Role of peptidylarginine deiminase in modulating macrophage functions in sepsis

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Project Summary Sepsis is a life-threatening organ dysfunction caused by a multifaceted dysregulated host response to an infection, including sepsis induced acute lung injury (sepsis-ALI). Sepsis is the most common cause of in- hospital deaths and costs more than $24 billion annually in the US. No specific therapy exists to prevent or treat the cellular dysfunction associated with sepsis and sepsis-ALI. While the complex mechanisms of sepsis have remained poorly defined, an emerging pathogenesis of sepsis and sepsis-ALI involves neutrophil and macrophage cell death causing inflammation and organ damage through the release of citrullinated histone 3 release (CitH3). Petidylarginine deiminase (PAD) 2 and 4 catalyze the formation of CitH3. PAD2 and CitH3 have been linked to sepsis with findings of elevated serum levels of PAD2 and CitH3 in septic patients. Mouse models have further demonstrated that knockout of Pad2 and utilization of pan-PAD inhibitors improved survival and prevented organ dysfunction in several different sepsis models. Throughout the past decade, Dr. Alam and Dr. Li have conducted extensive work on the PAD-CitH3 pathway in sepsis. This proposal is linked to the NIH R01 proposal (1R01HL155116-01A1) “PAD2 and CitH3 in Pathogenesis of Sepsis-induced ALI” by Dr. Alam and Dr. Li with a specific focus on elucidating the mechanisms of PAD and their physiologic impacts and modulation of macrophages in sepsis. In Aim 1, we will conduct experiments using Pad 2-/-, Pad4-/-, and Pad2-/-Pad4-/- mice to investigate the interaction of PAD2 and PAD4 in sepsis and sepsis-ALI outcomes. In Aim 2, we will use adeno-associated virus (AAV)- mediated delivery of Cas9/gRNA to perform targeted knockout and restoration of Pad2 in macrophages. Outcomes of mice with sepsis and their macrophage activity with somatic ablation and targeted RNA knockout will be compared to assess possible compensatory changes in mice with somatic ablation. For Aim 3, we will use RNA sequencing and proteomics to identify genes associated with the compensation of Pad2 ablation. The knowledge gained from this project will have a significant impact on our understanding of the PAD-CitH3 pathway in sepsis. This will contribute significant insight in to the pathogenesis and potential treatment of sepsis, sepsis-ALI, and sepsis induced multi-organ dysfunction.