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      • HARVEST
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      CHARACTERIZATION OF PORCINE NLRP3 INFLAMMASOME-MEDIATED INTERLEUKIN-1 BETA PRODUCTION IN RESPONSE TO INFLUENZA A VIRUSES

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      PARK-DISSERTATION-2019.pdf (4.313Mb)
      Date
      2018-12-19
      Author
      Park, Hong-su 1980-
      Type
      Thesis
      Degree Level
      Doctoral
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      Abstract
      Influenza A virus (IAV) causes respiratory infections in humans and animals. Pigs are infected not only by swine influenza virus (SIV), but also by human and avian influenza viruses. Pigs serve as the mixing vessel for the generation of novel reassortant viruses with pandemic potential as evidenced by the emergence of 2009 human pandemic IAV (pdm09). Therefore, IAV infection in pigs poses both an economic concern and a threat to public health. Virus-host interplay dictates the outcome of virus infection. The host innate immune system can sense the virus infection and produce multiple cytokines and chemokines to control inflammation as well as to mount antiviral responses. On the other hand, the host innate immune response can be antagonized by IAV-encoded proteins, one being the non-structural protein 1 (NS1). Thus, a better understanding of the porcine innate immunity to IAV infection and the innate immune evasion strategies by IAV is required for a rational design to combat IAV infections in pigs. Innate immune cells in lungs, particularly the alveolar macrophages, are indispensable for host protection against IAV infection. They produce pro-inflammatory cytokines including interleukin-1 beta (IL-1β) that recruits other immune cells and promotes phagocytic activities. IL-1β production is tightly regulated by a cytosolic multiprotein complex called the nucleotide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3) inflammasome. The NLRP3 inflammasome is comprised of NLRP3, apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC), and pro-caspase-1. The molecular mechanism underlying the NLRP3 inflammasome-mediated IL-1β production upon IAV infection in the swine host, and the mechanism by which IAV NS1 protein counteracts the porcine IL-1β response remain largely unknown. This PhD project was centered on the characterization of the NLRP3 inflammasome-mediated IL-1β production in response to IAV infection in the swine host; specifically, I first characterized the IL-1β production in primary porcine alveolar macrophages (PAMs) in response to different isolates of IAV. Next, I investigated the molecular mechanisms by which viral NS1 protein modulates the porcine NLRP3 inflammasome activity, and finally I examined the molecular pathways that are involved in inflammasome activation upon IAV infection in PAMs. This study showed that while various SIV strains could induce NLRP3 inflammasome-mediated IL-1β production in primary PAMs, the human pdm09 virus induced much less IL-1β than did SIVs. Subsequent analyses revealed that the NS1 C-terminus of pdm09, but not that of SIV, was responsible for the significant inhibition of the NLRP3 inflammasome-mediated IL-1β production. Mechanistically, the NS1 C-terminus of pdm09 disrupted the interaction between NLRP3 and ASC, a prerequisite for the NLRP3 inflammasome assembly; this led to an impaired formation of ASC specks, a hallmark of NLRP3 inflammasome activation. NS1 C-terminus of pdm09 also suppressed the ubiquitination of porcine ASC. Furthermore, two lysine (K) residues, K110 and K140, on porcine ASC were identified as the ubiquitination acceptor sites; mutation of these two lysine residues diminished the ubiquitination of ASC and significantly reduced the IL-1β production in response to the pdm09 virus with the NS1 C-terminal deletion. These results revealed that the NS1 C-terminus of pdm09 suppresses ASC ubiquitination to support the immune evasion by the virus. Further attempts were made to dissect the upstream mechanism of the NLRP3 inflammasome-mediated IL-1β production upon SIV infection, focusing on the mitochondrial dynamics regulated by dynamin-related protein 1 (DRP1). This study showed that SIV infection induced not only phosphorylation of DRP1 at serine 579 (S579) that is required for mitochondrial fission activity of DRP1, but also mitochondrial fission in PAMs. The reactive oxygen species produced from mitochondrial fission was also related to the IL-1β production. Phospho-mimetic mutation at S579 on DRP1 could upreguate the NLRP3 inflammasome activity, leading to an increased IL-1β production. Furthermore, the requirement of the kinase activity of receptor-interacting protein kinase 1 (RIPK1) for the IL-1β production and the association of RIPK1 with DRP1 suggested that RIPK1 is an upstream kinase for DRP1 phosphorylation. These results indicated an integral role of the RIPK1/DRP1 signaling axis in modulating the porcine NLRP3 inflammasome-mediated IL-1β production in SIV-infected PAMs. Taken together, this study defined the mechanism by which SIV induces porcine NLRP3 inflammasome-mediated IL-1β production and elucidated the strategies pdm09 employs to circumvent the host innate immunity. The obtained information will enhance our knowledge of the innate immunity to IAV infection in the swine host.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Veterinary Microbiology
      Program
      Veterinary Microbiology
      Supervisor
      Zhou, Yan
      Committee
      Hill, Janet E; Tikoo, Suresh K; Wilson, Joyce A; Zakhartchouk, Alexander
      Copyright Date
      May 2019
      URI
      http://hdl.handle.net/10388/11674
      Subject
      influenza A virus
      NLRP3 inflammasome
      interleukin-1 beta
      porcine alveolar macrophage
      non-structural protein 1
      ASC ubiquitination
      dynamin-related protein 1
      mitochondrial fission
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