ML364 ameliorates NLRP3-induced inflammatory response

The NLRP3 inflammasome is a critical mediator of innate immune responses and inflammatory diseases, and its activation is tightly regulated by post-translational modifications such as ubiquitination. While several E3 ligases have been shown to modulate NLRP3 activity, deubiquitinases are increasingly recognized as key regulators that control NLRP3 stability and activation. However, the identification of additional deubiquitinases and pharmacological inhibitors remains essential for a comprehensive understanding of NLRP3 inflammasome regulation. This study investigated the role of the deubiquitinase USP2 in NLRP3 inflammasome activation and evaluated the therapeutic potential of its inhibitor ML364 in inflammasome-driven inflammatory diseases.

Murine macrophage cell lines (J774A.1), HEK293 cells, and bone marrow-derived macrophages (BMDMs) were used to assess inflammasome activation through genetic knockdown, overexpression, and pharmacological inhibition of USP2. Protein interactions, ubiquitination status, ASC speck formation, caspase-1 activation, and IL-1β secretion were evaluated using immunoprecipitation, immunoblotting, microscopy, and ELISA. In vivo efficacy of the USP2 inhibitor ML364 was examined in mouse models of monosodium urate (MSU)-induced peritonitis and dextran sulfate sodium (DSS)-induced colitis by assessing inflammatory cell recruitment, cytokine production, disease activity, and histopathology.

USP2 was required for optimal NLRP3 inflammasome activation, as its knockdown or pharmacological inhibition significantly reduced caspase-1 activation, IL-1β release, and pyroptotic responses. Mechanistically, USP2 directly interacted with the NACHT domain of NLRP3 and promoted its stability by removing ubiquitin chains, whereas a catalytically inactive USP2 mutant failed to support inflammasome activation. USP2 deficiency increased NLRP3 ubiquitination and reduced protein abundance in macrophages. Importantly, ML364 effectively suppressed NLRP3 inflammasome activation in vitro and alleviated inflammatory responses in vivo, reducing neutrophil recruitment, IL-1β production, and tissue injury in MSU-induced peritonitis and DSS-induced colitis models.

USP2 acts as a critical deubiquitinase that promotes NLRP3 inflammasome activation by stabilizing NLRP3 through deubiquitination of its NACHT domain. Pharmacological inhibition of USP2 with ML364 effectively suppresses inflammasome activation and ameliorates inflammatory pathology in vivo. These findings identify USP2 as an important regulator of NLRP3 signaling and highlight ML364 as a potential therapeutic strategy for NLRP3 inflammasome-associated diseases.