Intestinal NOD2 and the microbiota enhance Coxsackievirus B3 pathogenesis - PROJECT SUMMARY Group B coxsackieviruses (CVB) are in the genus Enterovirus and Family Picornaviridae. CVBs are spread by fecal-oral transmission and can cause diseases including intestinal inflammation, pancreatitis, myocarditis and central nervous system diseases. Sensing of CVB3 RNA by melanoma differentiation-associated gene (MDA5) induces a Type 1 Interferon (T1IFN) response that is critical for reducing viral replication and dissemination within cells and the host. The pattern recognition receptor (PRR) Nucleotide Oligomerization Domain protein 2 (NOD2), which is critical for intestinal immunity, is classically known to detect bacterial peptidoglycan. Once stimulated, NOD2 requires RIP2 kinase (RIP2K) to activate NF-kB leading to a pro-inflammatory response. More recent data has demonstrated that NOD2 can also function as a cytoplasmic viral PRR by activating the production of T1IFNs in response to Influenza A virus. Interestingly, NOD2 has been implicated in a proviral role in CVB3-induced myocarditis, however, the underlying mechanism is not well understood. Because NOD2 is especially important in regulating the intestinal microbiota and maintaining intestinal immune homeostasis and CVB3 is an enteric virus that initially infects the intestinal tract leading to virus dissemination to other tissues, we sought to determine whether NOD2 expressed intestinal epithelial cells (IECs) would impact CVB3 infection. We observed that IEC NOD2 exacerbates CVB3 pathogenesis and fecal shedding in mice that were infected with CVB3. Additionally, we observed that the intestinal microbiota is required for CVB3 pathogenesis, suggesting that the microbiota may influence CVB3 infection by modulating host immunity through IEC NOD2. Furthermore, we demonstrate that IECs deficient for NOD2 have increased T1IFNs and Interferon Stimulated Genes (ISG) expression that limits CVB3 replication. CVB3 replication and ISG expression was rescued when IFIH1 (MDA5) expression was reduced with small interfering RNAs (siRNAs). In addition, NOD2-/- IECs have impaired mitochondrial health measured by increased ROS production and loss of mitochondrial membrane potential. In this proposal we will test our hypothesis that microbiota activation of IEC NOD2 induces mitophagy which blunts T1IFN responses and MDA5 expression resulting in exacerbated CVB3-induced pathogenesis. We will test key aspects of our hypothesis using the logical and innovative approach outlined in the following specific aims. Aim 1. Examine if CVB3 infection alters the microbiome causing dysbiosis that activates IEC NOD2 to contribute to CVB3-induced pathogenesis. Here we will define whether IEC NOD2 and the microbiota are initiators of CVB3-induced pathogenesis. Aim 2. Determine if NOD2/RIP2K expressed in IECs induces mitophagy to inhibit MDA5 activation and T1IFN responses. We will test whether NOD2/RIP2K signaling contributes to effective mitophagy leading to decreased MDA5-mediated antiviral responses and susceptibility to CVB3 infection. Characterizing and understanding the mechanisms of NOD2-dependent proviral responses are innovative new concepts that would markedly influence the current beliefs of NOD2 biology.
