Donor graft interferon regulatory factor-1 gene transfer worsens liver transplant ischemia/reperfusion injury

Abstract

Background: Liver ischemia and reperfusion (IR) injury is a phenomenon that leads to graft dysfunction after liver transplantation. Understanding the molecular mechanisms behind this process is crucial to developing strategies to prevent short- and long-term graft dysfunction. The purpose of this study was to explore the role of the transcription factor interferon regulatory factor-1 (IRF-1) in a model of orthotopic rat liver transplantation.

Methods: Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 3 hours preservation in cold University of Wisconsin solution. Adenovirus-expressing IRF-1 (AdIRF-1) or control gene vector (Adnull) was delivered to the liver by donor intravenous pretreatment 4 days before graft harvesting. Uninfected grafts also served as controls. Recipients were humanely killed 1-24 hours post-transplantation.

Results: Rats that underwent OLT with long-term preserved grafts (18 hours) displayed increased hepatic nuclear expression of IRF-1 protein at 1 and 3 hours. Rats pretreated with AdIRF-1 before transplantation had elevated alanine aminotransferase levels and increased expression of interferon (IFN)-beta, IFN-gamma, interleukin-12, and inducible nitric oxide synthase in the short-term period (3 hours) when compared with donor livers pretreated with Adnull. AdIRF-1 pretreated donor livers also exhibited increased susceptibility to early apoptosis in the transplanted grafts as shown by increased terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining and expression of cleaved caspase-3. Additionally, AdIRF-1 pretreated donor livers had increased activation of the MAP kinase Jun N-terminal kinase as compared with Adnull pretreated donor livers.

Conclusion: IRF-1 is an important regulator of IR injury after OLT in rats. Targeting of IRF-1 may be a potential strategy to ameliorate ischemic liver injury after transplantation to minimize organ dysfunction.

Figure 1. Ad-IRF-1 pre-treatment leads to over-expression of IRF-1 in donor livers

(A) Western blot of liver nuclear IRF-1 protein at different time-points following liver transplantation. After orthotopic liver transplantation with 18 hour cold preservation, IRF-1 is expressed strongly at 1 and 3 hours with a return to near baseline levels by 6 hours (B) Western blot analysis for nuclear IRF-1 shows a dose dependent increase in IRF-1 expression after AdIRF-1injection. IRF-1 expression is minimal at 1×10⁸ pfu, but is strongly expressed at 1×10⁹ and 3×10⁹ pfu. IRF-1 expression is sustained 4 days after treatment with a dose of 3×10⁹ pfu. (C) Time course of serum ALT levels after AdIRF-1 injection (3×10⁹ pfu) shows a mild increase in ALT levels 2 days after injection with normalization by 4 days. (D) H&E staining of liver sections from rats treated with AdIRF-1 or Adnull (3×10⁹ pfu) show no necrosis after 4 days.

Figure 2. Donor livers pre-treated with AdIRF-1 have worse IR injury

(A) Serum ALT levels from rats transplanted with donor livers 4 days after pre-treatment with AdIRF-1 (3×10⁹ pfu) show higher ALT levels than those pre-treated with Adnull (p<0.05) 6 hours after transplantation. Error bars show ±SD (n=4). (B) H&E staining 24 hours after liver transplantation demonstrate more necrosis (arrows) in donor livers pre-treated with AdIRF-1. Percentage of necrotic area was calculated in five high power fields for each animals using Adobe Photoshop, Version 7.0. Error bars show ±SD (n=4) (* p<0.05).

Figure 3. Donor livers pre-treated with AdIRF-1 have exaggerated expression of inflammatory mediators after transplantation

(A) Western blot of nuclear protein 1, 6, and 24 hours after transplantation shows low-level endogenous IRF-1 expression in untreated donor livers at early time-points as expected. However, donor livers pre-treated with AdIRF-1(3×10⁹ pfu) have high levels of IRF-1 protein expression at all time-points. (B) After transplantation mRNA levels of inflammatory mediators are greater in donor livers pre-treated with AdIRF-1. IFN-γ, IL-12, and IFN-β expression are highest 1 hour after transplant with AdIRF-1 pre-treatment. iNOS expression is also highest in AdIRF-1 pre-treated donor livers, and is sustained for 24 hours. (C) Western blot of iNOS protein from donor livers after transplantation shows sustained protein expression after 24 hours.

Figure 4. Donor livers pre-treated with AdIRF-1 have increased apoptosis 6 hours after transplantation

(A) TUNEL staining prior to transplantation shows no difference in livers pre-treated with AdIRF-1 vs. Adnull. (upper panels) However, 6 hours after transplantation there is increased TUNEL staining in donor livers pre-treated with AdIRF-1 (lower panels). (B) Western blot for cleaved caspase-3 and total caspase-3 using whole cell lysate isolated from the liver grafts 6 or 24 hours after reperfusion shows higher levels of cleaved caspase-3 in donor livers pre-treated with AdIRF-1. (C) Confocal immunofluorescence of liver tissue 6 hours after transplantation shows more intense staining of cleaved caspase-3 (red color) in donor livers pre-treated with AdIRF-1.

Figure 5. Increased IRF-1 expression after transplantation is associated with JNK activation

(A) Western blot of liver whole cell lysates for phosphorylated and total c-Jun NH₂-terminal kinase (JNK) prior to transplantation and 1 hour after transplantation shows that AdIRF-1 (3 × 10⁹ pfu) pre-treatment increases the amount of phosphorylated JNK (p-JNK) after transplantation. (B) Quantification of phosphorylated JNK bands demonstrates a several fold increase in JNK activation in donor rat livers pre-treated with AdIRF-1 when compared with controls.