Interferon regulatory factor-2 is protective against hepatic ischemia-reperfusion injury

Abstract

Interferon regulatory factor (IRF)-1 is a nuclear transcription factor that induces inflammatory cytokine mediators and contributes to hepatic ischemia-reperfusion (I/R) injury. No strategies to mitigate IRF1-mediated liver damage exist. IRF2 is a structurally similar endogenous protein that competes with IRF1 for DNA binding sites in IRF-responsive target genes and acts as a competitive inhibitor. However, the role of IRF2 in hepatic injury during hypoxic or inflammatory conditions is unknown. We hypothesize that IRF2 overexpression may mitigate IRF1-mediated I/R damage. Endogenous IRF2 is basally expressed in normal livers and is mildly increased by ischemia alone. Overexpression of IRF2 protects against hepatic warm I/R injury. Furthermore, we demonstrate that IRF2 overexpression limits production of IRF1-dependent proinflammatory genes, such as IL-12, IFNβ, and inducible nitric oxide synthase, even in the presence of IRF1 induction. Additionally, isograft liver transplantation with IRF2 heterozygote knockout (IRF2(+/-)) donor grafts that have reduced endogenous IRF2 levels results in worse injury following cold I/R during murine orthotopic liver transplantation. These findings indicate that endogenous intrahepatic IRF2 protein is protective, because the IRF2-deficient liver donor grafts exhibited increased liver damage compared with the wild-type donor grafts. In summary, IRF2 overexpression protects against I/R injury by decreasing IRF1-dependent injury and may represent a novel therapeutic strategy.

Fig. 1.

Interferon regulatory factor (IRF)-2 overexpression does not affect IRF1 levels. C57BL/6 mice (8–12 wk old) underwent adoptive transfer of IRF2 or control vector LacZ by tail vein injection of 1.5 × 10⁸ or 7.5 × 10⁸ plaque-forming units (pfu) of AdIRF2 or AdLacZ, and serum alanine aminotransferase (ALT) levels were determined at 24, 36, and 48 h. A: serum ALT levels at 36 h after treatment with 1.5 × 10⁸ or 7.5 × 10⁸ pfu of AdIRF2 or AdLacZ. Values are means ± SE (n = 3 per treatment). B: Western blots of liver nuclear extracts at 24, 36, or 48 h after injection of 1.5 × 10⁸ pfu of AdIRF2 or AdLacZ show expression of IRF2 and histone in the liver. Blots are representative of 3 per treatment.

Fig. 2.

IRF2 overexpression is protective in warm liver ischemia-reperfusion (I/R) injury. C57BL/6 mice (8–12 wk old) underwent partial warm liver I/R. A: untreated mice were subjected to 1 h of partial ischemia with predetermined euthanasia time points after 0, 1, 3, and 6 h of reperfusion. Liver nuclear protein extracts were subjected to Western blot analysis for IRF1 and IRF2 to determine a time course of activation. B: mice treated with AdIRF2 or AdLacZ control were subjected to 1 h of partial warm ischemia followed by 6 h of reperfusion, and serum ALT levels were determined. Values are means ± SE (n = 3 per treatment). *P < 0.05.

Fig. 3.

IRF2 overexpression results in decreased cytokine and IRF1 target gene production. C57BL/6 mice (8–12 wk old) underwent partial warm I/R. Animals were euthanized at 6 h of reperfusion, and liver tissue was sampled. Tissue was processed to recover mRNA and subjected to PCR for IL-12 (A), IFNβ (B), and inducible nitric oxide synthase (iNOS, C). *P < 0.05.

Fig. 4.

IRF2 overexpression does not alter nuclear IRF1 levels in vitro. A: liver nuclear protein extracts were analyzed by Western blot for expression of IRF1, IRF2, and histones to determine relative expression of these proteins after treatment with 1.5 × 10⁸ pfu of AdIRF2 or AdLacZ. B: primary cultured hepatocytes were subjected to gene transfer with 1.5 × 10⁸ pfu of AdIRF2. Cells were incubated for 24 h prior to nuclear protein extraction. Western blot analysis for IRF1 or IRF2 was performed on nuclear extracts. MOI, multiplicity of infection. C: primary cultured hepatocytes underwent gene transfer with AdIRF2. After incubation for gene transfer, cells were treated with IFNγ (100 U/ml). Nuclear protein extracts were analyzed for IRF1 and IRF2 by Western blot.

Fig. 5.

IRF2 overexpression decreases IRF1-dependent iNOS transcription in vitro. A: primary cultured hepatocytes were pretreated with AdIRF2 or AdLacZ at MOI 100, 10, 50, and 100 for 24 h. After incubation, cells were treated with IFNγ (100 U/ml) for an additional 2 h, and whole cell protein was isolated. Western blot analysis of cellular protein was performed for iNOS. B: primary cultured hepatocytes were treated with AdIRF1 or AdIRF2 for 24 h, and whole cell protein was isolated. Western blot analysis of cellular protein was performed for iNOS. C: RAW 264.7 cells were cultured and transfected with AdIRF2 or AdLacZ with MOI 100 for 24 h. Cells were then cultured in normoxia, hypoxia (1% O₂), or normoxia with LPS (100 ng/ml), IFNγ (100 U/ml), or both for an additional 8 h. Cells were harvested, and whole cell protein was isolated. Western blot analysis of cellular protein was performed for iNOS.

Fig. 6.

IRF2 heterozygote liver grafts result in increased injury after transplantation. A: wild-type (WT), IRF2 heterozygote, and IRF2 knockout mice were euthanized, and liver tissue was sampled. Nuclear protein extracts were obtained, and baseline levels of IRF1 and IRF2 were determined by Western blot analysis. B: serum ALT levels were determined following liver transplants in wild-type (WT-WT, n = 5) and IRF2 heterozygote (IRF2^+/− to WT, n = 3) liver transplants.