Phosphorylation of NLRP6 at Serine 27 site is critical for NLRP6 inflammasome activation and neurological impairment in ischemic stroke.

Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 6 (NLRP6) is a pattern recognition receptor that is highly expressed in microglia and forms a cytosolic inflammasome to aggravate neuroinflammation and neuronal injury during ischemic stroke. However, the post-translational mechanisms regulating the NLRP6 inflammasome activation are not well characterized. Herein, we demonstrated that the phosphorylation of NLRP6 was markedly increased in response to cerebral ischemia and was closely associated with NLRP6 inflammasome function. Serine 27 (Ser27) was identified as a conserved phosphorylation site in NLRP6, and NLRP6 inflammasome activity was tightly regulated by phosphorylation at this site. Moreover, the oligomerization and subsequent inflammasome assembly and activation of NLRP6 were positively regulated by Ser27 phosphorylation. Phenotypically, dephosphorylation at the Ser27 site blocked NLRP6 inflammasome activation in ischemic stroke, drastically reducing inflammasome-related neuroinflammation and neurological injury in vitro and in vivo. Our findings demonstrate a pivotal mechanism regulating NLRP6 inflammasome activation in ischemic stroke and provide evidence for the therapeutic targeting of NLRP6 Ser27 phosphorylation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02683-9.