*Purpose: Liver ischemia reperfusion injury (IRI) remains a significant clinical problem. Although the molecular mechanism of liver inflammatory response against IR has been studied extensively, the cellular basis remains poorly defined. This study aims to differentiate roles of liver resident (Kupffer cells, KCs) vs. infiltrating macrophages in liver IRI at both activation and resolution stages.

*Methods: In a murine liver partial warm ischemia model, we depleted KCs (F4/80⁺CD11b^low) by clodronate-encapsulated liposomes (CL), infiltrating macrophages (iMФs, F4/80⁺CD11b⁺) by diphtheria toxin in CD11b-DTR mice, and TIM-4+ KCs by anti-TIM-4 Abs. We also reconstituted mice in their KC or iMФ compartment with WT or TIM-4 deficient KCs or bone marrow-derived macrophages (BMMs). Liver IRI was evaluated at 6h (activation) and 7 days (resolution) post reperfusion. The TIM-4 expression and its relationship with liver IRI was also analyzed in 40 human liver allografts (20 IRI+ and 20 IRI-) pre- and post-Tx .

*Results: A subset of KCs, but not iMФs, expressed efferocytosis co-receptor TIM-4, which was downregulated by IR (at 6h) and recovered in the resolved livers (day 5-7). Depletion of KCs, or TIM-4+ KC, 48h prior to the onset of liver ischemia resulted in significant increases in liver IRI at 6h (sALT and histology) and delay in the inflammation resolution (14 days vs. 7 days in controls), leading to persistent liver inflammation and upregulation of pro-fibrotic gene expression (a-SMA, Col1A1). In contrast, the depletion of iMФs resulted in decreases in liver IRI and only slightly delayed the recovery process. While mice reconstituted with TIM-4 deficient BMMs were normal, those with TIM-4 deficient KCs were severely defective, in the resolution of liver IRI. Immunofluorescent staining of liver efferocytic macrophages isolated from IR-livers showed that anti-TIM-4 Abs inhibited liver macrophage efferocytosis in vivo. In vitro, anti-TIM-4 inhibited KC efferocytosis and diminished the immune regulatory effects (↓TNF-a, ↑IL-10) of co-incubating apoptotic cells. In human liver allografts, TIM-4 was predominantly expressed in CD68+cells and downregulated in IRI⁺, but not IRI^–, patients.

*Conclusions: These results document critical homeostatic roles of TIM-4⁺ KCs in the inflammation activation and resolution of liver IRI.

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