Background: Ischemia/reperfusion injury (IRI) stands as a prominent cause of primary-non function acute and chronic liver transplantation failure. IRI generates an inflammatory environment by activating resident Kupffer cells, leading to compromised allograft functionality and rejection. Over the past decade, iRhom2 has emerged as cardinal regulators of inflammatory and growth factor signaling[MC1] [ZG2] . Along these lines, our goal was to investigate iRhom2 function in IRI since no data are currently available on its potential role at the onset of the injury.
Method: In vitro IRI was tested on primary macrophages, hepatocyte cell line and iPSCs-derived hepatocytes using single or co-culture approaches. In vitro cold ischemia was performed under 2% O2 exposure in a preservation medium, followed by warm reperfusion at 1, 4 and 24 hrs. The effects of cold ischemia and reperfusion on wt and iRhom2 KD macrophages, as well as iPSCs-derived hepatocytes, were evaluated by ELISA, qPCR, and ATP analysis. Shotgun proteomics was performed to compare secretomes of wt and iRhom2 KD cells undergoing IRI. Hepatocyte senescence was evaluated via WB and β-galactosidase assay.
Results: Our findings demonstrate that iRhom2 is involved in liver IRI progression in transplanted patients[MP3] [ZG4] , and its in vitro downregulation in M1-like primary macrophages modulates the secretion of pro-inflammatory cytokines and danger-associated molecular patterns (DAMPs) associated with IRI damage. These findings prompted us to further dissect iRhom2 function in vitro IRI context. Interestingly, iRhom2 silencing accelerates macrophage recovery during reperfusion, as evidenced by increased cell viability and reduced cytotoxicity compared to control cells. Furthermore, iRhom2 activity in macrophages induces an IRI-associated senescence phenotype in injured iPSC-derived hepatocytes by modulating the secretion of the DAMP HMGB1, thus leading to the activation of the p21/p53 signaling pathway on target cells. Notably, we demonstrated that the senescence phenotype can be rescued by using anti-HMGB1 neutralization antibodies and not with anti-TNFα antibodies, the other major cytokine regulated by iRhom2. Altogether these findings suggest that HMGB1 is required to induce hepatocytes senescence, and further sustain the hypothesis that iRhom2 in IRI acts in a TNFα-independent fashion.
Conclusion: Taken together, these data suggest that iRhom2, in response to IRI, could regulate the secretion of cytokines and DAMPs responsible for the progression of the damage, by a mechanism that might be independent of TNF pathway. Thus, iRhom2 inhibition during IRI during IRI holds promise as a therapeutic target to improve recovery from IRI damage and potentially prevent late-onset allograft rejection due to hepatocyte senescence.