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Abstract / Cytokine 70 (2014) 28–79 113 Deacetylation-dependent regulation of RIG-I activation by the HDAc6 deacetylase mediates innate anti-viral immunity Helene Minyi Liu 1, Yueh-Ming Loo 2, Tien-Ying Hsiang 2, Fuguo Jiang 3, Joseph Marcotrigiano 3, Michael Jr. Gale 2, 1 National Taiwan University, Taipei City, Taiwan, 2 University of Washinton, Seattle, WA, United States, 3 Rutgers University, Piscataway, NJ, United States Retinoic-acid inducible gene I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the immune response against RNA viruses. Upon ligand binding, RIG-I undergoes a conformational change for homodimerization, and this RNA ligand-induced dimerization is a prerequisite of its induction of downstream innate immune signalling. The repressor domain (RD) of RIG-I governs RIG-I conformational change and RIG-I dimerization; however, how RIG-I dimerization is regulated beyond these processes is unclear. Here we report that reversible acetylation modifications of lysine residues in the RD controls RIG-I activation. Acetyl-mimetic mutants of RIG-I failed to signal downstream to activate IRF-3 in response to acute SenV infection, while HCV RNA recognition and binding activities of RIG-I were intact. Acetyl-mimetic mutants of RIG-I could not form virus-induced homodimers, suggesting that acetyl-lysine residues in the resting cells prevent RIG-I from homodimerization. During acute infection, deacetylation of RIG-I RD occurs and promotes RIG-I dimerization upon ligand binding. Our preliminary data indicated that the cytoplasmic deacetylase, Histone deacetylase 6 (HDAC6), may be involved in deacetylation of RIG-I during acute infection. HDAC6 knock-down cells were impaired in RIG-I-dependent IFN-beta promoter activity during SenV infection, and overexpression of HDAC6 accelerated the kinetics of RIG-I activation in response to infection. Our results define deacetylation as a key step in controlling RIG-I activation required for innate immunity. http://dx.doi.org/10.1016/j.cyto.2014.07.120

114 The incorporation of human recombinant tropoelastin improves the biocompatibility of high or low porosity polyurethane Hongjuan Liu, Steven G Wise, Anthony S Weiss, Shisan Bao, The University of Sydney, Sydney, NSW, Australia Polyurethanes (PU) are synthetic polymer and have been widely used in medical devices and artificial organs for tissue repair. High and low porous polymer scaffolds containing recombinant human tropoelastin (TE) and PU were generated by electrospinning technique; these scaffolds have favourable mechanical and biological properties used as implantable applications. This study was to evaluate the acute and chronic inflammatory response to implantation of low and high porosity PU scaffolds with/without TE in mouse model. The back of each C57B/L6 mouse (n = 32) was subcutaneously implanted with control PU, low porosity PU/tropoelastin (PU/TE) and high porosity PU/tropoelastin (HP PU/TE) implants. Mice (n = 8) were euthanized and harvested the tissue samples at 1 day, 1 week, 2 weeks and 4 weeks. Histopathology and immunohistochemistry were performed to assess inflammatory cell infiltration, collagen deposition and granulation formation of each sample. Compared to control PU, fewer neutrophils were found on PU/TE and HP PU/TE implant surface, associated with lower expression of GM-CSF at 1 day following implantation. After 1 weeks, compared the surrounding granulation tissue formation from PU, both HP PU/TE and PU/TE have been found that thinner capsule thickness with less total infiltrating cells and proliferating cells PCNA, fewer macrophage/FBGCs F4/80, less neovascularization VWF, down-regulation level of MMP-2, MMP-9 and cytokines, including GM-CSF, IL-6, IL-10. Compared the tissue ingrowth of low porosity PU/TE implant, high porous HP PU/TE scaffolds demonstrated a larger number of total infiltrating cells inside the implant, associated with larger collagen deposition, greater proliferation rate PCNA, higher level of cytokines GM-CSF, IL-10 and MMP-2 after 2 weeks. Overall results suggested that firstly, the incorporation of TE into either high or low porosity PU scaffold could improves in the biocompatibility of PU alone, uniquely measured here as a reduced foreign body inflammatory response and better wound healing response at whole implant sites. Secondary, high porosity PU/TE enhanced and accelerated the active cells proliferation and infiltration into the scaffolds compared to low porosity scaffold. http://dx.doi.org/10.1016/j.cyto.2014.07.121

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115 Phosphatase PP4 negatively regulates antiviral innate immune response by dephosphorylating TBK1 in macrophages Xingguang Liu 1, Zhenzhen Zhan 2, 1 Second Military Medical University, Shanghai, China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China

2

Effective recognition of viral infections and subsequent production of type I interferon are essential for the host antiviral innate immune responses. Type I IFN expression is tightly regulated by multiple intracellular regulators to prevent the development of immunopathological disorders and the harm to host. The phosphorylation and activation of serine/threonine kinase TBK1 plays an essential role in mediating type I interferon production. However, how the activated TBK1 is negatively regulated by its phosphatase(s) remains poorly understood. In this study, we report that PP4 expression was upregulated in Sendai virus (SeV)- and vesicular stomatitis virus (VSV)-infected macrophages. PP4 inhibited type I interferon production in macrophages triggered by SeV and VSV, thus enhanced RNA virus replication. Overexpression of a phosphatase-dead PP4 mutant (PP4-RL) abolished the inhibitory effects on type I interferon production. Accordingly, PP4 knockdown increased RNA virus-triggered type I IFN production and inhibited RNA virus replication. Moreover, PP4 directly interacted with TBK1 upon virus infection then dephosphorylated TBK1 at Ser172 and inhibited TBK1 activation, and subsequently suppressed IRF3 activation, resulting in suppression of virus-triggered innate immune response. Thus, PP4 functions as a feedback-negative regulator of RNA virus-triggered innate immune responses by function as a TBK1 phosphatase and suppress its activation. http://dx.doi.org/10.1016/j.cyto.2014.07.122

116 Regulation of STING via the RIG-I dependent RNA sensing pathway Yiliu Liu 1,2, Sidi Mehdi Belgnaoui 1, Alexander Sze 1,2, Marie-Line Goulet 1, Chunfu Zheng 3, Rongtuan Lin 1,2,4, 1 Lady Davis Institute, Montreal, Quebec, Canada, 2 Dept. of Experimental Medicine, McGill University, Montreal, Quebec, Canada, 3 Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China, 4 Dept. of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada The cytoplasmic pattern recognition receptor RIG-I is essential for recognizing RNA viruses with a 50 triphosphate (ppp) signature. Upon viral RNA recognition, RIG-I recruits adaptor protein MAVS to trigger the activation of IRF3 and NF-kB transcription factors through TBK1-IKK complexes, leading to the production of type I IFNs (a, b), pro-inflammatory cytokines, and antiviral factors. STING has been identified as an RIG-I signaling cofactor and a critical adaptor protein in a recently identified cGAS-mediated cytosolic DNA sensing pathway. In a recent functional study aiming to gain system-wide insight into downstream effector function of RIG-I, we identified STING among a plethora of differentially expressed genes induced by the RIG-I agonist 50 ppp RNA; in the present study, we further detail the mechanism of STING regulation. Our data shows that Sendai virus (SeV) infection induces STING expression at both the mRNA and protein levels in various cell types including A549, Huh7, PC3, and U87. Furthermore, by employing multiple RIG-I deficient or pathway-impaired cell lines, STING induction is shown to be dependent on functional RIG-I signaling. STING induction by the RIG-I agonist 50 ppp RNA was recognized as a delayed event resulting from an autocrine/paracrine mechanism. Indeed, co-treatment with TNFa and IFNa has a synergistic effect on the regulation of STING expression. Following SeV infection or TNFa–IFNa combined treatment, STING induction is partially decreased by siRelA or siIRF3; and is strongly diminished under combined siRelA and siIRF3 condition. Taken together, these observations demonstrate that STING expression is regulated via RIG-I signaling. http://dx.doi.org/10.1016/j.cyto.2014.07.123

117 IRF-3, IRF-7, and IPS-1 promote host defense against acute human metapneumovirus infection in neonatal mice Zhixuan Loh, Kirsten Spann, Jason Lynch, Ashik Ullah, Vivian Zhang, Engin Baturcam, Rhiannon Werder, Natthida Khajornjiraphan, Penny Rudd, Yeuh-Ming Loo, Andreas Suhrbier, Michael Gale Jr., John Uqham, Simon Phipps, University of Queensland, St. Lucia, QLD, Australia