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

炎性小体的活性控制炎症细胞因子的产生，炎症细胞因子将效应细胞招募到感染部位。炎性小体由模式识别受体（PRR）、受体和效应器组成。NLRP3是PRRs中的一种，与凋亡相关的斑点样蛋白含半胱天冬酶募集域（ASC）和原半胱天冬酶-1一起形成称为NLRP3炎症小体的细胞质蛋白复合物，在甲型流感病毒（IAV）感染时调节肺部炎症中起关键作用。我们之前的研究表明，猪流感病毒（IAV-S）感染诱导原代猪肺泡巨噬细胞（pam） NLRP3炎症小体介导的IL-1ß产生，IL-1ß水平与肺部病理相关。然而，IAV-S如何激活和调节NLRP3，以及NLRP3炎症小体如何参与猪感染IAV-S后的宿主免疫仍然是未知的。我的NSERC支持的研究项目的长期目标是了解猪对IAV-S感染的先天免疫反应的调节。5年的短期目标是了解猪NLPR3炎症小体在IAV-S感染中的激活和调控及其在猪IAV-S发病中的作用。我提出了三个具体目标：1)阐明IAV-S感染时NLRP3炎性体被细胞因子激活和调节的分子机制。我们将验证IAV-S感染PAMs诱导细胞激酶RIPK1（受体相互作用蛋白激酶1）磷酸化动力蛋白相关蛋白1 (DRP1)，导致线粒体分裂的假设。线粒体裂变释放活性氧（ROS）导致NLPR3炎性小体的激活。2)了解NLRP3炎性体是如何被病毒因子激活和调控的。我们询问病毒蛋白PB1-F2如何通过改变线粒体动态激活NLRP3炎性体；以及病毒NS1蛋白如何通过NLRP3和ASC的翻译后修饰调节炎性体活性。携带PB1-F2或NS1突变的突变病毒将通过反向遗传产生。他们激活炎性体的能力将被测试。3)探讨NLRP3炎性体在猪IAV-S发病中的作用。我们将研究哪些免疫细胞被招募到猪肺中以应对IAV-S感染；以及免疫细胞中NLRP3炎性体的激活动力学。具有不同激活NLRP3炎性体能力的突变病毒的发病机制将在猪中进行评估。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。该研究将获得IAV-S诱导猪炎症小体激活和调节机制的基础知识。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-1ß production in primary porcine alveolar macrophages (PAMs) and the IL-1ß 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.