PhospholipaseCγl/calcium-dependent membranous localization of Gsdmd-N drives endothelial pyroptosis, contributing to lipopolysaccharide-induced fatal outcome

Multiple organ perfusion is impaired in sepsis. Clinical studies suggest that persistent perfusion disturbances are prognostic of fatal outcome in sepsis. Pyroptosis occurs upon activation of caspases and their subsequent cleavage of gasdermin D (Gsdmd), resulting in Gsdmd-N (activated NH2-terminal fragment of Gsdmd) that form membrane pores to induce cell death in sepsis. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide (LPS). However, how Gsdmd-mediated pyroptosis occurs in endothelial cells and leads to impaired perfusion remain unexplored in endotoxemia. We used transgenic mice with ablation of Gsdmd and determined that mice lacking Gsdmd exhibited reduced breakdown of endothelial barrier, improved organ perfusion, as well as increased survival in endotoxemia. Phospholipase C gamma l (PLC gamma 1) contributed to Gsdmd-mediated endothelial pyroptosis in a calcium-dependent fashion, without affecting Gsdmd-N production. Cytosolic calcium signaling promoted Gsdmd-N translocation to the plasma membrane, enhancing endothelial pyroptosis induced by LPS. We used adeno-associated virus (AAV9) vectors carrying a short hairpin RNA (shRNA) against murine PLC gamma 1 mRNA under control of the tiel core promoter (AAV-tiel-shPLC gamma 1) to uniquely downregulate PLC gamma 1 expression in the endothelial cells. Here, we showed that unique inhibition of endothelial PLC gamma 1 attenuated breakdown of endothelial barrier, reduced vascular leakage, and improved perfusion disturbances. Moreover, unique downregulate endothelial PLC gamma 1 expression markedly decreased mortality of mice in endotoxemia. Thus, we establish that endothelial injury as an important trigger of fatal outcome in endotoxemia. Additionally, these findings suggest that interfering with Gsdmd and PLC gamma 1-calcium pathway may represent a new treatment strategy for critically ill patients sustaining endotoxemia. NEW & NOTEWORTHY Our study newly reveals that Phospholipase C gamma l (PLC gamma 1) contributes to gasdermin D (Gsdmd)-mediated endothelial pyroptosis in a calcium-dependent fashion. Cytosolic calcium signaling promotes activated NH2-terminal fragment of Gsdmd (Gsdmd-N) to translocate to the plasma membrane, enhancing endothelial pyroptosis induced by cytoplasmic LPS. Genetic or pharmacologic inhibition of endothelial PLC gamma 1 attenuated breakdown of endothelial barrier, reduced vascular leakage, improve perfusion disturbances, and decrease mortality of mice in endotoxemia.