Innate Immune Function in Influenza-Associated Myopathy

PROJECT SUMMARY / ABSTRACT Skeletal muscle myopathy has been reported following infection by influenza virus. In Influenza-Associated Myopathy (IAM), a systemic influenza infection can result in acute skeletal muscle damage that ranges from non- specific degeneration to extensive necrosis. IAM is associated with high serum creatine kinase (CK) levels. During the 2009 H1N1 influenza epidemic, 62% of hospitalized patients had increased serum CK levels. Given that an estimated 9-45 million individuals acquire influenza infections annually in the US, developing new strategies to reduce muscle damage are needed as influenza vaccines are difficult to design because of unpredictable changes in viral strains within and across populations. The severity of viral disease varies between individuals and depends on how the immune system responds to infection. One roadblock to understanding the pathogenesis of IAM is that the relative contributions of the virus and host factors in vivo are not well understood. Biopsy studies cannot show the temporal dynamics of viral invasion and subsequent recruitment of neutrophils and macrophages into muscle. The zebrafish is a powerful model to study host-pathogen interactions as genetic tools can be combined with in vivo imaging of transparent embryos. My laboratory uses a recently-developed zebrafish model of influenza A virus (IAV) infection where it was shown that: 1) IAV-infected zebrafish embryos exhibited mild muscle degeneration with sarcolemma damage and compromised extracellular matrix (ECM) adhesion; and 2) neutrophils localize to sites of muscle damage in IAV-infected embryos. Our specific goal in this proposal is to determine the mechanisms through which neutrophils influence the pathology of IAM. This project will test the novel hypothesis that overactivation of neutrophils during IAV infection triggers a damaging hyperinflammatory response that contributes to myopathy. In the first aim, we will test the hypothesis that reduction in reactive oxidative species (ROS) production following IAV infection will limit damage by strengthening muscle cell-ECM adhesion, and increase survival. This will be accomplished by examining how global ROS reduction and neutrophil-specific ROS reduction alters muscle degeneration, cell-ECM adhesion, and neutrophil localization in the muscle in vivo using IAV multi-spectral fluorescent reporter (Color-flu) strains. In the second aim, we will test the hypothesis that defects in neutrophil migration following IAV infection will increase muscle degeneration, and weaken ECM adhesion. To accomplish this, we will use in vivo confocal imaging to study two zebrafish mutants with defective neutrophil migration infected with Color-flu to test our hypotheses that: 1) neutrophil invasion into skeletal muscle is reduced, and 2) muscle degeneration is increased and cell-ECM adhesion is increased over controls. One of these mutants overexpresses miR-199 in neutrophils that disrupts neutrophil migration. Proposed RNA sequencing will allow us to determine microRNA gene regulatory networks thereby allowing us to establish a link between IAM and microRNA genetic regulation. Knowledge gained through the study of IAM may inform studies of other viral-associated myopathies.