Although the roles of the metabolic stress in organ ischemia‐reperfusion injury (IRI) have been well recognized, the question of whether and how these stress responses regulate innate immune ...activation against IR remains unclear. In a murine liver partial warm ischemia mode, we showed that prolonged ischemia triggered endoplasmic reticulum (ER) stress response, particularly, the activating transcription factor 6 (ATF6) branch, in liver Kupffer cells (KCs) and altered their responsiveness against Toll‐like receptor (TLR) stimulation. Ischemia‐primed cells increased pro‐, but decreased anti‐, inflammatory cytokine productions. Alleviation of ER stress in vivo by small chemical chaperon 4‐phenylbutyrate or ATF6 small interfering RNA (siRNA) diminished the pro‐inflammatory priming effect of ischemia in KCs, leading to the inhibition of liver immune response against IR and protection of livers from IRI. In vitro, ATF6 siRNA abrogated the ER stress‐mediated pro‐inflammatory enhancement of macrophage TLR4 response, by restricting NF‐κB and restoring Akt activations. Thus, ischemia primes liver innate immune cells by ATF6‐mediated ER stress response. The IR‐induced metabolic stress and TLR activation function in synergy to activate tissue inflammatory immune response.
This study demonstrates that ischemia promotes pro‐inflammatory innate immune activation by activating ATF6 signaling pathways in murine liver ischemia and reperfusion injury.
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.
Liver inflammation triggered by the insults of peri‐transplant ischemia and reperfusion is an innate immune‐dominated local response regulated by CD4+ T cells.
Liver transplantation is the gold standard of care in patients with end‐stage liver disease and those with tumors of hepatic origin in the setting of liver dysfunction. From 1988 to 2009, liver ...transplantation in the United States grew 3.7‐fold from 1713 to 6320 transplants annually. The expansion of liver transplantation is chiefly driven by scientific breakthroughs that have extended patient and graft survival well beyond those expected 50 years ago. The success of liver transplantation is now its primary obstacle, as the pool of donor livers fails to keep pace with the growing number of patients added to the national liver transplant waiting list. This review focuses on three major challenges facing liver transplantation in the United States and discusses new areas of investigation that address each issue: (1) the need for an expanded number of useable donor organs, (2) the need for improved therapies to treat recurrent hepatitis C after transplantation and (3) the need for improved detection, risk stratification based upon tumor biology and molecular inhibitors to combat hepatocellular carcinoma.
The authors discuss three critical obstacles that confront the field of liver transplantation today and identify solutions that can expand organ transplantation and improve care for a population in need.
Vascularized composite allotransplantation (VCA) is a viable reconstructive option for complex tissue defects. Although grafts with a large muscular component may be uniquely susceptible to ...ischemia–reperfusion (I/R) syndrome, the safe cold ischemia time in VCA has not been established. We investigated the effects of cold ischemia on innate immune response and recipient survival in a murine orthotopic hindlimb transplantation model. Surprisingly, mice receiving grafts exposed to 6 h or longer of cold storage demonstrated reduced survival and massive elevations in serum creatinine, blood urea nitrogen, and creatine kinase, compared with 1 h of cold storage recipients. This was accompanied by progressive increase in macrophage and neutrophil cell infiltration in muscle biopsy specimens, altered platelet endothelial cell adhesion molecule‐1 expression, and ultimate renal injury. Multiplex immunoassay analysis identified 21 cytokines in serum and 18 cytokines in muscle biopsy specimens that are likely essential in the complex response to I/R‐triggered injury in VCA. In conclusion, this study, in a mouse model of orthotopic hindlimb transplantation, is the first to document that prolonged cold ischemia triggers progressive I/R injury with vascular endothelial damage and may lead to irrecoverable local and remote organ damage. These experimental findings are important in guiding future therapies for human VCA recipients.
Murine orthotopic hindlimb transplantation with extended cold storage may result in progressive local ischemia–reperfusion injury, remote tissue dysfunction, and even recipient death.
Ischemia/reperfusion (I/R) injury is a multifactorial process detrimental to liver graft function. An understanding of the mechanisms involved in I/R injury is essential for the design of therapeutic ...strategies to improve the outcome of liver transplantation. The generation of reactive oxygen species subsequent to reoxygenation inflicts tissue damage and initiates a cellular cascade leading to inflammation, cell death, and ultimate organ failure. The accruing evidence suggests that Kupffer cells and T cells mediate the activation of neutrophil inflammatory responses. Activated neutrophils infiltrate the injured liver in parallel with increased expression of adhesion molecules on endothelial cells. The heme oxygenase (HO) system is among the most critical of the cytoprotective mechanisms activated during the cellular stress, exerting anti-oxidant and anti-inflammatory functions, modulating the cell cycle, and maintaining the microcirculation. The activation of toll-like receptors (TLR) on Kupffer cells may provide the triggering signal for pro-inflammatory responses in the I/R injury sequence. Indeed, dissecting TLR downstream signaling pathways plays a fundamental role in exploring novel therapeutic strategies based on the concept that hepatic I/R injury represents a case for host “innate” immunity.
Donor‐specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody‐mediated rejection. Intracapillary macrophage ...infiltration is a hallmark of antibody‐mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc‐independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody‐mediated rejection, in which B6.RAG1−/− recipients of BALB/c cardiac allografts were passively transferred with donor‐specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P‐selectin, which supports monocyte binding. In the experimental model, donor‐specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL‐1‐Ig abolished antibody‐induced monocyte infiltration in the allograft, but had little effect on antibody‐induced endothelial injury. Our data suggest that antagonism of P‐selectin may ameliorate accumulation of macrophages in the allograft during antibody‐mediated rejection.
The authors demonstrate that donor class I‐specific antibody elicits endothelial exocytosis and P‐selectindependent monocyte recruitment, and that the P‐selectin antagonist rPSGL‐1‐Ig curbs macrophage accumulation in a murine cardiac allograft in response to these antibodies.
Ischemia‐reperfusion injury (IRI), an innate immunity‐driven local inflammation, remains the major problem in clinical organ transplantation. T cell immunoglobulin and mucin domain (TIM‐3)–Galectin‐9 ...(Gal‐9) signaling regulates CD4+ Th1 immune responses. Here, we explored TIM‐3–Gal‐9 function in a clinically relevant murine model of hepatic cold storage and orthotopic liver transplantation (OLT). C57BL/6 livers, preserved for 20 h at 4°C in UW solution, were transplanted to syngeneic mouse recipients. Up‐regulation of TIM‐3 on OLT‐infiltrating activated CD4+ T cells was observed in the early IRI phase (1 h). By 6 h of reperfusion, OLTs in recipients treated with a blocking anti‐TIM‐3 Ab were characterized by: (1) enhanced hepatocellular damage (sALT levels, liver Suzuki's histological score); (2) polarized cell infiltrate towards Th1/Th17‐type phenotype; (3) depressed T cell exhaustion markers (PD‐1, LAG3); and (4) elevated neutrophil and macrophage infiltration/activation. In parallel studies, adoptive transfer of CD4+ T cells from naïve WT, but not from TIM‐3 Tg donors, readily recreated OLT damage in otherwise IR‐resistant RAG−/− test recipients. Furthermore, pre‐treatment of mice with rGal‐9 promoted hepatoprotection against preservation‐association liver damage, accompanied by enhanced TIM‐3 expression in OLTs. Thus, CD4+ T cell‐dependent “negative” TIM‐3 costimulation is essential for hepatic homeostasis and resistance against IR stress in OLTs.
TIM‐3–Gal‐9 negative T cell costimulation promotes hepatic homeostasis and cytoprotection against innate immune‐driven ischemia reperfusion injury in mouse liver transplants.
Ischemia/reperfusion injury (IRI) remains unresolved problem in clinical organ transplantation. We analyzed the role of Type‐I interferon (IFN) pathway in a clinically relevant murine model of ...extended hepatic cold preservation followed by orthotopic liver transplantation (OLT). Livers from Type‐I IFN receptor (IFNAR) knockout (KO) or wild‐type (WT) mice (C57/BL6) were harvested, preserved at 4°C in UW solution for 20 h and transplanted to groups of syngeneic IFNAR KO or WT recipients. Liver graft but not recipient IFNAR deficiency was required to consistently ameliorate IRI in OLTs. Indeed, disruption of Type‐I IFN signaling decreased serum alanine aminotransferase (sALT) levels (p < 0.001), diminished Suzuki's score of histological OLT damage (p < 0.01) and improved 14‐day survival (from 42%5/12 in WT to 92%11/12 in IFNAR KO; p < 0.05). Unlike in WT group, IFNAR deficiency attenuated OLT expression of TNF‐α, IL‐1β, IL‐6, MCP‐1, CXCL‐10, ICAM‐1; diminished infiltration by macrophages/PMNs; and enhanced expression of antioxidant HO‐1/Nrf2. The frequency of TUNEL+ apoptotic cells and caspase‐3 activity/expression selectively decreased in IFNAR KO group. Small interfering (si)RNA‐directed targeting of HO‐1 restored cardinal features of liver IRI in otherwise resistant IFNAR‐deficient OLTs. Thus, intact Type‐I IFN signaling is required for hepatic IRI, whereas HO‐1 is needed for cytoprotection against innate immunity‐dominated organ preservation damage in IFNAR‐deficient liver transplants.
Type‐I interferon signaling is required for liver ischemia/reperfusion damage whereas heme oxygenase‐1 is needed for cytoprotection.
Although pretransplant diabetes is a risk factor for mortality post–liver transplant, the underlying mechanism has not been fully defined. In a murine liver partial warm ischemia model, we addressed ...the question of how diabetes/hyperglycemia impacted tissue inflammatory injuries against ischemia reperfusion (IR), focusing on the advanced glycation endproduct (AGE) and its receptor (RAGE) pathway. Our results showed that hepatocellular injury was exacerbated in streptozotocin‐induced diabetic mice against IR, in association with hyper‐inflammatory immune activation in livers. Serum levels of AGEs, but not HMGB1, were increased in diabetic mice in response to liver IR. Both RAGE antagonist peptides and small interfering RNA alleviated liver injuries and inhibited inflammatory immune activation against IR in diabetic, but not normal, mice. Kupffer cells (KCs)/macrophages, but not hepatocytes, from diabetic mice expressed significantly higher levels of RAGE, leading to their hyper‐inflammatory responsiveness to both TLR ligands and AGEs. In vitro, hyperglycemia increased macrophage RAGE expression and enhanced their TLR responses. Our results demonstrated that activation of the AGE–RAGE signaling pathway in KCs was responsible for hyper‐inflammatory immune responses and exacerbated hepatocellular injuries in diabetic/hyperglycemic hosts against liver IR.
Hyperglycemia enhances liver inflammatory tissue injury against ischemia‐reperfusion by activating the advanced glycation end product and its receptor pathway.
The selectin antagonist known as recombinant P‐selectin glycoprotein ligand IgG (rPSGL‐Ig) blocks leukocyte adhesion and protects against transplantation ischemia reperfusion injury (IRI) in animal ...models. This randomized (1:1) single‐center double‐blind 47‐patient phase 2 study with 6‐month follow‐up assessed rPSGL‐Ig's safety and impact on early graft function at 1 mg/kg systemic dose with pretransplant allograft ex vivo treatment in deceased‐donor liver transplant recipients. Safety was assessed in all patients, whereas efficacy was assessed in a prospectively defined per‐protocol patient set (PP) by peak serum transaminase (TA) and bilirubin values, and normalization thereof. In PP patients, the incidence of poor early graft function (defined as peak TA >2500 U/L or bilirubin >10 mg/dL), average peak liver enzymes and bilirubin, normalization thereof and duration of primary and total hospitalization trended consistently lower in the rPSGL‐Ig group compared to placebo. In patients with donor risk index above study‐average, normalization of aspartate aminotransferase was significantly improved in the rPSGL‐Ig group (p < 0.03). rPSGL‐Ig treatment blunted postreperfusion induction versus placebo of IRI biomarker IP‐10 (p < 0.1) and augmented cytoprotective IL‐10 (p < 0.05). This is the first clinical trial of an adhesion molecule antagonist to demonstrate a beneficial effect on liver transplantation IRI and supported by therapeutic modulation of two hepatic IRI biomarkers.
Treatment with rPSGL‐Ig, a P‐selectin antagonist, improved early liver allograft function in a double‐blinded, placebo‐controlled, single‐center Phase II study, in association with decreased IP‐10 and augmented IL‐10 expression.
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