Alcoholic hepatitis (AH) affects disproportionally high numbers of veterans, and has a mortality rate that has not changed substantially over the last decade. As AH pathogenesis has distinct features and is characterized by very severe hepatocyte injury, sterile inflammation and neutrophil recruitment; our goal is to identify molecular targets that have significant roles in these processes. We found that dysregulation of the Src homology 2 domain containing (Shc) collagen-related proteins play important roles in calreticulin (CTR) exposure, and immunogenic cell death during AH, exacerbating sterile inflammatory signalling cascades. To study the regulatory role of Shc proteins in AH, we demonstrated that Shc was induced in patients with AH, and inhibition of hepatocyte Shc in an animal model with AH resulted in significantly attenuated inflammation and oxidative stress. In hepatocytes Shc played a role in CTR translocation and exposure, a critical DAMP that determines immunogenicity of cell death elicited by alcohol. Furthermore in neutrophils Shc/NOX2-dependent signals induced neutrophil extracellular trap formation thereby further augmenting inflammatory injury in AH. Based on these, we hypothesize that activation of Shc signals leads to immunogenic cell death and pro-oxidant sterile inflammatory pathways in AH. We propose three SPECIFIC AIMS to address the key areas generated by the main hypothesis:
Our first aim i s to determine the molecular mechanism by which Shc proteins are involved in redox signaling and calreticulin (CTR) exposure as DAMP in hepatocytes during alcoholic hepatitis. a) We propose to study the mechanism of redox-mediated p52Shc activation. To evaluate the mechanistic aspects of p52Sh signals, deletion mutants will be generated by site-directed mutagenesis to delineate the key phosphorylation sites. Stress signaling will be evaluated in conjunction with ROS production, and CTR translocation. b) We will define the signals eliciting CTR exit from the ER using both biochemical approaches and the novel real-time, live-cell confocal fluorescence microscopy. In our second aim we will focus on the role of neutrophil extracellular trap (NET) formation in alcoholic hepatitis. a) We propose studies that evaluate the key role of Shc/NOX2 in NET formation, and interrogate downstream signaling cascades. We discovered that p52Shc directly binds to the p47phox NOX2 subunit and directly plays a role in enzyme activation. The interaction will be assessed by biolayer interferometry assays and lucigenin/Amplex red assays for ROS production. We will study the mechanistic aspects of p47phox mobilization in correlation to p52Shc binding, activation of the NOX2 complex in lipid rafts and correlate to NET formation (by FRET microscopy, and by dynamic live cell imaging). b) As persistence of NETs can fuel further inflammation, we will investigate the mechanism of defective NET phagocytosis in AH.
Our third aim i s to study the in vivo effects of Shc signaling on inflammation, oxidative injury and NETosis in alcoholic hepatitis. We will test the hypothesis that reducing Shc improves alcoholic hepatitis by limiting immunogenic death and NET formation, by using conditional cell- specific ShcKO mice (ShchepKO and Shcneutko). We will complement these studies by using the PAD4-/- mice that are defective in NET formation, and adaptive neutrophil transfer experiments. To provide translational relevance to these studies, we propose to use Shc inhibitors or PAD4 inhibitors alone, or in combination in the mouse models of AH.
Alcoholic hepatitis is one of the most severe form of liver diseases, affecting an increasing number of patients at the Veterans Administrations, leading to hospitalizations with significant rate of mortality. Currently there are only limited treatment approaches available for alcoholic hepatitis that are only successful in select cases. Thus there is an urgent need to better understand the pathogenesis of redox stress and sterile inflammation in this disease and to develop rational treatment approaches. This proposal is focused on defining the link between novel sterile inflammatory pathways and NADPH oxidase-mediated oxidative stress that are both induced in alcoholic hepatitis. Based on the data generated we propose to use a novel therapeutic approaches to reduce mortality in alcoholic hepatitis.
