Gasdermin D in Bronchoalveolar Lavage Fluid Causes Cell Death Ex Vivo in Pulmonary Epithelial Cells

Abstract

RATIONALE: Acute respiratory distress syndrome (ARDS) is diagnosed in approximately 190,000 Americans annually and has a mortality rate up to 46%. The hallmark of ARDS is a dysregulated immune response, which leads to uncontrolled inflammation and endothelial and epithelial cell injury and death. Gasdermin D is a poreforming protein activated during this inflammatory cascade and induces pyroptosis within infected cells. Active gasdermin D has been found in extracellular vesicles, called microparticles, released from activated macrophages and can cause pulmonary vascular endothelial cell injury. This study investigated for the presence of active gasdermin D in bronchoalveolar lavage (BAL) fluid and the impact of gasdermin D-containing fluid on pulmonary epithelial cell death ex vivo. METHODS: Active gasdermin D levels were measured in BAL fluid collected from 44 hospitalized patients and three healthy volunteers via immunoblot and optical densitometry. A549 and primary pulmonary epithelial cells were exposed to patients' BAL fluid and evaluated for cell death. Cell death was measured in A549 pulmonary epithelial cells exposed to microparticles from LPS-stimulated THP-1 cas9 cells and LPS-stimulated THP-1 cas9/GsdmD knockouts. Finally, cell death was measured in primary pulmonary epithelial cells exposed to gasdermin D-immunodepleted BAL fluid. RESULTS: Significantly higher levels of active gasdermin D were found in the BAL fluid of patients with infection when compared to uninfected hospitalized patients and healthy volunteers. In A549 pulmonary epithelial cells, there was significantly greater cell death by LDH assay in cells exposed to BAL fluid from infected patients compared to fluid from healthy volunteers. There was a trend towards greater cell death by LDH assay in primary pulmonary epithelial cells exposed to BAL fluid from infected patients compared to fluid from healthy volunteers. Furthermore, in A549 pulmonary epithelial cells exposed to microparticles from LPS-stimulated THP-1 cas9 macrophages, there was significantly greater cell death by LDH assay compared to controls and cells exposed to microparticles from unstimulated THP-1 cas9 cells. This effect on cell death was abrogated when the epithelial cells were exposed to microparticles from LPS-stimulated THP-1 cas9/GsdmD knockouts. Finally, in primary pulmonary epithelial cells, the effect on cell death by BAL fluid from infected patients was significantly diminished by immunodepletion of active gasdermin D from the fluid. CONCLUSIONS: Microparticle gasdermin D, a pyroptotic protein, was found in the BAL fluid of hospitalized patients. Its presence was associated with infection in patients and with higher levels of pulmonary epithelial cell death ex vivo.