ROCK2 inhibition attenuates CTL-mediated platelet destruction by suppressing IL-6/STAT3-driven glycolysis in primary immune thrombocytopenia

Abstract

Abstract Abstract Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by excessive platelet destruction, in which cytotoxic T lymphocytes (CTLs) play a pivotal role. Although ROCK2 inhibition is known to modulate T cell function in diseases such as graft-versus-host disease, its role in ITP remains unclear. In this study, we investigated the effects and underlying mechanisms of the ROCK2 inhibitor KD025 on CTLs. In vitro, KD025 treatment significantly reduced CTL proliferation, degranulation (as indicated by decreased CD107a expression), and the production of cytotoxic effectors granzyme B and perforin at the protein and transcript levels. The frequency of CTL–platelet conjugates (CD8⁺CD61⁺) was also reduced. Moreover, platelets co-cultured with KD025-treated CTLs exhibited reduced activation and apoptosis, marked by a decrease in CD62P-positive platelets. Mechanistically, CTLs from ITP patients treated with KD025 showed impaired glycolytic activity, as reflected by a lower extracellular acidification rate (ECAR) and downregulation of glycolytic enzymes, including GLUT1, HK1, PFKM, PFKFB4, and PKM2. These changes were accompanied by reduced IL-6 production and decreased STAT3 phosphorylation. These in vitro findings were corroborated in an active ITP mouse model: daily KD025 administration reduced CTL cytotoxicity and significantly increased platelet counts in vivo. Importantly, co-treatment with colivelin, a STAT3 activator, reversed KD025-induced suppression of CTL cytotoxicity and glycolysis, confirming the involvement of the IL-6/STAT3 axis. In conclusion, ROCK2 inhibition by KD025 attenuates CTL-mediated platelet destruction in ITP by suppressing IL-6/STAT3-driven glycolysis and shifting CTL metabolism. These findings uncover a novel immunometabolic mechanism in ITP pathogenesis and suggest KD025 as a promising therapeutic strategy for this autoimmune disorder.