Grant: Discovery Grants Program - Individual

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.