Background: Macrophages are critical hematopoietic cells in IR-mediated liver inflammation and in the acquisition of hepatic homeostasis. Macrophage TIM-4 expression regulates the engulfment of apoptotic/necrotic bodies by binding to phosphatidylserine (PS) ligand. We hypothesized that modulation of TIM-4 can ameliorate innate immunity-driven hepatic IRI by ablation of TIM-4 mediated macrophage phagocytosis and TLR4 activation. Methods&Results: In our murine model of liver warm ischemia (90min) and reperfusion (0-24h), TIM-4 expression peaked at 6h of reperfusion, coinciding with the maximal hepatocellular damage. Indeed, TIM-4 KO or WT (B/6) mice treated with antagonistic TIM-4 mAb (RMT4-53; 0.5 mg i.v. at -1h) were resistant against liver IRI, as shown by sALT levels (984±403U/L, 548±244U/L vs. 7870±1958U/L in controls, p<0.001) and liver histology, compared with WT controls. In parallel, intrahepatic expression of TLR4, TNF-Α, IL-1Β, IFN-Β and CXCL-10 were decreased in TIM-4 KO and WT + anti-TIM-4 Ab. We then assessed intrahepatic phagocytic activity of macrophage/Kupffer cells. Twenty million apoptotic bodies (CFSE-labeled B/6 thymocytes) were injected i.v. into TIM-4 KO and WT mice +/- anti-TIM-4 Ab at 1h prior to 90min ischemia. At 6h of reperfusion, livers were harvested, non-parenchymal cell isolated, and CD11b macrophages were analyzed by FACS. CD11b⁺CFSE⁺ cells represented apoptotic body-phagocytosed macrophages, and the ratio of CD11b⁺CFSE⁺ frequency to total cells indirectly measured the phagocytic activity in liver IRI. Strikingly, CD11b⁺CFSE⁺ ratio was diminished in TIM-4 KO or anti-TIM-4 treated WT IR-livers, as compared with untreated WT controls (2.88±0.17%, 3.52±0.30% vs. 9.81±0.40%, p<0.001). To mimic in vivo settings, we used hydrogen peroxide (H₂O₂)-necrotic hepatocytes co-cultured with BMMs. Consistent with in vivo findings, H₂O₂-induced necrotic hepatocytes (green CMFDA) were efficiently captured and then engulfed by WT (TIM-4⁺) BMMs (red PE-CD11b), but not by TIM-4-deficient BMMs or by anti-TIM-4 mAb pretreated WT cells. Conclusion: This study provides new evidence that macrophage TIM-4 expression in hepatic IRI is required to: 1) phagocytose PS-liver necrotic cells; and 2) promote TLR4-driven inflammation response. Hence, modulation of physiological TIM-4 expression provides the basis for novel therapeutic strategies against liver IRI in transplant recipients.

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