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Abstract / Cytokine 70 (2014) 28–79 21 Essential function of type I IFN signaling in balancing inflammation during Streptococcus pyogenes infection Virginia Castiglia, Florian Ebner, Pavel Kovarik, University of Vienna, Wien, Austria Streptococcus pyogenes also known as Group A Streptoccocus (GAS) is a common human pathogen responsible of a variety of diseases that range from mild pharyngitis to rare but invasive and life-threatening infections. We have demonstrated that type I interferon (IFN) signaling is required for mice survival in a model of subcutaneous infection with S. pyogenes. However, it is not understood how these cytokines are induced and how they protect the host. Using mice deficient in the type I IFN receptor IFNAR1 we observed increased tissue damage at the site of infection despite similar bacterial loads in the absence of type I IFN signaling. IFNAR1-deficient mice exhibit hyperinflammation with increased levels of IL-1ß in the infected tissue and in the liver and spleen. Excessive IL-1ß production seems to promote organ injury as confirmed by the histology analysis and by the levels of transaminases and other parameters measured in the blood. On the other hand, IL-1ß signaling is essential for mouse survival during infection with S. pyogenes since mice lacking the IL-1 receptor or Caspase-1, the key IL-1ß-processing enzyme, are highly susceptible to S. pyogenesinfection. Together, our data provide evidence that type I IFNs are essential for balancing the immune response against S. pyogenes and that the absence of type I IFN signaling results in lethal systemic hyperinflammation accompanied by overproduction of IL-1b. http://dx.doi.org/10.1016/j.cyto.2014.07.028

the correlation between immune cell activation and insulin resistance? or metabolic inflammation? has revolutionized how obesity-linked diabetes is understood. Highfat diets (HFD), obesity, and saturated fats trigger pro-inflammatory pathways in adipose tissue and muscle macrophages, contributing to peripheral insulin resistance. In contrast, unsaturated fat consumption has beneficial effects on whole-body insulin sensitivity, but it is unknown if these effects are mediated through skewing antiinflammatory macrophage polarization. Methods: Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice were grown in culture and treated with BSA-conjugated saturated fatty acid palmitate (PA), unsaturated fatty acid palmitoleate (PO), or BSA. To evaluate dietary effects in vivo, BMDMs were isolated from C57BL/6 mice fed HFD (60% kcal) or low-fat diet (LFD) for 18 weeks. Results: Macrophages treated with PA displayed increased pro-inflammatory gene expression (Nos2, Ciita, Cxcl1) and cytokine secretion (KC, IL6, TNFaÞ relative to those given BSA, while PO up-regulated anti-inflammatory genes (Arg1, Chi3l3, Tgfb1). Interestingly, co-incubation with PO prevented PA-induced pro-inflammatory gene expression, cytokine secretion, and NFjB activation. To assess the in vivo effect of diet on macrophage polarization, we isolated bone marrow from high or low fat-fed mice and derived BMDMs over 7 days. Macrophages from HFD-fed mice showed elevated pro-inflammatory gene expression and cytokine secretion, revealing ex vivo memory of the in vivo environment. Moreover, a 6 h treatment with PO reversed this proinflammatory phenotype to resemble macrophages from LFD-fed mice. These results reveal the plasticity of macrophages in response to distinct fats. Discussion: Saturated and unsaturated fats differentially affect macrophage polarization, conferring a pro- or anti-inflammatory phenotype, respectively. Polarization appears to be reversible, as palmitoleate abolished inflammation resulting from palmitate treatment or high-fat diet. Our findings demonstrate a novel mechanism by which an unsaturated fat attenuates the metabolic inflammation of macrophages that in vivo precedes insulin resistance.

22 NOD2 immunity protects against obesity-induced inflammation and insulin resistance

http://dx.doi.org/10.1016/j.cyto.2014.07.030

Joseph F. Cavallari 1, Morgan D. Fullerton 1,2, Emmanuel Denou 1, Kevin P. Foley 1, Brandyn D. Henriksbo 1, Trevor C. Lau 1, Jonathan D. Schertzer 1, 1 Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada, 2 Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada

24 Interferon regulatory factor 4 and inflammation

Aims: Obesity is associated with immune cell infiltration and inflammation in metabolic tissues that contributes to insulin resistance. Pattern recognition receptors (PRRs) link obesity-induced inflammation and insulin resistance. Nucleotide oligomerization domain protein 2 (NOD2) is a PRR that detects muramyl dipeptide (MDP) present in the cell walls of all bacteria. NOD2 mutations are associated with inflammatory bowel disease, and NOD2 activation with MDP can ameliorate the inflammatory effects of experimental colitis in mice. Our lab has linked NOD2 immunity with metabolism and found that NOD2 deficiency exacerbates obesity-induced inflammation and insulin resistance. We sought to determine if NOD2 activation with MDP confers anti-inflammatory effects during acute bacterial or chronic dietary stress. We have found that MDP has anti-inflammatory and insulin sensitizing effects in mice during bacterial stress and diet-induced obesity. Methods: Mice were fed a chow or high fat diet (HFD; 60% kcal from fat) for 5 weeks. MDP (100 ug) or LPS (0.2 mg/kg) was administered i.p. as indicated. Glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamps were performed in 6 h-fasted conscious mice. Tissues were immediately collected and frozen in liquid nitrogen. Gene expression was determined by RT-PCR with TaqMan assays. Results: Mice that received MDP prior to LPS challenge had increased glucose tolerance, reduced glucose-stimulated insulin secretion, and increased suppression of hepatic glucose production during hyperinsulinemic-euglycemic clamps. Mice that received MDP throughout a HFD were more glucose and insulin tolerant, experienced less hepatic insulin resistance during clamps, and had lower expression of many inflammatory markers in adipose and liver tissues. Conclusions: NOD2 activation improves glucose homeostasis during acute bacterial stress and reduces inflammation and metabolic defects associated with diet-induced obesity.

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Melody W. Chang 1, Andrew D. Cook 1, Stephen L. Nutt 2, John A. Hamilton 1, Derek C. Lacey 2, 1 The University of Melbourne, Melbourne, VIC, Australia, 2 Walter and Eliza Hall Institute, Melbourne, VIC, Australia Interferon regulatory factor 4 (IRF4) was initially thought to be expressed only in cells of the lymphoid lineage, with IRF4 deficient (IRF4/) mice having increased numbers of immature T and B cells with impaired functions. More recently IRF4 has also been found to be expressed in myeloid cells. However, its function in macrophage development and regulation is not clear. It has been reported to be required for the generation of both the pro-inflammatory M1 and the anti-inflammatory M2 macrophage subpopulations. In the current study we have found that the expression of IRF4 was higher in the pro-inflammatory granulocyte macrophage-colony stimulating factor (GM-CSF)induced bone marrow-derived macrophages than in the macrophage-colony stimulating factor (M-CSF)-induced bone marrow-derived macrophages. We therefore hypothesised that IRF4 would be required for the regulation of pro-inflammatory genes and its deficiency would prevent mice from developing inflammatory responses. Unexpectedly, when the KRN serum-transfer arthritis model, which does not require T or B cells, was induced in wild type (WT) and IRF4/ mice, the latter showed a more severe disease phenotype. Significantly larger spleens were seen in the IRF4/ mice compared with the WT, comprising of more Ly6Clo monocytes. IRF4/ mice also had more macrophages than WT mice. RNA analysis of myeloperoxidase suggests that IRF4/ may have elevated numbers of neutrophils in the joints. Given these unexpected findings, experiments are currently being performed to further investigate the underlying functions of IRF4 in inflammation. http://dx.doi.org/10.1016/j.cyto.2014.07.031

http://dx.doi.org/10.1016/j.cyto.2014.07.029

23 Palmitoleate reverses palmitate-induced pro-inflammatory macrophage polarization

25 Hydroxychloroquine activates host antiviral innate immunity

Kenny L. Chan, Amira Klip, Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada, Department of Physiology, University of Toronto, Toronto, ON, Canada

Tsung-Hsien Chang 1, Li-Fong Wang 2, You-Sheng Lin 1, Chih-Shiang Yang 1, Chia-Yi Yu 3, Yi-Ling Lin 3, 1 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, 2 Division of Allergy, Immunology and Rheumatology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, 3 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Rationale: Insulin resistance is a major contributor to the development of type 2 diabetes, a multi-organ disease affecting >3  108 individuals. Over the last decade,

Hydroxychloroquine (HCQ) is an antimalarial drug also used in treating autoimmune diseases. It is also an autophagosome-lysosome fusion inhibitor broadly used in