48

Abstract / Cytokine 70 (2014) 28–79 genkwadaphnin doses also induced recovery of exhausted LCMV-specific CD4+ and CD8+ T cells. Furthermore, pretreatment of genkwadaphnin prior to LCMV infection prevent the generation of exhausted LCMV-specific CD8+ T cells. Such restoration of exhausted LCMV-specific CD4+ and CD8+ T cells by genkwadaphnin was closely associated with reduction of viral burden in sera as well as tissues. More intriguingly, genkwadaphnin treatment induced down-regulation of negative regulatory molecules such as PD-1 and Tim-3 in exhausted LCMV-specific CD4+ and CD8+ T cells with more apparent down-regulation of Tim-3, which suggesting that Tim-3 molecule is main target to restore exhausted T-cell responses with genkwadaphnin. Collectively, these results provide new valuable insight into the use of genkwadaphnin isolated form flos buds of Daphne genkwa to develop therapeutic strategy of chronic human diseases, such as hepatitis B and C virus (HBV and HCV), human immunodeficiency virus (HIV) as well as cancers. http://dx.doi.org/10.1016/j.cyto.2014.07.052

39

that Lymphotoxin? signals similar to TNF and suggested to be an alternative factor to induce inflammation in the absent of TNF 2 and both TNF and Lymphotoxin are not blockable by newly proposed inhibitor called Progranulin 3. These results encouraged us to investigate downstream regulator of TNF signalling pathway. Surprisingly, after 30 years of research on TNF, role of many components of TNF pathway are still unclear. TNF Receptor Associated Factor-2 (TRAF2) is one of those molecules which its role is controversial and not well defined yet 4. Since TRAF2 knock-out mice are lethal at birth, we have established various conditional and tissue specific TRAF2 knock-out to study the role of TRAF2 in different tissues. Interestingly, we found that loss of TRAF2 in keratinocytes disrupts TNF signalling and causes cell death, epidermal hyperplasia and skin inflammation. Surprisingly and contradictory to other models, additional loss of TNF did not completely rescue this phonotype. We have found that constitutive activation of non-canonical NFjB and excessive production of TNF and other cytokines are responsible for this TNF independent inflammation. We propose that this novel model of skin inflammation could be a better model and targeting non-canonical NFjB signalling might be a more effective target for treatment of inflammatory diseases. http://dx.doi.org/10.1016/j.cyto.2014.07.054

46 Distinct upstream role of type I IFN signal to hematopoietic stem cell-derived leukocytes and resident cells for connected recruitment of Ly-6Chigh monocytes and NK cells via CCL2–CCL3 cascade to restrict CNS-invasion of herpes simplex virus in mucosal tissues Seong Kug Eo, Erdenebileg Uyangaa, Ajit Mahadev Patil, Jin Hyoung Kim, Jin Young Choi, Seong Bum KIm, Department of Microbiology, College of Veterinary Medicine, Chonbuk, Republic of Korea Orchestrated mobilization of innate cells in inflamed tissues is believed to play a crucial role in the control of herpes simplex virus (HSV) replication for preventing CNS-invasion without immunopathology. Although type I interferons (IFNs-I) regulate the recruitment and activation of innate cells in infected sites, the crucial regulators and cell populations affected by IFNs-I that establish early orchestrated environment of innate cells in HSV-infected mucosal tissues is largely unknown. Here we found that highly enhanced susceptibility to HSV infection in the absence of IFNs-I signal was closely associated with reduced recruitment and activation of CD11b+ Ly6Chigh monocytes and NK cells. IFNs-I signal promoted the differentiation of CCL2producing CD11b+ Ly-6Chigh monocytes and IFN-g/granzyme B-producing NK cells, while the deficiency of IFNs-I signal induced CD11b+ Ly-6C[low] monocytes producing CXCL1 and CXCL2. Intriguingly, the recruitment of CD11b+ Ly-6Chigh monocytes preceded to those of NK cells with the levels peaked within 24 h pi, and the consecutive recruitment of monocytes and NK cells was kinetically associated with CCL2CCL3 cascade response. In bone marrow chimeric models, early recruitment of CD11b + Ly-6Chigh monocytes was regulated by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, while NK cell recruitment dominantly depended on resident cells. Also, IFNs-I signal in HSC-derived leukocytes appeared to suppress the recruitment of CD11b+ Ly-6G+ neutrophils to ameliorate severe immunopathology. Finally, tissue resident CD11b+ F4/80+ macrophages and EpCAM+ CD11c+ Langerhans cells appeared involved in producing initial CCL2 for self-migration amplification of early infiltrated CD11b+ Ly-6Chigh monocytes through stimulation by IFNs-I produced from infected epithelial cells. Collectively, these results define detailed IFNs-I-dependent pathway to establish orchestrated mobilization of CD11b+ Ly-6Chigh monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this implies that the cascade response of resident-to-hematopoietic-to-resident cells for cytokine-tochemokine-to-cytokine production to recruit orchestrated innate cells is critical to attenuate HSV replication in inflamed tissues. http://dx.doi.org/10.1016/j.cyto.2014.07.053

47 TRAF2 regulates NFkB transcription factors to inhibit cell death and inflammation in the skin Nima Etemadi 1,2, James Rickard 1,2, Holly Anderton 1, Sukhdeep Spall 1, Catherin Hall 1, David Vaux 1,2, Ueli Nachbur 1,2, John Silke 1,2, 1 Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia, 2 Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia Tumor Necrosis Factor (TNF) is an important factor in initiation and development of inflammation, and anti TNF therapy is well established for treatment of inflammatory diseases such as psoriasis, rheumatoid arthritis and Crohn’s disease.1 However, anti TNF drugs are not always efficient and developing new effective anti-inflammatory drugs is in the interest of many pharmaceutical companies.1 Therefore, We think that better understanding of TNF signalling pathway helps to discover new targets and design more effective drugs for inflammatory diseases. We have previously showed

48 IL-2 coordinates IL-2 producing and regulatory T cell interplay Inês F. Amado 1, Julien Berges 1, Rita J. Luther 2, Marie-Pierre Mailhé 1, Sylvie Garcia 1, Antonio Bandeira 1, Casey Weaver 2, Adrian Liston 3, Antonio A. Freitas 1, 1 Pasteur Institute, Paris, France, 2 University of Alabama, Birmingham, USA, 3 University of Leuven, Leuven, Belgium Many species of bacteria use quorum sensing to sense the amounts of secreted metabolites and adapt their growth according to their population density. We asked whether similar mechanisms would operate in lymphocyte homeostasis. We investigated the regulation of the size of Interleukin-2-producing CD4+ T-cell (IL-2p) pool using different IL-2-reporter mice. We found that in the absence of either IL-2 or regulatory CD4+ T-cells (Treg) the number of IL-2p-cells increases. Administration of IL-2 decreases the number of cells of the IL-2p-cell subset and pertinently, abrogates their ability to produce IL-2 upon in vivo cognate stimulation, while increasing Treg-cell numbers. We propose that control of the IL-2p-cell numbers occurs via a quorumsensing-like feedback loop where the produced IL-2 is sensed by both the activated CD4+ T-cell pool and by Treg-cells, which reciprocally regulate cells of the IL-2p-cell subset. In conclusion, IL-2 acts as a self-regulatory circuit integrating the homeostasis of activated and regulatory T cells as CD4+ T-cells restrain their growth by monitoring IL-2 levels thereby preventing uncontrolled responses and autoimmunity. J. Exp. Med. 2013 vol. 210(12) pp. 2707–2720 http://dx.doi.org/10.1016/j.cyto.2014.07.055

49 The pivotal role of interferon gamma (IFNc) in TLR9-mediated murine models of macrophage activation syndrome (MAS)/secondary hemophagocytic lymphohistiocytosis (sHLH) Vanessa Buatois, Laurence Chatel, Laura Cons, Cristina de Min, Marie H. Kosco-Vilbois, Walter G. Ferlin, NovImmune S.A., Geneva, Switzerland Aims: IFNc mediates disease in the murine model of primary HLH. Similar to HLH, patients with MAS/sHLH often present with a cytokine storm, overwhelming inflammation and multiorgan dysfunction. Thus, to assess the potential implication of IFNc in MAS, two variants of the murine MAS model were used. Administration of an antimouse IFNc monoclonal antibody (MAb) in both models allowed us to determine the level and pivotal role of this proinflammatory cytokine. Methods: C57BL/6 mice received repeated injections on days 0, 2, 4, 7 and 9 of the TLR9 agonist, CpG oligodeoxynucleotides (CpG ODN). To mimic a more severe, fulminant disease, along with CpG ODN, the anti-IL-10 Receptor mAb, 1B1.3A, was coadministered at 200 lg/mouse on days 0, 2, 4 and 6. To assess the significance of IFNc on disease, the mAb, XMG1.2, was injected 100 mg/kg) on days 1, 3 and 6. Levels of total IFNc werequantified by a fit-for-purpose ELISA. Results: In these models, IFNc neutralization lead to disease amelioration with parameters such as weight loss, splenomegaly, white blood cell counts, hyperferritinemia, hypercytokinemia and anemia successfully addressed. Post XMG1.2 administration, accumulation of immune-complexed IFNc reached a steady state level of 250 ng/ ml in both models. IFNc-induced inflammatory genes, found to be upregulated locally in the spleen and liver, were normalized by IFNc-blockade. Conclusion: Neutralization of IFNc improves clinical and laboratory features in even the more severe, fulminant model of MAS. These data suggest that IFNc, present as one of the cytokines in MAS, is central to disease and therefore provides a rationale