Recipient TIM4 signaling regulates ischemia reperfusion-induced ER stress and metabolic responses in liver transplantation: from mouse-to-human

Abstract

T-cell immunoglobulin and mucin (Tim)4 is expressed on APCs, including macrophages, as one of the main amplifiers in the mechanism of liver ischemia-reperfusion injury (IRI) following orthotopic liver transplantation (OLT). Though donor Tim4 selectively expressed on Kupffer cells serves as a checkpoint regulator of innate immune-driven IRI cascades, its role on cells outside the OLT remains unclear. To dissect the role of donor vs. recipient-specific Tim4 signaling in IR-induced stress and hepatocellular function, we employed a murine OLT model utilizing Tim4-knockout (KO) mice as either donor or recipient (WT → WT, WT → Tim4-KO, Tim4-KO → WT). In the experimental arm, disruption of donor Tim4 attenuated IRI-OLT damage, while recipient Tim4-null mutation aggravated hepatic IRI concomitant with disturbed lipid metabolism, enhanced endoplasmic reticulum stress, and activated pro-apoptotic signaling in the grafts. In the in vitro study, murine hepatocytes co-cultured with Tim4-null adipose tissue showed enhanced C/EBP homologous protein (CHOP) expression pattern and susceptibility to hepatocellular death accompanied by activated caspase cascade in response to TNF-α stimulation. In the clinical arm, liver grafts from forty-one transplant patients with enhanced TIM4 expression showed higher body mass index, augmented hepatic endoplasmic reticulum stress, enhanced pro-apoptotic markers, upregulated innate/adaptive immune responses, exacerbated hepatocellular damage, and inferior graft survival. In conclusion, although TIM4 is considered a principal villain in peri-transplant early tissue injury, recipient TIM4 signaling may serve as a savior of IR-triggered metabolic stress in mouse and human OLT recipients.

Keywords: endoplasmic reticulum stress; ischemia-reperfusion injury; liver transplantation; orthotopic liver tranplantation; t-cell immunoglobulin and mucin domain containing 4.

Figure 1

Donor Tim4-null mutation alleviates the hepatocellular damage and inflammatory response in IR-stressed mouse OLT: WT or Tim4-KO donor livers, stored in UW solution (4°C/18 h), were transplanted into WT recipients. OLT samples were collected at 6 h and 24 h post-reperfusion. (A) sAST and sALT levels (IU/L). (B) Representative H&E staining (original magnification, ×100) of sham livers and OLT (WT > WT, Tim4-KO > WT). (C) Suzuki's histological grading of liver IRI (n = 8/group). Data shown as mean ± SEM (*p < 0.05, 1-way ANOVA followed by Tukey's HSD test). (D) Representative TUNEL images and quantification of TUNEL + cells (n = 5/group). Data shown as mean ± SEM (*p < 0.05, Mann-Whitney U test). (E) qRT-PCR-assisted detection of mRNA coding for Il-1β, Mcp1, Cxcl1, Cxcl2 and Cxcl10. Data were normalized to Hprt gene expression. Sham: n = 4/group; OLT: n = 6–7/group. Data shown as mean ± SEM (*p < 0.05, Mann-Whitney U test). (F) Recipient mice after OLT were monitored for 14 days and cumulative survival was analyzed (Kaplan-Meier method). Dotted line: WT > WT; solid line Tim4-KO > WT (n = 10/group, *p < 0.05, log-rank test).

Figure 2

Recipient Tim4 disruption aggravates the hepatocellular damage and inflammatory response in IR-stressed mouse OLT: (A) sAST and sALT levels (IU/L). (B) Representative H&E staining (original magnification, ×100) of sham livers and OLT (WT > WT, Tim4-KO > WT). (C) Suzuki's histological grading of liver IRI (n = 8/group). Data shown as mean ± SEM (*p < 0.05, 1-way ANOVA followed by Tukey's HSD test). (D) Representative TUNEL images and quantification of TUNEL + cells (n = 5/group). Data shown as mean ± SEM (*p < 0.05, Mann–Whitney U-test). (E) qRT-PCR-assisted detection of mRNA coding for Il-1β, Mcp1, Cxcl1, Cxcl2 and Cxcl10. Data were normalized to Hprt gene expression. Sham: n = 3/group; OLT: n = 6–7/group. Data shown as mean ± SEM (*p < 0.05, Mann–Whitney U-test). (F) Recipient mice were monitored for 14 days and cumulative survival was analyzed (Kaplan–Meier method). Dotted line: WT > WT; solid line WT > Tim4-KO (n = 10/group, *p < 0.05, log-rank test).

Figure 3

Recipient Tim4-null mutation provokes hepatic lipid metabolism pathway and augments ER stress and apoptotic markers in OLT: (A) Western blot of CD36, SREBP1, PPARγ and MDA in sham and OLT livers (WT > WT, WT > Tim4-KO). The relative intensity to VCL was calculated. Data shown as mean ± SEM (*p < 0.05, ***p < 0.001, 1-way ANOVA followed by Tukey's HSD test). (B) Representative immunohistochemistry of CD36 and PPAR-γ in OLT livers (n = 2/group). (C) Serum FFA levels in sham and OLT mouse recipients. Data shown as mean ± SEM (*p < 0.05, 1-way ANOVA followed by Tukey's HSD test). (D) WB-assisted detection of ATF4, CHOP and VCL. The relative intensity to VCL was calculated. Data shown as mean ± SEM (***p < 0.001, Mann–Whitney U-test). (E) Representative staining for CHOP in OLT (n = 3/group). (F) WB of TNFR1, DR5, cleaved PARP, caspase 8, caspase 3 and VCL in OLT. The relative intensity to VCL was calculated. Data shown as mean ± SEM (*p < 0.05, Mann–Whitney U-test).

Figure 4

Tim4 deficient adipose tissue enhances hepatocyte SREBP1/CHOP and promotes TNF-α mediated cell death in vitro: (A) the scheme of the vitro experiment. (B) WB-assisted detection of SREBP1, caspase 8, CHOP and VCL. The relative intensity to VCL was calculated. Data shown as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, 1-way ANOVA followed by Tukey's HSD test). (C) Representative immunocytochemistry of F-actin (green), nucleus (blue) and CHOP (red) in murine hepatocyte cultured with AT-CM from WT or Tim4-KO mice (n = 2). Original magnification, ×400. (D) WB of cleaved PARP, caspase 8, cleaved caspase 3 and VCL in cultured hepatocytes. The relative intensity to VCL was calculated. Data shown as mean ± SEM (*p < 0.05, 1-way ANOVA followed by Tukey's HSD test).

Figure 5

Peri-transplant TIM4 gene levels are associated with the hepatocellular function and OLT outcomes: (A) Pre-transplant (post–cold storage) and post-transplant (2 h after reperfusion) liver biopsies (Bx) were collected from forty-one OLT patients and analyzed for TIM4 by qRT-PCR with normalization to GAPDH. The relationship between TIM4 and sAST (B) and sALT (C) at POD1. r: Spearman correlation coefficient. (D) Human OLT Bx (2 h post-reperfusion) were classified into low (n = 21) and high (n = 20) TIM4 expression groups. (E,F) Serum AST and ALT levels at POD1-7 (*p < 0.05, **p < 0.01, ***p < 0.001, 2-way ANOVA, Bonferroni post hoc test; data shown as mean ± SEM. (G) Incidence of early allograft dysfunction (EAD) (Fisher's exact test). (H) The cumulative probability of overall graft survival. The solid line indicates TIM4-high, while the dotted line depicts TIM4-low OLT patient cohorts (Kaplan–Meier method, log-rank test).

Figure 6

Augmented innate and adaptive gene activation in human OLT is accompanied by high TIM4 levels: qRT-PCR-assisted detection of mRNA coding for CD3, CD8, CD28, IL-17, Cathepsin G, CD80, CD86, CXCL-10, TLR2, and TLR4. Data normalized to GAPDH gene expression are shown in dot plots and bars indicative mean ± SEM (*p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney U-test).

Figure 7

TIM4 expression is positively correlates with hepatic ATF4, CHOP, MDA, caspase 8 and cleaved caspase 3 levels in human OLT: the relationship between peri-transplant TIM4 levels and ATF4 (A) CHOP (B) MDA (C) cleaved caspase 3 (D) and caspase 8 (E) expression in OLT at 2 h after reperfusion. r: Spearman correlation coefficient. (F) A simplified scheme of how TIM4 signaling may affect IR-stress and hepatocellular damage in OLT recipients.