Yuan Zhai

Cutting edge: TLR4 activation mediates liver ischemia/reperfusion inflammatory response via IFN regulatory factor 3-dependent MyD88-independent pathway.

The triggering molecular mechanism of ischemia-reperfusion injury (IRI), which in clinical settings results in excessive and detrimental inflammatory responses, remains unclear. This study analyzes the role of the TLR system in an established murine model of liver warm ischemia followed by reperfusion. By contrasting in parallel TLR knockout mice with their wild-type counterparts, we found that TLR4, but not TLR2, was specifically required in initiating the IRI cascade, as manifested by liver function (serum alanine aminotransferase levels), pathology, and local induction of proinflammatory cytokines/chemokines (TNF-α, IL-6, IFN-inducible protein 10). We then investigated the downstream signaling pathway of TLR4 activation. Our results show that IFN regulatory factor 3, but not MyD88, mediated IRI-induced TLR4 activation leading to liver inflammation and hepatocellular damage. This study documents the selective usage of TLR in a clinically relevant noninfectious disease model, and identifies a triggering molecular mechanism in the pathophysiology of liver IRI.

Ischaemia–reperfusion injury in liver transplantation—from bench to bedside

Ischaemia–reperfusion injury (IRI) in the liver, a major complication of haemorrhagic shock, resection and transplantation, is a dynamic process that involves the two interrelated phases of local ischaemic insult and inflammation-mediated reperfusion injury. This Review highlights the latest mechanistic insights into innate–adaptive immune crosstalk and cell activation cascades that lead to inflammation-mediated injury in livers stressed by ischaemia–reperfusion, discusses progress in large animal experiments and examines efforts to minimize liver IRI in patients who have received a liver transplant. The interlinked signalling pathways in multiple hepatic cell types, the IRI kinetics and positive versus negative regulatory loops at the innate–adaptive immune interface are discussed. The current gaps in our knowledge and the pathophysiology aspects of IRI in which basic and translational research is still required are stressed. An improved appreciation of cellular immune events that trigger and sustain local inflammatory responses, which are ultimately responsible for organ injury, is fundamental to developing innovative strategies for treating patients who have received a liver transplant and developed ischaemia–reperfusion inflammation and organ dysfunction.

Liver Ischemia and Reperfusion Injury: New Insights into Mechanisms of Innate—Adaptive Immune‐Mediated Tissue Inflammation

Ischemia and reperfusion injury (IRI) is a dynamic process that involves two distinctive yet interrelated phases of ischemic organ damage and inflammation‐mediated reperfusion injury. Although multiple cellular and molecular pathways contribute and regulate tissue/organ damage, integration of different players into a unified mechanism is warranted. The crosstalk between innate and adaptive immune systems plays a significant role in the pathogenesis of liver IRI. In this review, we focus on recent progress in the mechanism of liver innate immune activation by IR. Kupffer cells (KC), DCs, NK, as well as T cells initiate local inflammation response, the hallmark of IRI, by utilizing distinctive immune receptors to recognize and/or trigger various molecules, both endogenous and exogenous. The interlocked molecular signaling pathways in the context of multiple liver cell types, the IRI kinetics and positive versus negative regulatory loops in the innate immune activation process are discussed. Better appreciation of molecular interactions that mediate these intricate cascades, should allow for the development of novel therapeutic approached against IRI in liver transplant recipients.

Allograft Rejection by Primed/Memory CD8+ T Cells Is CD154 Blockade Resistant: Therapeutic Implications for Sensitized Transplant Recipients

We have shown that CD8+ CTLs are the key mediators of accelerated rejection, and that CD8+ T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8+ T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8+ T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8+ T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8+ T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8+ T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8+ T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.

Toll‐Like Receptor and Heme Oxygenase‐1 Signaling in Hepatic Ischemia/Reperfusion Injury

Ischemia/reperfusion injury (IRI) represents the major problem in clinical liver transplantation. We have shown that toll‐like receptor 4 (TLR4) signaling is specifically required in initiating antigen‐independent IRI leading to liver inflammation, whereas local induction of anti‐oxidant heme oxygenase‐1 (HO‐1) is cytoprotective. This study analyzes in vivo interactions between HO‐1 and sentinel TLR system in the pathophysiology of liver IRI. Using a 90‐min lobar warm ischemia model, wild type (WT), TLR4 KO/mutant and TLR2 KO mice were first assessed for the severity of hepatocellular damage at 6 h postreperfusion. Unlike in WT or TLR2‐deficient mice, disruption/absence of TLR4 pathway reduced IRI, as manifested by liver function (serum alanine aminotransferase levels), histology (Suzukis scores), neutrophil infiltration (myeloperoxidase activity) and local/systemic TNF‐α production (mRNA/protein levels). Moreover, defective TLR4 but not TLR2 signaling increased mRNA/protein HO‐1 expression. In contrast, tin protoporphyrin‐mediated HO‐1 inhibition restored hepatic damage in otherwise IRI‐resistant TLR4 mutant/KO mice. CoPP‐induced HO‐1 overexpression ameliorated hepatic damage in IRI‐susceptible TLR2 KO mice, comparable with WT controls, and concomitantly diminished TLR4 levels. In conclusion, this study highlights the importance of cross talk between HO‐1 and TLR system in the mechanism of hepatic IRI. Hepatic IRI represents a case for innate immunity in which HO‐1 modulates proinflammatory responses that are triggered via TLR4 signaling, a putative HO‐1 repressor.

CD154-CD40 T-cell costimulation pathway is required in the mechanism of hepatic ischemia/reperfusion injury, and its blockade facilitates and depends on heme oxygenase-1 mediated cytoprotection.

Background. Ischemia/reperfusion (I/R) injury remains an important clinical problem that affects both early and later allograft outcome. This study was designed to analyze the role of T cells and CD154-CD40 T- cell costimulation pathway in a mouse liver I/R model. Methods and Results. Ninety minutes of warm ischemia followed by 4 h of reperfusion in wild-type (WT) mice resulted in a significant hepatic damage, as assessed by liver function (serum alanine aminotransferase [sALT] levels), local neutrophil accumulation (myeloperoxidase activity), and histology (Suzuki’s score). In contrast, T-cell deficiency (in T-cell deficient [nu/nu] mice), disruption of the CD154 signaling (in knockout [KO] mice), or its blockade in WT recipients (after MR1 monoclonal antibody [mAb] treatment), virtually prevented hepatic I/R insult. Unlike CD154-deficient T cells, adoptive transfer of WT spleen cells fully restored hepatic I/R injury in nu/nu mice. Finally, the improved hepatic function in CD154 KO recipients, WT mice treated with CD154 mAb, or nu/nu mice infused with CD154-deficient cells resulted in consistently enhanced expression of heme oxygenase-1 (HO-1), a heat-shock protein with cytoprotective functions. Conclusion. This study confirms the importance of T cells, and documents for the first time the role of CD154 costimulation signals in the mechanism of hepatic I/R injury. We also show that CD154 blockade-mediated cytoprotection results and depends on HO-1 overexpression. Our data provide the rationale for human trials to target CD154-CD40 costimulation in hepatic I/R injury, particularly in the transplant patient.

Systemic Rather Than Local Heme Oxygenase-1 Overexpression Improves Cardiac Allograft Outcomes in a New Transgenic Mouse

Heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme catabolism, exhibits potent antioxidant and anti-inflammatory properties. We developed HO-1 transgenic (Tg) mice using a rat HO-1 genomic transgene under the control of the endogenous promoter. Transgene expression was demonstrated by RT-PCR in all studied tissues, and a modest HO-1 overexpression was documented by Western, ELISA, and enzyme activity assays. To assess the effect of local vs systemic HO-1 in the acute rejection response, we used Tg mice as organ donors or recipients of MHC-incompatible heart grafts. In the local HO-1 overexpression model, Tg allografts survived 10.5 ± 0.7 days (n = 10), compared with 6.5 ± 0.4 days (n = 6) for wild-type donor controls (p = 0.0001). In the systemic HO-1 overexpression model, Tg recipients maintained allografts for 26.8 ± 3.4 days (n = 10), compared with 6.3 ± 0.1 days (n = 12) in wild-type controls (p = 0.00009). Inhibition of HO activity by treatment with tin protoporphyrin blunted survival advantage in Tg mice and resulted in acute graft rejection (n = 3). Increased carboxyhemoglobin levels were consistently noted in Tg mice. Comparisons of grafts at day 4 indicated that HO-1 overexpression was inversely associated with vasculitis/inflammatory cell infiltrate in both models. Hearts transplanted into Tg recipients showed decreased CD4+ lymphocyte infiltration and diminished immune activation, as judged by CD25 expression. Thus, although local and systemic HO-1 overexpression improved allograft outcomes, systemic HO-1 led to a more robust protection and resulted in a significant blunting of host immune activation. This Tg mouse provides a valuable tool to study mechanisms by which HO-1 exerts beneficial effects in organ transplantation.

Absence of toll-like receptor 4 (TLR4) signaling in the donor organ reduces ischemia and reperfusion injury in a murine liver transplantation model

This study analyzes how toll‐like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild‐type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4°C for 24 hours. Unlike WT OLTs, TLR4‐deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzukis grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)‐γ‐inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM‐1), as well as tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐2, and IFN‐γ, yet increased IL‐4 and IL‐10 expression. The well‐functioning OLTs from TLR4 KO donors revealed attenuated activity of capase‐3, and enhanced heme oygenase‐1 (HO‐1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI‐dependent responses. Liver Transpl 13:1435–1443, 2007.

CXCL10 regulates liver innate immune response against ischemia and reperfusion injury

We have shown that activation of toll‐like receptor 4 (TLR4) and its interferon regulatory factor 3 (IRF3)‐dependent downstream signaling pathway are required for the development of liver ischemia/reperfusion injury (IRI). This study focused on the role of TLR4‐IRF3 activation pathway products, in particular, chemokine (C‐X‐C motif) ligand 10 (CXCL10). The induction of CXCL10 by liver IR was rapid (1 hour postreperfusion), restricted (ischemic lobes), and specific (no CXCL9 and CXCL11 induction). Functionally, CXCL10 was critical for IR‐induced liver inflammation and hepatocellular injury. CXCL10 knockout (KO) mice were protected from IRI, as evidenced by reduced serum alanine aminotransferase (sALT) levels and preserved liver histological detail. The induction of pro‐inflammatory genes, such as tumor necrosis factor alpha (TNF‐α), interleukin 1β (IL‐1β), IL‐6, and IL‐12β was diminished, whereas the induction of the IL‐10 gene remained intact in CXCL10 KO mice, indicating an altered liver response against IR. This was accompanied by selective down‐regulation of extracellular signal‐regulated kinase (ERK), but intact Jun N‐terminal kinase (JNK), activation in the KO IR livers. This altered liver inflammation response was (1) specific to IR, because lipopolysaccharide (LPS) induced a comparable pro‐inflammatory response in CXCL10 KO and wild‐type (WT) mice; and (2) responsible for liver cytoprotection from IR, because neutralization of IL‐10 restored local inflammation and hepatocellular damage. Conclusion: CXCL10 regulates liver inflammation response against IRI, and its deficiency protected livers from IRI by local IL‐10–mediated cytoprotection. Targeting CXCL10 may provide a novel therapeutic means to ameliorate liver IRI in clinics. (HEPATOLOGY 2008.)

Type I, but not type II, interferon is critical in liver injury induced after ischemia and reperfusion

We have documented the key role of toll‐like receptor 4 (TLR4) activation and its signaling pathway mediated by interferon (IFN) regulatory factor 3, in the induction of inflammation leading to the hepatocellular damage during liver ischemia/reperfusion injury (IRI). Because type I IFN is the major downstream activation product of that pathway, we studied its role in comparison with IFN‐γ. Groups of type I (IFNAR), type II (IFNGR) IFN receptor–deficient mice, along with wild‐type (WT) controls were subjected to partial liver warm ischemia (90 minutes) followed by reperfusion (1‐6 hours). Interestingly, IFNAR knockout (KO) but not IFNGR KO mice were protected from IR‐induced liver damage, as evidenced by decreased serum alanine aminotransferase and preservation of tissue architecture. IR‐triggered intrahepatic pro‐inflammatory response, assessed by tumor necrosis factor (TNF‐α), interleukin 6 (IL‐6), and chemokine (C‐X‐C motif) ligand 10 (CXCL‐10) expression, was diminished selectively in IFNAR KO mice. Consistent with these findings, our in vitro cell culture studies have shown that: (1) although hepatocytes alone failed to respond to lipopolysaccharide (LPS), when co‐cultured with macrophages they did respond to LPS via macrophage‐derived IFN‐β; (2) macrophages required type I IFN to sustain CXCL10 production in response to LPS. This study documents that type I, but not type II, IFN pathway is required for IR‐triggered liver inflammation/damage. Type I IFN mediates potential synergy between nonparenchyma and parenchyma cells in response to TLR4 activation. (HEPATOLOGY 2007.)