In vitro model of liver ischemia/reperfusion injury identifies iRhom2 as a novel disease target

Abstract

Background

Ischemia/reperfusion injury (IRI) is a leading cause of primary non-function and both acute and chronic failure after liver transplantation. IRI creates a pro-inflammatory environment through the activation of resident Kupffer cells, ultimately compromising allograft viability and promoting rejection. iRhom2 has recently emerged as a key regulator of inflammatory and growth factor signaling, suggesting its potential involvement in IRI. However, no data are currently available on its specific role at the onset of liver injury. Here, we aimed to investigate the function of iRhom2 in liver IRI.

Methods

We established an in vitro IRI model using primary macrophages and iPSC-derived hepatocytes in single and co-culture systems. Cold ischemia was simulated by culturing cells in preservation medium under 2% O₂, followed by warm reperfusion for 1, 4, and 24 hours. We compared wild-type (wt) and iRhom2 knockdown (KD) macrophages and evaluated the effects of IRI by ELISA, qPCR, and ATP assays. Shotgun proteomics was performed to analyze the secretome changes in wt and iRhom2 KD cells under IRI. Hepatocyte senescence was assessed by Western blotting and β-galactosidase staining.

Results

We show that iRhom2 contributes to IRI progression in transplanted patients, and its downregulation in M1-like primary macrophages leads to reduced secretion of pro-inflammatory cytokines and danger-associated molecular patterns (DAMPs) implicated in IRI. Remarkably, iRhom2 silencing promotes faster macrophage recovery during reperfusion, as indicated by increased viability and reduced cytotoxicity compared to control cells. Furthermore, iRhom2 activity in macrophages drives an IRI-associated senescence phenotype in injured iPSC-derived hepatocytes by modulating the secretion of the DAMP HMGB1, which in turn activates the p21/p53 signaling pathway in target cells. Importantly, hepatocyte senescence could be prevented by anti-HMGB1 neutralizing antibodies, but not by anti-TNFα antibodies, although TNFα is another major cytokine regulated by iRhom2. These findings support the notion that iRhom2-induced HMGB1 secretion, rather than TNFα, is critical in driving hepatocyte senescence during IRI.

Conclusion

Overall, our data suggest that iRhom2 modulates the release of cytokines and DAMPs in response to IRI through a mechanism that is at least partly independent of the TNF pathway. Targeting iRhom2 could thus represent a promising therapeutic strategy to reduce IRI-related injury, improve graft recovery, and prevent long-term rejection mediated by hepatocyte senescence.