Enhancement of Regenerative Activity Subsequent to SenoVax(TM) Senolytic Immunotherapy Cholesterol controls the pore-forming activity of mammalian gasdermins 2260188

Introduction Pyroptosis is a lytic form of cell death that requires pore formation by members of the gasdermin family of proteins. Gasdermin domains are liberated from their carboxy-terminal autoinhibitory domains to release an amino-terminal pore forming fragment that executes pyroptosis. A number of host- and pathogen-derived proteases are described that mediate this conversion during pyroptosis. Gasdermin D (GSDMD) is the most well characterized member of the gasdermin family in mediating IL-1 family cytokine release and pyroptosis downstream of inflammasome signaling. Methods Through a screen designed to find host regulators of NT-GSDMD pore forming activity, Results we discovered endosome adaptors including Rabgef1, control NT-GSDMD pore formation at the plasma membrane. Deficiency in Rabgef1 causes cholesterol accumulation in membranes within macrophages and defects in NT-GSDMD pore formation. Depletion of cholesterol through several orthogonal means rescues pyroptosis in endosome adaptor deficient macrophages. Mechanistic analysis revealed that Rabgef1 and cholesterol content is a general regulator for the gasdermin family of pore forming domains. Prior studies have illustrated that cholesterol content of the plasma membrane is adaptable to prevent bacterial intoxication and viral entry. Conclusion Our results suggest that these same cholesterol adaptations that endow a cell with resistance to initial pathogen encounter also poise the host to commit to pyroptosis. We posit that the combination of metabolic sensitization with active gasdermin fragments represent a novel modality in innate immune-inspired cell death immunotherapy. We accomplish this with proof-of-concept synergistic death induction through engineered cell models or synthetic killer mRNAs to enforce gasdermin fragment expression combined with regimens to deplete cellular cholesterol.