Withania somnifera extracts and withaferin A differentially regulate neutrophil pyroptosis, NETosis, and apoptosis

Neutrophils are short-lived, yet critical components of the innate immune system. These cells play crucial roles in host defense and inflammation. Their high turnover during inflammatory responses is often accompanied by inflammatory lytic cell death, such as NETosis and pyroptosis, which can amplify tissue damage. Thus, targeting neutrophil death pathways is an emerging therapeutic area. Withania somnifera (L.) Dunal (WS), a key Ayurvedic herb, shows immunomodulatory properties, but its effects on neutrophils remain underexplored. In this study, we investigated the impact of WS extracts and their major constituents, including withanolide A (WL), withanoside IV (WT), withanone (WO), and withaferin A (WFA), on the mechanisms of neutrophil death. Our findings reveal that WS extracts significantly attenuate interleukin-1β (IL-1β) release and pyroptotic cell death, with the alcoholic extract exhibiting greater efficacy than hydro-alcoholic and aqueous preparations. These effects are linked to the inhibition of NLRP3 inflammasome activation and caspase-1 signaling. Moreover, the alcoholic extract of WS suppressed phorbol 12-myristate 13-acetate (PMA)-induced NET formation, but did not affect ionomycin-induced NETosis. This selective inhibition was associated with a reduction in reactive oxygen species (ROS) generation, but had no effect on peptidyl arginine deiminase 4 (PAD4) activity. The WS alcoholic extract also conferred protection against neutrophil apoptosis, as evidenced by the downregulation of executioner caspase-7. Further analysis specified that WFA was primarily responsible for these effects. Together, these results provide novel insights into the role of WS and WFA in modulating neutrophil death pathways. These findings further highlighted the therapeutic potential of WS in inflammatory and neutrophil-mediated pathological conditions.