Dopamine Activates Dual Caspase and Granzyme B Pathways for Gasdermin-Mediated Inflammasome Signaling 2250456

Introduction Beyond its role in motor control, dopamine (DA) exhibits species-specific immunoregulatory functions controlling inflammation. We previously showed that DA induces inflammatory responses in primary human monocyte derived macrophages (hMDMs) through NF-κB-mediated activation of the NLRP3 inflammasome. Methods hMDMs from healthy individuals were exposed to DA (1 µM) for 5 hours, followed by BzATP (200 µM), a potent P2X7 receptor agonist, for 1 hour to stimulate NLRP3 activation. High-content imaging, immunoblotting and qPCR were then used to assess distinct stages of NLRP3 activation. Additionally, pharmacological inhibition studies were performed to determine the contribution of specific proteases to DA-mediated inflammasome activation. Results Our findings showed that DA-mediated inflammasome signaling involves gasdermin-D (GSDMD) cleavage, as well as the unique cleavage of gasdermin-B (GSDMB), a human-specific protein. Pharmacologic inhibition showed this dual cleavage occurs through parallel pathways: caspase-mediated GSDMD cleavage and novel granzyme B (GZMB)-driven GSDMB cleavage. Cleaved GSDMD translocates to the plasma membrane after initial mitochondrial localization, however GSDMB N-terminus persists at mitochondrial membranes, promoting sustained mtDNA release. This differential localization creates a positive feedback loop amplifying inflammasome signaling while maintaining low pyroptotic cell death. Conclusion These findings demonstrate that DA coordinates mitochondrial dysfunction with NLRP3-mediated GZMB-induced GSDMB cleavage, revealing potential therapeutic targets for inflammatory diseases characterized by elevated circulating dopamine.