Gossypol Induces Pyroptosis In Mouse Macrophages Via A Non-canonical Inflammasome Pathway

Aim:Gossypol(GOS), a polyphenolic compound isolated from cottonseeds, has been reported to possess many pharmacological activities including anti-fungal, anti-inflammatory and anti-cancer effects, but whether it could influence inflammasome activation has not been reported. In this study, the mouse peritoneal macrophages and RAW 264.7 cells primed with or without bacterial lipopolysaccharide(LPS) were used to examine the effect of GOS on the activation of inflammasome in these cells. The aim of this study is to clarify the action mechanism of GOS in the activation of the inflammasome pathway, which would provide an experimental basis for its pharmacological and toxicological actions in innate immune cells. Methods:1. Mice were injected with 1 ml of 3 % thioglycollate to elicit a large number of peritoneal macrophages after 4 days.2. Propidium iodide staining assays were used to detect the cytotoxicity of GOS on macrophages.3. Western blot analysis was used to determine the effect of GOS on the expression levels of the inflammasome pathway associated protein in the macrophages primed with LPS.4. Cytometric beads array was used to detect the effect of GOS on secretion of mature IL-1β from macrophages primed with LPS.5. Mice were injected intraperitoneally with live E. coli DH5α, and the effect of GOS on mouse survival was recorded.Results:1. GOS induced cell death in mice peritoneal macrophages with or without LPS stimulation in a dose- and time-dependent manner. GOS-induced cell death was characterized by rapid membrane rupture and robust release of HMGB1 and pro-caspase-11 comparable to ATP treatment, suggesting a pyroptotic cell death.2. GOS induced pyroptotic cell death in ASC-deficient RAW 264.7 cells.3. GOS induced high levels of HMGB1, pro-IL-1β and pro-caspase-11 released from LPS-activated macrophages, whereas it could only induce low-level secretion of mature IL-1β.4. Pan-caspase inhibitor and caspase-1 subfamily inhibitor, but not caspase-3 inhibitor, could sharply suppress GOS-induced cell death. Non-specific pore-formation inhibitor glycine could attenuate GOS-induced pyroptosis, whereas other canonical pyroptotic inhibitors, including potassium chloride and N-acetyl-L-cysteine, suppressed ATP-induced pyroptosis but failed to inhibit or even enhanced GOS-induced cell death.5. GOS treatment eliminated thioglycollate-induced macrophages in the peritoneal cavity with the recruitment of other immune cells. Intraperitoneal GOS administration markedly decreased the survival of mice in a bacterial sepsis model.Conclusion: Gossypol induces pyroptosis in mouse macrophages via a non-canonical inflammasome pathway, which raises a concern for its in vivo cytotoxicity to macrophages.