Interleukin-22: implications for liver ischemia-reperfusion injury

Abstract

Background: Ischemia-reperfusion injury (IRI) is common in general surgery and organ transplantation, and in the case of liver, it triggers proinflammatory innate immune cascade and hepatic necrosis, leading to increased incidence of early and late organ rejection. Interleukin (IL)-22, an inducible cytokine of T-cell origin and a member of the IL-10 superfamily, acts on target tissues through IL-22 receptor (IL-22R1).

Methods: Partial hepatic warm ischemia was induced in C57Bl/6 wild-type (WT) and type 1 interferon receptor-deficient (KO) mice for 90 min followed by 6 to 24 hr of reperfusion. WT mice were treated at 30 min before the ischemia insult with recombinant IL-22 or anti-IL-22 neutralizing antibody; phosphate-buffered saline and IgG served as respective controls.

Results: IL-22 was detected at 24 hr but not 6 hr of liver IRI. The expression of IL-22R1 was increased by 6 hr of reperfusion in WT but not type 1 interferon receptor KO mice that were protected from IRI. Treatment of WT mice with recombinant IL-22 decreased serum aspartate aminotransferase levels, ameliorated cardinal histological features of IR damage (Suzuki's score) and diminished leukocyte sequestration, along with the expression of IL-22R1 and pro-inflammatory cytokines. IL-22 antibody did not appreciably affect IRI but increased IL-22R1 transcription in the liver. Administration of IL-22 protein exerted hepatoprotection by STAT3 activation.

Conclusions: This is the first report investigating immune modulation by T-cell-derived IL-22 in liver injury caused by warm ischemia and reperfusion. Treatment with IL-22 protein may represent a novel therapeutic strategy to prevent liver IRI in transplant recipients.

Figure 1

IL-22 signaling in ConA and IRI models in WT mice at 6 h and 24 h of reperfusion. Quantitative RT-PCR-assisted analysis of (a) IL-22; (b) IL-22R1; and (c) IL-10R2 expression in mouse liver tissue subjected to ConA (N=6/group; 15 μg/g i.v.) or IR-triggered damage (90 min ischemia: N=6/group; Sham N=4/group). (Statistical comparisons between experimental and sham samples *: p<0.05, **: p<0.005). (a) Mice subjected to ConA injection expressed high levels of IL-22 at 6 h of infusion, before returning to baseline at 24 h, while IRI mice had low levels of IL-22 at 6 h and a modest increase by 24 h. (b) IL-22R1 expression was elevated in both models at 6 h, and returned to baseline at 24 h. (c) IL-10R2 was not affected by ConA or IRI.

Figure 2

Livers in WT and IFNAR KO mice at 6 h of reperfusion following 90 min of warm ischemia (Ischemia: N=8/group; Sham N=4/group). (a) Peripheral serum AST levels; (p<0.005). (b) Liver tissue histology (H&E staining; x4 or x40 magnification; representative liver sections shown). (c) Suzuki’s based grading of the hepatocellular damage. RT-PCR analysis of liver specimens subjected to ischemia of d) pro-inflammatory cytokines and (e) IL-22/IL-22 receptor complex genes. Experiment was performed twice with similar results (Statistical comparisons are between WT and IFNAR KO groups. *: p<0.05, **: p<0.005).

Figure 3

The effects of rIL-22 pretreatment (-30 min) in liver IRI model (6 h of reperfusion (PBS and rIL-22: N=8/group, Sham N=4/group). (a) Peripheral serum AST levels (p<0.05). (b) Liver tissue histology (H&E staining; x4 or x40 magnification; representative liver sections shown). (c) Suzuki’s based grading of the hepatocellular damage. RT-PCR analysis of liver specimens subjected to ischemia of (d) pro-inflammatory cytokines and (e) IL-22/IL-22 receptor complex. Experiment was performed twice with similar results (Statistical comparisons are between rIL-22 and PBS groups. *: p<0.05, **: p<0.005)

Figure 4

The effects of IL-22 neutralization (-30 min) in liver IRI (6 h of reperfusion, N=IgG and Anti-IL-22 Ab: 6/group, Sham N=4/group). (a) Peripheral serum AST levels. (b) Liver tissue histology (H&E staining; x4 or x40 magnification; representative liver sections shown). (c) Suzuki’s grading of the hepatocellular damage. RT-PCR analysis of liver specimens subjected to ischemia of (d) pro-inflammatory cytokines and (e) IL-22/IL-22 receptor complex (Statistical comparisons are between anti-IL-22 Ab and IgG groups. *: p<0.05)

Figure 5

The effects of rIL-22 pretreatment vs. IL-22 neutralization (6 h of reperfusion following 90 min of warm ischemia). Immunohistochemical staining of liver specimens for (a) neutrophils (Ly-6G); (b) macrophages (CD11b) and (c) T-cells (CD3). Left panels: representative liver sections (N=8/group for PBS and rIL-22, N=6/group for IgG and anti-IL-22 Ab) (x40 magnification). Right panels: Cell quantification /HPF±SEM. (*: p<0.05 for PBS vs. rIL-22 and IgG vs. anti-IL-22 Ab). Western blot-assisted detection of liver p-STAT3 and STAT3 in mice treated with PBS, rIL-22, IgG, or neutralizing IL-22 Ab (d). Beta-actin was used as internal control. Representative of two separate experiments is shown.