Human NLRC4 and CASP4 inflammasomes activate GSDMD-mediated killing of S. flexneri independently of macrophage pyroptosis and bacterial cardiolipin 2254736

Abstract Introduction Elimination of the infecting pathogen is a core function of the immune system. Macrophages are a crucial component of innate immunity against bacterial infections as they internalize and kill intracellular bacteria. Although macrophage restriction of some intracellular bacteria depends on inflammasomes, the specific mechanisms of inflammasome-mediated killing of bacteria are incompletely understood. Methods The role of inflammasomes in macrophage killing of Shigella flexneri is assessed by quantification of intracellular bacteria during infection of wild-type human-derived THP-1 macrophages and macrophages deficient in inflammasome components. Chloroquine resistance assay is used to assess IFNγ impact on vacuolar escape. Pyroptosis and GSDMD pore formation are inhibited with glycine and disulfiram. To assess GSDMD-mediated killing, GSDMD-/- macrophages are used and GSDMD is expressed ectopically in HEK293T cells. Results I found that S. flexneri is restricted in human macrophages and that macrophage restriction is enhanced by priming with IFNγ. IFNγ does not impact bacterial internalization and vacuolar escape, indicating that the intracellular restriction takes place in the cytosol. IFNγ priming results in killing of S. flexneri independently of pyroptosis and macrophage lysis. I found that macrophage restriction depends on NLRC4-mediated activation of CASP1 and GSDMD in unprimed macrophages. In contrast, I found that CASP4 is required for efficient IFNγ-dependent killing of S. flexneri and that IFNγ upregulates CASP4 and enhances bacterial restriction independently of GBP1 and NLRP11. Expression of GSDMD in HEK293T cells leads to killing of intracellular S. flexneri. GSDMD kills S. flexneri lacking cardiolipin and when oxidative stress is inhibited. Conclusion GSDMD is the executioner of inflammasome-mediated killing of S. flexneri. A new mechanism of GSDMD antibacterial activity is identified as GSDMD kills S. flexneri independently of cardiolipin and oxidative stress. Funding Source NIH T32 AI007061, NIH F32 AI188955 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)