A three day event discussing aspects of the innate immune system. With plenty of opportunity for networking and debate, this informal international meeting will bring you up to date with current research and thinking regarding an organisms first line of defence. Who Should Attend: Academic and biotechnology/biopharma research professionals interested in function and potency of immune response components against pathogens and tumours; also scientists interested in how understanding these mechanisms can inform the design of biological therapeutics and their mechanisms of action, with translational implications for therapy. This event has an open poster session. Posters can be submitted on any subject related to Innate Immunity This event has CPD accreditation. The deadline for abstract submissions for oral presentation is July 10th 2014. Abstracts for poster presentation only can be submitted up to two weeks before the event. You can download the instructions for authors at www.euroscicon.com/ABSTRACTSUBMISSIONS.pdf www.regonline.co.uk/innate2014

Table of Contents

Day 1: The Innate Immunity Interactions with Pathogens .................................................................................................. 5

Invited Speakers Abstracts ....................................................................................................................................................... 5

Exploring pathogen recognition in innate immunity using Drosophila as a model host ..................................................... 5

Is Immune Activation Necessary for HIV? ............................................................................................................................ 5

Non-specific Stressors: Under-appreciated Innate Defenses .............................................................................................. 5

HPV16 deregulation of the inflammasome response .......................................................................................................... 5

TRIF-mediated TLR3 and TLR4 signaling is negatively regulated by ADAM15 ..................................................................... 5

Purinergic signalling in controlling of intracellular parasites ............................................................................................... 6

The role of innate immune system on periodontal diseases ............................................................................................... 6

Soluble pattern recognition molecules and associated proteins – update on the lectin pathway of the complement

system .................................................................................................................................................................................. 6

Interaction of HIV with plasmacytoid dendritic cells and its relevance for HIV pathogenesis ............................................ 6

Lipid rafts influence NLR mediated immune responses against bacterial infections ......................................................... 6

Cross-talk between Fas and TLR signalling pathways - role in inflammation and cancer ................................................... 7

Paramyxovirus Inhibition of Human Complement Pathways .............................................................................................. 7

Oral Presentation Abstracts ..................................................................................................................................................... 7

THE HUMAN DEAD-BOX HELICASE 3 IN INNATE ANTIVIRAL IMMUNE SIGNALLING ........................................................... 7

INNATE IMMUNITY INTERACTIONS WITH PATHOGENSTHE ROLE OF EXTRACELLULAR GALECTIN-3 (GAL-3) IN HOST

RESPONSE TO HELICOBACTER PYLORI INFECTION. .............................................................................................................. 7

IL28B AND IL10 GENE VARIABILITY AND HUMAN PREDISPOSITION TO CHRONIC HEPATITIS C AND TICK-BORNE

ENCEPHALITIS, CAUSED BY RELATED VIRUSES ..................................................................................................................... 8

INNATE LYMPHOCYTE CELL ‘MEMORY’ AGAINST BACTERIAL INFECTION ........................................................................... 8

ROLE OF INNATE ANTIMICROBIAL MECHANISMS IN INTRACELLULAR SURVIVAL OF M. AVIUM SUBSPECIES AVIUM IN

CHICKEN CELLS ..................................................................................................................................................................... 9

Poster Presentation Abstracts ................................................................................................................................................. 9

PLAYING WITH FIRE: AN AGENT-BASED MODEL OF FEVER AND OTHER SYSTEMIC STRESSORS ......................................... 9

CHARACTERIZATION OF NATURAL AND ADAPTIVE FOXP3 REGULATORY T CELLS IN HUMAN VISCERAL LEISHMANIASIS . 9

EMR2 RECEPTOR LIGATION INDUCES MATURATION AND INFLAMMATORY ACTIVATION OF MACROPHAGE ................. 10

DECITABINE INHIBITS MDSC POPULATION IN TUMOR-BEARING MICE ............................................................................. 10

IMMUNE RESPONSE GENE EXPRESSION PROFILING IN A P. AERUGINOSA LIPOPOLYSACCHARIDE-INDUCED MODEL FOR

INFLAMMATION OF THE RAT TESTIS.................................................................................................................................. 11

VASOACTIVE INTESTINAL PEPTIDE MODULATES CYTOKINE PRODUCTION BY SALMONELLA-INFECTED HUMAN

MONOCYTES....................................................................................................................................................................... 11

PREBIOTIC AND PROBIOTIC AGENTS ENHANCE IMMUNE RESPONSES TO SALMONELLA TYPHIMURIUM INFECTION IN

PIGS .................................................................................................................................................................................... 11

ROLE OF NKT CELLS IN HUMAN VISCERAL LEISHMANIASIS ............................................................................................... 12

HUMAN THROMBOSPONDIN1 (TSP1) IS, A NEGATIVE REGULATOR FOR COMPLEMENT SYSTEM, PROMOTING DEFENCE

FOR BACTERIAL PATHOGENS FROM COMPLEMENT SYSTEM OF INNATE IMMUNITY in vitro .......................................... 13

EFFECT OF COMMERCIALLY AVAILABLE PLANT DEFENCE STIMULATORS (PDS) ON HUMAN INNATE IMMUNITY ........... 14

TLR4 PLAYS A PIVOTAL ROLE IN MONOCYTE ADHESION TO VASCULAR ENDOTHELIUM .................................................. 14

Day 2: Investigating interactions of the Innate and Adaptive Immune Systems .............................................................. 15

Invited Speakers Abstracts ..................................................................................................................................................... 15

The role of gamma delta T cells in melanoma and the effects of bisphosphonate therapy. ............................................ 15

B-cells at the interface between innate and acquired immunity to bacterial pathogens ................................................. 15

Sepsis as a Model of Impaired Communication between the Innate and Adaptive Immune Systems ............................. 15

Immunomodulatory networks controlling cancer metastatic dissemination ................................................................... 16

Understanding the roles of innate and adaptive immunity in the mechanisms of action of immunomodulatory

antibodies. .......................................................................................................................................................................... 16

Platelets as immune cells ................................................................................................................................................... 16

Oral Presentation Abstracts ................................................................................................................................................... 16

NATURAL ANTIBODIES ARE INNATE IMMUNE RESPONSIVE .............................................................................................. 16

SULFORAPHANE EPIGENETICALLY REGULATES THE LPS-INDUCED KINETICS OF INNATE IMMUNE RESPONSE IN PORCINE

MONOCYTE-DERIVED DENDRITIC CELLS ............................................................................................................................ 17

MODULATION OF IL-1 FAMILY CYTOKINES AND RECEPTORS DURING THE ACUTE AND CHRONIC INFLAMMATORY

RESPONSE OF HUMAN MONOCYTES ................................................................................................................................. 17

MODELING ANTIMICROBIAL PEPTIDE-INDUCED CHEMOKINE, CYTOKINE, AND BIOLOGICAL MEDIATOR RESPONSES .... 18

Poster Presentation Abstracts ............................................................................................................................................... 18

Day 3: Therapeutic applications of the Innate Immune system ........................................................................................... 19

Invited Speakers Abstracts ..................................................................................................................................................... 19

Regulation of autoimmune myocarditis and inflammatory cardiomyopathy by innate immunity ................................... 19

Talk to be confirmed ............................................................................................................. Error! Bookmark not defined.

M1/M2 Macrophages: A Copernican Revelation in Immunology ..................................................................................... 19

Oral Presentation Abstracts ................................................................................................................................................... 19

HYPOXIC-INDUCED PROTEOLYSIS OF MER TYROSINE KINASE AND CD36 BY ADAM PROTEASES REGULATES APOPTOTIC

CELL ACCUMULATION AND HIF2-REGULATED EFFEROCYTOSIS AND INFLAMMATION-RESOLUTION IN MACROPHAGES

TO SUPPRESS FIBROSIS AND CARDIAC FUNCTION. ............................................................................................................ 19

MECHANISMS BEHIND IMMUNOREGULATORY EFFECTS OF PROBIOTIC YEASTS .............................................................. 20

PHARMACOLOGICAL MODULATION OF THE AKT/MIR199A-5P/CAV1 PATHWAY INCREASES ENDOTOXIN TOLERANCE IN

CYSTIC FIBROSIS (CF)-AFFECTED MACROPHAGES AND DECREASES LUNG HYPER-INFLAMMATION IN CF MIC ................ 20

TARGETTING THE TRIPEPTIDE PRO-GLY-PRO DEMINISHES INFLAMMATORY EFFECTS OF CIGARETTE SMOKE ................ 21

Poster Presentation Abstracts ............................................................................................................................................... 21

ACTIVATED MONOCYTES PLAY AN IMPORTANT ROLE IN INNATE IMMUNITY AND INFLAMMATION IN OBESE CHILDREN21

EXPRESSION OF CYTOKINES AND MATRIX METALLOPROTEINASE 2 AND 9 BY INNATE IMMUNITY CELLS AS A POTENTIAL

BIOMARKER OF CARDIAC MORBIDITY IN CHAGAS DISEASE .............................................................................................. 22

THREE POTENTIAL PROBIOTICS OFFER PROTECTION TO ARTEMIA AGAINST V. ANGUILLARUM CHALLENGE BY

ENHANCING ITS IMMUNE SYSTEM AND ANTIOXIDANT STATUS ....................................................................................... 22

Day 1: The Innate Immunity Interactions with Pathogens Invited Speakers Abstracts Exploring pathogen recognition in innate immunity using Drosophila as a model host Dr Petros Ligoxygakis, Associate Professor of Genetics, University of Oxford, UK Pathogen recognition is the first step to immune activation. There are however, still many unanswered questions about how sensing in innate immunity works, when a limited number of receptors must sense an immense variety of pathogens. This is especially pertinent for Gram-positive bacteria where cell-wall heterogeneity has defied categorization in terms of host recognition. We are using the fruit fly Drosophila melanogaster as a model to address questions on recognition of infection on the bacterial cell wall, what happens to host sensing when the cell wall changes and what are the bacterial cell wall attributes, which contribute to immune system evasion. Is Immune Activation Necessary for HIV? Professor Wenzhe Ho, Dept. Pathology & Laboratory Medicine Dept. Anatomy & Cell Biology, Temple University School of Medicine, USA As an important indicator of HIV disease progression, chronic immune activation persistently provides new targets (activated CD4 T cells) for HIV, leading to AIDS. Therefore, to suppress immune activation is a key to slow down HIV disease progression. EGCG, the main component of green tea, not only has anti-inflammatory effect, but also can suppress HIV/SIV in vitro studies. Our ongoing investigations uisng SIV-infected rhesus macaques showed that EGCG administration could suppress the expression of inflammatory cytokines in SIV-infected rhesus macaques. In addition, when given intravaginally, EGCG reduced SIV infection of cynomolgus macaques. These findings support further studies on use of EGCG for treatment and prevention of HIV infection. Non-specific Stressors: Under-appreciated Innate Defenses Dr Edmund LeGrand, Adjunct Professor, Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA We used NetLogo computer simulations to show how host-induced non-specific stress can harm pathogens relatively more than the host. The pathogens are more vulnerable due to their rapid growth and replication, leaving them fewer resources to withstand stress. The model mimics the systemically harmful stressors of the acute-phase response such as fever, anorexia, hypoferremia, and anemia. Locally non-specific stress mimics the infection site’s nutrient deprivation, hypoxia, acidity, and free radicals. Regional stress simulates reduced blood flow due to coagulation and edema. We propose that hosts deliberately induce non-specific stress to help control pathogens locally, regionally, and systemically. HPV16 deregulation of the inflammasome response Dr Uzma Hasan, Oncoviruses and Innate Immunity Centre International de Recherche en Infectiologie, University Lyon, Hopital Lyon Sud, France HPV16: The human papillomavirus (HPV) high-risk mucosal types are 100% the causative agents of cervical and 30% in head and neck cancers. The development of cancers induced by HPV, particularly HPV16, is intimately linked to the persistence of viral infection. The innate immune response plays a key role in the early control of viral replication and are required for the orchestration of adaptive antiviral responses. We have demonstrated the loss of IL-1β and IL-18 secretion in HPV16 E6/E7 cells compared to normal primary keratinocytes treated with AIM2 and NLRP3 ligands. Protein levels of the proform of IL-1β are reduced in human keratinocytes expressing the viral oncoproteins and in cervical cancer biopsies. To date, there are limited studies on inflammasome activity in keratinocytes and its deregulation and / or possible activation by HPV16. Understanding how HPV16 regulates innate immune responses will help us to appreciate how to develop immune therapies against infection. TRIF-mediated TLR3 and TLR4 signaling is negatively regulated by ADAM15 Dr Sinead Miggin, Lecturer and Principal Investigator, National University of Ireland Maynooth, Ireland Toll-like receptors (TLRs) are a group of pattern-recognition receptors that play a crucial role in the recognition and induction of the innate immune response against bacterial and viral infections. The TLR adaptor protein, TRIF, facilitates TLR3 and TLR4 signaling and concomitant activation of the transcription factors, NF-B, IRF3 and IRF7 which ultimately leads to the production of proinflammatory cytokines and Type I Interferon. Herein, we opted to investigate the molecular mechanisms that regulate the TRIF signaling pathway. To this end, we purified the TRIF signalling complex by immunoprecipitation and identified the interacting partners by liquid chromatography mass spectrometry (LC-MS). We found that A Disintegrin and Metalloprotease (ADAM)15 interacted with TRIF in a TLR3 and TLR4 ligand dependant manner. Towards the functional characterization of the TRIF:ADAM15 interaction, we show that suppression of ADAM15 expression enhanced TLR3 and TLR4-mediated proinflammatory cytokine production via TRIF. Further, we show that

ADAM15 acts as a negative regulator of TRIF mediated NF-κB and IFNβ reporter gene activity. Mechanistically, we show that ADAM15 mediates the proteolytic cleavage of TRIF and therefore curtails TRIF-dependent TLR3 and TLR4 signaling. In conclusion, our study clearly shows for the first time that ADAM15 is a modulator of TLR signalling, acting as an anti-inflammatory molecule through impairment of TRIF-mediated TLR signalling. Purinergic signalling in controlling of intracellular parasites Dr Robson Coutinho-Silva, Extracellular nucleotides are newer danger signals that alert the immune system to loss of homeostasis. They activate P2 receptors responsible for a diversity of physiological response such as cell death, pro-inflammatory IL1β secretion, leukocytes migration, production of ROS, NO, and intracellular parasites killing. Purinergic Signalling mediated by activation of P2X7 receptor participates in innate and adaptive immune response against infection with L. amazonensis. In macrophages P2X7 receptors activation leads to PLA2 activation, production of ROS and secretion of inflammatory LTB4, and IL1-beta secretion culminating with parasite elimination. Thus, purinergic signaling could be a new cascade signaling to be explored in participating in control of a vast number of infections with intracellular parasites. The role of innate immune system on periodontal diseases Dr Sema Becerik, Ege University, Turkey Periodontitis is a common infectious disease characterized by the inflammatory destruction of tooth-supporting hard and soft tissues. It is becoming clear that most of the tissue damage that characterizes periodontal disease is caused indirectly by the host response to infection rather than directly by the infectious agent. Investigations into the mechanisms of the host-mediated response in periodontal disease have revealed that it involves the activation of the broad axis of innate immunity, specifically by upregulation of proinflammatory cytokines, and downregulation of growth factors. The pathogen-associated molecular patterns (PAMPs) may stimulate inflammatory host response via a family of membrane bound Toll-like receptors (TLRs). Recognition of PAMPs by TLRs leads to the induction of adhesion molecules, inflammatorycytokines and chemokines which regulate inflammatory and immune cell recruitment. TLR system was also shown to play role in bone loss in chronic periodontitis. Soluble pattern recognition molecules and associated proteins – update on the lectin pathway of the complement system Professor Steffen Thiel, Department of Biomedicine, Aarhus University, Denmark In the context of immunity, pattern recognition is the art of discriminating friend from foe and innocuous from noxious. The basis of discrimination is the existence of evolutionarily conserved patterns on microorganisms, which are intrinsic to these microorganisms and necessary for their function and existence. Such immutable or slowly evolving patterns are ideal handles for recognition and have been targeted by early cellular immune defence mechanisms such as Toll-like receptors, NOD-like receptors, RIG-I-like receptors, C-type lectin receptors and by humoral defence mechanisms such as the complement system. Complement is a proteolytic cascade system comprising around 35 different soluble and membrane-bound proteins. It constitutes a central part of the innate immune system, mediating several major innate effector functions and modulating adaptive immune responses. The complement cascade proceeds via controlled, limited proteolysis and conformational changes of constituent proteins through three activation pathways: the classical pathway, the alternative pathway and the lectin pathway, which converge in common effector functions. I will review the nature of the pattern recognition molecules involved in complement activation, as well as their close relatives with no or unknown capacity for activating complement. I will also discuss what kind of diseases may develop if an inflammatory generating mechanism like the complement system is out of control. Interaction of HIV with plasmacytoid dendritic cells and its relevance for HIV pathogenesis Dr Adriano Boasso, Principal Investigator - Research Fellow, Imperial College London, UK Activated plasmacytoid dendritic cells (pDC) produce large quantities of type I interferon (IFN-I) upon exposure to viruses. During HIV-1 infection, IFN-I may be beneficial during the initial stages of infection, but pDC overactivation may contribute to HIV-1-induced immune dysregulation and suppresses memory T cell responses. Our recent data suggest that the organization of the HIV-1 envelope into a functional substructure is critical for HIV-1-pDC interaction, and pDC maturation into IFN-I-producing cells (IPC) is reduced in response to the lees pathogenic HIV-2 compared to HIV-1. Lipid rafts influence NLR mediated immune responses against bacterial infections Associate Professor Sanjay Batra, Southern University and A&M College, Baton Rouge, Louisiana, USA While the role TLRs in host defense have been extensively studied, the role of intracellular (NOD)-like receptors (NLRs) against bacterial pathogens remain less explored. Studies highlighted the association of NOD1 and NOD2 with the plasma membrane following bacterial infection. In this context, NOD1 and NOD2 mutations interfere with their subcellular localization. Our studies showed that co-localization of NOD2, NLRP6 and NLRP12 in lipid rafts following Klebsiella challenge. Disruption of lipid rafts regulates NLR mediated responses in pathogen challenge models. Together these studies indicate that membrane recruitment of NLRs may be a general theme for pathogens to induce downstream host signaling.

Cross-talk between Fas and TLR signalling pathways - role in inflammation and cancer Dr Elizabeth Brint, Lecturer and Principal Investigator,University College Cork, Ireland Innate Immune receptors do not signal or exert immune effects in isolation. The level of cross-talk and interaction between different receptors alters the cytokine/chemokine profile produced by immune cells affecting a myriad of cellular responses. Here we have explored the interactions between Toll-Like Receptors and the Fas/Fas ligand pathways. Whilst Fas is more classically known as a death receptor we have shown that it plays an important role in modifying the TLR-driven inflammatory and immune responses in both macrophages and intestinal epithelial cells. These interactions play an important role in altering both responses to infection and cancer-associated inflammation. Paramyxovirus Inhibition of Human Complement Pathways Professor Griffith Parks, Dept. of Microbiology and Immunology, Wake Forest School of Medicine, USA Complement (C’) is an innate immune system that most animal viruses must face during natural infections. The ability of viruses to counteract this powerful system can impact viral replication, pathogenesis, and dissemination, as well as subsequent adaptive immunity. Likewise, there is intense interest in defining the role of C’ in control of emerging pathogens such as Nipah virus which have the potential for use in bioterrorism. This talk will highlight novel mechanisms by which paramyxoviruses activate and then inhibit C’ pathways, including how these viruses exploit host cell inhibitors to prevent neutralization. Oral Presentation Abstracts THE HUMAN DEAD-BOX HELICASE 3 IN INNATE ANTIVIRAL IMMUNE SIGNALLING L Gu, A Fullam, Y Höhn, R Brennan and M Schröder Institute of Immunology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland The human DEAD-box protein 3 (DDX3) has been implicated in different processes contributing to gene expression. However, more recently, it has been described to function in both innate antiviral immunity and viral replication; thus potentially finding itself at the centre of the molecular battle between viruses and the host. Importantly, DDX3 is required as an essential host factor for the replication of HIV and Hepatitis C Virus (HCV) and is therefore considered a potential drug target. On the other hand, DDX3 interacts with IκB-kinase-epsilon (IKKε) and TANK-binding kinase-1 (TBK1) and contributes to the induction of anti-viral type I interferons (IFNs). However, the molecular mechanism by which DDX3 contributes to IFN induction remains unclear. Our data demonstrates that DDX3 acts as a multifunctional adaptor molecule bringing together key signalling molecules in the RIG-I pathway. We show that DDX3 mediates phosphorylation of Interferon Regulatory Factor (IRF) 3 by the kinase IKKε. DDX3 directly interacts with IKKε and enhances its auto-phosphorylation and activation. IKKε then phosphorylates several serine residues in the N-terminus of DDX3. Phosphorylation of DDX3 at serine 102 (S102) is required for recruitment of IRF3 to DDX3, facilitating its phosphorylation by IKKε. Mutation of S102 to alanine disrupted the interaction between DDX3 and IRF3, but not IKKε. The S102A mutation failed to enhance ifnb promoter activation, suggesting that the DDX3-IRF3 interaction is crucial for this effect. In addition, we have recently shown that DDX3 also directly interacts with TRAF3, another crucial signalling molecule in IFN induction pathways downstream of RIG-I and other pattern recognition receptors. We have investigated in detail in which temporal order DDX3, TRAF3, IKKε and IRF3 are recruited to the adaptor molecule MAVS which mediates RIG-I signalling. It appears that DDX3 assembles both an early MAVS-bound complex and a subsequent soluble signalling complex that facilitates IRF3 phosphorylation. INNATE IMMUNITY INTERACTIONS WITH PATHOGENSTHE ROLE OF EXTRACELLULAR GALECTIN-3 (GAL-3) IN HOST RESPONSE TO HELICOBACTER PYLORI INFECTION. V. Vijayasubhash V. and B. Ho Department of Microbiology, Yong Loo Lin School of Mediciane, National University of Singapore, 5 Science Drive 2, Singapore 117545. Email: bow_ho@nuhs.edu.sg Galectin-3 (Gal-3) is a β-galectoside lectin which is upregulated and rapidly secreted by gastric epithelial cells in response to H. pylori infection. However, the role of extracellular Gal-3 and its functional significance in H. pylori-infected cells remain uncharacterised. Here we demonstrate that Gal-3 secretion is an initial host response to H. pylori infection. The molecular interaction between H. pylori and Gal-3 interferes with the binding of the bacteria onto the gastric epithelial cell surface, which prevents apoptosis, suggesting that extracellular Gal-3 may have a role in regulating H. pylori-induced apoptosis. Furthermore, the study also identifies extracellular Gal-3 as a probable chemoattractant which recruits THP-1 monocytes. The present study elaborates the significance of the binding of extracellular Gal-3 to H. pylori LPS has an impact on impeding the initial colonization process of H. pylori.

IL28B AND IL10 GENE VARIABILITY AND HUMAN PREDISPOSITION TO CHRONIC HEPATITIS C AND TICK-BORNE ENCEPHALITIS, CAUSED BY RELATED VIRUSES A. V. Barkhash1, G. V. Kochneva2, E. V. Chub2, M. I. Voevoda1,3, A. G. Romaschenko1

1 Institute of Cytology and Genetics SB RAS, 10 Lavrentyeva Ave., Novosibirsk, 630090 Russia; e-mail: barkhash@bionet.nsc.ru 2 State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region, Russia 3 Institute of Internal Medicine SB RAMS, Novosibirsk, Russia Chronic hepatitis C (HC) and tick-borne encephalitis (TBE) are common diseases in the territory of Russia. These diseases differ significantly in pathogenesis, although both of them are caused by single-stranded positive-sense RNA viruses with similar genome organization from the Flaviviridae family. It is still not clear whether molecular protective mechanisms against these two related viruses are similar or different; however, human genes that encode crucial components of antiviral immune response are most likely involved in these mechanisms. Previously, several single nucleotide polymorphisms (SNPs) located within interleukin 28B (IL28B) and interleukin 10 (IL10) genes were shown to be associated with human predisposition to chronic HC in several world populations. In this study, genotype frequencies for the IL28B and IL10 SNPs were compared in 119 HC patients, 132 non-immunized TBE patients (including 34 with fever, 60 with meningitis, and 38 with severe forms), and in the control Russian population (221 Novosibirsk citizens). PCR and RFLP analyses were used to determine the sample genotype. For the IL28B gene rs12980275 (A/G) SNP (located in 3’-flanking region), statistically significant increase in A/A homozygote frequencies for TBE patients (60.6%) (especially those with severe disease (71.4%)) as compared with the control group (47.5%) (P = 0.018 and 0.009, respectively) was detected. For the IL10 gene rs1800872 (C/A) SNP (located in the promoter region), no statistically significant differences in genotype frequencies were detected between TBE patients and control group; however, a significant increase in A/A homozygote frequencies for TBE patients with severe disease (10.5%) as compared with the control group (2.0%) was found (P = 0.007). Moreover, the frequency of this genotype was also significantly higher in HC patients (7.6%) than in the control group (P = 0.013). Thus, our data suggest that the A/A genotype of the IL10 gene rs1800872 SNP is associated with predisposition to both HC and TBE, caused by phylogenetically related viruses, in Russian population. This work was supported by the Russian Foundation for Basic Research (grant 14-04-00641a). INNATE LYMPHOCYTE CELL ‘MEMORY’ AGAINST BACTERIAL INFECTION A Monahan, A Dubois, R, Allen, R Ingram Queen’s University Belfast, Centre for Infection and Immunity, Health Science Building, 97 Lisburn Rd, Belfast, BT9 7AE. Recently, the definition of immunological memory has had to be re-addressed. The immune system has traditionally been compartmentalised into innate responses, constituting the early response to pathogens, which is non-specific and does not alter with subsequent exposures. Contrastingly, the T and B cells of the adaptive immune response take longer to respond on first exposure, but a pool of memory cells develop allowing enhanced specific responses on subsequent exposure. However, this dogma had to be revised following the demonstration that Natural Killer (NK) cells develop memory responses to viral infection. This phenomenon has yet to be investigated in anti-bacterial responses. Furthermore, innate lymphoid cells (ILCs) have recently been expanded beyond the archetypal NK cells and a diverse range of subsets of ILCs, paralleling the subsets of Helper T cells, have been identified. Whilst the full range of functions of these ILC subsets has not been fully elucidated it is clear they play a key role in limiting the expansion of microorganisms. The potential of ILCs to develop memory, emulating classical NK cells has not previously been investigated. To assess the ability of ILCs to develop enhanced ‘memory’ responses to bacterial infection, either ‘memory’ ILCs (from mice immunised with heat-killed Pseudomonas aeruginosa 10 days previously) or naive (sham immunisation) were isolated from the spleen by magnetic separation (>95% purity). The ILCs were transferred to unexposed hosts which were challenged with two times the lethal dose of P. aeruginosa. Transfer of ‘memory’ ILCs resulted in a significant reduction in bacterial CFUs in the peritoneal fluid (p = 0.006) and, notably, prevented systemic spread. Thus, primed ‘memory’ ILCs have augmented response against a bacterial pathogen. When ‘memory’ ILCs were purified from an IL-17 KO mouse their protective ability is lost. This suggests the rapid clearance is IL-17 dependent, indicating a role for ILC3 cells. In addition to reduced bacterial CFU, there are also suppressed levels of inflammatory cytokines in ‘memory’ ILC treated mice. This is presumably due to bacterial clearance and induction of resolution as increased neutrophil infiltration into the peritoneal cavity of ‘memory’ ILC treated mice was observed, consistent with the dependence on IL-17. To establish if this enhanced ‘memory’ response was simply as a result of activation of the ILCs, as has been previously reported for NK cells, ‘memory’ was induced by immunisation with either heat-killed Staphylococcus aureus or P. aeruginosa. Whilst the P. aeruginosa ‘memory’ cells were able to significantly reduce P. aeruginosa infection, the ‘memory’ cells induced with S. aureus were not. This demonstrates pathogen specificity in the ILC ‘memory’ response, opening up the potential to exploit this response for vaccine development. We have demonstrated that transfer of primed ‘memory’ ILCs into a previously unexposed host results in a significant enhancement of the innate immune system’s ability to clear bacterial infection. These findings have

the potential to have far reaching medical implications. Understanding the role of ILCs in protective memory responses to bacterial infection could revolutionise vaccine development and allow the design of novel vaccines ability to induce rapid immunity and induce effective immunity in typical difficult to immunise cohorts (neonates, elderly, immunocompromised). ROLE OF INNATE ANTIMICROBIAL MECHANISMS IN INTRACELLULAR SURVIVAL OF M. AVIUM SUBSPECIES AVIUM IN CHICKEN CELLS Nawzat A. Issa1, Mohammed shukur1, Sabine Tötemeyer1, Paul Barrow1, Michael A. Jones 1 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington LE12 5RD. Avian mycobacteriosis is a chronic infectious disease of poultry caused by different mycobacteria belonging to the M. avium-intracellular complex (MAC) and primarily M. avium subspecies avium. Data on the interaction between M. avium subspecies avium and host cells with which the pathogen interacts (chicken macrophages) at the level of innate antimicrobial mechanisms are extremely limited. Therefore, the present study aims to investigate the role of nitric oxide (NO) and reactive oxygen species (ROS) in intracellular survival of M. avium isolates from chicken and calf using cultured avian macrophage-like cells line HD11. By using NO and ROS stimulators and inhibitors agents this study showed that the activated HD11 cells with rchINF-y significantly inhibited the growth of the bacterial strains and enhanced the production of NO. However, reactive oxygen species have been reported to restrict the growth of bacterial organisms, in this study it has been seen that M. avium grow well in oxidizing environment and the bacterial infection causes the infected macrophages produce more ROS. Moreover, the antioxidants, N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI) inhibit the bacterial growth particularly at 48h PI. The present data described in detail the role of innate antimicrobial mechanisms in intracellular survival of M. avium in chicken cells. We found that intracellular survival of M. avium within in the host cells is greatly reduced by NO production and ROS inhibition. The project is funded by the Ministry of Higher Education and Scientific Research in Kurdistan region/ Iraq. Poster Presentation Abstracts PLAYING WITH FIRE: AN AGENT-BASED MODEL OF FEVER AND OTHER SYSTEMIC STRESSORS E. K. LeGrand, J. Day Department of Biomedical & Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, 37996 USA The mechanism by which fever, anorexia, and other costly components of the acute-phase response function in host defense has been contentious. The immune brinksmanship model states that these are host-initiated stressors that harm the pathogens relatively more than the host. Pathogens’ virulence is related to their high replication rate, but this rapid replication is also a basic vulnerability to stressors. Replication’s requirements for extra nutrients and energy, along with the vulnerability associated with synthesis, present a window of opportunity for host-derived non-specific stressors to serve as defenses. By only assuming that the pathogens require less energy to replicate than do host cells and that the host can monitor pathogens and its own status, we used the NetLogo computer simulation platform to develop a simple host-pathogen agent-based model. In our model we found that: 1) systemic non-specific stressors can preferentially harm and eliminate rapidly replicating pathogens, 2) application of the stress/harm should be applied rapidly (and relieved rapidly to prevent excessive self-harm), and 3) that the use of non-specific systemic stress as a defense is costly in terms of energy and risk. CHARACTERIZATION OF NATURAL AND ADAPTIVE FOXP3 REGULATORY T CELLS IN HUMAN VISCERAL LEISHMANIASIS A. C. A. SALMERON1; J. V. GALVÃO; G. R. MONTEIRO; S. M. B. JERONIMO; T. S. L KEESEN1

1- Biotechnology Centre of Federal University of Paraíba - Cidade Universitária- João Pessoa - PB – BRAZIL. Post Code: 58051-900 Correspondence author: tat.keesen@cbiotec.ufpb.br Leishmaniasis is an infectious disease caused by several species of protozoa of the genus Leishmania. Among the clinical forms of the disease, the most severe is visceral leishmaniasis (VL), whose main symptoms include fever, hepatosplenomegaly and anemia, and can be fatal if not treated correctly. The immune response to infection by Leishmania species may be associated to both exacerbation of the immune system and to infection control of the disease. These differences may be related to the existence of different sub-populations of immune cells of which monocytes and T cells may have a crucial role in this picture. In visceral leishmaniasis the severity of disease is associated with high IL-10 production by innate cells as monocytes. Regulatory T cells (Tregs) expressing CD4+CD25+FOXP3+ can also be an important source of cytokines, as IL-10, TGF-beta and IL-17 and these cytokines may modulate the innate and adaptive immune response in several diseases. However, the profile of the regulation and the possible mechanisms used by regulatory T cells have yet not been demonstrated in human visceral leishmaniasis. Thus, our goal was to characterize the natural (TregN)

and adaptive (TregA) regulatory T cells in human visceral leishmaniasis and to evaluate specific markers and cytokines produced by them. To reach this aim, whole blood from VL patients were collected and TregN and TregA subpopulations were evaluated after specific Leishmania antigen stimulation (SLA). Furthermore, Treg cells were labeled to differentiate Treg subpopulations (natural- CD4+CD25+FOXP3+CD45RA+ and adaptive -CD4+CD25+FOXP3+CD45RA-) and within each Treg subpopulations the level of IL-10, IL-17 and CTLA-4 were determined using flow cytometry. Our results showed that: 1) an increase in CD4+CD25+CD45RA- T cells after SLA stimulation as compared to CD4+CD25+CD45RA+ T cells in VL patients; 2) there is no difference in FoxP3 expression between Natural Tregs (TregN) and Adaptive (TregA), stimuli-independent; 3) Naturally, TregN produces more IL-10 than the TregA, stimuli-independent; 4) SLA no induced differences in the level of cytokine IL-17 in both subpopulatons; 5) Lastly, SLA induced a higher expression of CTLA-4 in TregN cells , but not induced changes in TregA. These data suggest that TregN cells may be involved in the modulation of innate immune mechanisms through cell-cell contact (CTLA-4 and IL-10 production) in VL patients and that this modulation may direct the immune response in this disease. Supported by: CNPq, Fapern-RN EMR2 RECEPTOR LIGATION INDUCES MATURATION AND INFLAMMATORY ACTIVATION OF MACROPHAGE Guan-Yu Yi, Yi-Shu Huang, Ching-Hsun Hu and Hsi-Hsien Lin Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan. Email: hhlin@mail.cgu.edu.tw. EMR2 is a myeloid cell-restricted member of the adhesion-class G protein-coupled receptors (adhesion-GPCRs) involved in the cellular migration and activation of human neutrophils. In addition to neutrophils, EMR2 is also highly expressed in human monocytes and macrophages. However, little is known of the function of EMR2 in monocytes/macrophages. In this study, we show that EMR2 expression in THP-1 cells is upregulated following macrophage differentiation and maturation. Interestingly, ligation of EMR2 receptor by a specific 2A1 mAb not only promotes THP-1 cell differentiation, but also induces THP-1 cell activation, leading to the production of inflammatory mediators such as IL-8, TNF-, and MMP-9. EMR2 gene knock-down by siRNA confirms the 2A1-induced production of inflammatory mediators is mediated specifically by EMR2. EMR2 activation by receptor ligation induces the activation of Akt, extracellular signal-regulated kinase (ERK), and NF-B in THP-1 cells. As expected, EMR2 receptor-induced signaling and inflammatory responses are inhibited specifically by relevant signaling inhibitors. Taken together, our results indicate that EMR2 receptor plays a role in the inflammatory activation of innate immune cells. DECITABINE INHIBITS MDSC POPULATION IN TUMOR-BEARING MICE

Jung-Ah Cho*1, Ho Park2, Tae-joo Kim, Young-Joo Kim, Hye-Kyung Yoon, Yong-Wook Lee, Kyung-Hwa Lee, Jung-Hee Lee, and Seung-Yong Seong 1#324, Biomedical Science Building, Seoul National University College of Medicine, 28 Yongon-dong, Jongno-gu, Seoul 110-799, Republic of Korea 2Sungkyunkwan University School of medicine, Kangbuk Samsung hospital, 101 Pyung-dong, Jongno-gu, Seoul 110-746, Republic of Korea Corresponding Author Details: Seung-Yong Seong (seongsy@snu.ac.kr), M.D., Ph.D., Professor (Department of Microbiology and Immunology, Department of Biomedical Sciences), Director (Wide River Institute of Immunology, Seoul National University)

Decitabine is a cytosine analog that is used as a chemotherapeutic agent for treatment of myeolodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Due to its well-known effect on myeloid lineage cell disorder, we examined a potential role of decitabine on myeloid-derived suppressor cells (MDSCs) in tumor-bearing mice. Tumor-bearing mice were intraperitoneally injected with the different doses of decitabine (0, 0.125 or 0.25 mg/kg). Analysis of immune cell population in spleen revealed the significantly reduced cell number of CD11b+Gr-1hi MDSCs in contrast to the significantly increased cell number of CD3+CD8+ T cells in mice injected with decitabine at a dose of 0.25 mg/kg. Further investigation for the subtype of the CD11b+Gr-1hi cells demonstrated that the decreased population was CD11b+Ly6g+ rather than CD11b+Ly6c+. Consistent with the in vivo data, in vitro assay using the different doses of decitabine (0, 10, 100 and 1000 nM) showed the specific reduction of CD11b+Gr-1hi cell number. To define whether the suppressive effect of decitabine on splenic MDSCs might arise from reduction of MDSCs in bone marrow or apoptosis induction, we performed annexin V staining for MDSCs from spleen and bone marrow of tumor-bearing mice and discovered that decitabine induced apoptosis of splenic MDSCs but not bone marrow MDSCs. These results demonstrate that decitabine eliminate MDSCs and increase T cells simultaneously in spleen at much lower dose than gemcitabine, suggesting that decitabine can greatly improve outcome of cancer immunotherapy.

IMMUNE RESPONSE GENE EXPRESSION PROFILING IN A P. AERUGINOSA LIPOPOLYSACCHARIDE-INDUCED MODEL FOR INFLAMMATION OF THE RAT TESTIS M.A. Palladino and G.A. Fasano Monmouth University, Department of Biology, Edison Science Hall Room E-154, 400 Cedar Avenue, West Long Branch, NJ, USA mpalladi@monmouth.edu Inflammation of the male reproductive tract by microbes contributes to male subfertility and infertility. Experimentally, bacterial pathogens are known to suppress androgen production as one example of the physiologic effects of inflammation on the testis. Members of the Toll-like receptor family (TLRs), defensin family and other receptors involved in innate and adaptive immunity are expressed in the testis and other male reproductive organs. Yet relatively little is known about molecular mechanisms involved in the detection and clearance of invading microbes and of genetic deficiencies that may render individuals susceptible to microbial insult and chronic inflammation of the male reproductive tract. The goal of this work was to determine the effects of lipopolysaccharide (LPS)-induced inflammation on innate and adaptive immune response gene expression pathways of the rat testis. Inflammation in Sprague-Dawley rats was accomplished via i.p. administration of LPS from P. aeruginosa (5 mg/kg body weight) for 3 or 6 hours. RNA was isolated from testes and cDNA synthesized for analysis by qPCR. The RT2 Profiler™ PCR Array Rat Innate and Adaptive Immune Responses Signaling Pathway (Qiagen) was used to evaluate expression of 83 genes. Only genes showing a 3-fold (up-regulated or down-regulated) change in expression with a p-value of <0.05 were categorized as statistically significant. Array results demonstrated that 11 genes (Ccl12, Cc13, Cd14, Cxcl10, Icam1, Il10, Il1b, Il6, Nfkbia, Tlr2, Tnf) were up-regulated after 3 hours of LPS-induced inflammation and expression of 6 genes (Ccl12, Cc13, Cd14, Nfkbia, Tlr2, Tnf) remained elevated after 6 hours. Five genes were up-regulated at 6 hours only (C3, Jak2, Nlrp3, Slc11a1, Tlr1). No genes were down-regulated after 3 or 6 hours following LPS administration. Genes involved in cytokine-mediated signaling comprised a major functional category of genes up-regulated in the testis following LPS-induced inflammation. In the future, specific functions of these genes will be investigated to elucidate the molecular mechanisms involved in the immune response to LPS in the testis. VASOACTIVE INTESTINAL PEPTIDE MODULATES CYTOKINE PRODUCTION BY SALMONELLA-INFECTED HUMAN MONOCYTES Basim Askar, Paul Barrow, and Neil Foster Corresponding author: Basim Askar, Animal Infection and Immunity, School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, LE12 5RD, UK. Telephone: +44-01159516469, E-mail: svxba@nottingham.ac.uk. Abstract Systemic salmonella infection is a frequent cause of Gram negative sepsis. Upon bacterial lipopolysaccharide (LPS) recognition, by CD14 and MD-2 molecules, mononuclear cells are activated via theTLR4 pathway. This results in down-stream activation of transcription factors such, as NF-κB, AP-1, MPAK and IFR-3, which transcribe inflammatory cytokine genes. In blood, LPS-activated monocytes can produce very high concentrations of inflammatory cytokines over prolonged periods of time. This can result in a so-called ‘cytokine storm’ which may, eventually, result in organ collapse and death. The mortality rate due to sepsis remains high and often occurs even after clearance of bacteria by anti-biotic intervention, due to sustained (un-regulated) production of inflammatory mediators. Therefore, anti-inflammatory therapy as an adjunct to antibiotics could reduce the mortality rate of sepsis. To date, several studies have assessed the therapeutic role of vasoactive intestinal peptide (VIP) both in vivo and in vitro since it possesses several desirable biological properties. VIP has been shown to suppress pro-inflammatory cytokines, alleviate histopathological changes and prevent mortality in mice rendered septic by LPS administration. However, nothing is known about the effect of VIP on of pro-inflammatory cytokine production in human monocytes challenged with virulent Salmonella (rather than LPS alone). The aim of the current study, therefore, was to investigate the effect of VIP on the production of inflammatory cytokines in human monocytes infected by S. Typhimurium 4/74. Our finding shows that freshly isolated human monocytes produce pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6 and also anti-inflammatory cytokines such as IL-10 and IL-4 following Salmonella challenge. However, incubation of infected monocytes with VIP (10-7 M) significantly decreased (P <0.05) production of IL-6, TNF-α, IL-1β but markedly increased production of anti-inflammatory IL-10. In conclusion, our study suggests that VIP may have potential as an anti-inflammatory adjunct during Gram negative sepsis PREBIOTIC AND PROBIOTIC AGENTS ENHANCE IMMUNE RESPONSES TO SALMONELLA TYPHIMURIUM INFECTION IN PIGS Ibrahim Naqid1, Ben Maddison2, Roberto La Ragione3 and Kevin Gough1

1School of Veterinary Medicine and Science. The University of Nottingham, Sutton Bonington Campus, Leicestershire, LE12 5RD. Mobile: +447517125549 Email: plxin1@nottingham.ac.uk 2ADAS UK, School of Veterinary Medicine and Science. The University of Nottingham, Sutton Bonington Campus, Leicestershire, LE12 5RD 3School of Veterinary Medicine, DK Building, University of Surrey, Guildford, Surrey. GU2 7XH. Abstract Background and Aim: Salmonellosis is one of the most important zoonotic bacterial pathogens in humans and Salmonella infections are often linked with consumption of contaminated pork. Salmonella enterica serovars Typhimurium and Enteritidis represent a major source of food poisoning in humans with the most important reservoirs of infection being contaminated meat and eggs. The aim of this study is to evaluate the effects of probiotic, prebiotic, and synbiotic diet regimes on antibody based immune responses to Salmonella Typhimurium infection in pigs. Materials and methods: Enzyme-linked immunosorbent assay (ELISA) was used in this study to examine the humoral immune response in 24 pigs challenged with S. Typhimurium. Piglets were weaned at 28 days of age and then given one of four different diets: a probiotic diet (Lactobacillus Plantarum B2984 strain, a prebiotic diet (lactulose) or synbiotic diet (both lactulose and Lactobacillus Plantarum) and control group (no addition to the diet) one week before challenge. Results: The data demonstrate that the inclusion of the probiotic Lactobacillus plantarum B2984 in the diet of piglets (~1 x 1010cfu/animal/day) enhanced serum IgG (p<0.001), IgM (p<0.001), and IgA (p=0.01) responses to S. Typhimurium. Similarly, inclusion of the prebiotic lactulose at 1% (w/w) of the feed on a daily basis in the diet enhanced serum IgG (p<0.01), IgM (p<0.01) responses to S. Typhimurium. Inclusion of both additives in the synbiotic diet, however, elicited a significant interaction when considering the immune responses to S. Typhimurium (IgM, p=0.004; IgA and IgG, p<0.001 for interaction); the benefits of isolated pre or probiotic administration with respect to immune responses were considerably reduced. Conclusions: The data support the use of Lactobacillus plantarum B2984 or lactulose as strategies to contribute to the protection of weaned piglets from zoonotic bacterial pathogens, but caution must be taken when combining dietary supplements as combinations can interact. ROLE OF NKT CELLS IN HUMAN VISCERAL LEISHMANIASIS Tatjana S.L. Keesen1; Joanna Gardel Valverde; Glória Regina Góis Monteiro; Bruna Leal Lima Maciel; Selma Maria Bezerra Jeronimo 1- Biotechnology Centre of Federal University of Paraíba - Cidade Universitária- João Pessoa - PB – BRAZIL. Post Code: 58051-900 Correspondence author: tat.keesen@cbiotec.ufpb.br

Although NKT cells are a small subpopulation in the peripheral blood, they are presumed to play a role in early stages of infection against various pathogens including protozoa. NKT cells are among cell types that link innate and acquired immunities and are involved early response to infection stimulating other cell types. Moreover, NKT cells are considered effectors cells due to cytokines and cytotoxic molecules produced by them sharing features with other conventional T lymphocytes. However, the role of NKT cells in visceral leishmaniasis (VL) is still not fully understood. Our goal was to determine activation status and cytokine profile of NKT cells in symptomatic VL due to Leishmania infantum infection. We studied VL patients (n=9) at diagnosis and compared them to positive (n=9) and negative (n=6) endemic controls (PosEC and NegEC) and non endemic controls (NonEC) (n=6). Furthermore, we also analyzed the same VL patients after 14 months of treatment (n=5). Whole blood assay was used and the frequency of NKT cells (CD3+6B11+), CD69 activation marker and the intracellular level of IFN- -17 and IL-10 were determined by flow cytometry. Ex vivo analyses showed that the total frequency of NKT cells in VL patients after 14 months of treatment was higher than all groups studied, and these cells presented a highly activated profile when compared to NegEC, PosEC and during symptomatic VL disease. Interestingly, we found that leishmanial antigens induced a decrease of IFN- VL patients and that NonEC presented the highest frequency of IFN-changes in IL-17 and IL-10 in NKT cells. However, after 14 months of treatment, VL patients presented higher frequencies of IL-17 than IFN- -10 within NKT cells. Lastly, the there was a negative correlation between parasite load and IFN-These findings suggest that NKT cells may be an important source of study in immune response of VL disease and this response seems to be impaired during symptomatic VL.

HUMAN THROMBOSPONDIN1 (TSP1) IS, A NEGATIVE REGULATOR FOR COMPLEMENT SYSTEM, PROMOTING DEFENCE FOR BACTERIAL PATHOGENS FROM COMPLEMENT SYSTEM OF INNATE IMMUNITY in vitro

Ameen S. S. Alwashmi1’2, Youssif M. Ali3, Nicholas J. Lynch1, Dimitrios Goundis4 and Wilhelm J. Schwaeble1 1Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK. ²Department of Medical Laboratories, College of Applied Medical Science, Qassim University, Buraidah, KSA. ³Department of Microbiology, Faculty of Pharmacy, Mansoura University, Egypt. 4The Medicines Company, New Jersey, USA Author’s Email: Ameen S. S. Alwashmi, asa31@le.ac.uk Author’s postal address 10 Mayflower Road, LE5 5QD, Leicester, Leicestershire, United Kingdom (UK) Abstract Recent research has highlighted physiologically meaningful links between Thrombospondin1 (TSP1), an extracellular matrix protein (ECM) essential for platelet activation and thrombus formation, and pathogen-host interactions with regards promoting adherence for Gram-positive pathogens to human endothelial and epithelial cell lines connecting the peptidoglycan of pathogens with host cell receptors (9). Here we report a novel role for TSP1 not only interact with host-pathogens but also exhibits negative regulation activity on alternative pathway (AP) and lectin pathway (LP) of complement activation. Since activation of inflamed cells caused by vessel injury or stress can trigger TSP1 release, the negative regulatory activity of TSP1 on the alternative pathway amplification loop may help to comprehend the mechanism of such pathogens in order to keep the infection under control, increasing bacterial opportunity to escape from innate immunity causing colonisation on host cells and dissemination to peripheral blood stream. Binding assay of ELISA was established to monitor TSP1 binding ability on such Gram-positive and Gram-negative bacteria including Streptococcus pneumoniae (capsulated D39), Staphylococcus aureus (DSM20233), capsulated and non-capsulated of Neisseria meningitidis group A (Z2491), B (MC58). Since peptidoglycan of Gram-positive bacteria found to adhere to TSP1 (9), it is also we demonstrate TSP1 to attach to capsulated and non-capsulated pathogens including Gram-negative bacteria and then provide protection from complement system via down-regulating the LP or AP activity by either interfering with C3 convertase complex (C3bBb) or competing Mannose-Binding-Lectin (MBL) by blocking N-acetyl-D-glucosamine presented in polysaccharide residual binding sites. Different ELISA were established to monitor complement activation initiated by either Mannan or Zymozan (polysaccharide-ligand) representing capsulated pathogens in vitro after adding recombinant exogenous TSP1 in dose-dependent manner in human serum. While incubating the exogenous TSP1 with serum onto coated mannan or zymozan on 96-well plate, TSP1 bound to the AP convertase complex formed of the complement activation products C3b and factor B and acted as a negative regulator of complement activation by competing for the binding of the complement component properdin, a positive regulator of the alternative pathway. When recombinant properdin was added, with or without TSP1, we demonstrated that the potent positive regulatory activity of properdin, which led to a significant increase of C3 deposition on mannan, was effectively blocked by TSP1 in a concentration dependent manner. We also demonstrate a direct binding of TSP1 to purified complement factor B suggesting TSP1 function to bind to C3 convertase of the alternative pathway. Interestingly, properdin is entirely composed of thrombospondin-like domains with a high degree of similarity to those of TSP1. This function of TSP1 led to a significant drop in C3 activation on mannan. In addition, TSP1 bound to mannan and down regulated MBL dependent activation of the LP. In conclusion, we propose a so far unknown physiological role of TSP1 as a negative regulator of properdin-mediated promotion of AP activity, adding a new example of how such ECM function on either bacterial surface or at the site of infected injury promoting the defence to self-inflamed cells or pathogens from complement system activation. Key Words: Lectin Pathway (LP), Alternative Pathway (AP), Thrombospodin1 (TSP1), Extracellular Matrix (ECM), Mannose-Binding-Lectin (MBL). Acknowledgements

I thank Dr Christopher D.Bayliss, Genetics department, University of Leicester, England to provide different strains of Neisseria meningitidis, and also I greatly appreciate with thank to both Dr Robert B Sim, MRC Immunochemistry Unit, Oxford University, England of providing purified human factor B, and Dr. Russel Wallis, Infection, Immunity and Inflammation department, University of Leicester of providing purified human MBL. This work granted and supported from Qassim University (Qassim region, Kingdom of Saudi Arabia, KSA) where granted my PhD scholarship at University of Leicester, and also from The Medicines Company, New Jersey, USA to grant support to W.J.S. Reference

1. N. BAENZIGER et al. Proc Natl Acad Sci, 68, 240-3. (1971) 2. J.W. Lawler et al. The Journal of biological chemistry, 253, 23, 8609. (1978) 3. D.D. Roberts et al. Cellular and Molecular Life Sciences, 65, 5, 667-671. (2008) 4. U. WIRTHMUELLER et al. J Immunol, 158, 4444-51. (1997) 5. GOUNDIS, D. & REID, K. B. Nature, 335, 82-5 (1988). 6. W. J.SCHWAEBLE et al. Immunol Today, 20, 17-21.(1999) 7. BONNEFOY, A., et al. Cell Mol Life Sci, 65, 713-27 (2008). 8. SWITALSKA, H. I., et al. J Lab Clin Med, 106, 690-700 (1985). 9. RENNEMEIER, C. et al. FASEB J, 21, 3118-32 (2007).

EFFECT OF COMMERCIALLY AVAILABLE PLANT DEFENCE STIMULATORS (PDS) ON HUMAN INNATE IMMUNITY Lény TEYSSIERa,c, Stéphanie DELEMASURE-CHALUMEAUb, Patrick DUTARTREb, David WENDEHENNEc, Olivier LAMOTTEc, Jean-Louis CONNATa,b a: INSERM U866 Lipide Nutrition Cancer, Université de Bourgogne, LPPCM, Facultés de Médecine et Pharmacie, 7 Bd Jeanne d’Arc, 21079 Dijon Cedex, France b: COHIRO Biotechnology, UFR de Médecine de Dijon, Facultés de Médecine et Pharmacie, 7 Bd Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France c: CNRS UMR 1347 Agroécologie, NO et réponse de défense des plantes, 17 Rue Sully, 21000 Dijon, France PDS (Plant defence stimulators) constitute a recent alternative to pesticides used for crop protection. These compounds called elicitors are of diverse nature, but they all act by stimulating innate immune system of plants. So, plants can better fight pathogens. Furthermore, there are many similarities in pathogen perception systems and cellular signalling in plants and animals. It is well established that many elicitors stimulate both human and plant innate immunity (Zipfel and Felix, 2005). Therefore, it is likely that human innate immunity could be modulated by PDS. The aim of this study is to evaluate pro/anti-inflammatory activity of five different commercially available PDS on human cell models. We studied the pro/anti-inflammatory effect of PDS (Bion® 50WG, Stifenia...) on human peripheral blood mononuclear cells (PBMC). These cells are exposed during twenty hours to various concentrations of PDS or their corresponding active molecules. Pro-inflammatory action is evaluated by measuring the quantity of the inflammatory cytokine IL-1β in the cells supernatants using ELISA test. To study anti-inflammatory effect, we used PBMC treated with LPS to trigger a basal inflammatory response. We then checked if PDS delivered at the same time as LPS modified IL-1β production. In addition, in all the experiments, the viability is evaluated with a XTT test. PDS, which were however used at equal or lower concentrations than in the fields, show different profiles in terms of cytotoxicity and inflammatory modulation. For example, Stifenia was slightly cytotoxic at 1 mg/ml and pro-inflammatory at 0,3 and 1 mg/ml concentrations. Conversely, Bion® 50WG at dosages from 0,3 mg/ml dose-dependently inhibited IL-1β production and proved to be anti-inflammatory. Interestingly, some active molecules have not the same inflammatory profile than their respective formulated PDS from the market. Our results indicate that PDS can differently interact with human innate immunity. We hope to use these particularities to better understand the innate immunity pathways that could be common in plants and animals. TLR4 PLAYS A PIVOTAL ROLE IN MONOCYTE ADHESION TO VASCULAR ENDOTHELIUM

Seung Jin Lee, Kyo Won Seo, Jin Ung Bae, Chi Dae Kim Department of Pharmacology, School of Medicine, Pusan National University, Yangsan, Gyeongnam 626-870, Rep of Korea

Toll-like receptor 4 (TLR4) is known to mediate monocyte adhesion to endothelial cells, however, its role on the expression of monocyte adhesion molecules is unclear. In the present study, we investigated the role of TLR4 on the expression of monocyte adhesion molecules, and determined the functional role of TLR4-induced adhesion molecules on monocyte adhesion to endothelial cells. When THP-1 monocytes were stimulated with Kdo2-Lipid A (KLA), a specific TLR4 agonist, Mac-1 expression was markedly increased in association with an increased adhesion of monocytes to endothelial cells. These were attenuated by anti-Mac-1 antibody, suggesting a functional role of TLR4-induced Mac-1 on monocyte adhesion to endothelial cells. In monocytes treated with MK886, a 5-lipoxygenase (LO) inhibitor, both Mac-1 expression and monocyte adhesion to endothelial cells induced by KLA were markedly attenuated. Moreover, KLA increased the expression of mRNA and protein of 5-LO, suggesting a pivotal role of 5-LO on these processes. In in vivo studies, KLA increased monocyte adhesion to aortic endothelium of wild-type (WT) mice, which was attenuated in WT mice treated with anti-Mac-1 antibody as well as in TLR4-deficient mice. Taken together, TLR4-mediated expression of Mac-1 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium, leading to increased foam cell formation in the development of atherosclerosis. Day 2: Investigating interactions of the Innate and Adaptive Immune Systems Invited Speakers Abstracts The role of gamma delta T cells in melanoma and the effects of bisphosphonate therapy. Dr. Katie Lacy, Consultant Dermatologist, Biomedical Research Centre/NIHR Clinical research consultant, Honorary Senior Lecturer KCL, UK Vγ9Vδ2 gamma delta T cells are unconventional T lymphocytes that exert anti-tumour cytotoxicity and are activated by bisphosphonates. Following recent advances in melanoma treatment with immunotherapy, we have investigated the role of Vγ9Vδ2 T cells in melanoma and the effects of bisphosphonate treatment. Although Vγ9Vδ2 T cells are present in the peripheral blood of all patients and in some cutaneous metastases, Vγ9Vδ2 T cells within metastases have reduced cytotoxicity. Despite evidence for in vitro efficacy, bisphosphonate treatment in patients appears to deplete theVγ9Vδ2 T cell subset. Further work is required to establish the potential for bisphosphonates in melanoma treatment. B-cells at the interface between innate and acquired immunity to bacterial pathogens Dr Pietro Mastroeni, Reader in Infection and Immunity, University of Cambridge, UK The engenderment and expression of T-cell and B-cell effector functions in bacterial infections is a complex process that involves subtle cellullar interactions, production of qualitatively adequate antibody responses and the cross talk between innate and acquired immunity. B-cells contribute to immunity to Salmonella via the production of opsonising antibodies that via FcRs enhance the anti-microbial functions of phagocytes. B-cells are also essential for the engenderment of acquired T-cell immunity to bacterial pathogens such a Salmonella. Both "innate" functions (i.e. TLRs and cytokines) and "acquired immunity" functions (e.g. antigen presentation, BCR engagement) are necessary for the activation of anti-Salmonella Th1 immunity by B-cells. The MHC class II-associated invariant chain controls innate and adaptive immunity in B cells Dr Bénédicte Manoury, PI, DR2, CNRS, Hôpital Necker; Unité INSERM U1151, Paris, France Intracellular Toll-like receptors (TLR3, 7 and 9) localize in endosomes and sense nucleotides from viruses and bacteria. Increasing evidences describe a cross talk between proteins that regulate both innate and adaptive responses. For example, UNC93B1, a chaperone essential for intracellular TLRs trafficking also interferes with the MHCII antigen presentation pathway and MHCII molecules were recently described to regulate TLR9 signaling. Here, we show, that in B cells, TLR7 resides in the lysosomes together with the MHCII molecule and the invariant chain (Ii). Following stimulation by TLR7 ligand, Ii specifically interacts with TLR7 and its adaptor molecule MyD88. Surprisingly, in the absence of Ii chain or when its expression is down regulated upon viral infection, TLR7 innate immune response but not adaptive is boosted. This suggests a new role for Ii chain by acting as a negative regulator of TLR7 signaling in B cells Sepsis as a Model of Impaired Communication between the Innate and Adaptive Immune Systems Dr James D. Faix, Director of Chemistry & Immunology, Director of Point-of-Care Testing, Department of Pathology, Stanford University Medical Center, Stanford, USA Sepsis is an unusual systemic reaction to what is sometimes an otherwise ordinary infection and it probably represents an abnormal pattern of response by the immune system to injury. An exuberant pro-inflammatory response on the part of the innate immune system is followed by immunosuppression of the adaptive immune

response, leaving the patient susceptible to nosocomial infection. Although the pathogenesis of sepsis is poorly understood, clues from experimental models and studies of the pattern of expression of cytokines and cell-surface markers in human patients point to a failure of appropriate communication between the two arms of the immune system. B cells and tumours: novel insights into tumour-induced immune escape Dr Sophia N. Karagiannis, Senior Lecturer in Translational Cancer Immunology, Head of Cancer Antibody Discovery and Immunotherapy, St John’s Institute of Dermatology, Division of Genetics and Molecular Medicine, King’s College London School of Medicine, UK The cross-talk between humoral immunity and tumours is largely unappreciated, but similarly to T cells, B cell and antibody responses are influenced by the presence of cancer and may be modulated to promote tumour growth or are rendered less potent in activating effector cells such as monocytes/macrophages. These mechanisms may prevent host immunity from mounting effective anti-tumoural responses. This talk will focus on the nature of B cells in tumour microenvironments and in the circulation and on the mechanisms of Th2-biased imflammation that may promote polarized production of antibodies. The findings will be discussed in the context of new opportunities for therapy approaches to counteract or bypass tumour-induced immune escape and for discovering novel biomarkers to inform patient stratification and treatment. Immunomodulatory networks controlling cancer metastatic dissemination Dr Victoria Sanz-Moreno, Head of the Tumour Plasticity lab, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, King's College, London, United Kingdom Our group is working on identifying molecular cues that will aid in tumour progression and metastatic dissemination, with a special interest in melanoma. Tumour cells use a family of proteins called Rho GTPases to regulate their cytoskeleton, therefore these proteins play a key role in regulating tumour initiation and tumour dissemination. We combine “OMICs”, state of the art microscopy in 3D matrices, molecular biology and patient information to identify molecular determinants driven by Rho GTPase signalling that correlate with melanoma progression and metastatic potential. At the same time, we are very focused on identifying novel therapeutic targets. Through OMICs approaches we have identified several Networks that drive tumour promoting inflammation and regulated by Rho GTPases. These networks impact tumour-stromal communication and offer attractive possibilities for cancer therapeutics. Understanding the roles of innate and adaptive immunity in the mechanisms of action of immunomodulatory antibodies. Dr Stephen A Beers, Antibody and Vaccine Group, Faculty of Medicine, University of Southampton, UK Recent clinical responses observed with immunomodulatory antibodies have reinvigorated the belief that the immune system holds the key to controlling cancer. Initial studies suggested that these agents directly target T cells or APCs to ‘release the brakes’ or ‘press the accelerator’ leading to productive immunity. It is now clear that this is too simplistic and the wide cellular distribution of immunomodulatory molecules provides for the activation of a range of innate and adaptive effectors. Understanding the relative contributions of innate and adaptive immunity to the mechanisms of action of immunomodulatory antibodies is key to unlocking their full potential as drugs. Platelets as immune cells Dr Fabrice Cognasse, Senior Scientist, Deputy Director for Scientific Affairs, the Auvergne-Loire Regional Branch of the French National Blood System EFS, Etablissement Français du Sang Auvergne-Loire, France Platelets could act as immune sensors, recognizing pathogens and therefore releasing immunomodulatory molecules. We demonstrated also that platelets could induce immune cell activation and modulate their functions. Our investigation discusses the extended role of platelets as immune cells to emphasize their interactions with infectious pathogens sensed as potentially dangerous. Furthermore my team investigate the platelet inflammatory response to transfusion context and to infectious triggering (HIV and SEPSIS). Finally, our work allows us to re-explore indications that platelets exert direct anti-infection immunity and we will present experimentally-driven arguments in favour of a role of platelet TLR in regulating certain immune activities. Oral Presentation Abstracts

NATURAL ANTIBODIES ARE INNATE IMMUNE RESPONSIVE Ding J. L., Panda S. and Ho B. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543. Email: dbsdjl@nus.ed.sg Natural antibodies such as natural IgG, which pre-exist in neonates and uninfected individuals, have remained a biological enigma due to our perception of their lack of antigen-specificity and low affinity for pathogens, when studied in isolation. The natural antibodies have been perceived to belong to the adaptive immune system. Although discovered five decades ago, their functional significance has remained unclear. Recently, we discovered that natural IgG plays a vital and immediate defense role by collaborating with plasma lectins such

as ficolin or mannose binding lectin. We describe how natural IgG crosstalks with ficolin-bound pathogen, bridging humoral to cellular immunity in an instant shortcut mechanism, to clear the invading pathogen via

-mediated phagocytosis. This process is non-specific to antigens; operates independently of the complement system, and it evokes a rapid pro-inflammatory response. AID knockout mice lacking natural IgG showed increased mortality during Pseudomonas infection. However, reconstitution of these mice with natural IgG conferred rapid and effective protection against systemic infection. We have characterized the molecular interaction between natural IgG:ficolin and delineated their contact points, revealing that infection-induced local acidosis and hypocalcemia conditions stimulate these two molecules to interact specifically. Bioactive peptides derived from the natural IgG and ficolin will prompt further development of immunomodulators, tunable to pH shifts in the infection-inflammation microenvironment. SULFORAPHANE EPIGENETICALLY REGULATES THE LPS-INDUCED KINETICS OF INNATE IMMUNE RESPONSE IN PORCINE MONOCYTE-DERIVED DENDRITIC CELLS Xueqi Qu1, Christiane Neuhoff1, Mehmet Ulas Cinar2, Maren Pröll1, Muhammad Jasim Uddin1, Dawit Tesfaye1, Ernst Tholen1, Christian Looft1 and Karl Schellander1

1 Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, 53115 Bonn, Germany 2 Departments of Animal Science, Faculty of Agriculture, Erciyes University, 38039 Kayseri, Turkey *Corresponding author: Prof. Dr. Karl Schellander Tel.: +49 228 732240 Fax: +49 228 732284 Email: ksch@itw.uni-bonn.de & xuqu@itw.uni-bonn.de Dendritic cell (DC) plays an essential role in detection of microbial attack in innate immune system. Activated DCs can induce acute inflammation and sepsis upon exposure to microbial if excessive signals occur. The epigenetic mechanisms involve in the development and differentiation of innate immune system, and consequently that of related pathologies (autoimmune diseases and hematological disorder), has advanced considerably in recent years. Histone acetylation is a key epigenetic modification controlling chromatin structure, DNA accessibility for transcription factor and gene expression. HDAC inhibitor sulforaphane (SFN) exhibits anti-oxidative, antimicrobial, anti-inflammatory, and anti-tumoral properties with inhibition of HDAC activity. In the present study we aimed to investigate the effects of SFN on the expression and kinetics of cytokine productions, we further analyzed the importance of SNF on the regulation of innate immune response with directly against the DCs maturation and enhancement of the phagocytosis in response to LPS stimulation through epigenetic modifications in porcine monocyte-derived dendritic cells (moDCs). The present work provides the first evidence that the SFN supplementation in vitro acts as the HDAC inhibitor in porcine moDCs with inhibition of HDAC activity. HDAC inhibitor SFN exerts profound dynamic administration on the moDCs innate immune antimicrobial against response in different pathogen challenging stages, down-regulated the expression of innate immune receptor, interfered with transcription factors remodeling and inhibited the expression of key antimicrobial cytokines and accessory molecules in the early stage after LPS stimulation. Conversely, SFN promoted the immune gene including cytokine expressions after 6 h LPS stimulation. Consistent with these dynamic immune-mediatory effects in LPS induction, we might suspect that SFN enhances the tolerance of moDCs and protects host cells from the inflammatory risk to bacterial infection. MODULATION OF IL-1 FAMILY CYTOKINES AND RECEPTORS DURING THE ACUTE AND CHRONIC INFLAMMATORY RESPONSE OF HUMAN MONOCYTES P. Italiani1, 2, E. Mosca1, R. Alfieri1, L. Milanesi1, and D. Boraschi2 1Institute of Biomedical Technologies (ITB), National Research Council (CNR), Segrate, Milano, Italy 2Institute of Protein Biochemistry (IBP), National Research Council (CNR), Napoli, Italy italianipaola@gmail.com IBP-CNR Via Pietro Castellino, 111 80131 Naples, Italy The IL-1 family encompasses eleven ligands and ten related receptors, all key players in different phases of acute and chronic innate/inflammatory response, both in the acute resolving inflammatory defence response, and in the persistent inflammation characteristic of chronic inflammatory diseases. Among IL-1 family ligands there are inflammatory and anti-inflammatory cytokines, including natural inhibitors/antagonists. Among IL-1 family receptors there are both activating, accessory and non-signalling “decoy” receptors, the latter being able to subtract the agonist ligands from interaction with activating receptors. The proper quantitative and temporal modulation of all these molecules underlies the physiological, resolving inflammatory defensive response, while an imbalance may result in the transition to chronic/pathological inflammation. The human innate/inflammatory response in a tissue can be modelled in vitro by culturing human normal monocytes isolated from blood in conditions that resemble the recruitment from blood into the inflamed site, then the encounter with the inflammatory agents, and eventually the conditions promoting tissue repair and homeostatic regulation upon resolution. In order to simulate all these events, CD14+ monocytes were exposed

sequentially in culture to CCL2 (from 0 to 2 h, 37°C, normoxia), LPS-displaying bacterial vesicles (from 2 to 14 h, 39°C, hypoxia), TNF-α (from 3 to 14 h, 39°C, hypoxia), IFN-γ (from 7 to 14 h, 39°C, hypoxia), IL-10 (from 14 to 24 h, 37°C, normoxia), and TGF-β (from 24 to 48 h, 37°C, normoxia). Moreover, by changing the culture conditions, we also reproduced the conditions of persistent/chronic inflammation, such as in rheumatoid arthritis, exposing CD14+ monocytes in culture to CCL2 (from 0 to 2 h, 37°C, normoxia), a cocktail of bacterial and viral stimuli (LPS, PDG, poly:IC, CpG, from 2 to 7 h, 39°C, hypoxia) and IFN-γ, ACPA-PA immune complexes, Survivin, M-CSF and GM-CSF (from 7 to 96 h, 39°C, hypoxia). The modulation of the IL-1 family members during the different phases of acute and chronic inflammatory responses of monocytes were profiled by transcriptome analysis using RNA-Seq. Expression of the genes for six cytokines (IL1B, IL1A, IL1RN, IL18, IL18BP, IL36G) and four receptors (IL1R1, IL1R2, IL1RAP, SIGIRR) are similarly regulated during the early phase of the acute and chronic inflammatory responses, with the peak of expression between 2 and 14 h. The expression of most of them returned to the low basal level during the resolution phase in the acute model (except IL18 that was lower than in basal conditions), while it was higher than in fresh monocytes in the chronic model from 24 h on. Exceptions are the genes of the two inhibitors IL18BP and SIGIRR. The former significantly increased from 24 to 96 h, while the latter decreased throughout the entire inflammatory reaction. The GSEA analysis revealed that in both models the expression of all the IL-1 family members is mainly modulated during the first fourteen hours of stimulation, a period in which it is possible to observe the largest variation of expression. Moreover, the analysis of differentially expressed genes between two consecutive time points confirmed that IL-1 family members are modulated during the entire course of the inflammatory reactions, except between 72 and 96 h in the chronic model where they remain practically unchanged. In conclusion, our results confirm the strong involvement of the IL-1 family genes, especially in the early phases of the monocyte inflammatory response. This work was supported by the EU FP7 project BioCog (GA 602461) and by the Cluster project Medintech of the Italian Ministry of University and Research. MODELING ANTIMICROBIAL PEPTIDE-INDUCED CHEMOKINE, CYTOKINE, AND BIOLOGICAL MEDIATOR RESPONSES K. A. Brogden1, C. Fischer1, S. Radhakrishnan2, S. A. Prasad2, R. Vidva2, and S. Vali2 1Dows Institute for Dental Research and Department of Periodontics, College of Dentistry, The University of Iowa, Iowa City, IA 52242, USA 2Cellworks Research India Pvt Ltd, Whitefield, Bangalore-560066, India and Cellworks Group, Inc., Saratoga, CA 95070, USA Abstract In the oral cavity, acute and chronic inflammation often results from physical damage and microbial infections and less frequently from immune reactions and malignant changes in oral tissues. All result in the production of antimicrobial peptides, chemokines, and cytokines that lead to cellular infiltrates, a vascular response, tissue destruction, and cellular proliferation. In our current work, we developed an in silico dendritic cell simulation model to better understand the effect of pro-inflammatory agonist concentration, the effect of temporal exposure of cells to both agonist and antimicrobial peptides, the primary and secondary signaling pathways involved, and the concentration and composition of the resulting chemokine, cytokine, and mediator responses. The model was validated by comparing the predicted responses of in silico stimulated dendritic cells to TLR agonists with the observed responses of cultured dendritic cells stimulated with the actual TLR agonists LPS and Porphyromonas gingivalis hemagglutinin B (HagB). With this model, we have accurately predicted the conditions in which HBD3 and histatin 5 regulate the chemokine and cytokine responses of human myeloid dendritic cells. Our observed results confirm the predicted findings that antimicrobial peptides act as both a stimulant (e.g., HBD3) and an attenuator (e.g., HBD3, histatin 5) of chemokine and pro-in

-induced chemokine and cytokines responses, attenuates seven chemokine and cytokine

-induced CCL3/MIP- -antimicrobial peptides have the ability to regulate the production of chemokines and pro-inflammatory cytokines. The concentration of histatin 5 and HBD3 and the temporal treatment of HBD3 to cells, with respect to microbial antigen treatment of cells, were all important in the magnitude of the overall chemokine and cytokine response. Such a mechanism may play an important role in the early events of oral inflammation that may be a target for the prevention or treatment of oral inflammatory conditions. Supported by NIH, NIDCR grant R01 DE014390. Poster Presentation Abstracts Poster abstracts will be finalised weeks before the event

Day 3: Therapeutic applications of the Innate Immune system

Invited Speakers Abstracts

Regulation of autoimmune myocarditis and inflammatory cardiomyopathy by innate immunity Dr Przemyslaw Blyszczuk, Junior Group Leader, Cardioimmunology, University of Zurich, Switzerland Inflammatory dilated cardiomyopathy refers to acquired cardiac dysfunction that often results from myocarditis. Infection-triggered heart-specific autoimmunity represents the most prominent cause of myocarditis. In my talk, I will present how innate immune response on various myeloid cells can promote survival of activated heart-reactive CD4+ T cells, but also can prevent uncontrolled expansion autoreactive T cells in mouse model of experimental autoimmune myocarditis. I will also present how innate signalling promotes pathological remodelling and aggravates development of typical end-stage heart failure phenotype in inflammatory dilated cardiomyopathy. High-intensity ultra-short electric pulse applications in modulating innate immunity Dr Sunil K. Joshi, Assistant Professor, School of Molecular Diagnostics & Translational Sciences, College of Health Sciences and Frank Reidy Research Center of Bioelectrics, Old Dominion University, Norfolk, VA, USA The development of specific adaptive immunity requires pathogen/tumor-specific antigen presentation to T cells by professional antigen-presenting cells (APCs). The most specialized APCs, known as Dendritic Cells (DCs), are critical for priming antigen-specific T cell responses. The antigen-presentation function of DCs strictly depends on functional maturation characterized by (a) high expression of MHC I/II and co-stimulatory molecules (CD40, CD80, and CD86) and (b) efficient antigen processing and presentation. The pathogens that cause Malaria, Tuberculosis, Flu, AIDS, cancer, and many other diseases suppress immune responses by inhibiting DC maturation leading to inefficient antigen processing, presentation, and dysfunctional CD8+ T cell priming. In the past several decades, many strategies have been developed to functionally mature and activate DCs in vivo. We recently tested an innovative approach to enhance DC maturation and antigen-presenting function without the use of cytokines, LPS or TLR ligands. We have used application of High-intensity ultra-short electric pulse to significantly enhance DC maturation and antigen-presenting function without inducing cell death, thus, triggering strong antigen-specific T cell priming and activation.

Toll-like receptors in atherosclerosis: opportunity for targeting Dr Claudia Monaco, Imperial College London, United Kingdom Atherosclerosis is now recognized as an inflammatory disease that stems from the involvement of the adaptive and innate immune response and is characterized by the upregulation of pro-inflammatory cytokines. Components of innate and adaptive immunity play both detrimental and beneficial roles in atherosclerosis. For instance, contrasting roles have been assigned to distinct T cell subsets in atherosclerosis, with Th1 being pathogenic, and T regulatory cells exerting protection. In my talk I will explore the concept that duality also exists in innate immune responses. Extracellular membrane-bound pattern recognition receptors such as Toll-like receptor (TLR) 2 and TLR4 are strong inducers of cytokine upregulation and play a detrimental role in atherosclerosis. In contrast, endosomal toll like receptors may have a protective role in the vasculature. M1/M2 Macrophages: A Copernican Revelation in Immunology Dr Charles Mills, BioMedical Consultants, United States M1 and M2 describes macrophage responses that Inhibit or Heal, and which also direct T cells into Th1 or Th2 like responses in higher animals. Most animals do not possess Adaptive Immunity. Instead, all animals rely on a rapid M1 response as the primary defense because one bacterium can become 8 X 1016 (the mass of a human) in 4 days. As animal anatomies became more complicated (e.g., the vasculature), T or B cell responses evolved for added protection. Much to slow to be a primary host defense, Adaptive Immunity is a secondary response directed by Innate Immunity/macrophages. Macrophages are the center of the immune solar system. Copernican Revelation. Oral Presentation Abstracts

HYPOXIC-INDUCED PROTEOLYSIS OF MER TYROSINE KINASE AND CD36 BY ADAM PROTEASES REGULATES APOPTOTIC CELL ACCUMULATION AND HIF2-REGULATED EFFEROCYTOSIS AND INFLAMMATION-RESOLUTION IN MACROPHAGES TO SUPPRESS FIBROSIS AND CARDIAC FUNCTION. Dr Edward Thorp, Department of Medicine, Columbia University, New York, USA and Department of Pathology and Feinberg Cardiovascular Research Institute, Northwestern University Medial School, Chicago, USA Following myocardial infarction (MI), myeloid phagocytes are recruited to sites of tissue injury, such as the heart, where they promote tissue repair and phagocytic clearance of necrotic and apoptotic cells. The molecular pathways required for apoptotic cell clearance (efferocytosis) in the heart are unclear, as well as the extent that efferocytosis per se affects cardiac remodeling, fibrosis, and function. MERTK and CD36 are receptors for apoptotic cells that are expressed on the surface of recruited and resident macrophage phagocytes in the heart. Given the evidence that clearance pathways are linked to resolution of inflammation

and ensuing tissue repair, we hypothesized that the efficiency of efferocytosis post MI contributes to the extent of subsequent heart repair. To test this, control, Mertk and CD36 deficient mice were subjected to MI by ligation of the left anterior descending (LAD) coronary artery, followed by molecular, cellular, histomorphometric, and functional indices of the heart and associated inflammation. In control animals, we discovered that Mertk mRNA and protein levels were significantly elevated in the heart post LAD ligation. By flow cytometry, MERTK protein was also detected specifically on the surface of Ly6cLOW phagocyte subsets. Relative to control, whole body Mertk knockout and myeloid Mertk deficiency from bone marrow chimeras, led to increased accumulation of TUNEL positive apoptotic cells in infarct border zones. In addition, elevated levels of pro-inflammatory cytokines such as TNFα and IL-6, and reduced levels of IL-10, were measured in knockouts. Initial infarct sizes as a percentage of ischemic area at risk were similar between both groups. However, infarct size in Mertk-/- mice subsequently expanded relative to control. At later time points, hearts from Mertk-/- mice had elevated scarring and ventricular wall thinning by histology. Echo-cardiography indicated that Mertk-/- hearts displayed exaggerated dysfunction, including reduced pumping capacity. Interestingly, MERTK and CD36 proteolysis in both human and mouse hearts was significantly elevated post MI, suggesting natural suppressive mechanisms of efferocytosis in the hypoxic heart. In-vitro, hypoxia promoted MERTK proteolysis and suppressed HIF2alpha-regulated efferocytosis pathways. Taken together, these findings suggest that efferocytosis pathways have the capacity to regulate cardiac remodeling and heart function after a heart attack. Strategies that target MERTK, ADAM17, and HIF molecular pathways to regulate optimal dead cell clearance and associated inflammation resolution and fibrosis pathways may be of benefit in helping to heal the heart and prevent exposure of cardiac self-antigen. MECHANISMS BEHIND IMMUNOREGULATORY EFFECTS OF PROBIOTIC YEASTS IM Smith1,2, A Baker1, N Arneborg2, L Jespersen2 Chr. Hansen A/S Health & Nutrition Division Bøge Allé 10-12 2970 Hørsholm Denmark 1. Health & Nutrition Division, Chr. Hansen A/S, Hørsholm, Denmark 2. Food Science, University of Copenhagen, Frederiksberg, Denmark The concept of individual microorganisms influencing the makeup of T cell subsets through interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. We evaluated the immune stimulating capabilities of a diverse selection of non-Saccharomyces yeasts by incubation with human monocyte-derived DCs followed by DC incubation with autologous naive T cells. Quantification of secreted cytokine levels revealed yeasts with highly reproducible and distinct DC and T cell cytokine induction profiles, as compared to the established probiotic S. boulardii. The observed differences in induced cytokine profiles, supported by T cell subset stains, indicate that certain yeasts are capable of inducing an immune response dominated by Treg cells, whereas others appear to induce a more complex adaptive immune response involving TH1, TH17, and Treg cells. To explore the mechanisms behind the observed cytokine induction, we blocked relevant DC pattern recognition receptors and investigated the cytokine inducing properties of yeast cell wall extracts. Our data identify the β-glucan receptor Dectin-1 as key for DC recognition of S. boulardii as well as non-Saccharomyces yeasts, initiating downstream signaling pathways leading to the observed DC cytokine profiles. In contrast, TLR2 and DC-SIGN do not appear involved in the recognition. As expected based on the identification of Dectin-1 as involved in yeast recognition, β-glucan containing yeast cell wall extracts induced robust DC cytokine secretion, an observation that parallels recent in vivo findings and appears to support a hypothesis that yeast cell wall components are responsible for the observed modulation of immune cell function. PHARMACOLOGICAL MODULATION OF THE AKT/MIR199A-5P/CAV1 PATHWAY INCREASES ENDOTOXIN TOLERANCE IN CYSTIC FIBROSIS (CF)-AFFECTED MACROPHAGES AND DECREASES LUNG HYPER-INFLAMMATION IN CF MIC Ping-xia Zhang 1,2, Jijun Cheng 3, Siying Zou 4, Anthony D'Souza 2, Jonathan L. Koff 5, Jun Lu 3, Patty J. Lee 5, Diane S. Krause 2,4, Marie E. Egan 1,6 and Emanuela M. Bruscia 1*

Departments of Pediatrics 1, Laboratory Medicine 2, Genetics 3, Cell Biology 4, Internal Medicine 5, and Cellular and Molecular Physiology 6, Yale University School of Medicine, New Haven Connecticut, USA *Presenting author

TARGETTING THE TRIPEPTIDE PRO-GLY-PRO DEMINISHES INFLAMMATORY EFFECTS OF CIGARETTE SMOKE M. Abdul Roda, X. Xu, T. Abdalla, M. Sadik, L. Viera, CM. Mc Nicholas, D. Sullivan, FA. Redegeld, G. Folkerts, PL. Jackson, A. Gaggar, JE Blalock. Utrecht University, Division of Pharmacology, Universiteitsweg 99, Utrecht The Netherlands. M.abdulroda@uu.nl Rational. Cigarette smoke (CS) exposure is the major risk factor for developing several lung diseases, such as chronic obstructive pulmonary disease (COPD) and eventually right sided heart failure. The extracellular matrix derived tripeptide proline-glycine-proline (PGP) has neutrophil activating and chemotactic properties. Prolyl endopeptidase (PE), a serine protease present in neutrophils, is the terminal enzyme in the PGP-generation pathway. We hypothesized that inhibition of PGP activity by the tripeptide arginine-threonine-arginine (RTR) would reduce the neutrophilic inflammation in the lungs in a cigarette smoke exposure model in mice. Methods. Two murine models were conducted. In a 6 weeks model, female 8-10 weeks old Balb/c mice were exposed to cigarette smoke one hour a day, five days a week. Fifteen minutes prior to each whole body smoke exposure the mice received either RTR, the non-active tripeptide alanine-serine-alanine (ASA) or vehicle (saline) via intratracheal instillation. During a 5 months model, mice were exposed to cigarette smoke or air one hour a day, five days a week as well, receiving RTR or saline (4 groups). After 10 weeks, half of the mice in the CS exposed saline treated group, were switched to RTR treatment to create a rescue phenotype (total of 5 groups). 16 hours after last air/CS exposure, the mice were sacrificed to collect bronchoalveolar lavage fluid (BALF), blood, heart and lungs. Results. In the 6 weeks model, RTR significantly reduced macrophage and neutrophilic influx into the lung (75%, and 79% versus saline respectively). Also, right ventricular hypertrophy (RVH) was significantly increased by 33% by CS and RTR treatment prevented RVH. PGP levels were increased in serum and BALF and reduced in RTR treated CS exposed mice. In all cases, ASA did not show any effect, suggesting that the effects seen of RTR are not just peptide effect but actual compound effects. Similar to the 6 weeks model, in the 5 months model a highly significant inhibition of macrophage and neutrophil influx was seen in RTR preventive and rescue model (p<0.0001). Interestingly, the RVH and PGP levels in BALF were significantly reduced (p<0.01) as well as the PE activity in BALF and serum in RTR preventive and rescue model. As a final end point, RTR completely blocked the development of emphysema, even in the rescue group. Saline treated CS exposed mice however, had an increased mean linear intercept compared to non-smoked controls (p<0.01). Conclusion In the two CS exposure murine models, RTR attenuates CS effects including: leukocyte influx and RVH development through the inhibition of PGP activity. These data suggest an important role for PGP in the development of smoking-related lung injury. Blocking PGP by RTR after 10 weeks of CS exposure and saline treatment seemed to reverse CS related effects to the lungs and partly to the heart. PGP plays an important role in the development of lung emphysema. Poster Presentation Abstracts

ACTIVATED MONOCYTES PLAY AN IMPORTANT ROLE IN INNATE IMMUNITY AND INFLAMMATION IN OBESE CHILDREN R.T. Mattos; C.A. Menezes; N.I. Medeiros; R.C.G. Fares, E.P. Franco; S.F.L. Oliveira; W.O. Dutra; R. Correa-Oliveira, F.R. Santos; J.A.S. Gomes

Obesity and its associated metabolic disorders are considered chronic low-grade inflammation characterized by elevated pro-inflammatory cytokines, infiltration of macrophages in the adipose tissue as well as in other metabolic organs. In mice and humans, circulating monocytes play an important role in cytokine/chemokine production; transmigration into inflamed adipose tissue and also contributes to the development of obesity related comorbidities. However, it is still clear whether these inflammatory changes occur in childhood obesity. The aim of this study was to evaluate the involvement of monocyte subsets in low-grade inflammation occurring during childhood obesity. Monocyte subtype numbers were determined and extensive monocyte phenotype (i.e. surface marker expression) was performed on whole blood samples. Monocytes were gated as M1 (CD14++CD16−) and M2 (CD14+CD16++) monocyte subsets, as described in previous studies. We also investigated the frequency of surface molecules such as Toll-like receptors (TLR-2, 4), CD36, HLA-DR, CD80, CD86, CD40, CD36 and intracellular cytokines such as IL-1, IL-8, IL-10, TNF- and TGF-β by multiparametric flow cytometry. Eleven obese individuals with Body Mass Index (BMI)

greater than 25kg/m2 (aged: 9.27 ± 1.27) and nine lean subjects (aged: 9.33 ± 1.58) with BMI lower than 23 kg/m2 and negative metabolic syndrome factors were recruited at the Treatment Service for obese children and adolescents with greater than 95 percentile called SEMPRE (Service of Preventive Medicine) in Aracaju – Sergipe/Brazil. The statistical analysis of monocytes subsets did not show differences between the groups. We also observed that M1 and M2 subsets exhibit high frequency of CD36, TLR 4 and IL-8 in obese children when compared with lean children. These differences were found to be statistically significant (p<0,05). We observed that M2 subsets exhibited high frequency of CD80, IL-1 and IL-6 in obese children when compared with lean children that exhibited high frequency of these molecules in M1 subsets. On the other hand lean individuals presented a high frequency of M1 and M2 monocytes expressing IL-10, TGF- β, TLR2 and CD86 when compared with the obese group. These data suggest that childhood obesity is associated with increased monocyte numbers expressing an activated phenotype and may be involved on the inflammatory process. In contrast with the lean group, that seems to develop a modulatory response caused by high expression of IL-10 and TGF- β, despite showing a high frequency of inflammatory molecules. Considering the results presented, we suggest that there is a fine balance in relation to cellular activation profile that is regulated by modulatory cytokine in the subsets of monocytes from lean children that is lost in obese individuals. Supported by: CNPq, FAPEMIG and CAPES EXPRESSION OF CYTOKINES AND MATRIX METALLOPROTEINASE 2 AND 9 BY INNATE IMMUNITY CELLS AS A POTENTIAL BIOMARKER OF CARDIAC MORBIDITY IN CHAGAS DISEASE

N.I. Medeiros; R.C.G. Fares; E.P. Franco; G.R. Sousa; R.T. Mattos; W.O. Dutra; R. Correa-Oliveira; M.O.C. Rocha; J.A.S. Gomes. Chagas disease is caused by infection with Trypanosoma cruzi and remains a serious public health problem and affects about 10 million of people in Latin America. After the acute phase, a chronic debilitating disease is established and different clinical forms may develop. The majority of patients are classified in indeterminate (IND) clinical form, without symptoms of the disease. Dilated cardiomyopathy (CARD), the most severe alteration in the chronic phase, affects about 30% of patients and is characterized by myocardial remodeling and interstitial fibrosis, resulting in extracellular matrix (ECM) changes. ECM remodeling is regulated by proteolytic enzymes, such as matrix metalloproteinases (MMP), and cytokines produced by immune cells, including phagocytes (macrophages and neutrophils). The distinct ability of phagocytes to present antigens, produce cytokines and provide co-stimulatory signals may contribute to the severity of the outcome of Chagas disease. In the current study, we used flow cytometry to measure the levels of MMP2, MMP9, TGF-TNF- -10 and IL-forms of Chagas disease and non-infected (NI) individuals, before and after in vitro stimulation with T. cruzi antigens. Our results show that neutrophils from IND patients present a higher frequency of MMP2 and IL-10 when compared with NI and CARD individuals after in vitro stimulation with parasite-derived antigens. On the other hand, neutrophils and monocytes from CARD individuals presented higher expression of MMP9 and inflammatory cytokines when compared with NI and IND groups after in vitro stimulation with parasite-derived antigens. Correlation analysis between MMPs and cytokines showed that MMP2 expression had a positive correlation with IL-10, an anti-inflammatory cytokine. MMP9 had a positive correlation with TGF- -process of fibrosis. These data suggest that MMP2 may have an important immunoregulatory role that leads to the maintenance of better cardiac function, while MMP9 seems to be involved in inflammation and cardiac remodeling in Chagas disease. Supported by: CNPq, FAPEMIG and CAPES

THREE POTENTIAL PROBIOTICS OFFER PROTECTION TO ARTEMIA AGAINST V. ANGUILLARUM CHALLENGE BY ENHANCING ITS IMMUNE SYSTEM AND ANTIOXIDANT STATUS A. Toufexi, E. Amanetidou, E. Giarma and M. Touraki* 230 Shepherds Bush Road, London, W67NL, U.K. * touraki@bio.auth.gr Lab. General Biology, Department of Biology, School of Sciences, AUTH, 54124 Thessaloniki Greece The crustacean Artemia, the live feed of aquatic organisms, is extensively used in aquaculture as a carrier of nutrients, antimicrobials and recently probiotics to its predators, namely fish. One of the most important diseases in fish culture is vibriosis, caused by the bacterium Vibrio anguillarum. In the present work Artemia franciscana cysts were used in order to study the potential effect of

probiotics on its immune and antioxidant system, following a challenge with the pathogen V. anguillarum. The probiotic bacteria studied were Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis subsp. lactis, administered at four doses (2x105 CFU/ml) to Artemia nauplii. In another set of experiments with separate groups for each probiotic, challenge with two doses (2x105 CFU/ml) of V. anguillarum followed probiotic administration. The effect of probiotics as well as of the challenge on Artemia was evaluated by the determination of the activity of phenoloxidase, superoxidase dismutase (SOD), glutathione reductase (GRed) and glutathione transferase (GSTs). The levels of lipid peroxidation (MDA) and survival of nauplii were also estimated in all the above experimental sets. All determinations were performed in triplicate. Challenge with the pathogen without any prior probiotics treatment, resulted in a significant decrease in the activities of the three antioxidant enzymes and of phenoloxidase, while lipid peroxidation dramatically increased compared to control. To the contrary, in the experimental groups employing probiotics followed by challenge, the corresponding levels of enzymatic activities and lipid peroxidation were not significantly different from control values. Survival of the nauplii decreased after challenge to about 15% while in the probiotic treated and challenged group amounted to 75%, a value not significantly different from control survival. Our results indicate that the probiotics studied enhance the immunological and antioxidant status of Artemia nauplii, thus rendering them able to withstand a challenge with the pathogen, without employing antibiotics that might lead to antibiotic resistant pathogens.

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