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

炎症体的活动控制炎性细胞因子的产生，这些细胞因子将效应细胞招募到感染部位。炎症体由模式识别受体(PRR)、转接子和效应器组成。核苷酸结合域和富含亮氨酸重复序列的蛋白3(NLRP3)是PRRs的一种，它与含有caspase募集结构域(ASC)和proaspase-1的凋亡相关斑点样蛋白一起形成一个胞浆蛋白复合体，称为NLRP3炎症体，在调节A型流感病毒(IAV)感染的肺部炎症中发挥关键作用。我们先前的研究表明，猪流感病毒S感染可诱导原代猪肺泡巨噬细胞(PAM)产生NLRP3炎症体介导的IL-1，IL-1水平与肺组织病理改变有关。然而，猪传染性支气管炎病毒S感染后，NLRP3炎性小体如何激活和调节NLRP3，以及NLRP3在宿主免疫中的作用仍不清楚。 本研究所资助的研究项目的长期目标是了解猪先天性免疫反应对S病毒感染的调节作用。本研究的短期目标是了解猪NLPR3型炎症体的激活和调控对猪新城疫病毒S感染的影响及其在新城疫病毒S致病中的作用。 我提出三个具体目标： 1)阐明细胞因子激活和调节NLRP3炎性小体感染IAV-S的分子机制。我们将验证这样一种假设，即IAV-S感染PAM后，通过细胞激酶RIPK1(受体相互作用蛋白激酶1)诱导动力蛋白相关蛋白1(DRP1)的磷酸化，导致线粒体分裂。线粒体裂解释放的活性氧(ROS)导致NLPR3炎症体激活。 2)了解病毒因子对NLRP3炎症体的激活和调节作用。我们询问病毒蛋白PB1-F2如何通过改变线粒体动态来激活NLRP3炎症体；以及病毒NS1蛋白如何通过对NLRP3和ASC的翻译后修饰来调节炎症体的活动。携带PB1-F2或NS1突变的突变病毒将通过反向遗传学产生。他们激活炎症小体的能力将得到测试。 3)探讨NLRP3炎症体在猪新城疫病毒S发病机制中的作用。我们将研究哪些免疫细胞被招募到猪肺以应对新城疫病毒-S的感染；以及免疫细胞中NLRP3炎性小体的激活动力学。具有不同激活NLRP3炎症体能力的突变病毒的发病机制将在猪身上进行评估。 意义：平衡的炎症体激活水平被认为有利于宿主对抗病毒感染。本研究将为猪传染性支气管炎病毒S诱导猪炎症小体激活及调控机制的研究提供基础知识。这些知识最终将有助于更好地控制猪的猪流感。

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.