review of the role of macrophages in inflammatory bowel disease

The role of macrophages in

inflammatory bowel diseases

Sigrid E.M. Heinsbroek1,* and Siamon Gordon2

The small and large intestine contain the largest number of macrophages in thebody and these cells are strategically located directly underneath the epitheliallayer, enabling them to sample the lumen. Such intestinal macrophages have adifferent phenotype from other tissue macrophages in that they ingest andmay kill microbes but they do not mediate strong pro-inflammatory responsesupon microbial recognition. These properties are essential for maintaining ahealthy intestine. It is generally accepted that tolerance to the intestinal flora islost in inflammatory bowel diseases, and genes involved in microbialrecognition, killing and macrophage activation have already been associatedwith these diseases. In this review, we shed light on the intestinal macrophageand how it influences intestinal immunity.

About130yearsago,ElieMetchnikoffput forwardthe idea of macrophage-like cells that, as part ofmammalian immunity, patrol the body and takeup foreign material (Ref. 1). Nowadays, themacrophage is known as a versatile cell playingcentral roles in inflammation, wound healing,tissue homeostasis and tissue remodelling.Macrophages are professional phagocytes,clearing dying cells and pathogens. They canalso function as antigen-presenting cells and areable to secrete a variety of cytokines thatmodulate migration and activation of otherimmune and nonimmune cells (Ref. 2).The intestinal immune system has to remain

tolerant to beneficial flora and encounteredfood antigens while dealing with invadingpathogens. This suggests that immuneresponses in the intestine need to be tightly

regulated. Together with dendritic cells, thestrategically located macrophages containpattern-recognition receptors (PRRs) torecognise conserved microbial molecules, andprovide immune-modulating functions that areof major importance in this regulation.Continuous activation of intestinal immuneresponses by normal intestinal flora is believedto cause inflammatory bowel diseases, whichare characterised by abdominal pain, diarrhoea,rectal bleeding, weight loss, malnutrition andfever (Ref. 3). The inflamed mucosal tissuesdisplay a large influx of granulocytes andmonocytes, and the inflammatory responseslead to further influx of inflammatory cells anddestruction of the mucosal structure.Two major forms of inflammatory bowel

disease are Crohn disease and ulcerative colitis,

1Department of Gastroenterology, Academic Medical Centre, University of Amsterdam,Amsterdam, The Netherlands.

2Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

*Corresponding author: Sigrid Heinsbroek, Department of Gastroenterology, University ofAmsterdam, 1105 AZ Amsterdam, The Netherlands. Tel: +31 20 5664109; Fax: +31 20 6917033;E-mail: [email protected]

expert reviewshttp://www.expertreviews.org/ in molecular medicine

1Accession information: doi:10.1017/S1462399409001069; Vol. 11; e14; May 2009

&2009 Cambridge University Press

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which are distinguished by the affected area:ulcerative colitis exclusively affects the colon,whereas Crohn disease can affect the wholegastrointestinal tract but is mainly localised tothe colon and ileum (Ref. 3). These two diseaseshave also been suggested to have differentcytokine profiles, with a T helper 1 (Th1)phenotype for Crohn disease and a Th2phenotype for ulcerative colitis. The incidence ofinflammatory bowel diseases in Europe isestimated at 2.2 million (Ref. 4). As a resultof limited understanding of the diseasepathogenesis, treatment is symptomatic andmainly relies on nonspecific immune suppression.Patients are thus chronically dependent onmedication, and have significant morbidity.The role for immune cells in inflammatory

bowel diseases has been extensively studied,and the influences on intestinal immuneresponses of dendritic cells, T cells, B cells andmast cells have been reviewed (Refs 5, 6, 7 and8, respectively). The role for macrophages ininflammatory bowel diseases is underlined bygenetic studies that link macrophage-specificmolecules with disease (Refs 9, 10), andexperimental colitis models where a lack ofmacrophages in the intestine increasesinflammation (Ref. 11), as discussed further inBox 1. In this review, we focus on the intestinalmacrophage, discuss its specific phenotype, andhighlight new insights into its role in intestinalinflammation.

Monocyte development into intestinalmacrophages: modulation of phenotypeThe intestine is lined with a layer of enterocytes(intestinal epithelium), which form thefirst barrier to the flora; some enterocytesspecialise into goblet cells that secretemucus. Macrophages lie strategically locatedunderneath the enterocytes, and their locationsuggests that they will be the first immune cellsin contact with invading pathogens (Fig. 1). Thelamina propria (loose connective tissue underthe epithelium) of the large and small intestinehas the largest number of resident macrophagesin the body (Ref. 12). Other cells in the laminapropria include mast and dendritic cells. T andB cells are mainly located within Peyerspatches, which also contain macrophages in thedome region. Peyers patches containspecialised epithelial cells, called M-cells, thattake up large samples of the intestinal lumen.

To control tissue homeostasis, the macrophageinteracts with epithelial, vascular and otherimmune cells as well as neurons.In general, macrophages develop from

granulocyte/monocyte precursors in the bonemarrow. These precursor cells develop intomonocytes under the influence of macrophagecolony-stimulating factor (M-CSF; also know asCSF-1), granulocytemacrophage colony-stimulating factor (GM-CSF; also known asCSF-2) and interleukin (IL)-3. Monocytes leavethe bone marrow, enter the bloodstream andafter approximately one day migrate intotissues where they differentiate intomacrophages. Little is known about howresident lamina propria macrophages becomelocalised to the intestine. Mucosal IL-8 andtransforming growth factor b (TGF-b) havebeen shown to be chemoattractants for bloodmonocytes (Refs 13, 14), but it is unclear if thisis sufficient to attract monocytes to the healthyintestine. Some tissue macrophages are able toproliferate locally, including Kupffer cells(Ref. 15) and alveolar macrophages (Ref. 16),but it has been shown that lamina propriamacrophages are unable to replicate (Ref. 14),which suggests intestinal macrophages arereplenished solely by blood monocytes.Interestingly, lamina propria macrophages are

phenotypically distinct both from bloodmonocytes and from other tissue macrophages(Table 1). Inflammatory macrophages and tissuemacrophages develop from distinct monocytesubsets: inflammatory cells develop fromCX3CR1loGR1 monocytes and tissuemacrophages from CX3CR1hiGR2 monocytes(Ref. 17). This supports the general idea thatresident lamina propria macrophages differ inphenotype and function from recruitedmacrophages during inflammatory boweldisease. Furthermore, development into tissuemacrophages is mediated by factors specific tothe local environment, which renders tissuemacrophages heterogeneous in function,receptor expression and inflammatoryresponses (Ref. 18).

The noninflammatory nature of laminapropria resident macrophagesDifferentiation into intestinal macrophagesresults in an anti-inflammatory phenotypealthough bactericidal activity is retained(Ref. 19). The exact factors involved in such




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differentiation are unclear, but cytokines such asTGF-b and IL-10 are able to induce at least some ofthe intestinal macrophage features (Ref. 20). Boththese cytokines are able to inhibit NF-kBactivation mediated by PRRs and downregulatesecretion of the inflammatory cytokine tumournecrosis factor a (TNF-a) (Refs 19, 21, 22, 23,24). Deficiency of TGF-b and IL-10 leads todevelopment of spontaneous inflammatorydiseases (Refs 25, 26). Unlike other residentmacrophages, the majority of lamina propriamacrophages lack TREM-1 (triggering receptorexpressed on myeloid cells 1), a receptorinvolved in amplification of inflammatoryresponses induced by microbial products. TGF-band IL-10 downregulate TREM-1 expression,supporting the idea that these cytokines areessential for stimulating a lamina propriaphenotype (Ref. 27). TGF-b alone downregulatesexpression of the immunoglobulin A (IgA) Fcreceptor (CD89) and the complement receptorCR3 (Refs 28, 29), while IL-10 affects expressionof C-type lectin PPRs (Refs 30, 31). Eventhough IL-10 and TGF-b render macrophages

anti-inflammatory, this seems not sufficient toproduce a complete intestinal macrophagephenotype, and other factors such as directinteraction with intestinal epithelial cells andinteractions with extracellular matrix proteinssuch as collagen have been suggested to play arole (Refs 32, 33). It is also likely that continuousexposure of lamina propria macrophages tomicrobial ligands influences the macrophagephenotype directly, via stimulation of PRRs; forinstance, prolonged muramyl dipeptide (MDP)stimulation via the PRR NOD2 (nucleotide-binding oligomerisation domain-containingprotein 2) mediates tolerance to subsequentstimulation with ligands for the Toll-likereceptors TLR2 and TLR4 (Ref. 34).Human lamina propria macrophages lack

expression of various PRRs, including thelipopolysaccharide (LPS) receptor CD14,the CR3 component CD11b and Ig receptorsCD16, CD32 and CD64, supporting a tolerantphenotype towards the intestinal flora(Refs 19, 35, 36). Furthermore, intestinalmacrophages also lack receptors for IL-2 and

Box 1. Methods for studying macrophage function in inflammatory bowel disease

Various tools are available to study mechanisms and cells involved in inflammatory bowel disease. Animalmodels have proved extremely useful. These can be divided into three categories depending on the cellsinvolved (Ref. 150): models that depend on a defect in the epithelial barrier function, models with a defectin innate immunity, and models caused by a defect in adaptive immunity. For most of these models,transgenic mice or mice with a specific gene deficiency are needed, which limits their use.Widely usedmodels are dextran sodium sulphate (DSS)- and trinitrobenzene sulphonic acid (TNBS)- induced

colitis, which are easy to use and low in cost. DSS damages the epithelial barrier, whereas TNBS haptenisesself-moleculesmaking them immunogenic. DSS-induced colitis is particularly interesting for the study of innateimmune function since T- and B-cell-deficient mice also develop disease in this model, suggesting it is mainlydriven by innate immune responses (Ref. 150). Using aDSS-inducedcolitismodel it was shown that depletion ofmacrophages causes more severe intestinal inflammation (Ref. 11). However, it has also been shown thatmacrophage depletion reduced spontaneous colitis in interleukin (IL)-10-deficient mice (Ref. 151). Thedifference in outcome of macrophage depletion is most likely due to the different mechanisms of inducedcolitis, and this may underline the dual function of intestinal macrophages. DSS-induced colitis disrupts themucosal barrier leading to microbial contact with the underlying lamina propria; depleting macrophageshere would lead to partial loss of tolerance and increased inflammation. IL-10-deficient mice developchronic colitis as a result of a lack of tolerance, which leads to an influx of inflammatory macrophages anddepleting these will reduce inflammation.The downside of using animal models of inflammatory bowel disease is that no one model represents all

aspects of the human disease, and so studies with patient material are needed to supplement and validatedata. Patient material is mainly used for genetic studies and many genes have been implicated ininflammatory bowel disease, as indicated in the main text and summarised in Table 2.Studying intestinalmacrophage function is complicatedby the fact that isolation andculturemethods change

macrophage phenotype and have low yields. Many researchers use blood-derived monocytes instead, whichalso lack the intestine-specific resident phenotype. Attempts to create intestinal macrophage cell lines havefailed because of a lack of knowledge of environmental factors needed to induce a lamina propria phenotype.




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IL-3 as well as the chemokine receptorsCCR5 and CXCR4, and the integrin LFA-1(leukocyte-function-associated antigen 1), whichprobably leads to a reduced response to certainimmune stimuli (Refs 19, 37). Lack of thecostimulatory molecules CD80 and CD86renders them unable to activate T-cell-mediatedimmune responses (Ref. 35). Together, thisphenotype results in a tolerant macrophage thatis highly phagocytic and bactericidal withoutsubsequent secretion of pro-inflammatorycytokines.The small and large intestine are distinct with

respect to function, amount of flora and themicrobial species they contain, and so it is notsurprising that there are differences betweenmacrophages isolated from these sites. Forinstance, jejunal macrophages express majorhistocompatibility complex class II antigen(MHCII), TLR2 and TLR4, but these seem to beabsent from colonic macrophages (Refs 19, 35,36, 38). Nevertheless, it is generally clear thatintestinal macrophages have an unusuallytolerant phenotype, although underlying

mechanisms are only starting to be clarified.Signalling components and macrophagereceptors that inhibit immune-stimulatoryresponses including SOCS (suppressor ofcytokine signalling) proteins, IRAK-M (IL-1-receptor-associated kinase), the CD200 receptor,and SHPS1 (SHP substrate 1; also known assignal-regulatory protein a, Sirpa) are stilllargely unexplored in the intestine, but arelikely to play important roles in maintainingintestinal tolerance (Ref. 39).

Inflammatory properties of recruitedmonocytes and macrophagesDuring intestinal inflammation, bloodmonocytesare recruited and differentiate into activatedmacrophages that produce pro-inflammatorycytokines (Refs 13, 40, 41, 42). This pro-inflammatory response has detrimental effects,leading to epithelial apoptosis, barrierdysfunction, necrosis, granuloma formationand fibrosis. Radiolabelled CD14 monocytesinjected back into patients with activeinflammatory bowel disease located

Localisation of macrophages within the lamina propriaExpert Reviews in Molecular Medicine 2009 Cambridge University Press

Intestinalepithelium

Mucus layer

Dendriticcell

Peyers patch

T cells B cells

LumenBacteria andfloral fungi

Lamina propria

Macrophage

Gobletcell

M cella

b c d

Figure 1. Localisation ofmacrophageswithin the lamina propria. Possible routes for microbial uptake in theintestine are indicated: (a) translocation throughM-cells; (b) endocytosis by enterocytes formicrobes,0.5 mm;(c) direct samplingof thegut lumenbydendritic cells that can formprotrusionsbetweenenterocytes; (d) invasionof the lamina propria by pathogenic micobes.




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predominantly to inflamed parts of the intestine(Ref. 42). Lamina propria macrophages frompatients with inflammatory bowel disease havebeen shown to upregulate expression of CD14,TREM-1, CD80 and CD86 (Refs 43, 44), andhave increased NF-kB activation (Ref. 45).Furthermore, a recent study of Crohn diseasepatients revealed an increased number of CD14-expressing macrophages in the intestine thatstimulate higher levels of interferon g (IFN-g)and IL-23 production (Ref. 46). These cells alsoproduced more TNF-a, in keeping withfindings from other studies of lamina propriamacrophages of Crohn disease patients (Refs47, 48).TNF-a is a key cytokine in inflammatory bowel

disease, and neutralising anti-TNF-a antibodiesare effective in treating many patients.However, besides its potent effect oninflammatory cells, TNF-a also regulatesepithelial cell growth and apoptosis, intestinalpermeability and matrix metalloproteinaseproduction. Therefore, it is not surprising that acomplete lack of TNF-a leads to more severeinflammation in colitis models (Ref. 49). Achemokine receptor antagonist that reducesinflux of inflammatory macrophages loweredpro-inflammatory cytokines and inflammationin experimentally induced colitis (Ref. 50), andsome drugs used to treat inflammatory boweldisease indeed reduce monocyte recruitment tothe inflamed intestine.

Interaction with microbesIntestinal floraCurrently it is still unclear if inflammatory boweldiseases are caused by changes in intestinal floraor by changes in immune responses towards theflora, but it is generally accepted that the intestinalflora plays a major role; this is supported byexperiments where IL-10-deficient mice, whichdevelop spontaneous colitis, stay healthy whenkept under sterile conditions, and, furthermore,antibiotics reduce inflammation in animalmodels and patients with inflammatory boweldisease (Ref. 51). Bacteria make up 99% of theintestinal flora; the remainder is mainly fungiand possibly protozoa. The highest number ofbacteria is found in the colon, which mayharbour more that 400 different, predominantlyanaerobic, species (Ref. 52). Besides theimportant role in protection against colonisationby pathogens and production of co-enzymes,the intestinal flora is also essential for intestinalhomeostasis (Ref. 53). Furthermore, a recentstudy has shown that bacterial ATP in thelumen could be responsible for the presence ofTh17 cells in the lamina propria (Ref. 54).According to the hygiene hypothesis, disorders

such as inflammatory bowel diseases andallergies, which are typical of the developedworld, arise as a result of a lack of microbes toshape the immune system; in particular, it hasbeen proposed that a lack of helminth infectionis related to predisposition to inflammatory

Table 1. Macrophage phenotype

Property Intestinalmacrophage

Inflammatorymacrophage

Refs

Phagocytic capability 1 1 20Toll-like receptors (TLR2, TLR4) 2 1 37C-type lectins (dectin-1, mannose receptor) 1 1 106Fc receptors (CD16, CD32, CD64) 2 1 20Complement receptors (CR3, CR4) 2 1 20Costimulatorymolecules (CD40,CD80,CD86) 2 1 41Chemotaxis (CCR5, CXCR4) 2 1 38Microbial killing 1 1 20Pro-inflammatory cytokine production

(TNF-a, IL-1, IL-6, IL-8)2 1 20

Activation inflammatory responses (TREM-1) 2 1 42

Abbreviations:CCR5,C-Cchemokine receptor type5;CXCR4,C-X-Cchemokine receptor type4; IL, interleukin;TNF-a, tumour necrosis factor a; TREM-1, triggering receptor expressed on monocytes 1.




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bowel diseases (Ref. 55). This is supported by asubstantial amount of data from studies in bothexperimental models and Crohn diseasepatients that suggest that helminth infectionshifts the predominantly Th1 immune responseto a Th2 response (Ref. 56). Macrophages havealso been shown to play a role in helminth-induced tolerance. Helminth infection increasedthe numbers of immunosuppressive intestinalmacrophages, which reduced experimentallyinduced colitis (Ref. 57). Together these dataform substantial evidence to suggest that theflora is essential for shaping intestinal immunity.Differences in flora composition and metabolic

activity have been found in patients withinflammatory bowel diseases compared withhealthy controls, including a reduction innumbers of potentially beneficial Bifidobacteriaand Lactobacillus species (Refs 58, 59). Differentviruses have been associated with inflammatorybowel disease, including Paramyxoviruses andherpes family viruses; however, available dataare largely contradictory (Ref. 39). The innateimmune system may also influence microbialflora composition as is shown by an elegantstudy using Rag22/2 T-bet-deficient mice: suchmice, lacking adaptive immune responses anddeficient in the inflammation-regulatingtranscription factor T-bet, develop spontaneouscolitis, and, interestingly, bedding materialtransferred disease to healthy wild-type mice(Ref. 60).In the normal healthy intestine, epithelial cells

form a barrier to the microbial flora; however,several ways have been described throughwhich the microbial flora are able to cross thisbarrier (Ref. 61) (Fig. 1). First, enterocytes areable to mediate endocytosis of antigens thathave diffused through a thick mucus layer, andthese antigens can then be presented tomacrophages and dendritic cells underneaththe basement membrane. Second, professionalendocytic and phagocytic M-cells take up largeamounts of microbial and food antigens, whichare thereby presented to underlying dendriticcells and macrophages. M-cells are solelyassociated with Peyers patches and the largenumber of internalised microbes is enhanced bya reduction in mucus covering these cells.Third, dendritic cells are able to formprotrusions through the epithelial layer to takeup luminal bacteria, although it is not knownwhether macrophages can also achieve this

(Ref. 62). Last, some pathogenic microbes havedeveloped ways to invade via intestinalepithelial cells, including Salmonella and Shigellaspecies; furthermore, functional alterations incommensal microbes may cause invasion byspecies generally seen as nonpathogenic (Ref. 63).

Pattern-recognition receptorsRecognition of microbial flora is essential forregulating immune responses and this ismediated by PRRs. PRRs recognise highlyconserved molecules expressed by microbesreferred to as pathogen-associated molecularpatterns (PAMPs). These PAMPs are generallyimportant for survival of microbes and includeLPS (part of the cell wall of Gram-negativebacteria), lipoteichoic acid (LTA) (part of Gram-positive bacteria), mannans and b-glucans(found on fungi), and viral double-strandedRNA or foreign DNA (Ref. 64). PRRs expressedby macrophages include TLRs (which aremembrane-bound molecules), C-type lectin(-like) molecules (which are mainly membranebound but can also be secreted), and cytosolicPRR-like molecules containing NODs, whichrecognise intracellular pathogens (Ref. 64).Recognition by PRRs can lead to phagocytosis,secretion of pro-inflammatory cytokines, andinduction of apoptosis and immunity.

Toll-like receptorsTheTollreceptor,previouslyshowntobeimportantin development, was first found to be involved inimmune defence against fungi in Drosophila(Ref. 65). Soon after, mammalian homologueswere characterised and to date the TLR family isknown to contain at least 11 members, with eachof them recognising specific ligands such as LPS,peptidoglycan, flagellin (a component of microbeflagella), DNA and RNA. TLRs are type Itransmembrane glycoproteins and are part ofthe IL-1 receptor family. The cytoplasmicsignalling domain is similar to the IL-1 receptorsignalling domain and is called the Toll/IL-1R(TIR) domain; it is characterised by threehomologous regions that are essential forsignalling. The extracellular domain of TLRs isvery different from the Ig-like domain of IL-1Rs,consisting of 1925 tandem-repeat copies ofleucine-rich repeats (LRRs), which mediateligand binding. Upon ligand recognition, TLRsdimerise and change conformation, whichrecruits downstream signalling molecules. The




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majority of TLRs signal via a pathway dependenton Myd88 (myeloid differentiation primaryresponse protein) that leads to NF-kB activationand translocation to the nucleus, where ittriggers expression of various genes that caninduce many processes, including cytokineproduction (e.g. TNF-a and IL-12), nitric oxide(NO) production, actin reorganisation,phagocytosis, phagosome maturation andinduction of apoptosis (Refs 66, 67, 68, 69). TLR3is the only TLR that is unable to signal viaMyd88 and instead signals via TRIF (Tir-domain-containing adaptor), while TLR4 is able tostimulate both Myd88- and TRIF-dependentpathways (Ref. 70).Monocytesandmacrophages expressmRNAfor

most TLRs, except perhaps TLR3 (Ref. 71). TLR1,TLR2 and TLR4 can be found on the surface ofcells, while TLR7, TLR8 and TLR9 are expressedon intracellular membrane compartments,including endosomes and endoplasmicreticulum (Refs 72, 73, 74, 75). It has beenproposed that intracellular compartments may bethe main site for TLR recognition of microbialcomponents, including surface-expressedmolecules (Refs 76, 77). TLRs are able tocollaborate with other PRRs, to produce aspecifically tailored microbial response.Mice deficient in TLR2, 4 or 9, which recognise

peptidoglycan, LPS and microbial DNA,respectively, have shown contradictory resultsin colitis studies, with some studies reportingsusceptibility to disease and others showingdisease protection (Ref. 78). Interestingly,patient studies have shown association only ofTLR4 with inflammatory bowel diseases(Refs 79, 80). TLR5 is the main receptor forflagellin, and since Crohn disease patients haveelevated levels of antiflagellin antibodies intheir serum it is likely this receptor plays a rolein colitis (Ref. 81). Consistent with this, TLR5-deficient mice develop spontaneous colitis(Ref. 82); however, currently no genetic linkagehas been found between TLR5 and Crohndisease in patients. The effects of various TLRagonists and antagonists on the course ofexperimental colitis have been studied but resultsremain controversial, showing that we still knowtoo little about this complex interaction (Ref. 78).

NOD-like receptorsThe NOD-like receptor (NLR) family comprisescytoplasmic PRRs recognising both microbial

and endogenous ligands. In humans the familycontains 23 members, which all contain C-terminal LRR domains for ligand recognition, acentral NACHT domain that mediatesoligomerisation, and an N-terminal effectordomain that generates downstream signalling.Two main groups of NLRs are the NODs andthe NALPs (NACHT/LRR/PYD-containingproteins).NOD1 andNOD2 are the best studied.NOD1 is

ubiquitously expressed,whileNOD2 is expressedby monocytes, macrophages, dendritic cells andPaneth cells (Ref. 83). NOD1 recognises meso-DAP (g-D-glutamyl-meso-diaminopimelic acid),which is found on all Gram-negative and someGram-positive bacteria as part of cell-wallpeptidoglycan. NOD2 recognises MDP, which ispresent on peptidoglycan of all bacteria. Uponligand recognition, both NOD1 and NOD2undergo conformational changes and form self-oligomers. Both have a caspase-recruitmentdomain (CARD) through which they recruit andactivate RIPK2 (receptor-interacting serine/threonine-protein kinase), which mediatessubsequent downstream signalling activatingNF-kB and mitogen-activated protein kinases(MAPKs), leading to production ofinflammatory mediators IL-1b and IL-8(Ref. 83). NOD2-deficient mice have been shownto be more susceptible to bacterial infection,especially via the oral route (Ref. 84).NOD2 lies on the IBD1 susceptibility locus (an

area on chromosome 16 that is strongly associatedwith inflammatory bowel disease), and aframeshift mutation, an insertion mutation andtwo missense mutations have been associatedwith Crohn disease (Refs 9, 85, 86). Thesemutations affect the LRRs and are thought toinfluence microbial recognition, subsequent NF-kB signalling and IL-1b secretion (Refs 87, 88,89, 90). Mice with the insertion mutationassociated with Crohn disease had higher levelsof NF-kB activation and IL-1b production uponMDP stimulation and showed more-severeinflammation in a colitis model, with increasedmacrophage apoptosis (Ref. 91). Polymorphismsin NOD1 have also been screened forassociation with inflammatory bowel diseases;one insertion/deletion polymorphism showsassociation although contradictory data exist(Refs 92, 93, 94, 95, 96).NALPs are expressed in immune cells but also

on mucosal epithelial cells and neurons (Ref. 97).




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These molecules contain a PYD (pyrine domain),which mediates signalling upon ligandrecognition. This leads to formation of theinflammasome, a molecular scaffold forcaspase-1 activation that mediates IL-1b andIL-18 signalling and can lead to programmedcell death. NALP1 and NALP3 are bothreported to form inflammasome complexes.NALP1 recognises MDP and Bacillus anthracistoxin (Refs 98, 99). NALP3 signalling isactivated by a variety of different ligands andstimuli including bacterial RNA, LPS, changesin potassium efflux, and uric acid from dyingcells associated with gout (Refs 98, 99, 100).IPAF (ICE protease-activating factor), which hasan N-terminal CARD domain, is also able toinduce caspase-1 activation upon recognition offlagellin in the cytoplasm (Ref. 101).Inflammasome-inducing NLRs have beenimplicated in autoimmune diseases includingMuckleWells syndrome and familial coldautoinflammatory syndrome (Refs 102, 103), andrecently it was shown that single-nucleotidepolymorphisms (SNPs) in NALP3 contribute toCrohn disease susceptibility (Ref. 104).

C-type-lectin-like receptorsThe C-type-lectin-like superfamily of receptorsrecognise proteins or carbohydrates and includemolecules with a classical carbohydrate-recognition domain that depends on calciumions (Ca2) for ligand binding and receptorswith similar structure that bind their ligandsindependently of Ca2 (Ref. 105). C-type lectinreceptors can have various functions and only afew play a role as PRRs; these include dectin-1(dendritic-cell-associated C-type lectin-1),dectin-2, DC-SIGN (dendritic-cell-specificintercellular-adhesion-molecule-3-grabbing non-integrin) and mannose receptor (MR) (Ref. 105).Dectin-1 was first identified on dendritic

cells (Ref. 106). This receptor is a type IItransmembrane receptor that consists of acarbohydrate-recognition domain, stalk region,transmembrane domain, and signalling domainthat contains an immunoreceptor tyrosine-based activation motif (ITAM)-like region.Dectin-1 binds b-glucans found on fungi, plantsand some bacteria, and binding is independentof Ca2 (Ref. 107). Dectin-1 is expressed bymonocytes, macrophages, neutrophils, dendriticcells, eosinophils (in humans), B cells (inhumans) and subpopulations of T cells, and its

expression is particularly high in tissues atportals of entry such as lung and intestine butlow or absent in other tissues, such as heart andbrain (Refs 108, 109). Dectin-1 expression isregulated by cytokines and immune stimuli(Ref. 30). Following b-glucan recognition, thedectin-1 ITAM motif is phosphorylated andsignalling pathways dependent andindependent of the tyrosine protein kinase SYKmediate phagocytosis and stimulate TNF-a,CXCL2 (MIP-2), IL-10 and IL-12 production(Ref. 109). Recently it has been shown thatdectin-1 signalling can induce dendritic cellmaturation and direct Th17 responses linkinginnate and adaptive immunity (Ref. 110).Dectin-2 is a type II transmembrane receptor

that contains a C-type lectin domain and asmall cytoplasmic domain (Ref. 111). Thereceptor is expressed by macrophages, dendriticcells and Langerhans cells and expression isupregulated during inflammation (Ref. 112).It recognises high-mannose structures in thepresence of Ca2 and has been shown tomediate fungal recognition, upon which itinteracts with the Fc receptor g chain forstimulation of TNF-a and IL-1ra (IL-1-receptor antagonist protein) production(Refs 113, 114).DC-SIGN was first identified as a lectin

able to bind intercellular adhesion molecule 3(ICAM-3) expressed by T cells and to supportT cell proliferation. This C-type lectin is a type IItransmembrane receptor with a cytoplasmictail (containing internalisation motifs and anincomplete ITAM), a transmembrane region, astalk that is required for oligomerisation, anda C-type lectin domain that mediatescarbohydrate-selective binding in a Ca2-dependent manner (Ref. 115). DC-SIGN isexpressed by dendritic cells and tissuemacrophages, and expression can be upregulatedby the Th2 cytokine IL-13. DC-SIGN recognisesmannose-containing oligosaccharides onendogenous ligands ICAM-2 (on endothelialcells) and ICAM-3, and exogenous ligandsincluding viruses, bacteria, parasites and fungi(Ref. 115). DC-SIGN has been shown to mediateendocytosis and phagocytosis, leading to ligandtargeting to endosomes and lysosomes (Ref. 115).It is thought to be used by pathogens to evadethe immune system. For instance, pathogenicMycobacterium tuberculosis enters dendritic cellsvia DC-SIGN and uses this receptor to




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downregulate dendritic-cell-mediated immuneresponses (Ref. 116). Furthermore, humanimmunodeficiency virus (HIV) can use DC-SIGNto infect T cells (Ref. 115). One polymorphism inthe gene encoding DC-SIGN has been shown tobe mildly associated with ulcerative colitis butnot with Crohn disease (Ref. 117).MR is a recycling receptor expressed by

macrophages and selected dendritic cellpopulations (e.g. inflamed-skin dendritic cells),but not monocytes. Expression is regulated bycytokines, growth factors and glucocorticoids(Ref. 118). MR is a type I transmembraneglycoprotein and contains a cytoplasmic tail,transmembrane domain, eight carbohydrate-recognition domains, a domain with fibronectintype II repeats, and a cysteine-rich domain.Both the carbohydrate-recognition domainand the cysteine-rich domain are able tobind ligands, enabling the MR to recognise alarge range of endogenous and exogenouscarbohydrate ligands, including fungi,protozoa, bacteria, parasites and viruses(Ref. 118). MR has been suggested to mediatephagocytosis and has been shown to stimulatepro-inflammatory cytokine secretion uponligand recognition (Refs 119, 120). Mannose-binding receptors may play a role ininflammatory bowel diseases since blockingexperiments with mannan reducedinflammation in colitis induced bytrinitrobenzene sulphonic acid (TNBS) (Ref. 121).

Microbial killingSome PRRs stimulate microbial uptake viaphagocytosis (Fig. 2), a process that mediatesuptake of particles larger than 0.5 mm. Uponreceptor ligation, receptors cluster and mediateparticle binding. Downstream signalling leadsto actin-based membrane motility that shapesthe membrane around the particle, forminga phagosome. Phagosomes mature by a seriesof fusion and fission events with endosomesand later lysosomes, creating a bacteriolyticenvironment in the phagolysosome (Ref. 122).The variety of phagocytic receptors expressedby macrophages enables phagocytosis of a largerange of particles and also provides signallingfor effector responses appropriate for theingested particle (Ref. 123).Phagosomematuration is regulated by calcium

flux and phosphoinositide signalling; it is alsothought to be regulated by phagosomal pH

(Ref. 124). During phagocytosis the pH falls,due to assembly of the ATPase complex thatmediates proton translocation. This acidicenvironment affects pathogen growth,stimulates NADPH oxidase assembly andcreates an optimal environment for hydrolyticenzyme activity. Superoxide anions producedby the NADPH oxidase complex can formhydrogen peroxide and hydroxyl radicals.Inducible NO synthase (iNOS) is anotherimportant enzyme for microbicidal activity,catalysing production of NO from arginine,oxygen and NADPH. Conversion into NOradicals leads to production of othermicrobicidal agents such as peroxynitrite(ONOO2), nitrogen dioxide (NO2) radicals,nitryl chloride (NO2Cl) and nitrogen trioxide(N2O3). Other killing mechanisms inphagosomes include enzymes such as proteasesand lysozyme, antimicrobial peptides such asdefensins, and metabolic competitors such aslactoferrin.Macrophages from patients with inflammatory

bowel disease show reduced phagocytosis andkilling ability (Ref. 125). By contrast, oxidativeburst activity of lamina propria macrophages isincreased during inflammatory bowel diseasecompared with macrophages in healthy mucosa(Ref. 126). A polymorphism in P40phox, anadapter protein of the NADPH protein complex,has been associated with Crohn disease(Refs 127, 128). Functional effects ofthe polymorphism are unclear but are thought toaffect microbial killing. Colitis was significantlyattenuated in iNOS-deficient mice, but no linkbetween iNOS and human inflammatory boweldiseases has been found yet (Ref. 129).Particularly interesting is the MST-1 gene,

encoding macrophage-stimulating protein(MSP; also known as hepatocyte growth-factor-like protein, HGFL), which is produced in theliver in an inactive form. It circulates in thebloodstream and is thought to be cleaved andactivated at sites of inflammation by tissueproteases and macrophage membraneproteases. Active MSP binds its receptor RON,through which it influences macrophagefunction, including chemotaxis, spreading andphagocytosis (Ref. 130). It also reducesmacrophage responses to LPS and cytokines bydownregulating IL-12, NO and cyclooxygenase(COX-2) production (Refs 130, 131). From thestudy of RON-deficient mice, it is clear that this




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mechanism is important in regulatinginflammatory responses (Ref. 130). MST-1 wassuggested as a gene involved in Crohn diseaseand recently it was shown that a SNP in MST-1,located in a region necessary for RON binding,is associated with the disease (Refs 132, 133).Together this suggests that MSP plays anessential role in macrophage-mediated tolerancein the intestine.

AutophagyAutophagy (Fig. 2) is a mechanism used by allcells of the body for homeostasis andproduction of nutrients during starvation. It isalso used for killing intracellular pathogens thathave escaped phagosomal killing (Ref. 134) andit is suggested to be a form of programmed celldeath (Ref. 135). An autophagosome ischaracterised by a double membrane

Phagocytosis and autophagy pathwaysExpert Reviews in Molecular Medicine 2009 Cambridge University Press

Actin

Bacteriuma Phagocytosis b Autophagy

Endosomal compartment

Phagosome

Autophagosome

Lysosomal compartment

Nucleus

Figure2.Phagocytosisandautophagypathways. (a)Phagocytosis is initiatedbyreceptorbinding,uponwhichtheactincytoskeletonshapestheplasmamembranearoundtheparticle.Onceaphagosomeis formed itmaturesvia fusion and fission with endosomes and lysosomes, creating a degradative and microbicidal environment.(b) An autophagosome is formed by enclosure of selected cytoplasmic content by a double membrane.The autophagsome matures by fusion with lysosomes, leading to degradation of the inner membraneand autophagsosomal content.




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surrounding selected organelles ormicrobes. Likephagosomes, autophagosomes mature duringfusion with endosomes and lysosomes, aprocess mediating degradation. TLR signallinghas been shown to enhance autophagy andinduce phagosome maturation usingcomponents of the autophagic pathway(Refs 136, 137). ATG16L1 and IRGM, two genesinvolved in autophagy, have been linked withCrohn disease (Refs 132, 138). Little is knownabout IRGM (immunity-related GTPase familyM protein), but human IRGM has been shownto induce autophagy and to reduce survival ofMycobacterium tuberculosis (Ref. 139). A deletionpolymorphism has also been linked with Crohndisease, resulting in altered IRGM expressionand reduced autophagy of Salmonellatyphimurium. It is still unclear if mutations inIRGM that have been linked with Crohn diseaseinfluence microbial killing. ATG16l1(autophagy-related protein 16-like 1) influencesS. typhimurium localisation and uptake byautophagosomes, but no role in microbialkilling has been shown (Ref. 140). Sinceautophagy increases access of the cell toantigens for MHC class II presentation, it ispossible that tolerance towards the microbialflora is influenced by ATG16l1 and IRGM. Cellsfrom ATG16l1-deficient mice show a block inautophagosome formation and subsequentIL-1b and IL-18 production (Ref. 141). Thesemice also had more severe inflammation aftercolitis induced by dextran sodium sulphate(DSS), which could be due to IL-1b-inducedchanges in epithelial barrier function. ATG16l1

has been shown to affect Paneth cell function,and Crohn disease patients with the risk alleleof ATG16l1 show similar Paneth cellabnormalities (Ref. 142).

Granuloma formationFormation of granulomata (dense structures ofimmune cells, particularly giant cells formed bymacrophage fusion) is common in Crohndisease patients. Little is known aboutgranulomata associated with Crohn disease butin other disorders they generally form inresponse to undegraded antigens (e.g.tuberculoid granulomata that form upon M.tuberculosis infection) or when antigen clearanceis deficient (e.g. in patients with a defect inP91phox, part of the NADPH oxidase complex).Although likely, it is still unclear if microbialantigens cause granuloma formation in Crohndisease (Refs 143, 144). Macrophage fusion canbe induced in the absence of microbialsubstance (Ref. 145) and electron microscopyhas not revealed the presence of microbeswithin granulomata, but in vitro studies haveshown that adherent-invasive Escherichia colifrom Crohn disease patients can inducegranuloma formation (Ref. 146). Underlyingmechanisms of giant-cell formation remain tobe unravelled; however, macrophage activationwith IL-4, a Th2 cytokine, is an important factor(Ref. 145). Crohn disease is suggested to be Th1driven, but it is possible that inflammation-induced tissue damage stimulates local woundhealing and Th2 responses. However, one studyhas associated granulomata in Crohn disease

Table 2. Genes linked to inflammatory bowel diseases that can influencemacrophage function

Gene Suggested functions Refs

NOD1 Bacterial recognition and regulation of subsequent antimicrobial responses 91NOD2 Bacterial recognition and regulation of subsequent antimicrobial responses 8, 9, 84NALP3 Induction of inflammasome 102TLR4 Bacterial recognition and regulation of subsequent antimicrobial responses 77, 78P40phox NADPH-oxidase formation 125, 126ATG16L1 Autophagosome formation 132, 133IRGM Autophagosome formation 125, 132MST1 Influences macrophage chemotaxis, spreading and phagocytosis 132, 150

Abbreviations: ATG16L1, autophagy-related protein 16-like 1; IRGM, immunity-relatedGTPase familyMprotein;MST1, macrophage-stimulating 1 gene (encoding MSP); NALP, NACHT/LRR/PYD-containing protein; NOD,nucleotide-binding oligomerisation domain-containing protein; TLR, Toll-like receptor.




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with Th1 cells (Ref. 147). It has been proposed thatinitial lack of adequate macrophage responses tointestinal flora leads to reduced neutrophilrecruitment and insufficient clearance ofmicrobes, which stimulates granulomaformation and chronic inflammation (Ref. 148).

This is supported by a recent study showingthat macrophage cytokine production is normalbut secretion is impaired in Crohn disease(Ref. 149). Macrophages of these patients wereshown to have a defect in vesicle traffickingleading to lysosomal degradation of

The role of macrophages in inflammatory bowel diseasesExpert Reviews in Molecular Medicine 2009 Cambridge University Press

Macrophage

Clearance of flora

Uncontrolledmicrobial growth

Influx of monocytesand macrophages

TNF- TNF-

Intestinal inflammationand tissue damage

a b

Gastricmucosa

Change in floraor defect in

microbial handling

Figure 3. The role of macrophages in inflammatory bowel diseases. (a) Macrophages in the lamina propriaclear encountered microbes without inducing strong pro-inflammatory responses. This maintains the healthybalance between microbial flora and the intestinal immune system. (b) A change in flora or a defect inmicrobial clearing processes could lead to uncontrolled microbial growth and activation of pro-inflammatoryprocesses. These processes mediate recruitment of inflammatory monocytes that develop into inflammatorymacrophages in the lamina propria.




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inflammatory cytokines instead of secretion.These Crohn disease patients had reducedneutrophil recruitment and microbial killing,compared with a healthy control group.

Concluding remarksIntestinal macrophages mediate microbialrecognition and killing but, unlike other tissuemacrophages, do not stimulate inflammatoryresponses upon microbial recognition.Mechanisms for microbial recognition (NOD2),microbial killing (NADPH oxidase, ATG16L1and IRGM) and macrophage activation (MST-1)have been shown to play essential roles ininflammatory bowel diseases, suggesting thatmacrophage function could be the origin ofthese disorders (Table 2). Interaction ofmacrophages with the intestinal flora steersimmune responses by activating varioussignalling cascades after binding of specificPRRs. A change in flora or a defect in microbialclearing processes can lead to uncontrolledmicrobial growth in the lamina propria, whichstimulates recruitment of inflammatorymacrophages (Fig. 3).Understanding how blood monocytes develop

into lamina propria macrophages may make itpossible to control this process in order todampen inflammation in patients withinflammatory bowel disease. The interactionsbetween the intestinal flora and macrophagesare also likely future targets for drugs, either inthe form of probiotics or by use of receptoragonists or antagonists that stimulate intestinalimmune responses to produce the requiredresponse.In the later stages of his life, Metchnikoff

became intensely interested in the gut flora andhow this might influence health, disease andageing. He advocated regular intake oflactobacilli, and although ridiculed by many inhis time, current knowledge shows that there isprobably more than a grain of truth in hisideas. However, there can be no doubt thatmacrophages and innate immunity contributeto major effector pathways in the pathogenesisof inflammatory bowel disease, providingattractive drug targets including, but notrestricted to, TNF-a.

Acknowledgements and fundingThe authors thank the anonymous peer reviewersfor their helpful suggestions.

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Further reading, resources and contacts

ReviewsStrober, W., Fuss, I. and Mannon, P. (2007) The fundamental basis of inflammatory bowel disease. Journal of

Clinical Investigation 117, 514-421This comprehensive review provides information on many aspects of inflammatory bowel disease.

Gordon, S. and Taylor, P.T. (2005) Monocyte and macrophage heterogeneity. Nature Reviews Immunology 5,953-964

Mosser, D.M. and Edwards, J.P. (2008) Exploring the full spectrum of macrophage activation. Nature ReviewsImmunology 8, 958-969

Martinez, F.O., Helming, L. and Gordon, S. (2008) Alternative activation of macrophages: an immunologicfunctional perspective. Annual Review of Immunology 27, 460-483

These reviews focus on macrophage plasticity, different macrophage activation states, and alternativemacrophage activation, respectively.

Akira, S. (2009) Innate immunity to pathogens: diversity in receptors for microbial recognition. ImmunologicalReviews 227, 5-282

This issue gives an excellent review of innate recognition receptors.

WebsitesEuropean Crohns disease and Colitis Organisation:

http://www.ecco-ibd.eu/index.php

Society for Mucosal Immunology:

http://www.socmucimm.org

Informative website on macrophages (developed by D. Hume and T. Freeman):

http://www.macrophages.com

Features associated with this article

FiguresFigure 1. Localisation of macrophages within the lamina propria.Figure 2. Phagocytosis and autophagy pathways.Figure 3. The role of macrophages in inflammatory bowel diseases.

TablesTable 1. Macrophage phenotype.Table 2. Genes linked to inflammatory bowel diseases that can influence macrophage function.

BoxBox 1. Methods for studying macrophage function in inflammatory bowel disease.

Citation details for this article

Sigrid E.M. Heinsbroek and Siamon Gordon (2009) The role of macrophages in inflammatory bowel diseases.Expert Rev. Mol. Med. Vol. 11, e14, May 2009, doi:10.1017/S1462399409001069




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ContentsAbstractMonocyte development into intestinal macrophages: modulation of phenotypeThe noninflammatory nature of lamina propria resident macrophagesBox 1Inflammatory properties of recruited monocytes and macrophagesFigure 1Interaction with microbesIntestinal floraTable 1Pattern-recognition receptorsToll-like receptorsNOD-like receptorsC-type-lectin-like receptorsMicrobial killingAutophagyFigure 2Granuloma formationTable 2Figure 3Concluding remarksAcknowledgements and fundingReferencesFurther Reading, resources and contactsFeatures associated with this articleCitation details for this article

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