achter Line aan te gaan naar Noorwegen.

Mijn liefste Sniegy, sorry voor alles dat je te voorduren hebt gehad met mij. Mijn stressmo-menten, het zeuren en de zenuwinzinkingen. Maar ik weet dat je trots op me bent, zoals ik ook trots ben op jou. Er zat ook een voordeel aan het harde werken, en dan vooral dat ik soms door werkte in de weekenden. Dit betekende dat jij je minder schuldig voelde als je zelf weg was voor een klus op zaterdag of de administratie nog moest doen op zondag. Dankjewel voor je oneindige steun en begrip!

Dank jullie allemaal! Thank you so much! Kiitos kovasti!

Curriculum

vitae

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Curriculum Vitae

Carian Eline Boorsma was born March 18th 1987 in Groningen, The Netherlands. Besides the Dutch nationality, she has the Finnish nationality as her mother is from Finland. In 2005, she stared studying Pharmacy at the University of Groningen. Already in the second year she decided to switch towards Pharmaceutical Sciences and received her Master in Medi-cal Pharmaceutical Sciences in the spring of 2011. Her first research project was conducted with Geny Groothuis at the department of Pharmacokinetics, Toxicology and Targeting. The second project was with Prof. dr. van Gilst at the Experimental Cardiology department of the University Medical Center Groningen. In 2011, she started as a graduate student at the lab of prof. dr. Barbro Melgert. During her project she investigated the role of macrophages in pulmonary diseases. Due to her research, she was awarded the Young Investigator Award by the Dutch Respiratory Society April 2014. On December 2nd 2016 she defends her PhD thesis.

As a student, she was an assistant at many different theoretical and practical classes. During her PhD, she was involved in the education committees for the PhD training- and education programs of the Graduate School of Medical Sciences (GSMS) and the GUIDE institution. She was also a member of the PhD Council of the GSMS as a representative for all PhD students of GUIDE for 3 years.

Aside her studies and her research project, Carian played basketball on a National level. The hard work payed off when she won the Championship of the “Promotie Divisie” in 2012 with her team “De Groene Uilen”.

In March 2016 she stared her job at IQ Products in Groningen as a Technical Sales Specialist.



Draijer C, Boorsma CE, Reker-Smit C, Post E, Poelstra K, Melgert BN. PGE2-treated mac-rophages inhibit development of allergic lung inflammation in mice. Journal of Leukocyte Biology, 2016.

Draijer C, Hylkema MN, Boorsma CE, Klok PA, Robbe P, Timens W, Postma DS, Greene CM, Melgert BN. Sexual maturation protects against development of lung inflammation through estrogen. American Journal of Physiology. Lung cellular and molecular Physiology, 2016.

Boorsma CE, Dekkers BG, van Dijk EM, Kumawat K, Richardson J, Burgess JK, John AE. Beyond TGF-beta: novel ways to target airway and parenchymal fibrosis. Pulmonary Phar-macology & Therapeutics, 2014.

Boorsma CE, Draijer C, Melgert BN. Macrophage heterogeneity in respiratory diseases. Medi-ators of Inflammation, 2013.

Draijer C, Robbe P, Boorsma CE, Hylkema MN, Melgert BN. Characterization of macro-phage phenotypes in three murine models of house-dust-mite-induced asthma. Mediators in Inflammation, 2013.

Manuscripts submitted and in revision

Boorsma CE, Putri KSS, Draijer C, Heukels P, van den Blink B, Cool RH, Brandsma CA, Nos-sent G, Brass DM,Olinga P, Timens W, Melgert BM. Exploring the Role of Osteoprotegerin in Pulmonary Fibro-sis. Submitted, 2016.

Boorsma CE, Putri KSS, de Almeida A, Draijer C, Mauad T, Brandsma CA, van den Berge M, Bossé Y, Sin D, Hao K, Olinga P, Timens W, Casini A, Melgert BN. A Potent Tartrate Resistant Acid Phosphatase Inhibitor to Study the Function of TRAP in Alveolar Macrophages. Sub-mitted, 2016.

Melgert BN, Boorsma CE, Postma D, van Geffen WH, Kerstjens HAM, Hylkema MN, Timens W, Brandsma CA. Current smoking is associated with fewer CD16+ monocytes in peripheral blood of male healthy controls and patients with COPD. Submitted, 2016.

Draijer C, Robbe P, Boorsma CE, Hylkema MN, Melgert BN. Dual role of YM1+ macrophages in allergic airway inflammation. Submitted, 2016.

Draijer C, Boorsma CE, Robbe P, Timens W, Hylkema MN, Ten Hacken NHT, van den Berge M, Postma DS, Melgert BM. Asthma is characterized by more IRF5+ M1 and CD206+ M2 mac-


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rophages and less IL10+ M2-like macrophages around airways compared to healthy airways. In revision for JACI, 2016.

Patents

Melgert BM and Casini A. Gold(III) compounds as Tartrate Resistant Acid Phosphatase inhib-itors, and therapeutic uses thereof. Filled 2015.

university of groningen macrophages: the …...carian eline boorsma noordelijke cara stichting the...

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