Neutrophil Inflammasome Activation and Pyroptosis Induced by a Metalloproteinase via HIF-1α and Gasdermin D Pathways

Bothrops jararacussu venom contains snake venom metalloproteinases (SVMPs) that contribute to inflammation and tissue damage. BjussuMP-II, a PI class SVMP, lacks hemorrhagic activity but retains proteolytic and immunomodulatory properties.

Here, we uncover a previously unrecognized function of BjussuMP-II in triggering NLRP3 inflammasome activation in human neutrophils, leading to IL-1β release and pyroptosis. Inflammasome activation was investigated through gene expression analysis by quantitative PCR and protein expression by immunoblotting. Caspase-1 activation and gasdermin D (GSDMD) cleavage were assessed to confirm pyroptotic signaling. Pharmacological inhibition assays were performed to dissect the involvement of specific signaling pathways, and IL-1β secretion was quantified by enzyme immunoassay. This discovery reveals a direct molecular link between SVMP activity and inflammasome-mediated inflammation, a fundamental mechanism with implications beyond snakebite pathology. It highlights inflammasomes as potential therapeutic targets to mitigate severe inflammatory responses in envenomed patients.

Mechanistically, BjussuMP-II increased expression of NLRP3, ASC, caspase-1, NEK7, and HIF-1α, as well as IL-1β and GSDMD cleavage, confirmed by immunoblotting and immunofluorescence. It promoted the release of IL-1β, LTB₄, LDH, and dsDNA, consistent with pyroptosis, and this release was reduced by MCC950 or disulfiram. Studies in Gsdmd-/- and HIF-1α-deficient neutrophils further demonstrated the requirement of these pathways for cytokine release.

Collectively, these results indicate that BjussuMP-II modulates inflammasome-associated signaling pathways in neutrophils, contributing to the inflammatory responses triggered by SVMPs.