*Purpose: Liver ischemia-reperfusion injury (IRI) is a major risk factor for early graft dysfunction and rejection in orthotopic liver transplantation (OLT). We and others have reported on pathogenic functions of T-cell immunoglobulin mucin 4 (TIM4) signaling in warm hepatic IRI models. Here, we analyzed mouse OLT and human liver transplant biopsies (Bx) to elucidate clinical relevance and underlining mechanisms of graft TIM4 signaling in OLT recipients.

*Methods: In the experimental arm, WT mouse recipients (BL6) were challenged with syngeneic livers from WT or TIM4 deficient (TIM4-KO) donors after 18h of cold storage (4C in UW solution), followed by liver/blood sampling at 6h/24h post-reperfusion. Primary mouse (WT) macrophage cultures were stimulated with HMGB1-enriched liver flush obtained from 18h cold-stored donor livers; while hepatocytes isolated from WT or TIM4-KO mice were challenged with macrophage culture medium. In the clinical arm, liver Bx collected at 2h after reperfusion (prior to abdominal closure) from fifty-five liver transplant patients, recruited under IRB protocol, were analyzed by RT-PCR.

*Results: Compared with WT mouse livers, TIM4 deficient grafts transplanted to WT recipients were resistant against IR-damage, as evidenced by: 1/ sAST levels(6h: 2885±341 vs 4563±681 IU/L, n=11/11, p<0.05; 24h: 930±182 vs 2740±835 IU/L, n=5/4, p<0.05); 2/ Suzuki's histological OLT grading (p<0.05); 3/ frequency of TUNEL+ cells (p<0.05); 4/ suppressed neutrophil/macrophage infiltration (p<0.05); 5/ decreased mRNA levels coding for CXCL10/MCP1/CXCL1 (p<0.05); and 6/ improved OLT survival (2-week: 75 vs 30%, n=8/10, p<0.05). Immunostaining revealed TIM4 was expressed by both hepatic parenchymal and non-parenchymal cells in naïve or IR-stressed WT livers. In comparison to WT counterparts, hepatocytes from TIM4-KO mice had decreased TIM4 expression (RT-PCR/flow cytometry) while exhibiting resistance against macrophage culture medium-triggered stress, as evidenced by decreased AST/ALT release (p<0.05). Post-reperfusion TIM4 levels in human liver graft Bx positively correlated with the expression of TLR4 (r=0.868, p<0.001), CXCL10 (r=0.459, p<0.001), CD68 (r=0.305, p=0.024), Cathepsin G (r=0.549, p<0.001) and sAST levels at POD1 (p=0.304, r=0.024). Patients with low-TIM4 expression levels in OLT (n=27) had improved hepatocellular function (lower sAST levels at POD1; 321±90 vs 748±342 IU/L, p=0.035) and shorter post-OLT ICU stay (15.9±2.4 vs 18.4±4.7 days) as compared with the high-TIM4 expression patient cohort (n=28).

*Conclusions: Our translation study: 1/ documents pathogenic function of liver-specific TIM4 signaling in promoting hepatocellular damage in IR-stressed OLT; 2/ unravels therapeutic potential of graft/hepatocyte TIM4 inhibition in liver transplant patients.

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