Regulation of NLRP3 inflammasome activity in response to swine influenza virus infection

炎性小体活性控制炎性细胞因子的产生，其将效应细胞募集到感染部位。炎性小体由模式识别受体、衔接子和效应子组成。含核苷酸结合结构域和富含亮氨酸重复序列的蛋白3（NLRP 3）是PRR之一，它与包含半胱天冬酶募集结构域（ASC）和半胱天冬酶原-1（procaspase-1）的肺水肿相关斑点样蛋白（speck-like protein，speck-like protein，speck-like protein）一起形成称为NLRP 3炎性体的胞质蛋白复合物，在调节甲型流感病毒（IAV）感染后的肺部炎症反应中起关键作用。我们的前期研究表明猪流感病毒（IAV-S）感染可诱导原代猪肺泡巨噬细胞（PAM）产生NLRP 3炎性体介导的IL-1，且IL-1水平与肺病理相关。然而，IAV-S如何激活和调节NLRP 3以及NLRP 3炎性体如何在IAV-S感染猪后促进宿主免疫仍然是未知的。** 我的NSERC支持的研究计划的长期目标是了解猪感染IAV-S后先天免疫反应的调节。在这5年期间的短期目标是了解猪NLPR 3炎性小体在IAV-S感染后的激活和调节及其在猪IAV-S发病机制中的作用。** 我提出了三个具体目标：**1）阐明IAV-S感染后NLRP 3炎性体被细胞因子激活和调节的分子机制。我们将检验IAV-S感染PAM诱导细胞激酶RIPK 1（受体相互作用蛋白激酶1）磷酸化动力蛋白相关蛋白1（DRP 1），导致线粒体分裂的假设。线粒体分裂释放的活性氧（ROS）导致NLPR 3炎性小体的激活。** 2)了解NLRP 3炎性体是如何被病毒因子激活和调节的。 我们问病毒蛋白PB 1-F2如何通过改变线粒体动力学激活NLRP 3炎性小体;以及病毒NS 1蛋白如何通过对NLRP 3和ASC的翻译后修饰调节炎性小体活性。将通过反向遗传学产生携带PB 1-F2或NS 1突变的突变病毒。他们激活炎性小体的能力将被测试。3)研究NLRP 3炎性体在猪IAV-S发病机制中的作用。我们将研究什么样的免疫细胞被招募到猪肺中以响应IAV-S感染;以及免疫细胞中的NLRP 3炎性小体激活动力学。 将在猪中评价具有不同激活NLRP 3炎性体能力的突变病毒的发病机制。** 意义：平衡的炎性小体激活水平被认为有利于宿主对抗病毒感染。本研究为深入了解IAV-S诱导猪炎症小体的激活和调控机制奠定了基础。这些知识最终将有助于更好地控制猪流感。**

The inflammasome activity controls production of inflammatory cytokines, which recruit effector cells to the site of infection. Inflammasome is comprised with pattern recognition receptors (PRR), adaptors and effectors. Nucleotide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3) is one of the PRRs, together with apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) and procaspase-1, the NLRP3 forms a cytosolic protein complex called NLRP3 inflammasome which plays a pivotal role in modulating lung inflammation in response to influenza A virus (IAV) infection. Our previous study showed swine influenza virus (IAV-S) infection induced NLRP3 inflammasome-mediated IL-1 production in primary porcine alveolar macrophages (PAMs) and the IL-1 level is associated with lung pathology. However, how IAV-S activates and modulates NLRP3 and how NLRP3 inflammasome contributes to the host immunity upon IAV-S infection in pigs remain elusive. ******The long-term objective of my NSERC supported research program is to understand the regulation of innate immune responses upon IAV-S infection in pigs. The short-term objective during this 5-year term is to understand porcine NLPR3 inflammasome activation and regulation upon IAV-S infection and its role in IAV-S pathogenesis in pigs. ******I propose three specific aims:******1) To elucidate the molecular mechanisms by which NLRP3 inflammasome is activated and regulated by cellular factors upon IAV-S infection. We will test the hypothesis that IAV-S infection of PAMs induces phosphorylation of dynamin-related protein 1 (DRP1) by a cellular kinase RIPK1 (receptor-interacting protein kinase 1), resulting in mitochondria fission. Release of reactive oxygen species (ROS) from mitochondria fission leads to the activation of NLPR3 inflammasome.******2) To understand how NLRP3 inflammasome is activated and regulated by viral factors. We ask how viral protein PB1-F2 activates NLRP3 inflammasome through changing mitochondria dynamic; and how viral NS1 protein regulates inflammasome activity through post-translational modifications on NLRP3 and ASC. Mutant viruses carrying mutations in PB1-F2 or NS1 will be generated by reverse genetics. Their ability to activate inflammasome will be tested.******3) To examine the contribution of NLRP3 inflammasome to IAV-S pathogenesis in pigs. We will study what immune cells are recruited to the pig lung in response to IAV-S infection; and the NLRP3 inflammasome activation kinetics in immune cells. Pathogenesis of mutant viruses with different ability to activate NLRP3 inflammasome will be evaluated in pigs. ******Significance: A balanced inflammasome activation level is believed to be beneficial to the host to combat virus infection. The research will gain the fundamental knowledge on the mechanisms underlying IAV-S induced inflammasome activation and regulation in pigs. The knowledge will eventually contribute to a better control of swine influenza in pigs.**