Activation of GSDME compensates for GSDMD deficiency in a mouse model of NLRP3 inflammasomopathy

Abstract

AbstractThe D301N NLRP3 mutation in mice (D303N in humans) causes severe multi-organ damage and early death driven by the constitutively activated NLRP3 (NLRP3ca) inflammasome. Triggered inflammasomes activate caspase-1 to process IL-1 family cytokines and gasdermin D (GSDMD), generating N-terminal fragments, which oligomerize within the plasma membrane to form pores, which cause inflammatory cell death (pyroptosis) and through which IL-1β and IL-18 are secreted. GSDMD activation is central to disease symptoms since spontaneous inflammation inNlrp3ca;Gsdmd-/-mice is negligible. Unexpectedly, whenNlrp3ca;Gsdmd-/-mice were challenged with LPS or TNF-α, they secreted high amounts of IL-1β and IL-18, suggesting an alternative GSDMD-independent inflammatory pathway. Here we show that GSDMD deficient macrophages subjected to inflammatory stimuli activate caspase-8, -3 and GSDME-dependent cytokine release and pyroptosis. Caspase-8, -3 and GSDME also activated pyroptosis when NLRP3 was stimulated in caspase-1 deficient macrophages. Thus, a salvage caspase-8, -3-GSDME inflammatory pathway is activated following NLRP3 activation when the canonical NLRP3-caspase-1-GSDMD is blocked. Surprisingly, the active metabolite of the GSDMD-inhibitor disulfiram, inhibited not only GSDMD but also GSDME-mediated inflammationin vitroand suppressed severe inflammatory disease symptoms inNlrp3camice, a model for severe neonatal multisystem inflammatory disease. Although disulfiram did not directly inhibit GSDME, it suppressed inflammasome activation in GSDMD-deficient cells. Thus, the combination of inflammatory signals and NLRP3caoverwhelmed the protection provided by GSDMD deficiency, rewiring signaling cascades through caspase-8, -3 and GSDME to propagate inflammation. This functional redundancy suggests that concomitant inhibition of GSDMD and GSDME may be necessary to suppress disease in inflammasomopathy patients.