Fig 2: TIM-4 in KCs is associated with liver fibrosis. A, KCs were isolated from mice with TIM-4 mAb (red fluorescence label) treatment or control mAb treatment and then examined using laser confocal microscopy to identify the successful blocking of TIM-4 function (n = 3 mice/ group, Scale bars: 50 µm). Negative control (NC) as negative control without any treatment. B, Liver tissues from TIM-4 mAb and control mice were processed for Masson's trichrome staining (n = 3 mice/ group, magnification, ×400). NC as negative control without any treatment. C, Hydroxyproline of livers removed from olive-induced and CCL4-induced mice with or without TIM-4 mAb treatment (n = 3 mice/ group). D, E, Mannose-conjugated polymers were used to deliver TIM-4 siRNA or its scrambled control siRNA in vivo, and the expression levels of TIM-4 in olive-induced and CCL4-induced liver were assessed using immunoblot and quantitative analysis (n = 3 mice/ group). F, Liver tissues from each group processed for Masson's trichrome staining (n = 3 mice/ group, magnification, x400). G, Hydroxyproline of livers removed from olive-induced and CCL4-induced mice with or without TIM-4 interference (n = 3 mice/ group). H, Inflammatory factors in livers were measured by enzyme-linked immunosorbent assay (ELISA) (n = 3 mice/ group). ***P < .0001. Values represent the mean ± SD of at least three independent experiments

Fig 3: TIM-4 in KCs mediates mitophagy via Akt1 during liver fibrosis. A, B, The expression levels of cleaved caspase-3 in KCs from olive-induced and CCL4-induced mice with or without TIM-4 interference were determined by immunoblot and quantitative analysis (n = 3 mice/ group). C, The number of apoptotic KCs from olive-induced and CCL4-induced mice with or without TIM-4 interference was determined with annexin V/PI staining and flow cytometric analysis (n = 3 mice/ group). D, Representative liver sections stained with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling assay (TUNEL) in sham and liver transplantation mice with or without TIM-4 interference (n = 3 mice/ group). E, Representative micrographs of the pathological damage observed in sham and liver transplantation mice with or without TIM-4 interference following staining with haematoxylin and eosin (n = 3 mice/ group, magnification, x400). F, The ratio of liver weight to body weight was measured in sham and liver transplantation mice with or without TIM-4 interference (n = 5 mice/ group). G, Serum concentrations of alanine aminotransferase (ALT) and aspartate transaminase (AST) in sham and liver transplantation mice with or without TIM-4 interference (n = 5 mice/ group). H, Hydroxyproline of livers removed from sham and liver transplantation mice with or without TIM-4 interference (n = 5 mice/ group). I, The expression levels of hepatocyte growth factor (HGF) in sham and liver transplantation mice with or without TIM-4 interference were assessed using immunohistochemistry (n = 5 mice/ group, magnification, x400). ***P < .0001. Values represent the mean ± SD of at least three independent experiments

Fig 4: TIM-4 blockade stimulates the differentiation of T cells into CD4+CD25+Foxp3+ T regulatory cells via the IL-4/STAT6/Gata3 pathway. (A) TIM-4+ and TIM-4- KCs were obtained from mice following liver transplantation and co-cultured with naive CD4+ T cells. The expression of p-STAT6 in T cells was determined using western blotting. (B) TIM-4 mAb was added to co-cultured cohorts as described in A, to analyze the expression of p-STAT6 in T cells. (C) The exogenous addition of IL-4 to TIM-4+ KCs co-cultured with naive CD4+ T cells (that received either no pretreatment or pretreatment with TIM-4 mAb) was performed and the expression of p-STAT6 in T cells was analyzed. (D) TGF-ß was added to TIM-4+ KCs co-cultured with naive CD4+ T cells (that received either no pretreatment or pretreatment with TIM-4 mAb) to determine the expression of Foxp3 and Gata3 protein in T cells. Data are presented as the mean ± standard deviation of the mean. aP<0.05 vs. control; bP<0.05 vs. TIM-4+ group. TIM-4, T cell immunoglobulin-domain mucin-domain-4; IL-4, interleukin-4; p-, phosphorylated; STAT6, signal transducer and activator of transcription 6; Gata3, transcription factor gata3; CD, cluster of differentiation; mAb, monoclonal antibodies; KCs, kupffer cells; TGF-ß, transforming growth factor-ß; Foxp3, forkhead box P3.

Fig 5: Orthotopic liver transplantation increases KC activation and TIM-4 expression. (A) KCs were isolated from mice in the sham and LT groups 24, 48 and 72 h following surgery. Cells were double stained with PE-CD14 and APC-CD163 and quantified using flow cytometry. (B) Liver samples were obtained from mice in the sham and LT groups and assessed using immunohistochemistry (magnification, ×400). Activated hepatic KCs are stained brown. (C) KCs were isolated from mice in the sham and LT groups 1, 3 and 7 days following surgery and TIM-4 expression was analyzed using western blotting. Data are presented as the mean ± standard deviation. aP<0.05 vs. sham group. KC, kupffer cells; TIM-4, T cell immunoglobulin-domain mucin-domain-4; CD, cluster of differentiation; APC, antigen presenting cell; LT, liver transplantation; PE, phycoerythrin; d, days.