Targeting TIM-1 on CD4 T cells depresses macrophage activation and overcomes ischemia-reperfusion injury in mouse orthotopic liver transplantation

Abstract

Hepatic injury due to cold storage followed by reperfusion remains a major cause of morbidity and mortality after orthotopic liver transplantation (OLT). CD4 T cell TIM-1 signaling costimulates a variety of immune responses in allograft recipients. This study analyzes mechanisms by which TIM-1 affects liver ischemia-reperfusion injury (IRI) in a murine model of prolonged cold storage followed by OLT. Livers from C57BL/6 mice, preserved at 4°C in the UW solution for 20 h, were transplanted to syngeneic recipients. There was an early (1 h) increased accumulation of TIM-1+ activated CD4 T cells in the ischemic OLTs. Disruption of TIM-1 signaling with a blocking mAb (RMT1-10) ameliorated liver damage, evidenced by reduced sALT levels and well-preserved architecture. Unlike in controls, TIM-1 blockade diminished OLT expression of Tbet/IFN-γ, but amplified IL-4/IL-10/IL-22; abolished neutrophil and macrophage infiltration/activation and inhibited NF-κB while enhancing Bcl-2/Bcl-xl. Although adoptive transfer of CD4 T cells triggered liver damage in otherwise IR-resistant RAG(-/-) mice, adjunctive TIM-1 blockade reduced Tbet transcription and abolished macrophage activation, restoring homeostasis in IR-stressed livers. Further, transfer of TIM-1(Hi) CD4+, but not TIM-1(Lo) CD4+ T cells, recreated liver IRI in RAG(-/-) mice. Thus, TIM-1 expressing CD4 T cells are required in the mechanism of innate immune-mediated hepatic IRI in OLTs.

Figure 1

B6 livers subjected to ex-vivo cold storage (20 h) were transplanted into syngeneic recipients. By 1 h, CD4 T cells, which infiltrated OLTs were (a) activated, evidenced by CD69 staining; and (b) expressed TIM-1 (representative of 4 experiments). By 6 h after OLT, the hepatocellular function was analyzed by (c) sALT levels, (d) liver histology (representative H&E staining; magnification x100 and x400), and (e) Suzuki’s histological score (*p<0.05, **p<0.01, n=8–10/group).

Figure 2

Quantitative RT-PCR-assisted detection of cytokine/transcription factors in OLTs (6 h post-transplant after 20 h of cold ischemia): (a) IFN-γ, Tbet; (b) IL-4, IL-10; (c) FoxP3, and (d) IL-17, IL-22, RORγt). Data normalized to HPRT gene expression (*p<0.05, **p<0.01, n=4–6/group).

Figure 3

Neutrophils and macrophages in OLTs following administration of anti-TIM-1 mAb or control Ig (6 h post-transplant after 20 h of cold ischemia). (a) MPO levels (n=4–6/group); immunohistochemical staining of (b) Ly-6G⁺ neutrophils; and (c) CD68⁺ macrophages; (d) immunofluorescence double staining of TIM-4-expressing macrophages (TIM-4⁺CD68⁺ cells -head arrow). Green – macrophage; red – TIM-4; blue – DAPI nuclear stain. Results scored semi-quantitatively by averaging number of positively-stained cells/field (400x magnification). Representative of 4–6 mice/group (b, c) and 2 mice/group (d).

Figure 4

Measurement of TIM-4 expression and associated cytokine/chemokine programs in OLTs by quantitative RT-PCR (6 h post-transplant after 20 h of cold ischemia): (a) TIM-4; (b) CXCL-1, CCL-2, CXCL-10; and (c) TNF-α, IFN-β, IL-1β, and IL-6). Data normalized to HPRT gene expression (*p<0.05, **p<0.01, n=4–6/group).

Figure 5

Necrosis/apoptosis events in OLTs (6 h post-transplant after 20 h of cold ischemia). (a, b) TUNEL-assisted detection of hepatic necrosis/apoptosis (dark arrows; magnification x100 and x400); (c) caspase-3 activity; (d) Western blot-assisted detection of Bcl-2/Bcl-xl; p-IκBα, and β-actin (**p<0.01, n=4–6/group).

Figure 6

TIM-1⁺ CD4 T cells are required to trigger liver IRI in RAG^−/− mice. Intrahepatic TIM-1 levels were elevated in livers subjected to 90 min of warm ischemia in RAG^−/− mice repopulated with syngeneic CD4 T cells (a). Adjunctive infusion of anti-TIM-1 mAb (day -2) restored IR-liver resistance in RAG^−/− mice repopulated with CD4 T cells, as evidenced by (b) decreased sALT levels; and (c) preserved liver histology (representative H&E; magnification x100 and x400) (n=4–6/group). Adoptive transfer of ConA-activated TIM-1^Hi but not TIM-1^Lo CD4+ T cells (−1 h) mediated IR-liver damage in RAG^−/− mice, as shown by (b) sALT levels; and (c) liver histology. (**p<0.01), n=2/group).

Figure 7

TIM-1⁺ CD4 T cells mediate IR-triggered (a) Tbet transcription; and promote (b) TNF-α, IL-6, and CXCL-10 expression in RAG^−/− recipients adoptively transferred with either syngeneic CD4+ T cells or ConA-activated TIM-1^Hi cells. (**p<0.01, *p<0.05, n=4–6/group).