Background & Aims Hepatic ischemia-reperfusion injury (IRI), characterized by exogenous antigen-independent local inflammation and hepatocellular death, represents a risk factor for acute and chronic rejection in liver transplantation. We aimed to investigate the molecular communication involved in the mechanism of liver IRI. Methods We analyzed human liver transplants, primary murine macrophage cell cultures and IR-stressed livers in myeloid-specific heme oxygenase-1 (HO-1) gene mutant mice, for anti-inflammatory and cytoprotective functions of macrophage-specific HO-1/SIRT1 (sirtuin 1)/p53 (tumor suppressor protein) signaling. Results Decreased HO-1 expression in human post-reperfusion liver transplant biopsies correlated with a deterioration in hepatocellular function (serum ALT; p <0.05) and inferior patient survival (p <0.05). In the low HO-1 liver transplant biopsy group, SIRT1/Arf (alternative reading frame)/p53/MDM2 (murine double minute 2) expression levels decreased (p <0.05) while cleaved caspase 3 and frequency of TUNEL + cells simultaneously increased (p <0.05). Immunofluorescence showed macrophages were the principal source of HO-1 in human and mouse IR-stressed livers. In vitro macrophage cultures revealed that HO-1 induction positively regulated SIRT1 signaling, whereas SIRT1-induced Arf inhibited ubiquitinating activity of MDM2 against p53, which in turn attenuated macrophage activation. In a murine model of hepatic warm IRI, myeloid-specific HO-1 deletion lacked SIRT1/p53, exacerbated liver inflammation and IR-hepatocellular death, whereas adjunctive SIRT1 activation restored p53 signaling and rescued livers from IR-damage. Conclusion This bench-to-bedside study identifies a new class of macrophages activated via the HO-1–SIRT1–p53 signaling axis in the mechanism of hepatic sterile inflammation. This mechanism could be a target for novel therapeutic strategies in liver transplant recipients. Lay summary Post-transplant low macrophage HO-1 expression in human liver transplants correlates with reduced hepatocellular function and survival. HO-1 regulates macrophage activation via the SIRT1–p53 signaling network and regulates hepatocellular death in liver ischemia-reperfusion injury. Thus targeting this pathway in liver transplant recipients could be of therapeutic benefit.

IntroductionOrthotopic liver transplant (OLT) is the primary therapy for end-stage liver disease and acute liver failure. Ischemia/reperfusion injury (IRI) occurs as an inevitable consequence of the transplant process, beginning with organ procurement and preservation and followed by reperfusion of the donor organ with recipient blood during transplant (1). Data from murine models have indicated that liver IRI has hypoxic cellular stress and inflammation-mediated injury components (2-5). Local circulatory damage first induces endogenous reactive oxygen species production causing hepatocyte death. This cellular damage initiates the second phase by recruiting and activating innate immune cells at the site of injury. IRI is then further exacerbated by the adaptive immune system; indeed, activated CD4 + T cells are essential in promoting IRI-related inflammation and hepatocyte damage in mice. IRI can lead to primary graft nonfunction and need for retransplantation (6) and predisposes the recipient to both acute and chronic rejection and graft loss as well as decreases the pool of transplantable organs. Although IRI is a signifi-BACKGROUND. Orthotopic liver transplant (OLT) is the primary therapy for end-stage liver disease and acute liver failure. However, ischemia/reperfusion injury (IRI) can severely compromise allograft survival. To understand the evolution of immune responses underlying OLT-IRI, we evaluated longitudinal cytokine expression profiles from adult OLT recipients before transplant through 1 month after transplant.

Induction of experimental autoimmune encephalomyelitis (EAE) in susceptible animals requires reactivation of encephalitogenic CD4+ T cells by antigen presenting cells (APCs) in the central nervous system (CNS). However, it has remained unresolved from where APCs in the CNS acquire myelin antigen (Ag) for T cell activation and under which conditions, i.e. whether only during EAE or also in the naïve CNS. Here, we investigated the kinetics of myelin Ag uptake by CNS APCs during EAE and in the naïve CNS. Our results show that during EAE CX3CR1+CD11b+ microglia were the first APCs in the CNS to contain myelin Ag upon induction of disease, albeit in very small numbers. Dendritic cells (DCs) arrived in the CNS in sizable numbers significantly later (day 5 post-immunization (p.i.)), without detectable myelin Ag, but acquired it by day 7 p.i. Furthermore, a sharp increase in neuroantigen-containing DCs coincided with the onset of EAE symptoms. Importantly, in naïve mice a low but consistent number of microglia contained myelin Ag, suggesting release by oligodendrocytes under steady state conditions. Although microglia isolated from naïve brain and spinal cord did not elicit a strong CD4+ T cell response in vitro, myelin Ag-containing microglia may still play a local role in modulating encephalitogenic CD4+ T cell responses in early EAE prior to the arrival of other professional APCs such as DCs. Finally, newly arriving DCs in the CNS not yet loaded with myelin Ag before the onset of EAE may be a potential therapeutic target.

Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase-1 (HO-1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin-1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO-1-mediated autophagy induction in human OLT and in a murine OLT model with extended (20 hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO-1. Fifty-one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO-1 levels correlated with well-preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO-1 overexpression by genetically modified HO-1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO-1-mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO-1 cytoprotection and identifies SIRT1-mediated autophagy pathway as a new essential regulator of HO-1 function in IR-stressed OLT.

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