Sepsis-Associated Immune Suppression and F-box Proteins

Sepsis is a devastating disorder and a leading cause of death in the US. Prior studies have focused mainly on its hyper-inflammatory state and yet anti-inflammatories for sepsis have not shown benefit. Mounting data suggest that immune suppression partakes in this disorder, the molecular mechanisms of which remain unclear. This application investigates a unique molecular model whereby we discovered an immunosuppressive protein called Fbxo24, that disposes of a mitochondrial synthetic protein, called DARS2, that is indispensable for critical cytoprotective functions of epithelia including innate immune function and preservation of cell proliferative activity. Our hypothesis is that Fbxo24, in part, mediates immunosuppression in experimental sepsis through ubiquitin-mediated degradation of DARS2, a secreted protein essential for host epithelial cell innate immune and reparative responses. Hence, in this application we will first elucidate if Fbxo24 targets DARS2 for its ubiquitin-mediated proteolysis in experimental sepsis (Aim 1). We will specifically elucidate the biologic relevance of reduced Fbxo24 levels in preclinical models of sepsis and organ injury as an immune suppressor using gene transfer in our Fbxo24 knockout mice and the mechanisms by which Fbxo24 mediates DARS2 ubiquitination and degradation in a site-specific manner. Next, we will determine if DAR2 is a crucial extracellular host defense protein in experimental sepsis (Aim 2). Here we will elucidate mechanistically how DARS2 is essential to maintain innate immune responses and cell proliferative activity through its regulated secretion from epithelia in vitro and in vivo. We will employ complementary murine and 2-hit models of immune suppression and an ex vivo isolated human lung system. These studies will provide a new pathobiologic model of immune dysregulation. This work serves as a platform for generating genetically engineered proteolytically stable DARS2 proteins that optimize epithelial cytoprotective functions in subjects with severe immune suppression in critical illness.