ADRIANO G ROSSI DRIVING INFLAMMATORY CELL APOPTOSIS TO RESOLVE INFLAMMATION MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UK. Host defence against invading organisms such as bacteria, parasites, fungi and viruses is orchestrated by leukocytes such as granulocytes (especially neutrophils and eosinophils) and macrophages. If the recruitment, activation and/or removal of such leukocytes from inflammatory sites is dysregulated, these cells also have the potential to elicit and contribute to tissue damage found in patients with chronic inflammatory diseases (e.g., asthma, rheumatoid arthritis, atherosclerosis, Alzheimer’s disease, inflammatory bowel disease, etc). Resolution of inflammation is an active and regulated physiological process that can be manipulated pharmacologically. Apoptosis and non-inflammatory phagocytosis of apoptotic cells by macrophages and other phagocytic cells (a phenomenon often termed efferocytosis) are key cellular processes that help co-ordinate inflammation resolution. Using a number of in vitro and in vivo model systems, evidence demonstrating that leukocyte apoptosis and the process of efferocytosis can be altered by pharmacological interventions to enhance the resolution of inflammation will be provided. It is proposed that such intervention will likely lead to novel therapeutic strategies for the treatment of inflammatory diseases.

MACROPHAGE PLASTICITY AND POLARIZATION AS THERAPEUTIC TARGETS ALBERTO MANTOVANI, MD Instituto Clinico Humanitas IRCCS, University of Milan, via Manzoni 113, 20089 Rozzano, Italy Macrophages are key orchestrators of chronic inflammation, in particular in the gastrointestinal tract and associated organs. They respond to microenvironmental signals with polarized genetic and functional programmes. M1 macrophages which are classically activated by microbial products and interferon-kill microorganisms and tumors . In contrast, M2 cells, tune inflammation and adaptive immunity; promote cell proliferation by producing growth factors and products of the arginase pathway (ornithine and polyamines); scavenge debris by expressing scavenger receptors; promote angiogenesis, tissue remodeling and repair. M1 and M2 cells represent simplified extremes of a continuum of functional states. Available information suggests that TAM are a prototypic M2 population. M2 polarization of phagocytes sets these cells in a tissue remodeling and repair mode and orchestrate the smouldering and polarized chronic inflammation associated to established neoplasia. Recent studies have begun to address the central issue of the relationship between genetic events causing cancer in the gastrointestinal tract and activation of protumor, smoldering, non resolving tumor-promoting inflammation. New vistas have emerged on molecules associated with M2 or M2-like polarization and its orchestration. Macrophage polarization has emerged as a key determinant of resolution of inflammation. Resident and recruited macrophages are key players in the homeostatic function of the liver and in its response to tissue damage. In response to environmental signals, macrophages undergo polarized activation to an M1 or M2 or M2-like activation states. These are extremes of a spectrum in a universe of activation states. Progress has been made in understanding the molecular mechanisms underlying the polarized activation of mononuclear phagocytes. Resident and recruited macrophages are a key component of diverse homeostatic and pathological responses of the hepatic tissue. Polarized macrophages interact with hepatic progenitor cells, integrate metabolic adaptation, mediate responses to infectious agents, orchestrate fibrosis in a yin yang interaction with hepatic stellate cells, and are a key component of tumor promoting inflammation. A better understanding of macrophage diversity and plasticity in liver homeostasis and pathology may pave the way to innovative diagnostic and therapeutic approaches. The role of negative regulators, including TIR8 (a member of the IL-1 receptor family) and D6 (a decoy and scavenger chemokine receptor) in the regulation of GI inflammation and carcinogenesis will be discussed. Innate immunity consists of a cellular and a humoral arm. We have used the long pentraxin PTX3 as a paradigm for the humoral arm of innate immunity and its interplay with cells. PTX3 is a multifunctional soluble pattern recognition receptor characterized by a C-terminal domain highly homologous to C-reactive protein and serum amyloid P component, associated to a N-terminal domain unrelated to other known proteins. PTX3 is produced upon stimulation with proinflammatory cytokines and Toll-like receptor engagement by different cell types, including endothelial cells, monocytes/macrophages, dendritic cells, fibroblasts and epithelial cells. The molecule binds with high affinity complement component, microbial moieties, growth factors and apoptotic cells. PTX3 plays non-redundant functions including innate immunity against selected microorganisms to regulation of inflammation. In addition PTX3 has a regulatory function. PTX3 serves as a paradigm for the interplay between the cellular and the humoral arm of innate immunity. Key references Bottazzi B, Doni A, Garlanda C, Mantovani A. An Integrated View of Humoral Innate Immunity: Pentraxins as a Paradigm. Annu Rev Immunol. 2010, 28:157-83. Deban L, Russo RC, Sironi M, Moalli F, Scanziani M, Zambelli V, Cuccovillo I, Bastone A, Gobbi M, Valentino S, Doni A, Garlanda C, Danese S, Salvatori G, Sassano M, Evangelista V, Rossi B, Zenaro E, Constantin G, Laudanna C, Bottazzi B, Mantovani A. Regulation of leukocyte recruitment by the long pentraxin PTX3. Nat Immunol. 2010, 11:328-34. Biswas S.K. and Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as paradigm. Nat Immunol 2010: 11, 889-896. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012, 122:787-95.

ALEXANDER CHERVONSKY COMMENSAL MICROBIOTA AND AUTOIMMUNITY The University of Chicago, Chicago Il, USA Microbial environment strongly influences development of autoimmune diseases. In genetically susceptible hosts the pathogenic and commensal microorganisms may contribute to disease progression. Genetically modified, microbiota-free and defined-microbiota colonized animals are important tools for understanding of microbial influences on autoimmunity. Using Type 1 diabetes (T1D) as a model for autoimmunity in susceptible strain of mice, we have determined that microbiota manipulates innate immune system affecting T1D development. In addition, dietary, metabolic and biologic insults can affect both microbiota and the pathogenesis of the disease.

IS IT POSSIBLE TO MODULATE TUMOR INFILTRATING MACROPHAGES IN VIVO? LESSONS FROM HUMAN PAPILLOMAVIRUS ASSOCIATED TUMORS. KARLA L. A. FERNANDEZ, RENATA A.M. ROSSETTI, SIMONE C. STONE, A. P. LEPIQUE. Department of Immunology, Institute of Biomedical Sciences, Universisty of Sao Paulo Infection by Human Papillomavirus, HPV, is the main cause for cervical cancer and a number of other malignancies. These tumors have a very long natural history, during which immune evasion, integration of the viral DNA into the host genome and cell immortalization are checkpoints in cancer development. Although HPV infections are very efficient in promoting peripheral tolerance towards viral antigens, most infected women eliminate tumor precursor lesions and infection spontaneously. The immune system is essential in these responses. Our laboratory has been working in the characterization of local and systemic effects of the tumor microenvironment in HPV associated tumors in experimental models and cervical lesion biopsies. In an isogenic mouse tumor model we have observed that the IL-6/JAK2/STAT3 and PI3K/Akt axis are constitutively active in both the tumor inflammatory

remains inactive with the consequent lack of co-stimulatory molecule expression and bias toward regulatory T cell activation. Similar results have been observed in the CD45

+ inflammatory population in in situ and invasive cervical

carcinomas. We have also described in both experimental models and cervical lesion biopsies the presence of M2 macrophages and increase in circulating leukocytes. As recently described, among the several factors that can induce M2 phenotype in macrophages, lactate, a product of tumor cell metabolism is one of them. Recent data from our laboratory shows that simultaneous inhibition of PI3K pathway and glycolysis inhibits tumor growth and converts tumor macrophages into M1 macrophages. By understanding the effects of tumor cells in the inflammatory infiltrate and immune cells in peripheral tissues, we are able to develop tools to inhibit tumor growth and activate anti-tumor responses in the host.

ANA MARIA CAETANO FARIA AGING ASSOCIATED INFLAMMATION (INFLAMMAGING) IN INDIVIDUALS LIVING IN ENDEMIC AREAS FOR SCHISTOSOMIASIS IN BRAZIL: RISKS AND BENEFITS. Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. There has been no systematic study of the immune response of individuals over 60 residing in área endemic for Schistosoma mansoni infection although senescence is reportedly associated with susceptibility to infection and progressive decline in immune function.We have shownpreviously, in two endemic areas in Minas Gerais, Brazil, that the frequency of individuals over 60 with chronic schistosomiasis is no longer negligible. Several aging-related immunological alterations are already described in medical literature, mostly in the T-cell compartment. Since aging is associated with a decline in T-cell function, it is not surprising that individuals over 60 would be more susceptible to infection. However, not all aged individuals in endemic areas have high intensity of infection; some of them display a negative stool-screening test for the presence of schistosome eggs and low levels of serum antibodies reactive with S. mansoni antigens indicating they are not infected. Non-infected, negative individuals may develop compensatory mechanisms to cope with immune dysfunction and to generate protective responses against the Constant threat of infection in these areas. Herein, we reviewed previous reports from our group showing that two mechanisms contribute to distinguish between infected and egg negative aged individuals. First, egg negative aged individuals develop innate immune responses to replace the decline in T-cell function that is observed with aging. Second, chronically activated regulatory T cells, that may impair protective immune responses, are more vigorous in infected aged individuals. We propose that egg negative individuals may be considered as an example of “healthy aging” in areas endemic for infectious disease. Our data also suggests that a higher inflammatory status (inflammaging) in older individuals living in these areas may represent a health benefit instead of a risk for developing chronic diseases.

THE ROLE OF PSYCHOLOGICAL STRESS IN CUTANEOUS WOUND HEALING ANDRÉA MONTE ALTO COSTA, TAÍS FONTOURA DE ALMEIDA, BRUNA ROMANA-SOUZA, State University of Rio de Janeiro, Brazil Stress is the process in which environmental demands exceed an individual’s ability to cope, resulting in affective, behavioral, and physiological changes. Stress stimulates the secretion of glucocorticoids and catecholamines through activation of the hypothalamic-pituitary-adrenal and sympathetic-adrenal medullary axes, respectively. Recent studies have shown that stress may also impair cutaneous wound healing. In our laboratory we used, for the first time, the rotational stress to investigate the effects of stress on cutaneous wound healing, and showed that rotational stress was associated with decreased wound contraction, reduced re-epithelialization, reduced MMP-2 and -9 activation, but with strongly increased nitrite levels. Furthermore, in the wound site, inflammatory cell infiltration, TNF-alpha expression, myofibroblastic differentiation, and angiogenesis were all delayed in the stress group. Blockage of beta 1 and beta 2 adrenoceptors reversed the deleterious effects of stress on wound contraction and re-epithelialization. In vitro experiments showed that high epinephrine concentrations increased murine skin fibroblast proliferation and nitric oxide synthesis, and strongly inhibited skin fibroblast migration and both pro- and active MMP-2. Leading us to conclude that rotational stress impairs cutaneous wound healing due to increased epinephrine levels. In another set of experiments, we treated animals with nicotine because nicotine addiction may modulate physiological response to stress. The stress+nicotine group presented a delay in wound contraction; these wounds showed a decrease in inflammatory cell infiltration and lower expression of TGF-beta, whereas there was an increase in angiogenesis and in TNF-alpha expression. In vitro fibroblast migration was also impaired by the nicotine treatment of stressed animals. So psychological stress delays cutaneous wound healing trough increased levels of cathecolamines and the administration of nicotine is not able to abolish this deleterious effect. Supported by: FAPERJ, CNPq and CAPES

ANNIE S. DE GROOT TREGITOPE: A NOVEL THERAPEUTIC AND TOLERANCE-INDUCING STRATEGY A.S. De Groot

1,2, L.P. Cousens

1, L. Moise

1,2, N. Najafian

3, F. Mingozzi

4, W. Elyaman

3, B. Mazer

5, S. Khoury

3, Y. Su

6,

D.W. Scott6, and W. Martin

1

1EpiVax, Inc., Providence, RI USA,

2University of Rhode Island, Providence, RI USA,

3Brigham and Women's

Hospital, Boston, MA USA, 4Children's Hospital of Philadelphia, Philadelphia, PA USA,

5McGill University Health

Center, Montreal, Canada, and 6Uniformed Services University of Health Services, Bethesda, MD USA

Purpose: Modulation of T cell responses provides new opportunities for the treatment of autoimmune and inflammatory diseases. Tregitopes are regulatory T cell (Treg) epitopes found in IgG that induce Tregitope-specific Tregs, provide beneficial immunomodulatory effects, paralleling effects attributed to intravenous immunoglobulin (IVIG). In this presentation, we will provide evidence that Tregitopes derived from human IgG can reproduce immunomodulatory effects of IVIG in vitro and in vivo. Methods: Six collaborating laboratories have evaluated the mechanisms of action and beneficial effects of Tregitopes in mouse models of MS (EAE), OVA-induced allergic airway disease, cardiac transplant, diabetes (NOD), and AAV-mediated gene transfer. Results: Tregitopes cause CD4+CD25+FoxP3+ Treg to expand and produce IL-10 in vitro, and iTreg to be induced in vivo. Induction and functions of Tregs have been examined in in vivo model systems such as D011.10 TCR transgenic mice, transplant of BM12 to C57BL/6, Allergy, AAV-mediated gene transfer and EAE. In OVA-induced allergic airway disease, we observed significant and reproducible expansion of Tregs in conjunction with decreased airway reactivity comparable to, if not greater than, IVIG. We will provide additional published and unpublished evidence demonstrating the antigen specificity of tolerance induction using Tregitopes in conjunction with target antigens, and discuss the relevance of Tregitopes to the treatment of human immune-mediated diseases. Additionally, Treg epitopes (Tregitopes?) have been identified in other common serum proteins, suggesting that peripheral control of inflammatory signals may in part be due to intrinsic self-protein “off switches". Conclusions: Tregitopes, by inducing Tregs and tolerance, are relevant the treatment of autoimmune and inflammatory disorders and may be a useful tool in the design of safer, more effective protein therapeutics, including monoclonal antibodies, novel scaffolds, replacement therapies or biosimilars. Supported by NIH SBIR Phase II R44 DK081261-03A1, SBIR Phase I R43AI102454-01, and SBIR Phase I 1R43HL114308-01.

ANTONIO LUCIO TEIXEIRA JR. A NOVEL ROLE FOR THE IMMUNE SYSTEM: COGNITION IEAT/UFMG, Brazil. The immune system is traditionally regarded as a host defense system. This view has a great heuristic value and fostered the development of the immunology field. Nevertheless this definition does not address a series of emerging data such as the increase of inflammatory mediators or defective immune response with aging. As a consequence, the immune system has been conceptualized as a homeostatic system, acting in close association with the central nervous system (CNS). In this scenario, the immune system influences several CNS functions, including cognition. This assumption is largely supported by clinical and experimental evidence. For instance, knockout mice for immune-related genes exhibit behavioral changes and cognitive deficits. Following inflammatory conditions, animals and humans experience a set of behavioral and cognitive symptoms known as a ‘sickness behavior’. This behavior can also be elicited by the therapeutic use of interferons (e.g. chronic hepatitis C). Major neuropsychiatric disorders, including bipolar disorder and schizophrenia, are associated with molecular and cellular immune changes, and some of these parameters are related with the severity of symptoms, illness state and prognosis. In Alzheimer’s disease, a neurodegenerative disease characterized by progressive cognitive decline, the immune system seems to play a pathogenic role, being the target of therapeutic strategies. In conclusion, the function of the immune system goes beyond host defense.

ARISTÓBOLO MENDES DA SILVA PROTEIN KINASES AND THEIR ROLE IN THE CELLULAR IMMUNE RESPONSE Protein kinases are enzymes that phosphorylate certain amino acid residues in specific proteins. They are key regulators of cell function by driving the activity, physical interaction and cellular localization of many proteins. The activity of protein kinases is tightly regulated by a number of molecular events such as the turning on or off by phosphorylation, and binding of activator proteins or small molecules. Protein kinases are involved in a myriad of cellular processes, including the immune cell function. In this module, the basic concepts in the protein kinases such as the phosphorylation of amino acids and proteins by protein kinases, how does phosphorylation regulate activity of proteins, and how the domains within protein structures regulate cell function will be presented. Yet, considerable attention will be given to specific protein kinases or groups constituted by them whose activities will determine the major outcomes in the cellular immune response. Activation of immune cells is triggered upon detection of stress stimuli that the organism receives. The outcome of these stimuli will be the activation of specific signaling pathways that control the expression regulation of inflammatory genes that are critical to cellular immune response. Therefore, protein kinases are essential cellular components required to modulate these pathways. Among these include the one which leads to the activation of the kinases responsible for nuclear factor kB (NF-kB) transcription factor activity regulation. This pathway involves the formation of NF-kB dimers as a result of phosphorylation-induced proteolysis mediated by the kinase responsible for NF-kB activation, IkB kinase (IKK). Other important protein kinases constitute a group called an mitogen-activated protein kinases (MAPKs). MAPK pathway is classically composed by a cascade of three kinases: (1) a MAPK that is responsible for the effector functions, (2) the kinase that activates the MAPK by phosphorylation (MAP2K or MKK), and (3) the kinase that activates the MKK (MAP3K or MEKK), which provides specificity in response to cell surface receptors upon stress recognition. Within the MAPKs family, the stress-activated protein kinase (SAPK) group has been defined as a group of kinases that are activated by stimuli that cause cell stress such as inflammation, which in turn control the expression of some proinflammatory genes such as those encoding cytokines. Finally, the function of a protein kinase with a role in viral immune responses will also be presented. Recently, emerging role for this protein kinase has been observed in bacterial and parasitic infections. The double-stranded RNA dependent protein kinase PKR is a host defense enzyme whose expression is up-regulated in response to Interferons (IFNs) and during viral infections. Increased levels of PKR result in its activation by auto-phosphorylation, which in turn phosphorylates the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2-alpha) resulting in the inhibition of global cellular protein synthesis. In summary, the basic concepts in the protein kinases and what is currently known about the aforementioned kinases in their overall contribution to the immune response will be discussed.

TYROSINE KINASES IN AUTOIMMUNE DISEASES ATTILA MÓCSAI Department of Physiology, Semmelweis University School of Medicine, Budapest, Hungary Myeloid leukocytes such as neutrophils or macrophages are critical components of innate immunity but their improper activation may also lead to tissue damage during autoimmune inflammation. We have previously shown that certain neutrophil responses require Src-family kinases, Syk and PLCγ2. Therefore, we tested the role of tyrosine phosphorylation pathways in in vivo inflammatory reactions. Src-family kinases, Syk and PLCγ2 were all found to be required for autoantibody-induced inflammatory reactions such as the K/BxN serum-transfer arthritis or autoantibody-induced skin blistering disease in experimental mice. The genetic deficiency of those signaling molecules also prevented accumulation of myeloid cells at the site of inflammation. Given the role of tyrosine kinases in β2 integrin-mediated leukocyte activation, we hypothesized that Src-family kinases, Syk and PLCγ2 are also required for β2 integrin-mediated leukocyte migration. Surprisingly, neutrophil migration in a conventional Transwell assay did not require Src-family kinases, Syk or PLCγ2 even though it was strongly reduced by the genetic deficiency of the β2 integrin-chain CD18. In addition, the Src-family kinase inhibitor dasatinib did not affect in vitro neutrophil migration. In vivo competitive migration assays (in which wild type and knockout cells are allowed to migrate to the site of inflammation within the same animal) also revealed that Src-family kinases, Syk and PLCγ2 were not required for neutrophil or monocyte migration in sterile peritonitis or autoantibody-induced arthritis models. On the other hand, tyrosine kinases were required for immune complex-induced cytokine production by neutrophils and macrophages. Taken together, Src-family kinases, Syk and PLCγ2 are required for neutrophil activation and cytokine production but do not play any direct role in CD18-mediated migration of myeloid cells to the site of inflammation.

ROLE OF IL-33 IN LUNG INFLAMMATION BERNARD RYFFEL Tissue damage of different origin may release danger signals and several biochemical responses including oxidative stress causing inflammation and repair. Endogenous danger signals include proteins (HMGB1, heat shock proteins, hyaluronan), lipids (oxidized phospholipids, eicosanoids), DNA fragments (ATP, uric acid), which may activate innate immune receptors such as TLR and NLR receptors leading to inflammasome activation with the production of IL-1beta. IL-33 belongs to the IL-1 family, but is another kind of alarm signal, which is probably released upon cell death such as IL-1alpha. IL-33 is activated by different protease and activates type 2 innate lymphoid cell (ILC2) and Th2 T cell responses through signaling the IL-33R/ST2. Here I review the role of the alarmin IL-33 in protease and injury models of inflammation and repair.

UNCOVERING A PHOSPHATASE/MICRORNA NETWORK TO CONTROL MACROPHAGE ACTIVATION CARLOS HENRIQUE SEREZANI Macrophages are pleiotropic cells involved in tissue injury as well as generation and maintenance of immunosuppressive state. Thus, strategies to modulate macrophage function represent an attractive therapeutic approach to prevent overwhelming inflammatory response or to improve host defense in settings of host vulnerability. Macrophage activation is dictated by pathogen recognition receptors, including Toll like receptors (TLRs), which results in the generation of proinflammatory programs, leading to leukocyte recruitment and increased microbial ingestion and killing. TLR family members and the IL-1β receptor (IL-1βR) share a conserved cytoplasmic Toll–IL-1R (TIR) domain that binds adaptor proteins including myeloid differentiation factor 88 (MyD88) and elicits inflammatory responses. We have shown that the phosphatase and tensin homolog PTEN inhibits phagocytosis of Candida albicans, a pathogen that is recognized by different pathogen recognition receptors including TLR2. PTEN is a lipid and protein phosphatase conventionally known as a tumor suppressor, but this phosphatase is also activated in many inflammatory diseases, such as diabetes and atherosclerosis. Here, we speculate that PTEN is an endogenous brake that inhibits TLR responsiveness in macrophages. Initially, we investigated whether PTEN controls the expression of TLRs and its adaptors. Silencing PTEN expression in vivo or in vitro enhances the expression of MyD88 but not other adaptors and TLR receptors. Conversely, PTEN overexpression in primary macrophages inhibits MyD88 expression. These data correlated with enhanced TLR4 and TLR2-induced NFkB p65 phosphorylation in macrophages. Next, we decided to investigate the molecular programs involved in PTEN-mediated MyD88 inhibition. Neither PTEN deletion nor overexpression changed the expression/activation of the MyD88 transcription factor STAT-1, which suggest a post-transcriptional effect of PTEN in controlling MyD88. Thus, we sought to investigate whether PTEN controls the expression of microRNAs (non-coding RNAs that increase mRNA degradation) involved in MyD88 levels. A focused microRNA array revealed that PTEN silencing increased the expression of 15 microRNAs and decreased the expression of other 22 microRNAs involved in inflammatory responses, including the MyD88-targeting microRNAs miR-125b and miR146b. These data suggest that PTEN is a global regulator of microRNA expression in macrophages. We performed add-back experiments in PTEN-/- macrophages transfected with miR-125b, miR-146b and miR-21, followed by determination of MyD88 expression. Our data showed that miR-125b, but nor other microRNAs, impaired PTEN-mediated MyD88 inhibition and decreased TLR-induced TNF-PTEN in controlling TLR responses in vivo was determined in a model of polymicrobial sepsis. In vivo treatment with PTEN siRNA or with a specific inhibitor enhances mouse lethality, bacterial load and decreases neutrophil recruitment to the site of infection. Together, our work shows that PTEN is an endogenous brake by controlling the levels of homeostatic MyD88 in a miR-125b dependent manner. PTEN activation could be an important target to control overwhelming inflammatory response found in severe sepsis. These studies provide a foundation for new therapeutic strategies to improve immune responsiveness in settings of host vulnerability and to dampen it in settings of exaggerated inflammation.

PROTEOLYTIC HYPERACTIVITIES ASSOCIATED WITH INFLAMED EPITHELIA FROM SKIN AND INTESTINE DERAISON CÉLINE

1, 2, 3

1 Inserm, U1043, Toulouse, F-31300, France 2 CNRS, U5282, Toulouse, F-31300, France 3 Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, F-31300, France Impaired barrier function is a hallmark of many inflammatory diseases affecting epithelia. We will document proteolysis as a key regulatory mechanism of epithelial barrier integrity and inflammation. Uncontrolled proteinase activity is detrimental to cellular homeostasis, and this activity can be regulated at two different levels: activation of precursor zymogens and blockade by endogenous inhibitors. The role of the balance between proteases and protease inhibitors in the maintenance of barrier function is well illustrated in the skin and in the bowel, two organs in direct contact with the environment. We will discuss our understanding of the interlinked “inside-out” and “outside-in” theories of inflammatory epithelial diseases. In the skin, we will exemplify how skin barrier defects, inflammation, and allergy are interlinked but not successive. Experimentally induced barrier injuries initiate cytokine release by keratinocytes, which in turn activate inflammatory cells. On the opposite, studies demonstrated that independently to the epithelial failure, in absence of a particular serine protease inhibitor, keratinocytes secrete pro-inflammatory and pro-allergic mediators in a cell-autonomous manner through kallikrein protease hyperactivities. The gastrointestinal tract is the organ the most exposed to proteases. Several studies have shown that the balance between proteases and their inhibitors is disrupted towards increased proteolytic activity in inflammatory bowel disease (IBD). Massive immune cell infiltration explains in part high level of protease activity but we will illustrate a new emerging concept showing that epithelial cells are directly involved in this hyper proteolytic activity and play a major role in maintaining inflammation. We will also demonstrate that restoring the proteolytic balance using a protease inhibitor targeting all types of protease-producing cells represents a promising therapeutic approach to treat inflammatory diseases. Enhance barrier recovery by blocking proteases may be better than using immune suppressors to break the vicious cycle in case of chronic inflammatory diseases.

FIBRE, SHORT-CHAIN FATTY ACIDS AND GUT MICROBIOTA REGULATE INFLAMMATORY RESPONSES CHARLES R. MACKAY Monash University, Clayton, VIC, Australia The incidence of ‘western-lifestyle’ diseases have increased in recent decades, particularly asthma, allergies, certain autoimmune diseases, and metabolic diseases with an inflammatory basis. The hygiene hypothesis has been the prevailing model to account for this increase, however epidemiology studies also support the contribution of diet. Diet affects the composition of the gut microbiota, and recent studies have identified various molecules and mechanisms that connect diet, the gut microbiota and immune responses. We will show how diets high or low in fibre impact on inflammatory disease in mice, and the cellular and molecular mechanisms involved. In particular, we demonstrate a role for ‘metabolite sensing’ GPCRs, epithelial integrity through inflammasome pathway activation, and induction of regulatory T cells. We propose that lack of dietary fibre, rather than hygiene, underpins western lifestyle diseases.

CHRISTOPHER K GLASS A GENOME-WIDE VIEW OF MACROPHAGE ACTIVATION Sven Heinz, Chris Benner, Minna Kaikkonen, Michael Lam, Casey Romanoski Depts. of Medicine and Cellular and Molecular Medicine, University of California, San Diego, La Jolla CA Our recent studies have utilized a combination of genome-wide and genetic approaches to define the molecular mechanisms that underlie the development of classically activated and alternatively activated macrophage phenotypes. These studies suggest a relatively simple model of hierarchical interactions between lineage-determining and signal-dependent transcription factors that are required to build functional cis-active regulatory elements. The majority of these interactions occur at enhancer-like elements that are distant from target promoters. The discovery that these enhancer elements themselves generate transcripts referred to as ‘eRNAs’ raises a number of interesting new questions regarding the mechanisms underlying enhancer function and therapeutic approaches to modulate macrophage phenotypes.

PLASTICITY OF HELPER T CELLS IN THE INTESTINE DANIEL MUCIDA, PH.D., assistant professor and head, Laboratory of Mucosal Immunology, The Rockefeller University The gut mucosa hosts large numbers of activated lymphocytes, exposed to stimuli from diet, microbiota and pathogens. Although CD4 (helper) T cells are crucial for immune defense, intestinal homeostasis precludes exaggerated response towards luminal contents, harmful or not. We investigated intrinsic mechanisms used by CD4 T cells to avoid excessive activation within the intestinal tissue. Using genetic tools to label and interfere with T cell development transcription factors, they show that CD4 T cells acquire cytotoxic function while losing CD4-helper activity, including inflammatory potential, in a intestine-specific, microbiota-dependent manner. Their results show a remarkable plasticity in the CD4 T cell lineage that allows chronic exposure to luminal antigens without pathological inflammation.

NON-HEALING INFECTION WITH LEISHMANIA MAJOR IS CONTROLLED BY IL-ß AND INFLAMMASOME DEPENDENT MECHANISMS CHARMOY M., ROMANO A., MAYER-BARBER K., SHER A., AND SACKS D. Laboratory of Parasitic diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America Infection of C57BL/6 mice with most L. major strains, such as L. major Friedlin (Fn), results in a healing lesion with minimal pathology at the site of inoculation in the skin. By contrast, infection of C57BL/6 mice with the West African Lm Seidman strain (Sd), isolated from a patient with chronic cutaneous lesions, despite a polarized Th1 response and comparable growth of parasites during the first weeks of infection, results in a non-healing lesion, poor parasite clearance, and severe pathology, with complete destruction of the ear dermis. In contrast to infection with Lm Fn, infection with Lm Sd is associated with an early and persistent recruitment of neutrophils to the site of inoculation. The pathology was preceded by an upregulated expression of multiple cytokines and chemokines, including IL-17, CXCL1, IL-27, IL-10, and IL-1β. Whereas no phenotype was observed in IL-17 or CXCL-1 deficient mice, IL-1R deficient mice, lacking the receptor for both IL-1α and IL-1β, as well as mice deficient in IL-1β, ASC and caspase-1/11, showed minimal pathology and completely healed their infections with Lm Sd. Altogether, these data suggest that early parasite driven IL-1β upregulation and secretion, dependent on inflammasome assembly and caspase-1 activation, plays a crucial role in the development of non-healing infection and severe pathology. Further understanding of this model may help to reveal the mechanisms underlying severe cutaneous pathology following Leishmania infection in humans.

"HITTING THE ACCELERATOR FOR TUMOR IMMUNITY" GITR IMMUNOTHERAPY ABROGATES TUMOR IMMUNE SUPPRESSION THROUGH LOSS OF REGULATORY T CELL LINEAGE STABILITY DAVID SCHAER, Taha Merghoub & Jedd Wolchok, Ludwig Collaborative Laboratory, Memorial Sloan-Kettering Cancer Center, NY, NY. Ligation of the co-stimulatory TNF family receptor GITR by agonist antibody has recently entered early phase clinical trials for the treatment of advanced malignancies. However, even though the preclinical ability of GITR modulation to activate immune responses and induce tumor regression is well documented, the underlying mechanisms, particularly its effects on CD4+ Foxp3+ regulatory T cells (Tregs), have not been fully elucidated. As we translate this immunotherapeutic approach into the clinic, it is important to fully understand its mechanism(s) of action. Our previous preclinical research has demonstrated that in vivo GITR ligation by agonist antibody (DTA-1) causes a >50% reduction of intra-tumor Tregs as a consequence of Treg Foxp3 expression down modulation. We now demonstrate that Treg instability induced by GITR ligation is tumor dependent and alters the expression of various transcription factors and cytokines important for Treg function. Foxp3 loss is preceded by a drop in expression of the transcription factor Helios, with a corresponding increase in expression of inflammatory transcription factors T-bet and Eomes. GITR Modulated intra-tumor Tregs acquire an inflammatory effector phenotype, up regulating IFNγ and down regulating IL-10. The resultant loss of lineage stability causes Tregs to lose their intra-tumor immune suppressive function, making the tumor susceptible to killing by tumor specific CD8 T cells. The enhanced understanding of GITR immunotherapy gained from this research is currently being used to inform clinical biomarkers and facilitate the most effective and safe use of GITR therapy in humans.

THE ROLE OF CYTOKINES IN INFECTION AND INFLAMMATION EDDY F Y LIEW Institute of Immunology, Infection and Inflammation, University of Glasgow, Glasgow G12 8AT, UK Cytokines are hormones of the immune system. Cytokine-targeting represents a major triumph in immunology scientifically, clinically and commercially. There is therefore considerable interest in discovering novel cytokines. I will illustrate the pleiotropic role of some of the novel cytokines by focusing on interleukin (IL)-33. IL-33 is the latest member of the IL-1 family. It is the ligand of ST2, which is expressed mainly on activated Th2 cells, epithelial cells, neuronal cells and mast cells. IL-33 can skew a predominantly Th1 cell population to Th2 cells phenotype in vivo. Furthermore IL-33 potently induces alternatively activated macrophages (M2). IL-33 signals via MyD88, the canonical pathway shared by all members of the IL-1 family. Additionally, IL-33 activates type II cytokines via mTOR (mammalian target of rapamycin). IL-33 mRNA is expressed early during infection of the intestinal-dwelling nematode Trichuris muris in mice and IL-33 treatment enhances resistance to Trichuris infection by inducing Th2. Furthermore, IL-33 reduces the pathology and mortality of experimental cerebral Malaria infection in mice by activating M2. Importantly, IL-33 also effectively attenuates sepsis by mobilising the innate cells neutrophils, to the site of infection and helps to clear the pathogens. Thus IL-33 is evolutionally preserved for the host defence against infections. However, IL-33 can also induce Type 2 innate lymphoid cells (ILC2), which mediate airway inflammation. Furthermore, IL-33 contributes to the severity of mucocytis accompanying chemotherapy (Irinatecan). Blocking IL-33 prolongs the treatment and effectiveness of chemotherapy in colorectal cancer. In contrast, IL-33 appears to restore the long-term potentiation in a murine model of Alzheimer’s disease. Therefore, IL-33 is a double-edged sword and targeting IL-33 should be approached with caution.

INN ATE IMMUNITY PLAY A PIVOTAL ROLE IN THE PATHOGENESIS OF L.BRAZILIENSIS INFECTION EDGAR M. CARVALHO Immunology Service, Federal University of Bahia, Salvador-Bahia, Brazil

National Institute of Science and Technology of Tropical Diseases (INCT-DT) CNPq/MCT, Salvador-Bahia, Brazil The pathogenesis of leishmaniasis have been predominantly linked to adaptive immune response. Despite macrophage activation by IFN-γ be considered the major mechanism of leishmania killing, patients with cutaneous (CL) and mucosal leishmaniasis (ML) due to L. braziliensis have an exaggerated inflammatory response that associated to pathology. In contrast, subjects exposed to L. braziliensis but able to control parasite growth, subclinical infection (SC), have a very weak type 1 immune response. In this study, monocyte and macrophage functions were determined in patients with early CL, CL, ML, SC and health subjects (HS). Monocyte subpopulations and TLR were determined by FACS, cytokine production by Elisa and L. braziliensis killing by microscopy. Intermediate monocytes were increased in E-CL and CL in comparison to HS. This subpopulation also expressed more TLR/2 and TLR/4 and was the major source of TNF-α upon L. braziliensis infection. Production of chemokines (CXCL9 and CXCL10) as well as TNF-α were evaluated in six days monocyte derived macrophages. While macrophages from CL and ML produce high levels of TNF-α, CXCL9 and CXCL10, these pro-inflammatory molecules were produced at low levels in subjects with subclinical infection. In a cohort study enrolling house hold contacts of CL patients without previous history of leishmaniasis development of CL after 2 years follow up was associated with high production of CXCL10. While macrophages from SC subjects produce very little pro-inflammatory cytokines, these cells had a greater ability of leishmania killing than macrophages from CL and ML patients. This data indicated that while monocytes and macrophages from CL and ML patients participate in the pathology of leishmaniasis, these cells by the increased ability to kill leishmania participate in the control of parasite growth in subjects with SC L. braziliensis infection.

EDWARD A. DENNIS LIPIDOMICS OF THE MACROPHAGE REVEALS REGULATORY MECHANISMS FOR INFLAMMATORY MEDIATOR PRODUCTION Departments of Chemistry/Biochemistry and Pharmacology School of Medicine, University of California at San Diego La Jolla, California, 92093-0601 USA The omics evolution began at the end of the 20

th century with the cloning of the human genome. The 21

st century has

already seen the development of comprehensive proteomics analyses, but the emerging evolution is to metabolomics, the definition of which is the identification and quantification of all of the molecular constituents of the cell including its nucleic acids, amino acids, sugars, and fats. But by far, the largest number of distinct molecular species in cellular metabolism lies in the fats (or lipids) where tens of thousands of distinct molecular species exist in cells and tissues [Proc.Natl.Acad.Sci.U.S.A.,106, 2089-2090 (2009)]. We have applied novel liquid chromatographic-mass spectrometric based lipidomics techniques termed “CLASS” [Ann Rev of Biochem, 80, 301-25 (2011)] generally in the context of an overall omics analysis of immunologically-activated macrophages integrating transcriptomics, proteomics, and metabolomics of lipid metabolites [J Biol Chem, 285, 39976-85 (2010)]. As part of the LIPID MAPS Consortium [www.lipidmaps.org], our laboratory has developed a robust and comprehensive approach to the lipidomics analysis of hundreds of fatty acids, acylethanolamines and inflammatory eicosanoids, including their numerous metabolites arising from an array of cyclooxygenases, lipoxygenases, cytochrome P450s and non-enzymatic oxidation producing isoprostanes, as well as combinations thereof [J. Lipid Res. 50, 1015-1038 (2009)]. Building on our previous application of lipidomic analysis to characterize cellular lipid signaling of Toll-like (TLR) and purinergic receptors and the discovery of their “synergy” in stimulated macrophages as models for inflammation and infection [J. Biol. Chem., 282, 22834-22847 (2007)], we have compared eicosanoid formation in various primary macrophages [J Leukocyte Biology, 90, 563-74 (2011)] and analyzed the fluxes of metabolites using the results of transcriptomics and “directed proteomics” [Mol Cell Proteomics, 11, M111.014746, 1-9 (2012)] approaches. Also lipidomic analysis of cells supplemented with small amounts of the omega-3 fatty acids eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) provides information on the overall effects of EPA and DHA on the inflammatory eicosadome [Proc Natl Acad Sci U S A, 109, 8517-22 (2012)]. To elucidate an in vivo model of infection and inflammation, we previously reported on the progression from inflammatory to resolution phases in Lyme Disease [J Biol Chem, 284, 21599-612 (2009)] and have now explored the effect of influenza infection by lipidomic profiling of bioactive lipid species in a mouse influenza model using virus strains of both low and high pathogenicity. Integration of the lipid measurements with targeted gene expression data demonstrate that different metabolites correlate with the pathogenic phase than with the resolution phase of infection. Findings from the animal models were extended to human nasopharyngeal lavages obtained during the influenza season [Cell, 154, 213-27 (2013)]. Human plasma has also been profiled to quantify almost six hundred distinct lipid molecular species present across all mammalian lipid categories and the implications for the future of clinical medicine and the understanding of the mechanisms of disease has been discussed [Quehenberger & Dennis, New Eng J Med, 365, 1812 (2011)]. [Supported by LIPID MAPS Glue Grant U54 GM069338, R01 GM020501, R01 GM064611, and NIAID HHSN272200800058C]

FABIANA SIMÃO MACHADO “CONSEQUENCES OF CONTROLLING INFLAMMATION DURING TRYPANOSOMA CRUZI INFECTION” Biochemistry and Immunology Department – Institute of Biological Science – Federal University of Minas Gerais – ICB/UFMG Trypanosoma cruzi is a protozoan responsible for the American tripanosomiasis, a high neglected illness known as Chagas’ disease. The infection can lead digestive and/or cardiac complications; however, there are not effective treatments against it after the acute phase. 5-lipoxygenase (5-LO) enzyme synthesizes lipidic mediators as lipoxins (LXs) and leukotrienes (LTs) that exert strong influence in immune response modulation. LTs have pro-inflammatory while LXs have anti-inflammatory/resolutive actions. Because the “power” of these mediators in the immune system, and, since 5-LO is activated during the majority of inflammatory processes, 5-LO is a possible target for therapeutic actions in infectious diseases. Ours studies demonstrated that manipulation of 5-LO activity by 5-LO inhibitor (i5-LO) revealed to be a great strategy for therapeutic use against T. cruzi infection improving several parameters as parasitemia, inflammatory infiltrate and cardiac function. Moreover, our data demonstrated that aryl hydrocarbon receptor (AhR), a LXs nuclear receptor, activity is responsible to modulate innate and adaptative immune response and development of myocarditis during experimental T. cruzi infection. Finally, we demonstrated that LXs/AhR pathway induces Suppressor of cytokine signaling 2 (SOCS2) that modulates immune response, cardiomyocytes and heart function during Tc infection. Thus, 5-LO/AhR/SOCS2 play a biologically important role in modulate the effective immune responses to Tc and mediated counter-regulatory activity in cardiac function/protection. In summary, the manipulation of these molecules, mainly 5-LO activity by i5-LO, could be a great strategy for therapeutic use against Chagas’ disease acting as a “time machine” tool capable to control “where and when” the effectively immune response must acts to avoid pathology development due to T. cruzi infection. CNPq and FAPEMIG

PATHOGENESIS OF ACUTE CORONARY SYNDROMES FILIPPO CREA, CATHOLIC UNIVERSITY, ROMA, ITALY When primary prevention of atherosclerosis fails, the progression of coronary atherosclerosis can remain clinically silent for years, decades, or even for life, as indicated by the high prevalence of coronary atherosclerosis in subjects dying of noncardiac causes. In contrast, some patients, at a certain point in their life, exhibit an acute coronary syndrome (ACS), followed by a period of stability that can be short or last for years or decades. These simple clinical observations suggest that the mechanisms responsible for plaque growth and for plaque instability are different and that the causes and mechanisms of plaque instability are multiple. Accordingly, the paradigm that implies a single type of culprit coronary plaque as a cause for instability does not adequately fit the findings of postmortem studies. Indeed, on the one hand, plaque fissure is frequently asymptomatic and contributes to stepwise, clinically silent plaque growth rather than precipitating an abrupt coronary occlusion; conversely, it is not observed in 30% to 50% of patients who have ACS. Furthermore, the scientific community promptly accepted the notion that inflammatory cell activation plays a key role in the pathogenesis of ACS, and it is now commonly believed that activation of inflammatory cells in the culprit stenosis is the cause of coronary instability in all patients. However, this notion is in sharp contrast with the observation that about 40% of patients with ACS have low or very low levels of C-reactive protein, a very sensitive marker of inflammation. Finally, coronary angiography fails to demonstrate obstructive atherosclerosis in up to one-third of patients with symptoms suggestive of ACS and raised troponin levels and/or ischemic-like STsegment changes, thus suggesting that functional alterations of epicardial arteries and/or of coronary microcirculation play an important pathogenetic role. The complexity of postmortem and clinical observations suggests that it is unlikely to identify a common cause for the phenotype of ACS. To better understand the multiple causes of coronary instability, it is desirable to construct a pathogenetic classification of ACS based on simple clinical descriptors. The multiple causes of coronary instability can be more easily assessed in 3 homogeneous groups of patients with a similar clinical presentation: 1) patients who have obstructive atherosclerosis and systemic inflammation; 2) patients who have obstructive atherosclerosis without systemic inflammation; and 3) patients without obstructive atherosclerosis (see figure).

"INTERPLAY BETWEEN THE ENDOPLASMIC RETICULUM AND OXIDATIVE STRESS IN VASCULAR DISEASE" FRANCISCO RM LAURINDO The endoplasmic reticulum (ER) is an emerging player in redox pathophysiology, given its role in redox protein folding and stress responses. The ER oxidoreductin (Ero1) flavoenzyme is a major source of oxidants and the ER redox balance is tightly controlled by yet unclear mechanisms that may involve the productive consumption of hydrogen peroxide for protein folding. In addition, a particular advance in this context is the increasingly evident interplay between the ER and Nox NADPH oxidases, the main enzymatic source of reactive oxygen with signaling purposes. Understanding isoform- and context-specific subcellular Nox compartmentalization allows relevant functional inferences. Catalytic/regulatory Nox transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Duox maturation also involves regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox calcium signaling regulation of via ER targets such as SERCA. Growing evidence suggests Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, while Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation and redox-dependently interacts with p47phox. Together, results implicate PDI as a possible Nox organizer. We propose such convergence between Noxes and the ER may have evolutive roots given ER-related functional contexts which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights into Nox-related (patho)physiology and allow the identification of mechanistical networks central to the regulation of cell signaling and inflammation. (Research supported by: FAPESP, CNPq – INCT Redoxoma)

FRANKLIN ALAN SHER REGULATING COLLATERAL DAMAGE IN TH1 RESPONSES TO INTRACELLULAR PATHOGENS Alan Sher, David Kugler, Paul R. Mittlestadt, Jonathan D. Ashwell and Dragana Jankovic Immunobiology Section, Laboratory of Parasitic Diseases, NIAID, and Center for Cancer Research, NCI. National Institutes of Health, Bethesda, MD 20892 If not properly regulated, Th1 responses launched by the host to control intracellular pathogens can cause life-threatening immunopathology. Since these negative regulatory functions are themselves host protective , they constitute a critical aspect of immune anti-microbial defense. An excellent example of host-protective negative regulation of CD4 T cell function occurs during the Th1 response to Toxoplasma gondii, an intracellular protozoan parasite. This IL-12-driven response results in production of high levels of IFN-parasite replication in both hematopoetic and non-hematopoetic cells . Nevertheless, the exuberant cytokine production occurring in T. gondii infection can also be host detrimental, an outcome first documented in acutely infected IL-10

-/- mice that, while successfully controlling parasite growth, succumb to cytokine-storm mediated

immunopathology. Subsequent studies have revealed similar pathological sequelae in T. gondii-infected IL-27R-/-

animals and in chronically infected mice deficient in 5-lipoxygenase .The common feature of these three genetic deficiencies is that each results in the over-expression of IL-12 by APC, which in turn leads to uncontrolled Th1-type responses., Recently, we have defined an additional non-redundant pathway by which CD4+T cells self-regulate their Th1 function during T.gondii infection through the induction of glucocorticoids (GC). The immunomodulatory circuit involved is independent of IL-10 and involves direct suppression of T cell function by GC rather than indirect modulation through an APC intermediate. As described there, this self-regulatory pathway, involving co-ordination with the neuroendocrine systems represents a novel mechanism preventing collateral tissue damage by anti-microbial Th1 responses. This work was supported by the intramural research programs of the NIAID and NCI, NIH

CONTROL OF PATHOGEN COLONIZATION BY VIRULENCE FACTORS AND THE MICROBIOTA GABRIEL NUÑEZ

Department of Pathology and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, U.S.A. The mechanisms that allow pathogens to colonize the intestine and the indigenous microbiota to inhibit pathogen colonization remain unclear. We found that that germ-free animals are unable to eradicate Citrobacter rodentium, a model for human infections with attaching/effacing (A/E) bacteria. These Gram-negative bacteria are food- and waterborne non-invasive pathogens which attach to and colonize the intestinal tract by inducing characteristic A/E lesions on the intestinal epithelium, leading to transient enteritis or colitis in humans. The genome of A/E pathogens including Enterohemorragic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC) and C. rodentium harbor the locus of enterocyte effacement (LEE) that is critical for bacterial colonization and the ability to cause pathology. We found that early in infection, LEE virulence genes were expressed and required for pathogen growth in conventionally raised but not germ-free mice. LEE virulence gene expression was downregulated during the late phase of infection, which led to relocation of the pathogen to the intestinal lumen where it was out-competed by commensals. The ability of commensals to out-compete C. rodentium was determined, at least in part, by the capacity of the pathogen and commensals to grow on structurally similar carbohydrates. Moreover, dietary carbohydrates can influence the ability of members of the gut microbiota to out-compete the pathogen in the intestine. Our studies indicate the members of the microbiota use metabolic pathways to out-compete pathogens. Furthermore, intestinal pathogens have developed strategies to avoid competition with commensals based on the expression of virulence factors during the early phase of the infection.

MICROBIOTA, INFLAMMATION AND CANCER GIORGIO TRINCHIERI, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, NIH, Frederick, MD 21702. Mammals live in partnership with a rich commensal microbiota on the body epithelial surfaces. This partnership is critical for tissue formation, metabolism and the development and function of the innate and adaptive resistance. The intestinal microbiota regulates the physiological host-microbial mutualism while allowing protective immunity in the case of mucosal infections or damage. The cross-talk between the microbiota and the immune system involves innate cell types such as macrophages and dendritic cells. Similarly, the skin microbiota has an autonomous function in controlling the local inflammatory milieu and tuning resident T lymphocyte functions. Independently of gut commensals, normal skin microbiota establishes an immunological tone that lowers the threshold of activation for protective skin immune responses. The gut microbiota plays a role not only in local tissue formation and the development of mucosal immunity, but quantitative or qualitative alteration of its composition (by antibiotics treatment or other causes of dysbiosis) affects systemic immunity. The gut microbiota affects autoimmunity at distance (joints, central nervous system), resistance to viral lung infections, hepatic chronic and autoimmune diseases, mammary carcinogenesis, and inflammation-dependent metabolic syndrome. The local effects of the microbiota are explained by the interaction of bacteria or their products with innate receptors or other sensors in epithelial cells or associated hematopoietic cells. However, the cellular and molecular mechanisms by which the gut commensal microbiota regulates the threshold of activation of systemic innate and adaptive immunity are only beginning to be characterized. Alteration in the gut commensal microbiota also modifies the microenvironment of tumors. Although the role of septic or aseptic inflammation in cancer is well documented, how commensal bacteria affect inflammation in the sterile tumor microenvironment remains unclear. We show that specific alterations of therapy-induced inflammation in antibiotics-fed or germ-free mice impair the response of sub-cutaneous cancers to CpG-oligonucleotide-immunotherapy or platinum chemotherapy. In microbiota-depleted mice, decreased cytokine production from tumor-infiltrating monocyte-derived cells following CpG-OGN treatment reduced tumor necrosis whereas deficient chemotherapy-induced production of reactive oxygen species by tumor-infiltrating myeloid cells impaired tumor destruction. Thus, optimal response to cancer immunotherapy and chemotherapy and survival requires an intact commensal microbiota.

GIUSEPPE CIRINO Cardiovascular inflammation and L-cysteine/H2S pathway Giuseppe Cirino & Mariarosaria Bucci Dipartimento di Farmacia Università di Napoli Federico II, Via Domenico Montesano 49, 80131, Napoli, Italy In the mammalian cardiovascular system, H2S joins carbon monoxide (CO) and endothelial derived relaxing factors, (EDRFs)-nitric oxide (NO), as the third gasotransmitter. In the vasculature, cystathionine-γ-lyase (CSE) is the main enzyme responsible for H2S biosynthesis starting from the substrate e.g. L-cysteine. There is a growing body of evidence that supports a role for H2S in regulating the vascular homeostasis. H2S (NaHS) is known to induce a concentration-dependent relaxation of large conduit arteries. Interestingly, H2S also relaxes peripheral resistance vessels such as mesenteric arteries suggesting a role for H2S also in the regulation of vascular resistance and systemic blood pressure. This vasodilatory effect is dependent on the activation of KATP channels. However, a cross-talk exists between the L-Argine/NO and L-cysteine/H2S pathways. Furthermore, it has been shown that H2S acts as an endogenous non-selective inhibitor of phosphodiesterase activity. Compelling evidence links H2S to regulation of erectile function while it remains unclear whether the L-cysteine/H2S pathway plays a pathogenetic role in erectile dysfunction. Despite the rapid growth of the field, it should be noted that several aspects of H2S physiology in the cardiovascular system remain unsolved and the lack of reliable inhibitors and donors remains a major limitation. The presentation will mainly focus on the role of the L-cysteine-H2S pathway in cardiovascular inflammation.

GLAUCIA FURTADO TNFa-DEPENDENT DEVELOPMENT OF LYMPHOID TISSUE IN THE ABSENCE OF RORgt

+ LYMPHOID TISSUE

INDUCER CELLS. Immunology Institute, Icahn School of Medicine at Mount Sinai, 1425 Madison Ave, New York NY 10029 Lymphoid tissue often forms within sites of chronic inflammation. Here we report that expression of the pro-inflammatory cytokine TNFa drives development of lymphoid tissue in the intestine. Formation of this ectopic lymphoid tissue was not dependent on the presence of canonical RORgt

+ lymphoid tissue inducer (LTi) cells, because animals

expressing increased levels of TNFα but lacking RORgt+ LTi cells (TNF/Rorc(gt)

-/- mice) developed lymphoid tissue in

inflamed areas. Unexpectedly, such animals developed several lymph nodes that were structurally and functionally similar to those of wild type animals. TNFα production by F4/80

+ myeloid cells present within the anlagen was

important for activation of stromal cells during the late stages of embryogenesis and for the activation of an organogenic program that allowed development of lymph nodes. Our results show that lymphoid tissue organogenesis can occur in the absence of LTi cells and suggest that interactions between TNFα-expressing myeloid cells and stromal cells have an important role in secondary lymphoid organ formation.

GUSTAVO B MENEZES MECHANISMS OF LEUKOCYTE RECRUITMENT HOW DO DEAD CELLS TELL THE IMMUNE SYSTEM WHERE THEY ARE? Neutrophils are recruited to sites of infection where they help to kill and clear pathogens. However, sterile cell death or injury can also trigger robust neutrophil migration and accumulation, and there is enough evidence to support that these cells may also contribute to additional damage. In this sense, to understand the mechanisms involved in the recruitment of these cells may aid to development of more effective therapies. Here we discuss how new imaging techniques may be used to visualize and quantify neutrophil migration to sterile injury.

PLATELETS AS INFLAMMATORY CELLS: NOVEL ACTIVITIES IN INFECTION AND THROMBOSIS GUY A. ZIMMERMAN, M.D. Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah, USA and Laboratory of Immunopharmacology, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil There is clear and evolving evidence that human platelets, which are best known as major effectors of hemostasis, have activities across the innate and adaptive limbs of the immune continuum and are major inflammatory cells. Platelets from mice and other surrogate experimental animals also have inflammatory activities that are similar to those of human platelets. Key inflammatory functions of human and murine platelets include interaction with and signaling of leukocytes of several classes (myeloid leukocytes, lymphocytes, dendritic cells, macrophages), release of preformed chemokines and other inflammatory mediators, activation-dependent synthesis of the pleiotropic cytokine interleukin 1β (IL-1β) and delivery of IL-1β and other immune modulators to target cells, regulation of endothelial permeability and inflammatory phenotype, and, in the case of murine platelets, regulation of lymphangiogensis and lymph node topography. In addition, platelets are key sensors of pathogens and respond to microbes with activities that can initiate or amplify inflammation and thrombosis. Platelets likely evolved as multi-tasking host defense cells with progressive specialization for hemostatic, immune, and microbe-sensing functions. New discoveries and ongoing analysis of platelet activities in bacterial, viral, and plasmodial diseases and disease models demonstrate previously-unrecognized pathogen-sensing and inflammatory capabilities, and that the depth of the inflammatory repertoire of platelets is substantial and unexpected.

HAMIDA HAMMAD CROSSTALK BETWEEN AIRWAY EPITHELIAL CELLS AND DENDRITIC CELLS IN ASTHMA Department of Molecular Biomedical Research, Zwijnaarde, Gent, and University of Ghent, Belgium Allergic asthma is characterized by accumulation of eosinophils, mast cells and Th2 cells that lead to goblet cell hyperplasia, bronchial hyperreactivity and airway wall remodeling. Dendritic cells are crucial not only in the initiation of T cell responses, but also for their maintenance. Different DC subsets seem to perform different tasks in the process of allergic sensitization. Studies in Flt3L-/-, CCR2-/-, CD11cDTR and langerin-DTR mice have revealed an important division of labour between DC subsets in the lung

10. Inflammatory type (CD64

+, MAR-1

+) DCs and CD11b+ cDCs

present allergens best, whereas CD103+ cDCs, pDCs and non-professional APCs like B cells and macrophages are

poor APCs. We also found that the interaction between allergens, lung epithelial cells (ECs) and dendritic cells (DCs) leads to asthma development. Lung DC activation by airway epithelial cells is induced by IL-1. Indeed, mice lacking IL-1R on radioresistant cells, but not hematopoietic cells, failed to mount a Th2 immune response and did not develop asthma to HDM. Experiments performed in vivo and in isolated air–liquid interface cultures of bronchial ECs showed that TLR4 signals induced the release of IL-1α, which then acted in an autocrine manner to trigger the release of DC-attracting chemokines, GM-CSF, and IL-33. Consequently, allergic sensitization to HDM was abolished in vivo when IL-1α, GM-CSF, or IL-33 was neutralized. These findings put IL-1α upstream in the cytokine cascade leading to epithelial and DC activation in response to inhaled HDM allergen. We are now trying to understand the molecular mechanisms through which epithelial cell activation induces asthma in mice and in patients.

HEPING XU INFLAMMATION IN RETINAL AGEING AND AGE-RELATED RETINAL DEGENERATION Centre for Vision and Vascular Science, Queen’s University Belfast Inflammation is an adaptive response of the host to noxious insults. The magnitude of inflammation may vary from low-grade to overt aggressive inflammation depending on the level of insults. Although the retina is considered as an immune “privileged” tissue, inflammation does occur at all levels. During normal ageing, oxidative stress accumulates in the retina resulting in a low-grade para-inflammation in the forms of mild microglial activation and incomplete complement activation. The physiological role of para-inflammation is to maintain tissue homeostasis and functionality. Dysregulation in the para-inflammatory response may result in sustained chronic inflammation, which may contribute to the pathogenesis of age-related retinal degeneration. The nature of the retinal immune response in the ageing eye and in eyes with age-related retinal degeneration will be discussed.

RANKL SIGNAL TRANSDUCTION AND OSTEOCLAST DIFFERENTIATION HIROSHI TAKAYANAGI Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan RANKL is a TNF family cytokine that is essential for osteoclast ifferentiation. In addition, recent reports revealed RANKL to play various roles in multiple tissues. RANKL signals the cell through numerous immunomodulatory molecules such as TRAF6, NF- -Fos and NFATc1. NFATc1 is the master transcription factor for osteoclast development and its expression is autoamplified during osteoclastogenesis. NFATc1 activation requires calcium signaling, which is mediated in osteoclasts by immunoreceptor tyrosine-based activation motif (ITAM) in dual

-like receptors such as OSCAR and TREM-2, which function as costimulatory receptors for RANK. Here I will introduce the recent advance in the understanding of molecular mechanisms underlying osteoclast differentiation by focusing on intracellular signal transduction of RANKL.

OSTEOIMMUNOLOGY HIROSHI TAKAYANAGI Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo ERATO, JST Bone is a part of the skeletal system which supports the body and enables the locomotion, while the immune system protects the host from the pathogens. The bone and the immune system thus have totally different functions. However, the bone marrow harbors the immune cells including hematoipoietic stem cells and B cells, and it is often observed that the bone homeostasis is influenced by the activated immune responses. Therefore, osteoimmunology, the research on the interactions and shared molecules of the bone and immune systems, has attracted much attention. Rheumatoid arthritis (RA) is an autoimmune disease in which activated immune responses causes the severe bone destruction, the studies on which have been the driving force for the field of osteoimmunology. Th1 and Th2 cells inhibit osteoclastic bone resorption through IFN- -4, respectively. In contrast, Th17 cells which infiltrate into RA synovium produce IL-17 which induces RANKL in synovial fibroblasts and inflammatory cytokines such as TNF- d IL-1. These cytokines increase RANKL further and stimulate the osteoclast precursor cells, synergistically contributing to the bone destruction. Here I review the history and recent advances of osteoimmunology, which provided molecular basis for understanding the pathogenesis and developing new strategies against RA. References (1) Nat Rev Immunol 7, 292-304, 2007 (2) Nat Rev Rheumatol 5, 667-76, 2009

HUGO CASTRO FARIA NETO COGNITIVE IMPAIRMENT IN EXPERIMENTAL MALARIA Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection. Previous work has detected a persistent cognitive impairment in children who survived an episode of CM. Potential therapeutic interventions to this condition have not been investigated and are urgently needed. HMG-CoA reductase inhibitors (statins) have been widely prescribed for the treatment of cardiovascular diseases. In addition to its effect on the inhibition of cholesterol synthesis, statins have been associated with pleiotropic anti-inflammatory effects. We investigated the effect of treatment with statins during experimental cerebral malaria and its possible therapeutic effect on cognitive impairment. Plasmodium berghei ANKA (PbA) infected mice displayed clear signs of CM and were treated with lovastatin in addition to chloroquine. Intravital examination of pial vessels of infected animals showed a decrease in functional capillary density and an increase in the rolling and adhesion of leukocytes to the endothelium that were reversed by treatment with statins. Brain levels of MDA, IL- F- -1 and IL-12 levels were also increased in PbA-infected mice, but reduced after treatment with statins. Fifteen days post-infection cognitive dysfunction was detected by different memory and cognition tests in animals rescued from CM by chloroquine treatment, but cognitive dysfunction was absent in animals treated with lovastatin- chloroquine combination. A similar effect was observed in a model of bacterial sepsis, suggesting that statins may have neuro-protective effects in other severe infectious syndromes than CM. Statins may be a valuable adjuvant therapeutic tool to prevent cognitive impairment in children surviving an episode of CM.

PROTEASE SIGNALLING IN ARTHRITIS JASON J. MCDOUGALL, Departments of Pharmacology, and Anaesthesia, Pain Management & Perioperative Medicine, Dalhousie University, Halifax, Nova Scotia. B3H 4R2. Canada. During arthritis, serine proteases are released into the joint leading to tissue remodelling and degeneration. In addition to these classical enzymatic effects, proteases can also cleave and activate specific G protein-coupled receptors called protease-activated receptors (PARs). PAR1 is cleaved by thrombin which has been shown to cause proliferation of synovial fibroblasts in explants taken from rheumatoid arthritis patients (1). Among the serine proteases known to cleave PAR2 in joints are mast cell tryptase and matriptase. Preclinical studies have determined that PAR2 activation leads to pro-inflammatory responses such as vasodilatation and increased leukocyte trafficking (2). Furthermore, the localisation of PAR2 on joint afferent nerves suggests that these receptors may play a role in neurogenic inflammation and pain. Indeed, selective PAR2 agonists cause peripheral sensitization of articular nociceptors leading to the generation of joint pain which can be blocked by transient receptor potential vanilloid-1 (TRPV1) and neurokinin-1 receptor antagonists (2). The most recent PAR to be discovered (PAR4) has also been implicated in joint inflammation and pain where selective agonists cause joint oedema, enhanced leukocyte trafficking and peripheral nerve sensitization (3). These pro-inflammatory effects of PAR4 activation occur through a bradykinin B1 receptor pathway and involve the degranulation of connective tissue mast cells in the synovium (4, 5). Recent studies in my laboratory have shown that cathepsin G, which typically cleaves PAR4, leads to a reduction in nociception suggesting that in the joint it may disarm PAR4. Preliminary data showing that neutrophil elastase evokes a pain response in joints and the possibility that this protease activates PAR2 will also be discussed. REFERENCES 1. Furuhashi I, Abe K, Sato T, Inoue H. Thrombin-stimulated proliferation of cultured human synovial fibroblasts through proteolytic activation of proteinase-activated receptor-1. Journal of pharmacological sciences. 2008;108(1):104-11. Epub 2008/09/13. 2. Russell FA, Schuelert N, Veldhoen VE, Hollenberg MD, McDougall JJ. Proteinase-activated receptor-2 (PAR(2) ) activation sensitises primary afferents and causes leukocyte rolling and adherence in the rat knee joint. Br J Pharmacol. 2012. Epub 2012/08/02. 3. McDougall JJ, Zhang C, Cellars L, Joubert E, Dixon CM, Vergnolle N. Triggering of proteinase-activated receptor 4 leads to joint pain and inflammation in mice. Arthritis and Rheumatism. 2009;60(3):728-37. Epub 2009/02/28. 4. Russell FA, Veldhoen VE, Tchitchkan D, McDougall JJ. Proteinase-activated receptor-4 (PAR4) activation leads to sensitization of rat joint primary afferents via a bradykinin B2 receptor-dependent mechanism. Journal of Neurophysiology. 2010;103(1):155-63. Epub 2009/11/06. 5. Russell FA, Zhan S, Dumas A, Lagarde S, Pouliot M, McDougall JJ. The pronociceptive effect of proteinase-activated receptor-4 stimulation in rat knee joints is dependent on mast cell activation. Pain. 2011;152(2):354-60. Epub 2011/01/18.

IMMUNOSURVEILLANCE MECHANISMS BY CENTRAL VERSUS EFFECTOR MEMORY T CELLS JENS V. STEIN New imaging techniques including twophoton microscopy (2PM) and mesoscopic imaging have uncovered the dynamic interactions between lymphocytes and antigen-presenting cells and the large-scale rearrangements of lymphoid tissue during inflammation. Here, we will discuss factors orchestrating central versus effector T cell migration in vivo, with a focus on G-protein-coupled receptors and their intracellular effectors. To this end, we use 2PM to follow the dynamic behavior of antigen-specific T cells in lymph nodes (“central memory T cells”) and salivary glands (“effector memory T cells”) during the memory phase of an antiviral immune response. In both tissues, central and effector memory T cells show robust migration, albeit at slower speeds and more complex cellular shape in salivary glands. Furthermore, both effector and central memory T cells maintain their ability to rapidly engage with CD11c

+

antigen-presenting cells. The absence of the Cdc42 guanine exchange factor DOCK8 and pharmacological inhibition of the Rho effector ROCK resulted in impaired effector memory T cell migration in salivary glands, presumably owing in part to its greater stromal tissue complexity as compared to lymph nodes. Taken together, these findings suggest tissue-specific roles for intracellular signaling modules, which control tissue-specific T cell function.

JESUS G. VALENZUELA PRO- AND ANTI-INFLAMMATORY PROPERTIES OF SAND FLY SALIVARY MOLECULES. Vector Molecular Biology Section, LMVR, NIAID, NIH/Rockville, MD, USA During feeding, sand flies deposit in the skin salivary components that assists in the acquisition of blood by modulating the hemostatic and inflammatory system. On the other hand, animals that have been exposed to the bites of these insects develop immune responses to some of these salivary proteins. Furthermore, in animals not previously exposed to insect bites or saliva, insect saliva favors Leishmania establishment and infection, however, in sand fly-exposed animals, immunity to saliva prevents parasite establishment and infection. We have focused our efforts in identifying and characterizing these salivary components by transcriptomic and functional genomic approaches to understand the dual effect of sand fly saliva. In this presentation I will discuss the recently characterized salivary molecules that can suppress the inflammatory system by acting on key immune cells or affecting key molecules of the hemostatic system and the recently identified salivary proteins that have the opposite effect, the recruitment of pro-inflammatory cells or the induction of pro-inflammatory cytokines. The consequences of these responses to parasite establishment will be discussed.

JING X. KANG OMEGA-6/OMEGA-3 FATTY ACID RATIO AND INSULIN SENSITIVITY Laboratory for Lipid Medicine and Technology, Department of

Medicine, Massachusetts General Hospital and Harvard Medical School,

Boston, Massachusetts 02114, USA Omega-6 and omega-3 polyunsaturated fatty acids are essential nutrients that function as important modulators of multiple biological pathways that affect health and disease. They are the precursors of key lipid mediators that modulate inflammation, which is associated with insulin resistance. Generally, omega-6 fatty acids promote inflammation whereas omega-3 fatty acids have anti-inflammatory properties. However, modern Western diets are deficient in omega-3 fatty acids but have excessive levels of omega-6 fatty acids, resulting in omega-6/omega-3 fatty acid ratios of >10:1, which is contradictory with our genetic profile that was established on a 1:1 ratio. Our current research aims to address whether this fatty acid imbalance is linked to the growing prevalence of insulin resistance, diabetes, and obesity. The results from our studies using the fat-1 transgenic mouse model and dietary intervention approaches demonstrate that altering the tissue omega-6/omega-3 fatty acid ratio has significant effects on various stages of development of obesity and diabetes, including eating behavior in young mice, insulin sensitivity in aging animals, inflammation in adipose tissue, and the expression and signaling of genes involved in glucose metabolism. Our studies have also shown that reducing the tissue omega-6/omega-3 fatty acid ratio can effectively prevent chemical-induced type 1 diabetes and diet-induced type 2 diabetes in animals. Our findings implicate that a high tissue omega-6/omega-3 fatty acid ratio may be a risk factor for insulin resistance, and that balancing the omega-6/omega-3 ratio may be a safe and effective strategy for the prevention and treatment of insulin-resistant disorders.

TOWARDS THE DEVELOPMENT OF GM-CSF AS A TARGET IN INFLAMMATION/AUTOIMMUNITY JOHN A HAMILTON University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia

GM-CSF was originally defined in vitro as a haemopoietic growth factor for the development of granulocytes and macrophages from bone marrow precursor cells following their proliferation and differentiation. However, it can also act as a pro-survival, activating and/or differentiation factor for more mature myeloid populations and to drive inflammation/autoimmunity in many animal models. Clinical trials have commenced in rheumatoid arthritis (RA), multiple sclerosis and asthma with some success reported in RA patients. How GM-CSF functions in inflammation will be discussed as will the issues surrounding its targeting.

HYDROGEN SULFIDE IN RESOLUTION OF INFLAMMATION JOHN L. WALLACE MCMASTER UNIVERSITY, HAMILTON, ON, CANADA Hydrogen sulfide (H2S) is a gaseous mediator produced throughout the gastrointestinal tract and by many species of commensal and infectious bacteria. Like nitric oxide and prostaglandins, H2S is an important mediator of mucosal defence; that is, suppression of its synthesis leads to increased susceptibility to ulceration. On the other hand, administration of H2S donors, including compounds derived from garlic, can greatly increase the resistance of the mucosa to injury induced by nonsteroidal anti-inflammatory drugs or ischemia-reperfusion injury. H2S also exerts a number of anti-inflammatory effects, including inhibition of leukocyte adherence to the vascular endothelium. Chronic suppression of H2S synthesis leads to a significant increase in the ‘basal’ level of inflammation in the digestive tract, as well as decreased cyclooxygenase-2 expression and prostaglandin synthesis. When inflamed or injured, a marked increase in the capacity of gastrointestinal tissue to synthesize H2S is observed. There is solid evidence that in such circumstances, H2S contributes significantly to promoting the healing of the damaged tissue and to resolution of the inflammatory response. Thus, inhibition of H2S synthesis results in significant delays in ulcer healing and exacerbation of tissue inflammation. Administration of H2S donors accelerates healing of ulcers. The ability of H2S to enhance mucosal defence, healing and resolution of inflammation has been exploited in the development of several novel anti-inflammatory drugs. H2S-releasing derivatives of mesalamine have been shown to exert markedly enhanced anti-inflammatory effects, compared to the parent drug, in models of colitis. One of the key mechanisms for the enhanced anti-inflammatory activity appears to be suppression of pro-inflammatory cytokine production. H2S-releasing derivatives of cyclooxygenase inhibitors exhibit comparable anti-inflammatory activity to the parent drugs, but with markedly reduced injurious effects in the digestive tract.

JULIO SCHARFSTEIN INTRACARDIAC EDEMA AND TRYPANOSOMA PARASITISM: NOVEL VIEWS ON THE PATHOGENIC ROLES OF THE KALLIKREIN-KININ SYSTEM Increased vascular permeability is a common manifestation of inflammation caused by tissue injury in various settings, including parasitic infections. In a recent review (Scharfstein et al., Frontiers Immunol, 2013), we have advanced the hypothesis that extracellular forms (trypomastigotes) of Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, may take advantage of proteolytic generation of bradykinin and endothelin-1 in intramyocardial inflammatory exudates to persistently infect cardiovascular cells through the cooperative activation of bradykinin receptors (BKRs) and endothelin receptors (ETRs) (Scharfstein et al., JEM 2000; Andrade et al., BJP 2011). While our studies progressed, independent investigations in animal models of allergic inflammation linked activation of the kallikrein-Kinin system (KSS) to mast cell secretion of heparin or polyphosphates, both of which tentatively defined as endogenous activators of the contact system (Oschatz et al. Immunity 2011; Moreno-Sanchez et al., JBC 2012). Inspired by these break-through discoveries, we then resorted to intravital microscopy (hamster cheek pouch model) to determine whether trypomastigotes (Dm28 strain) evoke plasma leakage through the activation of the mast cell/KKS axis. Combined to supportive evidences in mast cell deficient mice (B6-kitW-sh/W-sh) infected with T. cruzi, we found that plasma extravasation through post capillary venules depends on functional integration between innate immunity (TLR2/CXCR2-dependent initiation of inflammation) and cruzipain/kinin-driven proteolytic circuits (propagation of the inflammatory edema) (Monteiro et al., JI 2006; Schmitz et al., JLB 2009; Andrade et al. Adv Parasitol 2012). Collectively, these findings laid the groundwork for the studies in the intracardiac model of infection, described herein. Guided by echocardiograph, we inoculated Dm28c trypomastigotes (tissue culture derived) in the left ventricle of naïve B6 mice pretreated with a single-dose of cromoglycate (mast cell stabilizer) or pretreated with BKR or ETR antagonists. In a second series of experiments, we measured parasite tissue load in the heart of mast cell deficient (B6-KitW-sh/W-sh) versus cardiac tissues from the control strain. Our results (see accompanying abstract by Oliveira et al.) revealed that heart parasitism was markedly reduced either in cromoglycate-treated WT mice or mast cell-deficient B6-KitW-sh/W-sh mice. Adding weight to these findings, we found that intracardiac parasite load was decreased in WT mice pretreated with a single-dose of (i) HOE-140 (B2R antagonist) (ii) B9858 (B1R antagonist) (iii) FXII inhibitor (blocker of contact phase/KKS activation) and (iv) bosentan (blocker of ETaR/ETbR). We then measured intracardiac leakage of TRITC-dextran by confocal microscopy and found evidence of (subtle) tracer extravasation soon after parasite injection in the heart of naïve mice. Notably, the microvascular leakage reaction in the heart was abolished in mice pretreated with BKR and ETR antagonists. We then checked whether these early pharmacological interventions could have cardioprotective effects as the infection proceeded. Indeed, histopathological analysis (30 d.pi.) showed that myocarditis and collagen deposition were drastically reduced in WT mice pretreated either with HOE-140, B9858 or bosentan. Given the artificial nature of the intracardiac model, we then infected BK1R-deficient mice intraperitoneally and assessed pathogenic outcome in the heart during sub-acute and chronic stages of infection. Consistent with the findings obtained in the intracardiac model of infection, BK1R-deficient mice exhibited reduced intracardiac parasitism (early in infection) and showed markedly reduced myocarditis/fibrosis at the chronic stage (90 d p.i.). Collectively, our studies suggest that intermittent flares of plasma leakage, forged through the intracardiac activation of the mast cell/KKS axis, may propitiate a window of opportunity for parasite invasion of cardiovascular cells expressing BK1R. Additional studies are required to verify whether drugs that preserve/restore the integrity of endothelium barrier function in the chagasic heart, such as BK1R antagonists, might reduce inflammation/fibrosis while at the same time limiting T. cruzi ability to invade heart cells through the signaling of BK1R/BK2R/ETaR/ETbR, as we originally hypothesized (Scharfstein et al, Frontiers in Immunol 2013). Research supported by CNPq, CAPES, INBEB/INCT and FAPERJ.

KIRILL ZYKOV THE NEW APPROACH TO INFLAMMATORY PATHWAYS BLOCKADE Asthma and COPD are the most common inflammatory pulmonary diseases. The most widespread anti-inflammatory drugs in asthma are corticosteroids (CS), but in severe forms of disease in some patients there is CS insensitivity and in COPD the role of CS is uncertain. This makes it necessary to find out new strategies for the control of inflammatory process in chronic obstructive lung diseases. Alkylating drugs (AD) were used as CS-sparing agents in asthma but its use is limited because of serious side-effects. In therapeutic doses ADs lead to DNA-DNA cross-linkage with cytotoxic effect, but, previously it has been shown that AD in the very low dose (100-fold lower than cytotoxic ones) affect the cytokine receptors without DNA damage. The blockade of cell signal transduction by IL-2b and TNF- α was observed with such ultra-low doses of ADs. It was previously shown, that the application of non-cytotoxic doses of an AD melphalan reduces the severity of murine experimental colitis. In asthmatic patients in placebo-control study it was demonstrated, that the inhalation of ultra-low doses of melphalan resulted in bronchial epithelium regeneration and clinical improvement (reduction of exacerbation rate and rescue medication use). Dynamics of IL-5, IL-8 and IFN-γ concentrations in BAL and peripheral blood were associated with clinical improvement. Thus, the ultra-low doses of ADs have beneficial effect both in acute and chronic inflammatory diseases. We hypothesized that such anti-inflammatory activity is the result of blocking of signal transduction through various cell surface receptor including IL-2R and TNFaR. This phenomenon can be the base for the creating of new approach for inflammatory diseases treatment, using cell signaling blockade with low doses of ADs.

DELAYED AND ABERRANT IMMUNOLOGICAL RECONSTITUTION IN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION KOUJI MATSUSHIMA Department of Molecular Preventive Medicine, Graduate School of Medicine, TheUniversity of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, JapanDelayed and aberrant reconstitution of immune system in allogeneic hematopoietic stem cell transplantation KEY WORDS: BM-GVHD, osteoblastic HSC niche, B cell insufficiency, anti-CD4 antibody, LN-GVHD, chronic GVHD Kouji Matsushima, Yusuke Shono, Fumiko Suenaga, Yong Wang, Mizuha Kosugi, Jun Abe and Satoshi Ueha Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Allogeneic-hematopoietic stem cell transplantation (allo-HSCT) is the most effective therapy for hematologic malignancies, although its five-year survival rate is still 50%. Disrupted bone marrow (BM) hematopoiesis and delayed immune reconstitution are life-threatening complications of allo-HSCT. One of the major risk factors for BM insufficiency is graft-versus-host disease (GVHD) in which donor T cells recognize the recipient as “foreign” and attack the tissues of recipient. However, how GVHD impairs BM hematopoiesis has been largely unknown. Using several mouse models of major histocompatibility antigen (MHC)-mismatched allo-HSCT, we explored the effects of GVHD on the possible targets in BM, HSCs and hematopoietic niches. By using hematopoietic reconstitution in MHC I- and MHC II-double mismatched GVHD model, we found that the BM hematopoiesis, especially the B-lymphopoiesis, was severely impaired in the GVHD mice. The B-lymphopoiesis was impaired in CD4 T cell-dependent GVHD model, regardless of the type of mismatch (i.e. major or minor). Recipients of FasLgld/gld CD4 T cells partially restored the B lymphopoiesis, suggesting that donor CD4 T cells play a key role in the defective B-lymphopoiesis partially through FasL-dependent cytotoxicity. Re-transplantation experiments revealed that GVHD-affected BM contains substantial number of HSCs that reconstitute all hematopoietic cell lineage. In contrast, hematopoietic niche in GVHD-affected BM failed to support the B-lymphopoiesis, which was potentially due to the loss of osteoblasts in GVHD-affected BM. Osteoblasts were completely lost within 7 days after allo-HSCT. Accordingly, expression of mRNA for osteoblast markers and the bone formation was severely decreased in the GVHD mice. Administration of anti-CD4 antibody in the early time largely prevented BM-GVHD, while preserving GVL/T effects. Furthermore, Allo-HSCT patients with a history of GVHD often fail to reconstitute class switched memory B cells and serum IgGs/ IgA even after full recovery of mature B cells in the periphery blood, thereby refractory to vaccination. We hypothesized that such impairment of humoral immunity could be caused by the effects of GVHD on lymph nodes (LNs), pivotal induction sites for humoral immune responses. tThe number of T and B cells was much lower in GVHD-affected LNs, despite the moderate yet readily detectable recovery of these cells in BM, thymus and spleen. GVHD-affected LNs displayed atrophy, fibrosis, and loss of high endothelial vessels and stromal integrity. Conscequently, GVHD mice failed to exhibit antigen-specific antibody responses after subcutaneous immunization. In conclusion, our study proposes an unprecedented concept of BM- and LN-GVHD, mediated by allogeneic CD4 T cells and CD8 T cells, respectively. We believe that GVHD of these lymphoid organs predisposes the patients to prolonged immune dysfunction after allo-HSCT. Importantly, aberrant reconstitution of immune system is also suggested to be associated with chronic GVHD in which donor-derived newly generated CD4 T cells are pathogenic to cause autoimmune-like disease. We are currently investigating chronic GVHD with particular interest in its association with the effect of GVHD on lymphoid organs. Reference: 1, Shono Y, Ueha S, Wang Y, Abe J, Kurachi M, Matsuno Y, Sugiyama T, Nagasawa T, Imamura M, Matsushima K. Bone marrow graft-versus-host disease: early destruction of hematopoietic niche after MHC-mismatched hematopoietic stem cell transplantation. Blood. 2010. 115(26):5401-11.

MOLECULAR AND CELLULAR BASES FOR CHRONIC INFLAMMATION-ASSOCIATED ORGAN FIBROSIS KOUJI MATSUSHIMA The Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Organ fibrosis is an intractable, progressive condition that arises in multi-factorial chronic inflammatory diseases in which excessive deposition of extracellular matrix (ECM) severely impairs tissue architecture and function, eventually resulting in organ failure. The cellular origin and molecular bases for the accumulation of Col I-producing fibroblasts and myofibroblasts, which are responsible for the excessive deposition of ECM during the fibrotic process remain elusive. After brief introduction on the current understanding of the cellular origin of myofibroblasts, I will present our recent study on the dynamics of fibroblasts in an experimental fibrosis. Qualitative change, rather than quantitative change, is a hallmark of activated fibroblasts in bleomycin-induced lung fibrosis: Collagen I-producing fibroblasts were isolated from transgenic mice harboring enhancer/promoter sequences of α2(I) collagen gene linked to EGFP. Freshly isolated fibroblasts from bleomycin-treated mice had activated phenotype with the increase of collagen I expression and intracellular organelle complexity. These fibroblasts up-regulated expression of α-smooth muscle actin at day 7 and 14 but not 21 after bleomycin treatment. The number of fibroblasts in the whole lung did not increase even at the peak of fibrosis. Both proliferation and apoptosis of fibroblasts slightly increased when the inflammation progressed. We also analyzed gene expression profiles of normal and activated fibroblasts by a second generation DNA sequencer, identifying important signatures of activated fibroblasts including novel activation markers. These findings suggest that qualitative change, rather than quantitative change, is a hallmark of activated fibroblasts in bleomycin-induced lung fibrosis.

LEO OTTERBEIN IMMUNE MODULATION BY CARBON MONOXIDE Ischemia-reperfusion injury (IRI) remains a challenging clinical problem, and the predominant injury in organ transplantation. Carbon monoxide (CO), a bioactive product of heme oxygenase catabolism of heme is an accepted protective molecule capable of attenuating the inflammatory process and accelerating tissue recovery. The mechanisms of action have been ascribed to the ability of CO to influence inflammation, cell death and proliferation. In a model of kidney ischemia/reperfusion injury, we describe an intricate relationship between CO and purinergic signaling axes that results in activation of the circadian rhythm protein Period2 (Per2), stabilization of hypoxia-inducible factor 1a (HIF1a), increased mitochondrial biogenesis and upregulation of erythropoietin (EPO) that ultimately attenuates kidney IRI. Absence of CD39, EPO or Per2 completely abrogated the protection afforded by CO. Collectively, these studies identify adenosine-elicited stabilization of Per2 in the control of HIF-dependent metabolism and ischemia tolerance and implicate Per2 stabilization as a potential new strategy for treating kidney ischemia

NUCLEAR TRANSCRIPTION FACTORS AND THEIR FUNCTION IN IMMUNE SYSTEM LIRLÂNDIA P. SOUSA Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Brazil. When innate immune cells detect infectious or sterile injury, their surveillance receptors recognize molecular patterns associated to pathogens (PAMPs) or host-derived (DAMPs) triggering a signaling cascade that leads to activation of transcription factors with consequent gene transcription. This so called inflammatory response is characterized by coordinated activation of various signaling pathways that regulate expression of enzymes, chemokines, cytokines, adhesion molecules and extracellular matrix proteins, leading to recruitment and activation of leukocytes, which will do the task - eliminate the injurious stimulus and also restore tissue homeostasis. There are many transcription factors

such as NF-B, AP1, CREB, c/EBP and IRF, which convey the signal from the stimulated receptors to the nucleus and transcribe it to proteins that will instructs the immune system to produce the efficient response. Particularly, the nuclear factor-κB (NF-κB) family plays important roles in different aspects of immune system including apoptosis, stress response, host defense against pathogens and inflammation. A prominent role of NF-κB in both immune cell development and function is based on the regulation of inducible transcription of several genes whose products are essential components of the immune response such as cytokines, chemokines and adhesion molecules. In this class we will discuss about the broad range of NF-κB activities in the development and functional divergence of lymphocytes as well as in the controlling of infectious disease and resolution of inflammation in addition to the recent involvement in neuronal plasticity.

PRO- AND ANTITHROMBOTIC RESPONSE OF PLATELETS DURING INFLAMMATION BURYACHKOVSKAYA L., UCHITEL I.,SUMAROKOV A Russian Cardiology Research complex, Moscow, Russian Federation 121552, 3

rd Cherepkovskaia str., 15A,

Introduction: Platelets play a crucial role in thrombosis and inflammation. Different platelets properties are determinate during megakaryopoiesis. Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways and leads to different platelets birth. The aim of our study was to analyze platelets heterogeneity during inflammation and to describe pro- and antithrombotic activity of different subpopulation. Methods: Platelets spontaneous and induced by 0.1, 1.0 and 5.0μM ADP aggregation (PA) of 18 healthy subjects (HS), 26 meningococcemia (MCC) with DIC patients (pts) and 36 CHD pts 1 day and two weeks after percutaneous coronary intervention (PCI) was studied by simultaneous analysis of the mean aggregate size changes and light transmission using an aggregation analyzer BIOLA (Russia). Platelet morphology, number of different platelet forms and leukocyte-platelet aggregates (LPA) were estimated by scanning electron microscopy. The levels of IL-1, IL-2, IL-6, IL-11, TNF-α were measured in serum by immune-enzyme analysis. Results: In all pts with inflammation there was 66.9±14.7% of LPA and 5.4±1.9% of large reticulated platelets (LRP), which are normally absent in HS. The number of LRP was higher than 3% and that correlated with the level of spontaneous PA (2.9±7.7ru vs 1.2±0.1ru in HS, r=0.376) and only in 32% pts increased 5.0μM ADP PA was revealed. In all pts the level of IL-1, IL-2, IL-6, IL-11, TNF-α, was significantly increased, which are responsible for megakaryocyte polyploidization and their maturation. At the same time only in pts with MCC the number of proplatelets with impaired aggregation activity raised. The IL-1, IL-2, IL-6 addition to HS platelets in vitro didn’t lead to their transformation to proplatelets or LRP. Dual antiplatelet therapy in 2 weeks decreased the number LRP and LPA but had no influence on proplatelets. Conclusion: Inflammatory cytokines promote the polyploidization of megakaryocytes and their ability to produce large platelets with high prothrombotic activity. Simultaneously, the high intravascular platelets consumption leads to increased production of non-active proplatelets as defense mechanism. Financial support: We acknowledge for financial support Rfbr: grant 11-04-01332.

LUKE O'NEILL NEW FRONTIERS IN INFLAMMATION RESEARCH O’Neill, Luke A.J. School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland. Research into the inflammatory process has a long and distinguished history, with observations and discoveries spanning from ancient times to today’s world of data accumulation, analysis of complex systems and a bewildering array of pro-inflammatory and pro-resolving processes. A key goal has always been the identification of cell types and inflammatory mediators involved, and then the attempt to implicate these cells and/or mediators in the pathogenesis of inflammatory diseases such that new therapies might emerge. There has been remarkable success in the targeting of pro-inflammatory cytokines such as TNF, IL-1 and IL-6, with more recent successes in blocking cytokines such as IL-17. Other avenues have proved less fruitful clinically, notably in attempts to limit chemokines or signalling pathways activated in inflammatory cells. We still however have a long way to go to truly understand the complexities of the inflammatory process. Newer advances include the uncovering of multiple pathways involved in innate immunity. The best characterised involve the Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Genetic variation in several of these components has been linked to inflammatory diseases, notably in the TLR system and in the NLR protein Nlrp3 and associated proteins. From work on Nlrp3 there has also been a resurgence of interest in the IL1 system as a key driver of inflammation in diseases such as gout and diabetes (both Type I and Type II). Particles such as uric acid, beta amyloid, hydroxyapatite and IAPP, all known to activate macrophages, are now known to be sensed by Nlrp3, leading to caspase-1 activation and the processing of pro-IL-1beta, although the exact mechanism has yet to be uncovered. Nlrp3 is therefore an important drug target, and we have found that an agent called Nlrp3 is a potent and selective Nlrp3 inhibitor, active both in vitro and in vivo. Other areas to emerge recently include the description of metabolic changes triggered by innate immune receptors. Inflammation involves a shift towards so-called ‘Warburg metabolism’ and new insights to emerge include elevations in metabolites that act as signals for inflammation, including the Kreb’s cycle intermediate succinate which activates the transcription factor HIF1alpha as well as being able to activate macrophages via the succinate receptor SUCRN1. Other metabolites such as NAD impact on the epigenome by regulating histone deacetylases, and an interesting link between metabolism and epigenetics in inflammation is emerging. Also of interest is a greater understanding of circadian regulation of inflammation, via clock proteins such as Bmal1. The inflammatory process has long been known to have a circadian component and these newer molecular insights may help in our understanding of the control of inflammation and it’s dysregulation in diseases. Inflammation therefore remains an intellectually challenging aspect of biomedicine, with great potential for the development of new therapies to ease the suffering caused by the many inflammatory diseases.

EICOSANOIDS SHAPE THE MACROPHAGE INNATE IMMUNE RESPONSE TO PAMPS MARC PETERS-GOLDEN, M.D., DIVISION OF PULMONARY AND CRITICAL CARE MEDICINE, UNIVERSITY OF MICHIGAN MEDICAL SCHOOL, ANN ARBOR, MICHIGAN, USA Eicosanoids are bioactive metabolites of arachidonic acid, the best known of which are prostaglandins and leukotrienes. Two specific eicosanoids, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), are synthesized in abundance by macrophages upon encountering microbes. Our laboratory has shown previously that, acting via the

- -coupled receptors BLT1 and EP2, respectively, LTB4 enhances while PGE2 suppresses classic phagocytic functions of macrophages such as microbial ingestion and killing. Here we have extended this work to examine effects of these two eicosanoids on macrophage expression of pattern recognition receptors (PRRs) as well as the signaling and cytokine responses generated upon PRR engagement by pathogen-associated molecular patterns (PAMPs). First, we demonstrate that LTB4-BLT1 enhances the expression of the important fungal PRR dectin-1. This occurs via increased expression of the transcription factor PU.1, and results in increased macrophage binding of yeast as well as increased cytokine responses to dectin-1 engagement in vitro and in vivo. Second, we show that LTB4-engagement of PRRs that depend on the adaptor protein MyD88 for signaling. This was explained by the fact that LTB4 promotes expression of MyD88 via activation of its transcription factor STAT-1, which in turn is the consequence of its ability to reduce expression of the STAT-1 inhibitor, suppressor of cytokine signaling-1 (SOCS1). Third, we show that PGE2-EP2 reduces expression of Toll-like receptor (TLR) 4 on macrophages, while increasing expression of SOCS1. Finally, we show that PGE2-EP2 shapes the nascent response to TLR4 engagement by dampening LPS-induced generation of the pro-inflammatory cytokine TNF- the anti-inflammatory cytokine IL-10. Together, these data indicate that LTB4 and PGE2 exert powerful and opposing actions which influence both the amplitude and the nature of protective host responses to pathogens. Since many states of immunosuppression are characterized by underproduction of LTB4 or overproduction of PGE2, administration of LTB4 or blockade of PGE2 synthesis or signaling present therapeutic opportunities to enhance antimicrobial defenses.

MARCELO A. MORI, Ph.D METABOLIC HOMEOSTASIS: SIGNALS FROM THE ADIPOSE TISSUE Federal University of São Paulo, São Paulo, Brazil Aging and obesity are risk factors for multiple diseases, including type 2 diabetes, cancer and neurodegeneration. However, this risk strongly depends on the genetic background of the individual. My laboratory uses animal models to elucidate genetic pathways that modulate the risk for the development of aging/obesity-related diseases. Our recent data points to a primary role of the immune system and the miRNA processing pathways in adipose tissue to determine the risk of type 2 diabetes in response to aging and obesity.

MARCELO NICOLÁS MUSCARÁ, PHD “H2S, SYNOVITIS AND BONE LOSS” Dept. of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, SP, Brazil. Along the last decade it has been largely demonstrated that the noxious gas hydrogen sulfide (H2S) is endogenously produced and that physiological (nM – microM) concentrations of H2S are able to modulate pain and inflammation through different mechanisms. We thus decided to evaluate the ability of endogenous and exogenous H2S to modulate carrageenan (CGN)-induced synovitis in the rat knee. We observed that pre-treatment with either the H2S donor – Lawesson’s reagent (LR) or the non-steroidal antiinflammatory drug (NSAID) indomethacin significantly attenuated the CGN-induced pain responses evaluated as gait score or tactile allodynia, as well as the inflammatory response characterized as joint swelling, inflammatory cell infiltration, increased synovial myeloperoxidase activity, protein-tyrosine nitration, inducible NOS (iNOS) activity and NO production. On the other hand, inhibition of endogenous H2S with propargylglycine potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. These results suggest that exogenous H2S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, while locally produced H2S exerts little immunomodulatory effect, and support the development and use of H2S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms. In fact, NSAIDs are the most commonly prescribed agents for arthritic patients, although gastric effects limit their long-term use. Considering the reported gastric safeness of H2S-releasing NSAIDs, in addition to the anti-inflammatory effects of H2S administration to rats with synovitis, we decided to evaluate the effects of the H2S-releasing naproxen derivative ATB-346 in this animal model. We observed that both naproxen and ATB-346 reduced edema, pain score, tactile allodynia and leukocyte infiltration despite the significantly lower naproxen bioavailability from orally administered ATB346, thus suggesting that H2S may compensate for this deficiency In addition, we have also observed that the presence of an H2S-releasing moiety in the ATB-346 structure confers additional anti-inflammatory activities to those of the parent naproxen compound in the alveolar bone loss process secondary to ligature-induced periodontitis in rats. In fact, ATB-346 treated rats had significantly diminished bone loss, lower defect bone volume and area which are secondary to the presence of periodontal disease, in addition to the lack of gastric damage. As a whole, these findings support the potentially therapeutic usefulness of H2S donors, either alone or as adjuvants, for the safe treatment of different inflammatory conditions, although deeper studies on the underlying mechanisms and side effects still remain to be performed.

MARINA A. FREUDENBERG GM-CSF TREATMENT OF FETAL LIVER CELLS GENERATES IMMORTAL, NON-TRANSFORMED ALVEOLAR MACROPHAGE-LIKE CELLS AND ALLOWS NEW INSIDES TO MACROPHAGE PROPERTIES. FREUDENBERG MA

1,2, WEGNER M

1, ENGELHARD P

1, GALANOS C

3 AND FEJER G

4

1University Medical Center Freiburg,

2BIOSS, Freiburg University,

3Staatliches Weinbauinstitut Freiburg, Germany and

4School of Biomedical and Biological Sciences, University of Plymouth, UK

Macrophages are diverse cell types in the first line of anti-microbial defense and according to subset they can promote or dampen inflammation. Only a limited number of primary models exist to study macrophage functions. Mouse bone marrow-derived, M-CSF-induced cells (BMM) with a limited lifespan are the most common in vitro model. We developed a simple method yielding self-renewing, non-transformed, GM-CSF/STAT5-dependent macrophages (MPI cells) from mouse fetal liver. Unlike other types of primary macrophages, MPI cells from various wild type, gene-deficient or transgenic mice strains can be propagated indefinitely in unlimited quantities and can be easily manipulated genetically. The cells reflect the innate immune characteristics of alveolar macrophages (AM) and in many ways they differ from BMM. Thus, MPI cells are much more sensitive to selected microbial agents, including LPS, lipopeptide, Mycobacterium tuberculosis, cord factor and adenovirus, and show a unique pattern of innate responses. Unlike BMM they mount very high pro-inflammatory but no anti-inflammatory IL-10 responses and exhibit differential LPS sensing. Moreover, they show an as yet unknown regulation of IL-1α production upon LPS exposure, likely to play a key role in lung inflammation in vivo.

THE INFLAMMATORY HOST RESPONSE AS AN AMPLIFICATION LOOP OF SHIGA TOXIN TOXICITY MARINA PALERMO Shiga-toxin (Stx) producing Escherichia coli (STEC) induce a broad spectrum of pathologies like watery diarrhea, bloody diarrhea or Hemolytic Uremic Syndrome (HUS), the most severe systemic and life-threatening complication. We demonstrated that inflammation is a central component of the pathogenic mechanism, and in this way that the innate and inflammatory responses define the evolution of STEC infections to HUS. The mouse model by intravenous injection of Stx alone or together with LPS reproduce not all, but several hallmarks of human HUS. Particularly tubular renal damage and the activation of inflammatory response. Thus, from the mouse model we had the first evidence that both major cellular components of inflammatory response, which are PMN and monocytes are involved in the pathogenic mechanism of Shiga toxin. As soluble mediators, eNOS physiologically protects against endothelial injury and toxicity induced by STx2. In addition, we recently demonstrated that Stx2 treatment induces in mice a strong oxidative stress and that the simultaneous treatment with synthetic antioxidants significantly reduced mortality rates induced by Stx2. Both of them reduce the oxidative damage in cells by direct neutralization of free radicals and/or through the enhancement of biosynthesis of intracellular GSH. However, inflammation and thrombosis are systemic processes, so we studied if the specific damage in the capillary beds of kidney involves chemokines and their receptors. Particularly, we specifically addressed the contribution of CCR1 in the murine model of HUS because CCR1 participates in the pathogenesis of several renal diseases by orchestrating myeloid cell kidney infiltration. We showed that Stx-treated CCR1 KO mice have an increased survival rate associated with less renal dysfunction, seen as lower levels of urea and creatinine; as well as less histological damage compared with controls. These data demonstrate that CCR1 participates in cell recruitment to the kidney, and in the amplification of the inflammatory response that contributes to HUS development. Blockade of CCR1 could be important to the design of future therapies to restrain the inflammatory response involved in the development of HUS.

MARK HOGARTH ANTIBODY DEPENDANT EFFECTOR SYSTEMS IN HUMANS AND PRIMATES. IMPLICATIONS FOR THE DEVELOPMENT OF THERAPEUTIC MONOCLONAL ANTIBODIES. Halina M. Trist*, Peck Szee Tan*, Bruce D. Wines*, Paul A. Ramsland*, E Gardiner†, Geoffrey A. Pietersz*, Janine Stubbs*, Stephen J. Kent§, Ivan Stratov§, Dennis R. Burton|| and P. Mark Hogarth*. Correspondance to pmhogarth@burnet.edu.au *Centre for Biomedical Research, Burnet Institute Melbourne. §Department of Microbiology and Immunology, University of Melbourne †Australian Centre for Blood diseases Monash University ||Department for Immunology and Microbial Science and IAVI Neutralizing Antibody Centre and Center for HIV/AIDS Inflammation induced by the antibodies and their Fc receptor-based effector functions is a major factor in the effectiveness of vaccines, therapeutic antibodies and pathology associated with may autoimmune diseases. The use of nonhuman primates (NHP) is often a critical step in the preclinical development of vaccines and therapeutic antibodies targeting inflammation cancer, or infection. Also NHP are useful models of human inflammatory and infectious diseases. We have undertaken a analysis of human and NHP Fc receptor function, structure and genetics. We found that there are marked differences between NHP Fc receptor functions and the human equivalent in their capacity to interact with human IgG. The NHP Fc receptors show distinct differences in the hierarchy of IgG binding compared to human activating and inhibitory receptors. Moreover we show that polymorphisms of the receptors can profoundly affect function. Mutagenesis studies combined with IgG binding analysis as well as molecular modelling identify the basis of these functional differences. Our studies suggest that antibody dependant responses that might be anticipated in humans may not be faithfully recapitulated in NHP. This has implications for the interpretation of preclinical studies of vaccines and biological drugs particularly monoclonal antibodies and may also have implications for the use of for NHP as a model for understanding human immunity in infection or inflammation.

MAURO MARTINS TEIXEIRA ENDOGENOUS CONTROLLERS OF THE RESOLUTION OF INFLAMMATION Teixeira MM and Pinho V Imunopharmacology, ICB, Universidade Federal de Minas Gerais The resolution of inflammation is a tightly controlled active process essential to maintain the balance of cellular homeostasis after stimuli that cause tissue dysfunction or damage. This process of resolution involves key events including leukocyte apoptosis, recognition and phagocytosis of dying cells (efferocytosis) and leads to the return to the pre-inflammatory states. We have demonstrated that the treatment with SOD (that increases production of endogenous H2O2) or exogenous H2O2 accelerated resolution of inflammation resulting in increased number of apoptotic leukocytes in models of arthritis and asthma. Moreover, resolution was delayed in gp91

phox-/- mice that lack

the ability to assemble the NADPH oxidase and are, hence, incapable of generating ROS. Interestingly, ROS production was diminished in gp91

phox-/- mice. Moreover, the inhibition of ROS production by apocynin prolonged

inflammatory response. Thus, our data demonstrate a critical role for ROS as an endogenous pro-resolutive mediator of inflammation. Financial support: FAPEMIG, CNPq,

STRATEGIES TO EXPLOIT PRO-RESOLVING GPCRS FOR DRUG DISCOVERY. MAURO PERRETTI William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom. Recent appreciation of the existence of endogenous pro-resolving pathways that control the outcome of experimental inflammation brings a fresh approach on devising new strategies for the treatment of inflammatory pathologies. Furthermore, the continuous definition of inflammatory molecular and cellular mechanisms operative in pathologies previously un-appreciated for their inflammatory etiology amplifies these opportunities. Resolution of acute inflammatory episodes is assured by the engagement of specific mediators that act on given targets to inhibit cell trafficking, dispose safely of migrated cells and orchestrate tissue repair with return to homeostasis (see Ortega-Gómez 2013; PMID: 23592557). Thus, the receptor targets of the effectors of resolution are truly endowed with a novel opportunity for drug discovery. In this presentation I will focus on the receptor for Annexin A1 and Lipoxin A4, termed FPR2/ALX (or Fpr2/3 in the mouse) both in terms of definition of its molecular functioning (homo- and hetero-dimerization), its central role in controlling host response in complex experimental settings such as sepsis or myocardial infarct, with a novel twist towards tissue protection. Finally, new tools acting at FPR2/ALX will be discussed, including highly selective and potent peptides (Dalli 2013; PMID: 23686496) together with novel small molecules. It is an exciting era for those who have pioneered the concept of the resolution of inflammation, defining pathways and processes, since the next decade will address the real translation potential for this innovative area of research, establishing whether the philosophy behind this approach to inflammatory mechanisms and targeting is a valid one. Funds Acknowledged: Wellcome Trust, British Heart Foundation, Medical Research Council UK, William Harvey Research Foundation, Nuffield Foundation.

ROLE OF FOXP3

+ REGULATORY T CELLS RECRUITED AFTER TREATMENT WITH BCG KILLED BY

EXTENDED FREEZE DRYING (EFD BCG) IN THE CONTROL OF INFLAMMATORY DISEASES. MICHELINE LAGRANDERIE

1, GILLES MARCHAL

1, OLGA OVCHINIKOVA

2 AND B.BORIS VARGAFTIG

3

Foxp3

+ expressing regulatory T cells (Tregs) play a crucial role in maintaining immune tolerance and homeostasis. A

number of molecules have been reported to contribute to Treg suppressive function, however in highly inflammatory environments (microbial infections) activated Tregs could suppress effector T cells and thus exacerbate the infectious disease. Thus, we have developed a novel product EFD BCG that induced the recruitment of Tregs and reduced inflammation without increasing microbial or viral infections. In the various mouse models tested (chronic asthma, colitis, atherosclerosis) EFD BCG treatment increased the number of IL-10-producing Foxp3

+ Tregs and reduced inflammation. The mechanisms by which EFD BCG controls

the various diseases studied, involved reduction of inflammatory cytokines (IL-5, IL-6, IL-13, IL-17, TNF- -transcription factors (GATA-activated PPAR- - ssion in the sites of inflammation: lungs (asthma), colon (colitis) and aortas (atherosclerosis). Moreover, EFD BCG treatment had no measurable side effects and sustained efficient immuno-competence of the host, as attested by the unaltered immune defence against Mycobacterium tuberculosis (Th1 mediated protection), Neisseria meningitidis (Th2 mediated protection) and influenza virus in mice. All the properties of EFD BCG suggest that it could be a tolerable and efficient new therapeutic treatment strategy for inflammatory diseases and enter in the clinical phase rather rapidly. 1 Immunotherapix, Institut Pasteur, Paris France

2 Pharmacologie Cellulaire, Institut Pasteur, Paris France

3 Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden.

TISSUE DAMAGE CONTROL: UNCOUPLING RESISTANCE TO INFECTION FROM DISEASE SEVERITY. MIGUEL P. SOARES, PHD Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal. E.mail: mpsoares@igc.gulbenkian.pt Web page: http://www.igc.gulbenkian.pt/research/unit/43 The immune system allows for immediate recognition, containment and cytotoxic targeting of pathogens. While central in providing host resistance to infection this defense strategy is not sufficient per se to confer protection from disease. The pathogenesis of infectious diseases is driven, to a large extent, by the degree of tissue dysfunction and/or damage imposed directly by pathogens or indirectly by the host immune response conferring resistance to infection. Depending on the extent of tissue damage imposed, organ dysfunction might develop, underlying the pathologic outcome of infectious diseases. Therefore, host protection from infectious diseases must incorporate an additional defense strategy that limits tissue damage. This defense strategy, referred as disease tolerance, does not exert a negative impact on pathogens. Instead, it acts via a series of evolutionary conserved mechanisms that confer tissue damage control, that is, limit tissue damage and organ dysfunction associated with infection. As illustrated in this talk, tissue damage control relies on the expression of stress-responsive genes regulating metabolic adaptation, cytoprotection and/or tissue regeneration in response to specific forms of stress associated with infection. Presumably, tissue damage control acts as an inherent component of inflammation and immunity that uncouples resistance to infection from disease severity.

“CURRENT AND NOVEL THERAPIES FOR OCULAR DISEASE” MILES STANFORD Ocular inflammation in its various guises may account for 10% of blindness in developed countries and commonly affects young patients with socio-economic consequences. The pathophysiology of the conditions that cause these diseases is ill understood and, because they are chronic, treatment has to be carried out over many years with an increasing risk of side effects. However, most will burn out with time. The goal of therapy is therefore to protect vision until such time as quiescence occurs. Whilst the majority of cases can be controlled with topical treatment alone, severe, sight threatening disease requires either control with peri- or intraocular therapy (which may need to be frequently repeated) or with systemic immunosuppression. Classically, disease is controlled with systemic corticosteroids with their known complications or other secondary immunosuppressive agents. There is little in the way of RCTs to guide the clinician and most new trials of agents compare the effects to standard of care treatment rather than placebo. Recent advances in ocular imaging have enabled the fine structure of the retina and, in particular, the macula to be evaluated better allowing earlier intervention than was previously possible. Over the last few years there has been an increasing reliance on biological therapies (anti TNF, interferon, etc) but, whilst undoubtedly effective, their role long term is uncertain and they are expensive making their use in resource poor settings untenable: new agents are therefore required. One major trend has been in the development of long acting local therapies (eg intravitreal triamcinolone, osurdex, retisert, iluvien, etc) and these hold great hope as they do not cause unwanted systemic effects although the local management of complications can be challenging. Future therapies using peptide tolerance, iontophoresis of agents (avoiding ocular puncture) and tailored biological therapy are being considered or are in early trial

MOMTCHILO RUSSO REGULATORY T CELLS: WHERE DO THEY WORK? Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, Brazil We have previously shown that regulatory T (Treg) cells that accumulate in the airways of allergic mice upregulate CC-chemokine receptor 4 (CCR4) expression. These Treg cells suppressed in vitro Th2 cell proliferation but not type 2 cytokine production. In my talk I will show, using a well-established murine model of allergic lung disease or oral tolerance, the in vivo activity of Treg cells in allergic airway inflammation with special focus on CCR4 function. I will outline that allergic, but not tolerant, mice treated with anti-CD25 Ab show an increased airway eosinophilia and IL-5– or IL-4–producing Th2 cells when compared with untreated mice. Notably, mice with CCR4 deficiency display an augmented airway allergic inflammation compared with wild-type or CCR2 knockout (KO) mice. The allergic phenotype of CCR4KO mice was similar to that observed in anti-CD25–treated mice. The exacerbated allergic inflammation of CCR4KO mice was directly associated with an impaired migration of Treg cells to airways and augmented frequency of pulmonary Th2 cells. Adoptive transfer of CD25+CD4+ T cells expressing high levels of CCR4, but not CCR4KO CD25+CD4+ T cells, attenuated the severe airway Th2 response of CCR4KO mice. My talk outline that CCR4 is critically involved in the migration of Treg cells to allergic lungs and that, in the lung, Treg cells attenuate airway Th2 activation and allergic eosinophilic inflammation.

WHAT ROLE FOR H2S IN PAIN TRANSMISSION? DR. NATHALIE VERGNOLLE Like nitric oxide, H2S is a biological gas that can be synthesized and released by tissues. H2S release is modulated by inflammation and a number of studies have investigated its role in the cardinal signs of inflammation. Of particular interest is the role of H2S in the transmission of the nociceptive messages and in inflammatory pain. Conflicting results report that exogenous administration of H2S induces pain or reduces nociceptive responses to stimuli. These contradictory results seem to depend on the dose of H2S that is delivered, but also on the type of tissues and the models that have been investigated. A number of studies have investigated the effects of H2S on primary afferents and latest results implicate an effect of H2S on ion channels. In particular, some of the anti-nociceptive effects of H2S can be explained by the inhibition of calcium channels of the transcient-receptor potential family. This lecture will review all those results and will discuss the relevance of using H2S-releasing drugs for the treatment of pain and visceral hypersensitivity.

PROTEASE-ACTIVATED RECEPTORS AND TRANSIENT RECEPTOR POTENTIAL CHANNELS: SUBSTRATES FOR SIGNALING INFLAMMATION AND PAIN NIGEL W. BUNNETT, MONASH INSTITUTE OF PHARMACOLOGICAL SCIENCES, 318 ROYAL PARADE, PARKVILLE, VICTORIA 3052, AUSTRALIA Injury and inflammation induce the activation of proteases from the circulation and immune, neuronal and epithelial cells that can regulate cells by cleaving protease-activated receptors (PARs). Activation of PARs, notably PAR2, on nociceptive neurons stimulates the release of neuropeptides that mediate neurogenic inflammation and pain. PAR2 sensitizes or activates transient receptor potential (TRP) ion channels, notably TRPV1, TRPV4 and TRPA1, which amplify protease-induced inflammation and pain. In order to localize and identify proteases that are activated during inflammation, we have used activity-based probes comprised of a warhead protease inhibitor group, which covalently binds activated proteases, and a near-infrared tag for detection. Probes for cysteine cathepsins and serine proteases were administered to mice with inflammatory diseases, and activated proteases were detected using non-invasive fluorescence molecular tomography imaging and cellular confocal imaging. We identified activated proteases in macrophages and microglial cells during inflammation and pain states, including cathepsins and trypsins. Studies of mice lacking PARs and TRP channels indicate that these proteases cause neurogenic inflammation and pain that requires expression of PAR1, PAR2 and TRPV1, TRPV4 and TRPA1. We examined the signalling mechanisms by which proteases control activity of PARs and TRPs using model cell lines (HEK) and primary nociceptive neurons. Some proteases (e.g. trypsin, tryptase) cleave PAR2 at the canonical activation site to induce Gq-mediated calcium

- -arrestin-mediated receptor endocytosis and MAP kinase signalling. Other proteases (e.g. elastase, cathepsin S) cleave PAR2 at distinct sites and act as biased agonists that induce distinct mechanisms of receptor signalling and trafficking. Activated PAR2 promotes influx of Ca

2+ ions in HEK

cells and neurons that requires expression of TRPV4, suggesting that PAR2 couples to this channel. This mechanism depends in part on PAR2-induced generation of TRPV4 agonists such as 5’6’-EET, and also requires TRPV4 phosphorylation (Y110) by unidentified tyrosine kinases. Thus, inflammation triggers the activation of diverse proteases that regulate the activity of nociceptors by via PARs and TRP channels. Unravelling the mechanisms by which PARs couple to TRP channels provides new insights into protease regulation of inflammation and pain, with therapeutic implications.

THERAPUTIC IMPLICATIONS OF VASCULAR INFLAMMATION NIKITA LOMAKIN Acute coronary syndrome is a multifactorial disease. Contemporary data prove that inflammation plays a key role in the process of plaque destabilization and development of life-threatening conditions. Despite substantial progress of recent decades in development of modern approaches to ACS management, there is a share of patients resistant to standard ACS treatment, which form a group of patients with the worst cardio-vascular outcomes. Individualization of patient management is one of the most promising research trends with focus on identification of inflammation biomarkers, which are linked with plaque destabilization and clot formation. Such biomarker should be used in routine practice to predict cardiovascular outcomes and provide a ground for practical decisions in individual cases of anti-inflammatory therapy. In the presentation we review currently available inflammation markers. Additionally, we will compare two existing anti-inflammatory approaches in ASC therapy - indirect or pleyotropic and specific.

ANNEXIN A1 MIMETIC CONTROLS THE INFLAMMATORY AND FIBROTIC EFFECTS OF SILICA PARTICLES PATRÍCIA GONÇALVES TRENTIN(1); TATIANA PAULA TEIXEIRA FERREIRA(1); ROD FLOWER(2); MAURO PERRETTI (2); MARCO AURÉLIO MARTINS (1); PATRÍCIA MACHADO RODRIGUES E SILVA(1). (1) Laboratory of Inflammation, FIOCRUZ, RJ/Brazil; (2) Department of Biochemical Pharmacology, William Harvey Institute, London, UK. The time dependency and spatial characteristics of the inflammatory response are regulated by endogenous anti-inflammatory mediators, which prevent the exacerbation of this response. Endogenous glucocorticoids are pro-resolving mediators an example of which is the glucocorticoid-regulated protein Annexin-A1 (AnxA1). Since silicosis is characterized by unabated inflammation, we tested the therapeutic properties of an AnxA1- derived peptide in experimental acute silicosis. The classical steroid, dexamethasone, was used as control. Silicosis was induced in mice by intranasal instillation of silica (10 mg/mouse). Pharmacological treatment with the AnxA1 derivative, peptide Ac2-26 (dose range 50 - 200 µg intranasal), or dexamethasone (25 µg intranasal; DEX) started 6 h post-silica and was carried on for 7 consecutive days, with experimental analyses being conducted at day 7. Lung function (resistance and elastance) and hyperreactivity to methacholine (3 - 27 mg/ml) were obtained from whole-body invasive plethysmography. Morphological alterations in the lung were analyzed by histologically (hematoxylin-eosin and Picrus sirius to assess granuloma and collagen deposition, respectively), whilst cytokine and chemokine levels, as well as total collagen, were quantified in lung extracts. In vitro experiments with lung fibroblasts prepared from mice lacking

the AnxA1 receptors termed Fpr1-/-

and Fpr2-/-

mice were conducted, using IL-13 or TGF as stimulants, testing Ac2-26 effects upon, collagen and MCP-1 release. Silicotic animals showed increased baseline of airway resistance and elastance, as well as hyperreactivity to the bronchoconstrictor methacholine. These responses were abolished by treatment of mice with either Ac2-26 or DEX. At the top dose of 200 µg intranasal daily, peptide Ac2-26 abolished leukocyte infiltrate, collagen deposition and granuloma formation in silicotic mice, readouts that were partially affected

by DEX. Of interest, the generation of cytokines (TNFα and TGF) and chemokines (KC and MCP-1) was inhibited by Ac2-26 but not by DEX. To identify potential targets for the effects of Ac2-26, we used lung fibroblasts which responded to Ac2-26 with reduced production of collagen and MCP-1, but not on cell proliferation, as assessed in

response to IL-13 or TGF. The formyl-peptide receptor Fpr1 was a major determinant for the inhibitory properties of Ac2-26 on lung fibroblasts and mediated inhibition of collagen deposition and MCP-1 release. Collectively, these results unveil novel protective properties of the AnxA1 derivative Ac2-26 peptide on the inflammatory and fibrotic responses promoted by silica, and suggest that AnxA1 mimetics might be a promising strategy for innovative anti-fibrotic approaches, at least in silicosis.

PATRICIA RIEKEN MACEDO ROCCO MD, PHD Professor of Respiration/Federal University of Rio de Janeiro UNDERSTANDING THE MECHANISMS OF ACTION OF STEM CELL THERAPY IN ACUTE LUNG INJURY Acute respiratory distress syndrome (ARDS) is a multifaceted syndrome with a wide-ranging clinical phenotype, making it a challenge to translate experimental results into feasible therapies in the clinical setting. Several studies have addressed the therapeutic benefits of bone marrow-derived cells, such as bone marrow-derived mononuclear cells (BMDMCs) and mesenchymal stromal cells (MSCs), in murine models of ARDS. Bone marrow-derived cells have been shown to mitigate systemic and pulmonary inflammation, as well as decrease lung edema and enhance bacterial clearance, resulting in lower mortality. Nevertheless, there are controversies regarding the mechanisms of action of stem cells in ARDS. We will discuss the beneficial effects of cell therapy in different models of ARDS, focusing on the different mechanisms of action.

LEUKOCYTE RECRUITMENT IN DIFFERENT ORGANS- NO UNIVERSAL PARADIGM PAUL KUBES There is a growing body of evidence that the innate immune system recognizes sterile injury and in a systematic process, responds and helps to repair injury. In a model of sterile injury where a thermal probe is brought near the surface of the liver and kills approximately 200 cells, the sinusoids around the injury site are rapidly constricted and filled with platelets. The platelets are essential for the subsequent neutrophil infiltration. The neutrophils use multiple gradients of chemoattractants to enter the injury site. Monocytes are recruited next, first the CCR2, inflammatory monocytes and subsequently the CX3CR1 positive alternative monocytes appear at the site of injury. Each of these cell types play a key role in the effective repair of the injury site. Interestingly, not all cells are recruited into the injury site; resident iNKT cells are restricted from the site early on but with time they also infiltrate the injury site. This is different from infections in liver where platelets are patrolling the vasculature and adhere rapidly to Kupffer cells to help localize and eradicate bacteria. Platelets also bind neutrophils and induce neutrophil extracellular traps (NETS). NETs are not seen in our sterile injury model.

PEDRO MUANIS PERSECHINI IMMUNOMODULATION BY EXTRACELLULAR NUCLEOTIDES AND NUCLEOSIDES: LESSONS FROM MACROPHAGES. Pedro Muanis Persechini (1, 2) and Julieta Schachter (1, 2) (1) Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, RJ, Brasil. (2) Instituto Nacional de Ciência e Tecnologia de Pesquisa Translacional em Saúde e ambiente da Região Amazônica – INPeTAm Macrophages are multi-functional and highly plastic cells that display important roles in the immune-inflammatory system and in homeostasis of most of mammalian tissues. They mediate their activity by the production and secretion of pro- and anti-inflammatory mediators and the expression of a diversified panel of receptors and transport systems in their plasma membrane. These membrane components may change according to their specific differentiation state and microenvironment milieu. Macrophages have many subtypes and names. Modulation of its activity is a challenge to our ability to modulate/control inflammatory diseases, cancer, vaccination, virus and parasite infection, autoimmune and degenerative diseases, and other situations of immune-modulatory interest. Cytokine secretion, lipid metabolism, phagocytosis, antigen presentation, hosting of intracellular virus and parasites, and production reactive oxygen and nitrogen species are amongst the most studied issues. Receptors, ion channels and transport systems related to extracellular purines and pyrimidines has gained increased attention in recent years. Extracellular ATP, other nucleotides, and adenosine can bind to one or more of the nucleotide (P2) and adenosine (P1) receptors and induce the secretion of cytokines, the production of reactive oxygen species, the synthesis and secretion of bioactive lipid derivatives, and modulate the intracellular levels of Ca

2+ and

cAMP. These receptors are part of a complex extracellular network that couples to the intracellular milieu and includes ecto-enzymes such as CD39 and CD73, adenosine uptake, nucleotide secretion, and the control of the intracellular concentration cAMP. This extracellular network extends to other cells and multiple intercellular interactions. Treg lymphocytes, Dendritic Cells, vascular endothelial cells, red blood cells, platelets, B cells, and other leukocytes, all participate. Some virus, bacteria and protozoa developed special strategy to cope with this network by expressing and/or modulating the expression of some of its components in a way that we are only beginning to understand. Conversely, the immune system can use this network to control infection. Decoding the immune-modulatory role of this vast and pleiotropic network is major task for immunologists and cell biologists. We have been studying ion channels and other transport mechanism of macrophages. Particularly, the P2X7 receptor is an ATP-gated cation-selective channel (Na

+, K

+, and Ca

2+) that is connected with an intracellular array of signaling

pathways that goes beyond the efflux of K+, the increase in free intracellular Ca

2+ concentration and the well-known

phenomena of induction of cell death, activation of inflammasome, and secretion of IL1-β. Its discovery and initial characterization was related with the phenomenon known as ATP-induced “permeabilization” of the plasma membrane, characterized by the fast (1-20 min) uptake of fluorescent dyes such as the cation ethidium and the anion Lucifer yellow. This transport mechanism frequently described as a large non-selective pore was recently associated with pannexin channels. We have shown that this is an oversimplified view. The ATP-induced, P2X7-associated “permeabilization” phenomenon is the result of a complex transport mechanism that is coupled to the P2X7 receptor through a poorly understood signaling pathway and involves at least two distinct transport mechanism, one for cations and another one for anions. We will review the evidence leading to these conclusions and discuss its implications for the immunomodulation mediated by extracellular nucleotides. FINANCIAL SUPORT: CNPq, FAPERJ, CAPES

CHEMOKINES: TAILS FROM THE CLINIC PHILLIP MURPHY Knowledge about the chemokine system has advanced from molecules to mice to man, with CCR5 and CXCR4 antagonists maraviroc and plerixafor now on the market for HIV/AIDS and hematopoietic stem cell mobilization, respectively. CXCR4 blockade is also a rational therapeutic strategy in WHIM syndrome, a rare congenital immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (neutropenia due to impaired egress from the bone marrow) that is caused by gain-of-function mutations in CXCR4. We have developed a Phase I clinical trial to repurpose plerixafor in WHIM syndrome. In addition to defining parameters of safety and efficacy in this disease, the trial has identified new patient groups and a potential role for CXCR4 in G6PC3 deficiency, a newly discovered inherited disorder of glucose homeostasis that like WHIM syndrome is characterized by severe congenital neutropenia and myelokathexis. This trial is providing basic new insights about the role of CXCR4 in leukocyte trafficking in man, as well as information about the utility of plerixafor in WHIM syndrome and the feasibility of chronic blockade of this essential receptor in any disease where it may play a role in pathogenesis.

PEROXYNITRITE, A POTENT OXIDIZING AND NITRATING CYTOTOXIN PRODUCED BY INFLAMMATORY CELLS RAFAEL RADI Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay Peroxynitrite, the diffusion-controlled reaction product of nitric oxide ( NO) and superoxide radicals (O2

-), participates

in oxidation and nitration reactions in vitro and in vivo. In particular, inflammatory cells such as macrophages and neutrophils can lead to substantial amounts of peroxynitrite which can exert cytotoxic effects on invading pathogens and promote tissue oxidative damage. The right metabolic condition for optimal peroxynitrite formation rates requires the prior immunostimulation of cells for the induction of nitric oxide synthase (iNOS) followed by activation of membrane- associated NADPH oxidase (NOX-2) by processes such as phagocytosis that trigger the “oxidative burst” and O2

- formation. For immunostimulated macrophages, phagocytosis promotes the intraphagosomal release of

peroxynitrite for a period of 60-90 minutes, which is sufficient to cause substantial oxidation and nitration of microbial biomolecular targets (e.g. protein tyrosine nitration) and eventually cell death. On the other hand, peroxynitrite formation by inflammatory cells can modify host tissue components and participate in immunomodulatory and/or tissue damaging processes. The oxidizing capacity of peroxynitrite can be modulated by potent endogenous antioxidant mechanisms, most notably the enzymes of peroxiredoxin family that readily decompose peroxynitrite via fast reaction with active site thiols or by exogenous compounds that can be supplied as peroxynitrite-decomposition catalysts (e.g. MnPorphyrins) to provide pharmacological protection. In the presentation, we will analyze the biological chemistry of peroxynitrite in terms of the oxidative pathways that promote its cytotoxic effects, provide an overview of its role in inflammatory processes and how its steady-state concentrations and actions can be modulated in vitro and in vivo. The appreciation of the biological chemistry of peroxynitrite helps to reveal how redox-based mechanisms participate in inflammatory disease conditions.

RITHWIK RAMACHANDRAN NEUTROPHIL PROTEINASES AND AGONIST BIASED SIGNALING THROUGH PARS Proteinase Activated Receptors (PARs) are G-protein coupled receptors activated by proteolytic processing of the receptor exodomain. We have identified that PAR signaling triggered by neutrophil proteinases is distinct from that triggered by classical PAR activating enzymes such as thrombin or trypsin. We will present data that describes the molecular basis for the biased signaling profile and distinct receptor dynamics that are triggered by neutrophil proteinases. We will also describe a role for these biased signaling events in regulating endothelial barrier function.

HERPES STROMAL KERATITIS CAN RESOLVE IN THE BLINK OF AN EYE Robert L. Hendricks*, Hongmin Yun*, and Alexander M. Rowe* Department of *Ophthalmology, Immunology, and Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213 Mice provide a popular model of herpes simplex type 1 (HSV-1) keratitis (HSK), a chronic immunoinflammatory response to HSV-1 corneal infection. In humans, HSK typically results from reactivation of HSV-1 that is retained in a latent state in sensory neurons of the trigeminal ganglion. Axonal transport and release of reactivated virus induces recurrent bouts of infection and inflammation leading to progressive corneal scarring and visual impairment. Patients with HSK gradually loose corneal sensation in association with a loss of nerve fibers in the cornea. It is unclear if nerve loss is due to viral or inflammatory damage to corneal nerves. While the immunological mechanisms of HSK-associated inflammation and tissue damage have been thoroughly studied in mice, a possible role of nerve damage in regulating these immunological mechanisms has been largely ignored. Noting that HSK in mice is consistently associated with loss of corneal blink reflex, we demonstrated that clinically apparent inflammation promptly resolved when corneal desiccation was prevented by performing tarsorrhaphy (stitching eyelids closed) on mice with severe HSK. Performing tarsorrhaphy on infected eyes before the onset of HSK results in a mild and transient form of HSK with vascularization and mild opacity leading to a cornea that is only slightly clouded by scar tissue. CD4

+ T cell-

deficient mice differ from wild type mice in that their HSK and nerve damage are transient, with recovery of blink reflex slightly preceding resolution of corneal opacity. We conclude that chronic HSK in mice is due to persistent, but reversible CD4

+ T cell-mediated damage to corneal nerves, suggesting that controlling neurotrophic damage might

represent a useful therapeutic adjunct to current immunology and virology based therapy for HSK in humans.

ROGER CHAMMAS GALECTIN-3 INTEGRATES CELLULAR AND TISSUE RESPONSES TO HYPOXIA AND SERUM DEPRIVATION IN SELECTED TUMOR MICROENVIRONMENTS Roger Chammas and the Cell Adhesion and Cancer Group Center for Translational Cancer Research Instituto do Câncer do Estado de São Paulo and Faculdade de Medicina da Universidade de São Paulo Galectin-3 is an animal lectin that is found in diverse cellular compartments, depending on the cellular context and functional state of cells. Normal cells tend to accumulate galectin-3 in the nucleus. Upon malignant transformation, galectin-3 may concentrate in the cytoplasm and can be secreted to the extracellular matrix, where it acts as a matricellular protein, interfering with integrin function among other cell surface glycoproteins. We had shown a focal accumulation of galectin-3 in glioblastomas. Galectin-3 expression was associated to a very specific tumor microenvironment, the pseudopalisades- areas of viable tumor cells surrounding necrotic areas that are commonly found in advanced stages of glioblastomas. Galectin-3 expression in hypoxic and nutrient deprived cells is associated the activation of HIF-1a and it was inhibitable by NF-kB antagonists. In glioblastoma cells, galectin-3 expression was associated with its translocation to mitochondria, suggesting its involvement in mitochondrial homeostasis. Inhibition of galectin-3 expression led to increased cell death, indicating that galectin-3 acts as a prosurvival factor in very specific tumor microenvironments associated with glioblastoma progression. The protumoral role of galectin-3 was also observed in murine melanomas, engrafted in both wild type and galectin-3-deficient mice. Absence of galectin-3 from both tumoral and stromal compartments was associated with attenuated tumor growth, as compared to an experimental condition where both tumor and stromal cells expressed galectin-3. Intriguingly, de novo expression of galectin-3 was frequently observed in tumors derived from cells that did not express galectin-3 when these cells were engrafted in galectin-3 deficient mice. This observation prompted us to investigate whether galectin-3 expressing cells would favor tumor growth when admixed with galectin-3 negative cells. Indeed, when cells are admixed, tumors grew much more efficiently than those engraftments of either cell alone, indicating cooperation between galectin-3 expressing cells and galectin-3 negative cells. Further experiments indicated that galectin-3 expression is related with the induction of a proangiogenic tumor environment. We will further discuss mechanistic details of the proangiogenic roles of galectin-3 within tumors, its interface with innate immunity and the evidence that could support the notion that its targeting in selected tumors may be an effective strategy to control tumor growth. Support: FAPESP (Center for Cell-based Therapy Research), Instituto Nacional de Ciência e Tecnologia- Redoxoma and UICC-Yamagiwa Yoshida Grant

INFLAMMATION, HOMEOSTASIS AND DISEASE RUSLAN MEDZHITOV Inflammation is an adaptive response to infection, tissue injury and other alterations of normal tissue function and homeostasis. The inflammatory response consists of four universal components: inflammatory triggers, sensors, mediators and targets. The targets of the inflammatory response are the tissues responsive to a particular set of inflammatory mediators under a given condition. Depending on the nature of the inflammatory trigger, different combinations and quantities of the inflammatory mediators are produced by sensor cells. The inflammatory pathway is regulated at the level of all four components of the pathway. The principles of inflammatory regulation and their implications for normal physiology and pathology will be the focus of this presentation.

REDOX SIGNALING IN INFLAMMATORY PROCESS OF CHRONIC DEGENERATIVE PULMONARY DISEASES SAMUEL SANTOS VALENCA

1, MANUELLA LANZETTI

2, TATIANA VICTONI

3, VINCENT LAGENTE

3 AND LUÍS

CRISTÓVÃO PORTO2.

1 Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro - Brasil.

2 Programa de Pós-graduação em

Biologia Humana e Experimental, Universidade do Estado do Rio de Janeiro - Brasil. 3

INSERM 991, Université de Rennes 1 – France. Oxidative stress is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and pulmonary emphysema, which represents the alveolar lesion in COPD. Increased oxidative stress may result from the burden of oxidants in cigarette smoke and air pollutants, but may also be generated by activated inflammatory cells, providing a mechanism which accounts for why oxidative stress remains high in patients with COPD even after quitting smoking. Nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor that controls antioxidant response element (ARE)-regulated antioxidant and cytoprotective genes, has an essential protective role in the lungs. The major consequence of cigarette smoke-induced oxidative stress is the activation of central signaling pathways such as MAPK and the proinflammatory transcription factor NFκB, which activates multiple inflammatory genes contributing to inflammation. In addition, oxidative stress also contributes to a proteinase-antiproteinase imbalance, both by inactivating anti-proteinases, such as α1-antitrypsin and by activating proteinases, such as MMPs. On the other hand, oxidants also promote inflammation by activating AP1, which orchestrates the expression of multiple inflammatory genes recognized to be important in COPD, such as TNF-α. In contrast, our recent study focused on evaluating the redox imbalance response in the lungs after porcine pancreatic elastase stimulation at various time points of COPD establishment. We also investigated the participation of the nitrosative stress pathway as an aggravator of protease-induced pulmonary emphysema. We hypothesized that peroxynitrite synthesis, from the nitric oxide and superoxide anion reaction, contributes to the development of pulmonary emphysema, disturbing the antioxidant defenses and generating oxidative damage at the cellular level. Therefore, nitrosative stress as well as oxidative stress plays a key role in regulating COPD formation. Footnote This study was granted by CAPES-COFECUB project (680/10).

SANDRO JOSÉ DE SOUZA "BEYOND REASON AND BORDERS: THE RISE OF GLOBAL CREATIONISM” Science teaching is under serious risk worldwide. Until recently, there was a general sense that creationism was a reason of concern only in the United States. Nowadays, several facts indicates that the influence of creationism is affecting science teaching in several countries including Darwin's England. In my talk I will discuss how this happened and also how American creationist organizations are crossing borders and acting worldwide. I will also discuss the situation in Brazil and draw comparisons with the situation in other countries."

SERGIO LIRA INTERPLAY OF THE HOST LOCAL MICROBIOTA AND GENETIC PERTURBATIONS DICTATES SITE-SPECIFIC DEVELOPMENT OF INTESTINAL NEOPLASMS IN MICE Immunology Institute, Icahn School of Medicine at Mount Sinai, 1425 Madison Ave, New York NY 10029 The preferential localization of some neoplasms, such as serrated polyps (SPs), in specific areas of the intestine suggests that non-genetic factors may be important for their development. Mice expressing HB-EGF throughout the intestine (HBUS mice) develop SP exclusively in the cecum. Here we show that a specific set of taxa was associated with SPs and infiltrated the lamina propria of SP. This was associated specific innate inflammatory response characterized by IL-17 producing TCRgd cells and a neutrophilic infiltrate. Alteration of the cecal microbiota by antibiotic treatment or by embryo-transfer rederivation using different mothers dramatically attenuated SP development. Together, these results point to a crucial role for the microbiota in the localized development of SP in a genetically susceptible host.

COORDINATED CHANGES IN DNA METHYLATION IN ANTIGEN-SPECIFIC MEMORY T CELLS. SHINICHI HASHIMOTO (1,2,); KOUJI MATSUSHIMA (1) (1). Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo; (2). Faculty of Medicine, Kanazawa University Abstract: Memory T cells are central regulators of both humoral and cellular immune responses. T cell differentiation results in specific changes in chromatin structure and DNA methylation of cytokine genes. A lack of methylation at the appropriate loci in T and B lymphocytes is associated with transcription and rearrangement of immunoglobulin and TCR genes, and with cell lineage-specific expression of CD4, CD8, and CD21. When naive T cells differentiate to Th1 cells, but not to Th2 cells, DNase hypersensitive sites appear in the IFN-γ gene. Furthermore, the IFN-γ gene is methylated to a lesser extent in human and murine Th1 and CD8 effector cells than in naive and Th2 cells. Additionally, methyl-CpG-binding domain protein 2-deficient mice display reduced memory CD8+ T cell differentiation following acute viral infection. While the methylation status of a limited number of gene loci in T cells has been examined, the genome-wide DNA methylation status of memory T cells remains unexplored. In order to further elucidate the molecular signature of memory T cells, we conducted methylome and transcriptome analyses of memory T cells generated using T cells from TCR transgenic mice. The resulting genome-wide DNA methylation profile revealed >1,000 differentially methylated regions (DMRs) across the murine genome during the process of T cell differentiation. These DMRs included genes, which are associated with cytokine production and immune responses. Methylation changes in memory T cells exposed to specific antigen appeared to regulate enhancer activity rather than promoter activity of immunologically relevant genes. By comparing DMRs between naive and antigen-specific memory T cells, this study provides new insights into the functional status of memory T cells. Financial support: This work was supported by Core Research for Evolution Science and Technology of the Japan Science and Technology Agency.

SONIA JANCAR CENTRAL ROLE OF PAFR IN THE INDUCTION OF REGULATORY MACROPHAGES AND DENDRITIC CELLS Sonia Jancar and Francisco José Oliveira Rios Dept. Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil. Macrophages and dendritic cells (DCs) present membrane receptors (PRRs) that interact with microbial structures (PAMPs) and with altered self components such as oxidized particles and molecules expressed in dying cells. (DAMPs). Stimulation of these receptors generates lipid mediators (prostanoids, leukotrienes and PAF). We will present evidence that autocrine activation of PAFR during PRRs engagement shifts macrophages and dendritic cells towards a regulatory phenotype. Stimulation of macrophages with oxLDL recruits CD36 and PAFR into membrane lipid rafts and co-stimulation of these receptors leads to increased oxLDL uptake and establishment of a regulatory phenotype. Upon activation with oxLDL, CD36 and PAFR co-localize in human monocytes/macrophages. These receptors co-immunoprecipitate with the lipid raft markers, flotilin and GM1. Stimulation with oxLDL induce high levels of IL10 which is dependent on PAFR/CD36 association since only HEK293T cells transfected with both receptors were able to produce IL10. CD36/PAFR association induces the expression of markers of regulatory phenotype, IL10high/IL12low, arginase and manose receptor. Moreover, CD36/PAFR complexes were detected in human atherosclerotic plaques but the significance of this finding remains to be determined. Clearance of apoptotic cells plays a central role in tissue homeostasis, and impaired clearance has been associated with autoimmunity and chronic inflammatory diseases. We found that phagocytosis of apoptotic cells by macrophages also requires PAFR/CD36 association in lipid rafts and that this induces a regulatory phenotype. In tumor microenvironment, the presence of apoptotic cells and oxidized molecules, could shift macrophages towards suppressor phenotype favoring tumor growth. Indeed, PAFR antagonists reduced melanoma growth in mice and, associated to chemotherapy, increased mice survival. Also, addition of apoptotic cells to a sub-tumorigenic dose of melanoma cells promotes tumor growth and this was reversed by PAFR antagonists. Dendritic cells (DCs) also express PAFR and its activation during maturation induced by LPS, directs the DCs towards a regulatory phenotype by inducing PGE2 and IL10. PAFR antagonists by disrupting this suppressor pathway, significantly increased their capacity to induce antigen-specific lymphocyte proliferation. Studies are in progress to investigate if PAFR antagonists could increase the intensity or type of immune response in vivo. These results suggest a central role for PAFR in macrophages and dendritic cells phenotype with implications in atherosclerosis, clearance of apoptotic cells, tumor growth and in the adaptive immune response.

TOLEROGENIC AND ANTI-INFLAMMATORY USES OF BIODEGRADABLE PLG NANOPARTICLES FOR TREATMENT OF (AUTO)IMMUNE DISEASES STEPHEN D. MILLER, D. R. Getts, A. Martin, D. McCarthy, R. Terry, Z. Hunter, W. Yap, M. Getts, M. Pleiss, X. Luo, N. King, and L. Shea. Feinberg School of Medicine, Northwestern University, Chicago, IL, USA Ag-specific tolerance is the desired therapy for immune-mediated diseases. We have previously shown that i.v. infusion of antigens covalently linked with ECDI to apoptotic syngeneic splenic leukocytes (Ag-SP) efficiently induces T cell tolerance for the prevention and treatment of both the relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) model of multiple sclerosis (MS) and T1D in the NOD mouse. Apoptotic Ag-SP accumulate in the splenic marginal zone and are engulfed by F4/80

+ marginal zone macrophages and CD8

+ DCs inducing

upregulation of the immunoregulatory costimulatory molecule PD-L1 in an IL-10-dependent manner. Tolerance results from the combined effects of PD-L1/PD-1-dependent T cell anergy and the activation of regulatory T cells (Tregs) recapitulating how tolerance is normally maintained in the hematopoietic compartment in response to uptake of senescing blood cells. A recently published phase I clinical trial in MS patients has shown that infusion of autologous myelin peptide-coupled PBMCs (Ag-PBMC) is safe and induces dose-dependent suppression of myelin-specific T cell responses. However, widespread clinical use of tolerance therapy induced by Ag-PBMC will be hampered by its cost and complexity. Up until this time, antigen-containing nanoparticles have been studied for their ability to stimulate immune responses for purposes of vaccination against infectious diseases or to activate the immune system against tumour antigens. In contrast, we hypothesized that nanoparticles administered by the i.v. route might serve as surrogates for apoptotic cells, simplifying the induction of tolerance for the treatment of autoimmune diseases, allergic diseases or tissue transplants. Indeed we have shown that i.v. administration of CNS myelin peptides covalently linked to 500nm carboxylated nanoparticles (Ag-NP), either composed of polystyrene or biodegradable poly(lactide-co-glycolide) (PLG), effectively abrogated development of EAE when used prophylactically and ameliorated progression of clinical disease relapses in R-EAE when administered therapeutically. Ag-NP treatment suppressed myelin-specific Th1- and Th17-mediated autoimmune responses, infiltration of inflammatory cells to the CNS, and CNS demyelination. Ag-NP-induced tolerance is mediated by the combined effects of cell-intrinsic anergy and Treg activation. Ag-NP tolerance is dependent on PLG particle size (500nm diameter particles are optimal), uptake by marginal zone macrophages via a pathway mediated by the MARCO scavenger receptor, and can be induced either by particles encapsulating the autoantigen or carrying covalently linked antigen on their surface. Additionally, ‘naked’ carboxylated PLG nanoparticles were found to target inflammatory monocytes/macrophages in a MARCO-dependent fashion leading to their sequestration in the spleen and eventual apoptosis and were effective in ameliorating acute inflammatory diseases. Our findings demonstrate the feasibility of using Ag-NP as a novel, safe and cost-effective means for antigen-specific therapy of MS and other (auto)immune-mediated diseases using an FDA-approved material, PLG, which can be easily manufactured under GMP conditions. Supported by grants from the Myelin Repair Foundation and NIH grants EB013198 and NS046543

STEPHEN HOLDSWORTH DIRECTOR CENTRE FOR INFLAMMATORY DISEASE MONASH UNIVERSITY DEPARTMENT OF MEDICINE MONASH HEALTH - CLAYTON VICTORIA - AUSTRALIA SELECTING NOVEL THERAPEUTIC TARGETS IN INFLAMMATION Anti Neutrophil Cytoplasmic Antibody (ANCA) associated Vasculitis (AAV) is a form of vasculitis first described in the 1990s. It is a major cause of kidney failure (causing crescentic rapidly progressive glomerulonephritis RPGN) but also involves the upper airways , lung and other vascular beds. Untreated it has a poor prognosis but immunosuppression with cyclophosphamide and steroids significantly improves outcomes but with considerable morbidity. New less toxic therapies are urgently needed. This is an autoimmune disease with two major target autoantigens, both are neutrophil lysosomal enzymes. Myeloperoxidase (MPO) and Proteinase 3 (PR3).There are two forms of the disease each associated with a different pattern of autoimmunity. PR3 AAV is associated with granulomatous small vessel vascultis with mainly pulmonary and upper airways involvement (although GN can also occur) and a chronic relapsing course. It is also called Granulomatous Polyangiitis (GPA). MPO AAV also called Microscopic Polyangiitis (MPA) mainly causes RPGN and is less likely to relapse after remission has been achieved. These differences together with differences in geographic distribution and GWAS HLA associations only with GPA make it likely that these are two related, but different diseases. There are animal models of MPO AAV but not of GPA so the immunopathogenesis of MPO AAV is better understood . This presentation will focus on autoimmune human anti MPO AAV and what we have learned from pathological human and experimental studies. Finding new therapeutic targets is critically linked to understanding the immunopathogenesis of this disease. Little is known of the events that initiate anti MPO autoimmunity or the factors predisposing to it, but it begins with loss of MPO tolerance and the development of circulating anti MPO ANCA and measurable anti MPO CD4 T cells. The target autoantigen (MPO) is in neutrophils but these must be activated by infection, to allow MPO membrane translocation before ANCA can bind thereby activating complement which then induces neutrophil activation. Activation changes the neutrophil phenotype such that is “sticks” in the glomerular capillary bed where degranulation and NET formation occur releasing MPO and other proinflammatory and injurious molecules. Our human renal biopsy study reveals that this induces injury directly but by making MPO extracellular it becomes revealed to circulating anti MPO CD4 cells that direct injurious DTH type effectors (macrophages, neutrophils and fibrin) that induce crescentic GN. Development of a murine model has allowed the definition of the immunodominant nephritogenic peptide of the CD4 epitope of MPO. We have confirmed its capacity to induce nephritogenic immune responses. US colleagues have defined the dominant B cell MPO epitope recognized by sera from patients with acute anti MPO GN. This epitope has high sequence homology with the mouse T cell nephritogenic epitope and the human dominant B cell epitope can induce GN in mice. Nasal insufflation of the immunodominant MPO peptide induces nasal tolerance that can prevent and treat anti MPO GN in our mouse model. Finally we have assessed the key cytokines mediating acute anti MPO induced GN. Our studies show IL 17A and IL 23 as key mediators of anti MPO autoimmunity and GN and potential therapeutic targets .

METABOLOMICS IN THE ANALYSIS OF OCULAR AND INFLAMMATORY DISEASE STEPHEN P YOUNG Rheumatology Research Group, Centre for Translational Inflammation Research, College of Medical and Dental Sciences, University of Birmingham Research Laboratories, Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB U.K Inflammation is a critical component of normal responses to infection and injury, but substantial systemic changes to host organs and cells can result. Energy expenditure can be significant, resulting in widespread modulation of metabolism. Resting non-proliferative tissues have distinctive metabolic activities and requirements, which differ considerably from those in infiltrating immune cells, since these are proliferating and differentiating. Immune responses in tissues may therefore be modulated by the availability of substrates in the inflamed site and host tissue metabolism affected in the inflammatory environment. We have been exploring the potential for metabolomic analysis to identify disease-specific alterations in metabolism to provide novel insights into the inflammatory process. We have used NMR-based metabolomic fingerprinting to identify unique metabolic profiles in the eye in different forms of uveitis, in CSF in neurological conditions, but also in blood and urine in trauma and inflammatory arthritis. This suggests that the site-specific metabolic changes are reflected in systemic metabolic shifts. We have found that muscle metabolites, probably resulting from sarcopenia, are found in urine and can be used to predict responses to anti-TNF therapeutics in rheumatoid arthritis patients. In vitro culture of differentiated macrophages and synovial fibroblasts identifies significant metabolic differences in macrophage subtypes and unique metabolic profiles in fibroblasts derived from rheumatoid arthritis. This suggests that both host tissue cells and infiltrating immune cells contribute to alterations in the profile of systemic metabolites. Metabolomics is probably the systems biology approach most readily applicable to the study of inflammatory disease. Such studies have already provided insights into interaction between inflammatory cells and organs, energy substrate use, tissue breakdown, the microbiome and drug metabolites, and will find widespread application in the investigation into the regulation of inflammatory diseases.

Elucidating mechanisms by which TRPV1 and TRPA1 are involved in joint inflammation and pain. SUSAN D BRAIN, Section of Vascular Biology and Inflammation, Cardiovascular Division, King’s College London, SE19NH, UK There is a need for more effective treatments of the inflammatory hyperalgesia associated with arthritis. This group has been studying the role of transient receptor proteins transient receptor potential (TRP) ligand-gated non-selective ion channels. We and others have shown that the vanilloid 1 (TRPV1) channel is involved in the pain and to a lesser extent the inflammation. The blockade of the TRPV1 channel can be approached through two main ways, either through desensitization of the TRPV1 receptor (e.g. through repeated application of agonists such as capsaicin) or through development of TRPV1 antagonists that do not possess adverse effects. The role of other TRP channels is also under investigation. Recently, we have shown that the TRP ankyrin (TRPA1) channel is involved in mediating pain (e.g. increased sensitivity to mechanical stimuli) in a distinct manner to TRPV1, through studies in murine models of joint inflammation. TRPA1 agonists mediate increased blood flow in a range of tissues including the skin and synovial membranes. We are currently trying to understand how this may influence inflammatory hyperemia. This downstream activity following TRPA1 activation may become more important in a cold environment, as a relationship between cold exposure and increased pain in arthritic joints has been suggested to exist. These studies are supported by Arthritis Research UK.

ROLE OF JAK/STAT AND ERK SIGNALING PATHWAYS IN THE ACTIVATION OF EPITHELIAL CELLS BY CIGARETTE SMOKE EXTRACT. TATIANA VICTONI

1, Florence GLEONNEC

1, Marion DAUDE

1, Samuel S. VALENÇA

2, Luis Cristovão PORTO

2,

Vincent LAGENTE1,

Elizabeth BOICHOT1.

1: UMR991 INSERM, Faculté de Pharmacie, Université de Rennes 1. Rennes, France. 2: Laboratório de Reparo Tecidual, DHE/IBRAG/UERJ, Rio de Janeiro, Brasil. Chronic obstructive pulmonary disease (COPD) is a pathology characterized by an abnormal inflammatory response and associated with a destruction of lung parenchyma and loss of lung elasticity, leading to an airway limitation not fully reversible. Tobacco smoking continues to be a major cause of COPD. Cigarette smoke activates epithelial cells and resident macrophages by releasing proteases and chemokines. This phenomenon is responsible of the migration of inflammatory cells in the lung tissue such as neutrophils, macrophages and lymphocytes. These cells are able to release proteolytic enzymes leading to the degradation of components of the extracellular matrix. Strong evidence shows that recurrent bacterial infections contribute to the inflammatory process and consequently to the worsening of COPD. Based on these observations, we studied the early events in the development of COPD associated with recurrent bacterial infection. We showed that the combination of a cigarette smoke extract associated with low doses of LPS is able to synergistically increase the release of chemokines, by alveolar epithelial cells through the activation of MAP kinase signaling pathways ERK1/2 and JAK/STAT. We also demonstrated that the phosphodiesterase 4 inhibitor, roflumilast N-oxide (RNO) inhibits the secretion of these cytokines, thereby inactivating pathways JAK/STAT and ERK1/2. Moreover the treatment of the cells with roflumilast N-oxide significantly reduces the production of these chemokines. This in vitro model demonstrates the synergistic effect of CSE associated with LPS on the release of cytokines and activation of signaling pathways. This effect could be responsible for the progression and exacerbation of COPD. Moreover, we have demonstrated that neutrophil élastase can lead to the release of chemokines by alveolar epithelial cells by activating the p38 signaling, but not by JAK/STAT pathways. Our study also shows the effect of RNO on the release of cytokines induced by NE or by the combination CSE/LPS. These results highlight other mechanisms by which Roflumilast N-oxide exerts its anti-inflammatory effect in COPD. Ackowledgements: This study was supported by CAPES-COFECUB project (680/10).

GLIOMA STEM CELL SURVIVAL STRATEGY IN VIEW OF NICHE INVOLVING IMMUNE/INFLAMMATORY CELLS KOUICHI TABU, NOZOMI MURAMATSU, AND TETSUYA TAGA Department of Stem Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan “Cancer stem cells” (CSCs), a functional subset of cancer cells, are characterized by radio- and chemo-resistance and have been postulated as key drivers of cancer relapse and progression. CSCs reside in a specialized microenvironment, known as the ‘niche’, which is a complex architecture composed of, for instance, various cells. However, to date, very little is known about the identity of niche components. C6 rat glioma cell line contains a sub-population of CSCs, which is enriched using the Hoechst 33342 side population (SP) technique. C6 SP cells are tumorigenic, but main population (MP) cells are not. We recently found that a portion of C6 SP cells differentiate into vascular endothelial cells (VECs) to compose a vascular niche. In mice brain intracranially transplanted with SP cells, it is revealed that the vessels are composed of approximately equal numbers of SP-derived and host-derived VECs. SP-derived VECs mostly have accumulative and resistant properties for anti-cancer drugs, suggesting that SP-derived VECs may confer some survival advantages as a drug barrier for cancer cells. Various types of cells, including VECs and stromal fibroblasts, have been reported to function as CSC niche. Tumor-associated macrophages (TAMs) in large quantities are also observed in many solid tumors, including glioma. However, it remains unclear how the interaction between CSCs and TAMs could contribute to tumor development. cDNA microarray analysis of highly tumorigenic C6 glioma SP and non-tumorigenic MP cells indicated that SP cells show significant upregulation of immune/inflammatory genes, including the ones for some cytokines related to monocyte recruitment and differentiation, suggesting that SP cells may have essential roles in TAM behaviors. Interestingly, when C6 SP cells were transplanted together with “SP cell-educated” macrophages into the NOD/SCID brain, elevated tumor formation was observed compared to the transplantation together with “MP cell-educated” macrophages. We are in the process of elucidating “selfish strategies” that cancer stem cells take. Our further study will provide important insights into the fundamental aspects of cancer development from CSCs, which should help develop improved therapeutic strategies against cancer. 1. Kondo T, Setoguchi T, and Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA, 101: 781-786, 2004. 2. Tabu K, Bizen N, Kokubu Y, Muramatsu N, Nobuhisa I, Kagawa T and Taga T. Cellular heterogeneity contributes to tumor stem cell maintenance in C6 glioma. The 10th Annual Meeting of International Society for Stem Cell Research, 2012.

THIAGO MATTAR CUNHA ARTHRITIC PAIN: BEYOND THE PERIPHERAL SENSITIZATION It has been well accepted that inflammatory pain, such as arthritic pain, is caused mainly by the release of local inflammatory mediators, which mediate the sensitization of primary nociceptive neurons (nociceptors). In this talk, it will be discussed recent data regarding the mechanisms occurring at the level of the spinal cord and dorsal root ganglions that participate in the induction and maintenance of inflammatory pain. Among these processes, it will be focused on the activation of glial cell, such as microglia, astrocytes as well as satellite glial cells.

TIMOTHY JOHN WILLIAMS MAST CELL TRAFFICKING TIMOTHY J WILLIAMS; SARAH J COLLINGTON & CHARLOTTE L WELLER. Leukocyte Biology Section, NHLI Division, Faculty of Medicine, Imperial College London, South Kensington, London, SW7 2AZ, United Kingdom The interaction between specific types of leukocytes and cells of the affected tissues determines the symptoms of allergic reactions, but the exact nature of the relationship is the subject of considerable debate. Tissue mast cells have a key role, not only in acute reactions, but in determining more chronic events by releasing proteases, cytokines and growth factors. Our interest is in the chemoattractants released during allergic reactions and how these mediators induce the movement of leukocytes from the blood to the tissue. We have evidence that both human and murine mast cell progenitors express the BLT1 receptor

1. As activated mature mast cells produce the ligand for this receptor,

leukotriene B4, this provides a possible mechanism to increase tissue mast cell numbers at sites of allergic inflammation. This may, for example, contribute to the mast cell hyperplasia associated with allergic rhinitis. We found that immature mast cells also express the chemokine receptor CCR2 and that this couples to chemotaxis under certain conditions

1. Once recruited, committed mast cell precursors proliferate and mature under the control of local

factors such as stem cell factor (SCF). During maturation, mast cells lose their BLT1 receptors1 and other receptors

appear to be involved in their movement along local chemotactic gradients to guide the cells to their final destination in the tissue. CCR3, the receptor for the eotaxins, has been reported to be expressed by mast cells, but we have not as yet found a role for this receptor in this context. However, mouse immature mast cells express the EP3 receptor and surprisingly prostaglandin E2 is highly chemotactic for these cells

1. Thus, leukotriene B4 and CCR2 ligands may be

important for recruiting mast cell progenitors from the blood to the tissues and other mediators such as prostaglandin E2 and SCF may have a role in localising cells within the tissue, e.g. to the epithelium. 1: Collington SJ, Williams TJ, Weller CL (2011) Mechanisms underlying the localisation of mast cells in tissues. Trends in Immunology. 32(10):478-85 Research funded by Asthma UK

Defect of oral tolerance in neonates : a role for physiological gut inflammation ? Valérie Verhasselt Univ. Nice Sophia Antipolis, TIM, EA 6302, 06200 Nice, France Pioneer studies by Medawar have suggested that the neonatal immune system is immunologically immature and prone to tolerance induction. This was shown in a mouse model of systemic administration of an alloantigen in utero or during the perinatal period. Later works showed, that neonates are biased for Th2 responses as compared to adults, but also that any kind of immune response could be induced in neonates under appropriate conditions; this was also shown when antigen was given by systemic route. The few studies in rodents that studied immune response in neonate upon oral antigen administration demonstrated that oral tolerance is more difficult to induce in neonates than in adults. In humans, defect of early oral tolerance induction is exemplified by food allergy onset preferentially in early childhood while frequent spontaneous remission are observed due to a progressive shift from Th2 immune response towards Treg or Th1 induction. Currently, basic and clinical research is necessary to establish which factors govern immune response at the gut interface in the neonate to insure both efficient protocol of oral vaccination for infectious disease and optimal oral tolerance induction for allergic disease prevention. In a mouse model using Ovalbumin (OVA) as antigen, we found that antigen transfer during the 3 first weeks of lactation resulted in tolerance induction. However, our more recent data indicate some limits to oral tolerance in neonates: (1) oral tolerance induction reached full effectiveness only when OVA transfer occurs the third week of life (2) the transfer of antigen with intrinsic adjuvant activity such as antigen from house dust mite results in immune sensitization. We will present here our recent advances in understanding the reason of defective oral tolerance in early life and pinpoint the possible role of physiological inflammatory response in gut draining lymph node of the neonate.

SIGNALLING FOR APOPTOSIS AND RESOLUTION OF INFLAMMATION VANESSA PINHO The inflammatory response is a physiological process that has the major role of restoring tissue homeostasis. However, uncontrolled or unresolved inflammation may cause tissue damage and contribute to the pathogenesis of chronic inflammatory and autoimmune diseases. Thus, apoptosis of leukocyte plays a major role in the resolution of acute neutrophilic and eosinophilic inflammation in vivo. Apoptotic cells dampen inflammatory signals by sequestering chemokines, releasing molecules that inhibit further granulocyte influx and attract monocytes, and by reprogramming macrophages. Several molecular pathways regulate leukocyte survival and death during inflammatory responses. However, definite evidence for the participation of intracellular signaling pathways for the resolution of inflammation in animal models exists only for a few pathways. Understanding mech- anisms that regulate apoptosis in vivo is vital in providing clues at which molecular pathways one should focus to develop novel therapeutic strategies

INVOLVEMENT OF INFLAMMASOME PATHWAY AND PURINERGIC RECEPTORS IN THE DEVELOPMENT OF FIBROSIS VINCENT LAGENTE

1, Thomas GICQUEL

1, Sacha ROBERT

1,

Alain FAUTREL1, Isabelle COUILLIN

2 and Elisabeth BOICHOT

1

1 : UMR991 INSERM/Université de Rennes 1, Faculté de Pharmacie, 2 avenue du Prof Léon Bernard, 35043 Rennes cedex, France. 2 : INEM, CNRS UMR7355/University of Orleans, France Several inflammatory diseases have been reported to be linked with the activation of NLRP3-inflammasome. The best-characterized inflammasome consists of three main components, the Nod-like receptor (NLR)-family protein, NLRP3, pro-caspase-1, and the ASC (Apoptosis speck-like protein containing a CARD) adapter, which bridge interactions between the former proteins. It has also been previously demonstrated that pulmonary fibrosis is closely associated with the activation of NLRP3-inflammasome pathway and production of IL-1β. Adenosine triphosphate (ATP) has been described also as a danger signal activating NLRP3-inflammasome leading to the pro-inflammatory cytokine IL-1β release in lung. ATP mainly activates the P2X7 purinergic receptor, leading to trigger ASC-caspase-1 complex in a NLRP3-dependent manner and induces the production of IL-1β. We recently showed that ATPγS and BzATP, two analogs of ATP are able to potentiate the release of IL-1β from human monocyte-derived macrophages induced by low concentration of LPS. In the same conditions no increase in IL-1α and IL-6 was observed. We also observed that P2X7R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP strongly suggesting the involvement of the P2X7R/NLRP3 pathway in the secretion of IL-1β from ATP-stimulated human macrophages. Monosodium Urate (MSU) has also been described as a danger signal activating NLRP3-Inflammasome pathway. We showed that MSU elicited an increased production of IL-1β, IL-1α, IL-6 at 6h and 24h from human macrophages. Antibody arrays confirmed induction of IL-1β secretion in supernatant and also showed an increased production of ET-1, GM-CSF and MMP-8 production at 24h. Immunohistochemistry showed that P2X7R and NLRP3 protein were expressed in macrophages. These results showed the involvement of NLRP3 pathway and P2 receptor leading to cytokine release from macrophages in inflammatory process. Supported by Agence Nationale de la Recherche (ANR) project (ANR2010 MIDI01202), CAPES-COFECUB project (680/10) and INSERM

ROLE OF MATRIX METALLOPROTEINASES IN THE DEVELOPMENT OF INFLAMMATORY PROCESS AND TISSUE REMODELING VINCENT LAGENTE, SACHA ROBERT, THOMAS GICQUEL, ALAIN FAUTREL AND ELISABETH BOICHOT UMR991 INSERM/Université de Rennes 1, Faculté de Pharmacie, 2 avenue du Prof Léon Bernard, 35043 Rennes cedex, France Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. Pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components mainly collagen in the lung interstitium. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. We previously demonstrated a significant inhibition of bleomycin-induced pulmonary fibrosis in mice by the MMP inhibitor batimastat. We also reported a correlation of the differences in collagen deposition in the lungs of bleomycin-treated mice with a reduced molar pro-MMP-9/TIMP-1 ratio in broncholaveolar lavage fluid, beginning as early as the inflammatory events at day 1 after bleomycin administration. The differences in TIMP-1 level, particularly at early events after bleomycin administration, suggest that early altered regulation of matrix turnover may be involved in the further development of bleomycin-induced pulmonary fibrosis. We also demonstrated that IL-1R/MyD88 signaling is required in the bleomycin-induced increased TIMP-1 level and pulmonary fibrosis in mice. In vitro, using LX2 fibroblasts, we showed an increase in mRNA for MMP-1 and MMP-9 but no differences were observed for MMP-2, TIMP-1, 2 and 3. In contrast, TGFbeta is able to increase mRNA for MMP-2 but reduces MMP-1. This suggests that MMP/TIMP imbalance may occur respectively to the activation and the cellular microenvironment. Finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of tissue remodeling and fibrosis.

WIM VAN DEN BERG ROLE OF TH17 AND BACTERIAL FLORA IN ARTHRITIS Rheumatology research and advanced therapeutics, Radboud University Nijmegen Med Centre, Nijmegen, The Netherlands Rheumatoid arthritis is characterized by chronic joint inflammation and concomitant destruction of cartilage and bone. It is considered as an autoimmune disease and cytokines play a major role, including the T cell derived IL-17. IL-17 alone is inflammatory , but displays limited erosive character. However, it can greatly exaggerate joint erosion, when overexpressed in a joint already bearing an immune complex mediated arthritis. In addition, IL-17 can shift the cytokine dependency of an arthritic process, with complete loss of TNF and IL-1 dependency. Il-17 is a dominant cytokine in T cell driven flares of arthritis. Apart from IL-17 itself, mediators involved in Th17 cell generation such as IL-21, as well as Th17 cell derived IL-22 appear intriguing therapeutic targets. Since anti-TNF treatment causes upregulation of IL-17 in a subset of patients , concomitant suppression of TNF and IL-17 is warranted. Efficacy of combined treatment is obvious in murine collagen arthritis. Environmental triggers are major drivers of Th17 development. Arthritis is absent under germ free conditions and the TLR4 pathway is crucial in TH17 generation and arthritis expression. Apart from the gastrointestinal tract, oral flora might contribute. Concomitant periodontal disease, driven by P gingivalis or P Negrescens can exaggerate arthritis through induction of Th17 and suppression of IL-4.

XAVIER NOREL ROLE OF H2S IN HUMAN BLOOD VESSELS: INTERACTION WITH THE CYCLOOXYGENASE PATHWAY Ingrid GOMEZ

1,2; Thomas ROGER

3; Chabha BENYAHIA

1,2; Erwan GALARDON

3; Dan LONGROIS

1,4; Xavier

NOREL1,2

1Inserm U698, CHU X. Bichat, Paris, France;

2Paris 13 Univ, Villetaneuse, France;

3Paris Descartes Univ, UMR 8601

CNRS, France; 4CHU X. Bichat, ICU, Paris, France.

Introduction: Recent investigations have shown that hydrogen sulfide (H2S, produced by CSE enzyme) can down regulate prostaglandin (PG)E2 synthesis in human keratinocytes and articular chondrocytes. Furthermore, in the cardiovascular system of rodents a protective role for H2S has been shown. To our knowledge, there is no study on human vessels about H2S and metabolites of the cyclooxygenase pathway like PG. Varicose vein is one of the most common diseases in western country. Nevertheless, its pathogenesis is still poorly known. Our recent study has demonstrated that PGE2 synthesis was decreased in varicose veins, when compared with healthy saphenous veins (SV) and leads to the reduction of metalloproteinase (MMP)-1 activation. The aim of this study is to investigate the role of H2S and PGE2 on the vascular wall remodelling of varicose veins. Methods: Human SV (n=15) and varicose veins (large (LDv) and small (SDv) diameter varicosities from the same patients, n=16) were obtained at Bichat hospital (Paris). Endogenous H2S release was measured by a polarographic method. The CSE activity was demonstrated using L-Cystein stimulation (10

-5 to 10

-3 M with or without a CSE

inhibitor, beta cyano-L-alanine (BCA). Measurements for PGE2, MMP and TIMP concentration in varicose veins and SV supernatant were realised by ELISA after a 24h incubation with various treatments (H2S 10

-5 to 10

-2 M and BCA

10-5

to 10-3

M). Results: Endogenous H2S was significantly increased in SDv and even more in LDv when compared to SV. After L-Cystein stimulation, the production was also significantly greater in varicose veins than in SV, and this production was inhibited by BCA. A significant decrease of PGE2 concentration in supernatant of varicose veins was detected, and PGE2 production in all tissue was significantly inhibited by H2S. The BCA incubation restored PGE2 concentration in varicose veins at the same level than in SV. Furthermore, the MMP-1 activity was reduced in varicose veins when compared with SV, and their activity was significantly reduced in presence of H2S. In contrast, TIMP-1 / -2 are significantly higher in varicose veins, their expression were increased with H2S and reduced in presence of BCA. Conclusion: In human varicose veins, the increase of the endogenous H2S content leads to a decreased PGE2 production, which is correlated to a reduced MMP-1 activity. Therefore, our results suggest that H2S could play an important role in the vascular wall remodelling observed during varicose veins pathogenesis, through the down regulation of PGE2 levels and of MMP-1 activity.

THE TGF-B SUPERFAMILY CYTOKINE MIC-1/GDF15: A MOLECULE LINKING INFLAMMATION, CANCER AND METABOLISM. DAVID A BROWN

1,2; YASMIN HUSAINI

1; VICKY WW TSAI

1,2; LELE JIANG

1,2; SAMUEL N BREIT

1,2.

1St Vincent’s Centre for Applied Medical Research, St. Vincent's Hospital Sydney, NSW. 2010. Australia

2University of

New South Wales, Sydney, NSW. 2052. Australia Whilst there is a wealth of epidemiological data linking the inflammation, cancer and obesity/metabolism, many of the molecules linking these processes are still to be identified. One molecule participating in all these processes is MIC-1/GDF15, a divergent member of the TGF-b superfamily that we first cloned some years ago, based on its increased expression with macrophage activation. In the last decade, studies from many groups, including ours, have contributed to the understanding of MIC-1/GDF15 biology, its role in disease pathogenesis and potential therapeutic and diagnostic applications. Whilst there is a lot still to be discovered, some features suggest an important role in physiology and disease. MIC-1/GDF15: - - is powerfully induced in most cell types by stresses such as injury, inflammation and malignancy. - Is present in the circulation of all individuals and increases in serum MIC-1/GDF15 serum levels predict all cause, as well as specific cardiovascular and cancer mortality a finding that can be harnessed for disease diagnosis and management. - When overexpressed in experimental animals protects from development of a number of inflammatory disease including models of arthritis and atherosclerosis. Treatment with recombinant protein may be useful for therapy of inflammatory diseases. - Its serum levels also predict insulin resistance, impaired glucose control and worsening albuminuria in type 2 diabetics - Is a centrally acting appetite regulator, a process that is subverted in diseases like cancer when its overexpression leads to anorexia/cachexia, a common medical problem and an important area of unmet medical need, that might be treatable with monoclonal antibodies. - Further the action of MIC-1/GDF15 as a powerful appetite suppressant indicate that recombinant MIC-1/GDF15 might be harnessed for treatment of severe obesity. This talk will focus on the role played by MIC-1/GDF15 in normal biology and multiple, disparate disease processes.