iRhom2 regulates HMGB1 secretion to modulate inflammation and hepatocyte senescence in an in vitro model of ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) represents a major challenge in liver transplantation, driving acute dysfunction and contributing to long-term allograft rejection. This process triggers a robust inflammatory response, leading to hepatocyte damage, senescence, and impaired liver regeneration. While the underlying mechanisms remain incompletely understood, increasing evidence highlights macrophage-derived signaling as a pivotal driver of hepatocyte fate during IRI. Here, we identify iRhom2 as a key regulator of immune-mediated liver injury, orchestrating macrophage-driven inflammation and hepatocyte senescence. iRhom2 is known to modulate the secretion of multiple cytokines by macrophages, yet its specific contribution to IRI-driven hepatocyte senescence has not been fully elucidated. We reveal a significant upregulation of iRhom2 in IRI+ reperfused allografts, particularly in Kupffer cells and monocyte-derived macrophages. Functional characterization in iRhom2-deficient macrophages revealed reduced ER stress, preserved mitochondrial function, and attenuated apoptosis, indicating a protective role against IRI-induced cellular damage. Proteomic profiling further uncovers iRhom2-dependent secretion of inflammatory mediators, with HMGB1 emerging as a critical damage-associated molecular pattern (DAMP) molecule in this context. Notably, HMGB1 release occurs independently of TACE catalytic activity, suggesting an alternative unexplored regulatory mechanism. Furthermore, co-culture experiments confirm that macrophage-derived HMGB1 directly induces senescence of human induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps) under in vitro IRI condition, driving the up-regulation of key senescence markers and disrupting cell cycle dynamics. Strikingly, HMGB1 neutralization enhances hepatocyte viability and mitigates senescence, underscoring its pathogenic role. Additionally, HMGB1 knockdown in macrophages protects hepatocytes, though p21 expression remains unaffected, hinting at additional senescence pathways. Our findings establish iRhom2 as a central orchestrator of macrophage-driven hepatocyte dysfunction in IRI and suggest that targeting the iRhom2-HMGB1 axis could represent a promising therapeutic strategy to improve post-transplant liver recovery and long-term graft survival.