S. Yue

KEAP1-NRF2 complex in ischemia-induced hepatocellular damage of mouse liver transplants

BACKGROUND & AIMS The Keap1-Nrf2 signaling pathway regulates host cell defense responses against oxidative stress and maintains the cellular redox balance. METHODS We investigated the function/molecular mechanisms by which Keap1-Nrf2 complex may influence liver ischemia/reperfusion injury (IRI) in a mouse model of hepatic cold storage (20h at 4°C) followed by orthotopic liver transplantation (OLT). RESULTS The Keap1 hepatocyte-specific knockout (HKO) in the donor liver ameliorated post-transplant IRI, evidenced by improved hepatocellular function and OLT outcomes (Keap1 HKO→Keap1 HKO; 100% survival), as compared with controls (WT→WT; 50% survival; p<0.01). By contrast, donor liver Nrf2 deficiency exacerbated IRI in transplant recipients (Nrf2 KO→Nrf2 KO; 40% survival). Ablation of Keap1 signaling reduced macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while simultaneously promoting anti-apoptotic functions in OLTs. At the molecular level, Keap1 HKO increased Nrf2 levels, stimulated Akt phosphorylation, and enhanced expression of anti-oxidant Trx1, HIF-1α, and HO-1. Pretreatment of liver donors with PI3K inhibitor (LY294002) disrupted Akt/HIF-1A signaling and recreated hepatocellular damage in otherwise IR-resistant Keap1 HKO transplants. In parallel in vitro studies, hydrogen peroxide-stressed Keap1-deficient hepatocytes were characterized by enhanced expression of Nrf2, Trx1, and Akt phosphorylation, in association with decreased release of lactate dehydrogenase (LDH) in cell culture supernatants. CONCLUSIONS Keap1-Nrf2 complex prevents oxidative injury in IR-stressed OLTs through Keap1 signaling, which negatively regulates Nrf2 pathway. Activation of Nrf2 induces Trx1 and promotes PI3K/Akt, crucial for HIF-1α activity. HIF-1α-mediated overexpression of HO-1/Cyclin D1 facilitates cytoprotection by limiting hepatic inflammatory responses, and hepatocellular necrosis/apoptosis in a PI3K-dependent manner.

ATF6 Mediates a Pro-inflammatory Synergy between ER Stress and TLR Activation in the Pathogenesis of Liver Ischemia Reperfusion Injury

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.

Myeloid PTEN Deficiency Protects Livers from Ischemia Reperfusion Injury by Facilitating M2 Macrophage Differentiation

Although the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in regulating cell proliferation is well established, its function in immune responses remains to be fully appreciated. In the current study, we analyzed myeloid-specific PTEN function in regulating tissue inflammatory immune response in a murine liver partial warm ischemia model. Myeloid-specific PTEN knockout (KO) resulted in liver protection from ischemia reperfusion injury (IRI) by deviating the local innate immune response against ischemia reperfusion toward the regulatory type: expression of proinflammatory genes was selectively decreased and anti-inflammatory IL-10 was simultaneously increased in ischemia reperfusion livers of PTEN KO mice compared with those of wild-type (WT) mice. PI3K inhibitor and IL-10–neutralizing Abs, but not exogenous LPS, recreated liver IRI in these KO mice. At the cellular level, Kupffer cells and peritoneal macrophages isolated from KO mice expressed higher levels of M2 markers and produced lower TNF-α and higher IL-10 in response to TLR ligands than did their WT counterparts. They had enhanced Stat3- and Stat6-signaling pathway activation, but diminished Stat1-signaling pathway activation, in response to TLR4 stimulation. Inactivation of Kupffer cells by gadolinium chloride enhanced proinflammatory immune activation and increased IRI in livers of myeloid PTEN KO mice. Thus, myeloid PTEN deficiency protects livers from IRI by facilitating M2 macrophage differentiation.

β‐catenin regulates innate and adaptive immunity in mouse liver ischemia‐reperfusion injury

Dendritic cells (DCs) are critical mediators of immune responses that integrate signals from the innate immune system to orchestrate adaptive host immunity. This study was designed to investigate the role and molecular mechanisms of STAT3‐induced β‐catenin in the regulation of DC function and inflammatory responses in vitro and in vivo. STAT3 induction in lipopolysaccharide (LPS)‐stimulated mouse bone marrow‐derived DCs (BMDCs) triggered β‐catenin activation by way of GSK‐3β phosphorylation. The activation of β‐catenin inhibited phosphatase and tensin homolog delete on chromosome 10 (PTEN) and promoted the phosphoinositide 3‐kinase (PI3K)/Akt pathway, which in turn down‐regulated DC maturation and function. In contrast, knockdown of β‐catenin increased PTEN/TLR4 (Toll‐like receptor 4), interferon regulatory factor‐3 (IRF3), nuclear factor kappa B (NF‐κB) activity, and proinflammatory cytokine programs in response to LPS stimulation. In a mouse model of warm liver ischemia and reperfusion injury (IRI), disruption of β‐catenin signaling increased the hepatocellular damage, enhanced hepatic DC maturation/function, and PTEN/TLR4 local inflammation in vivo. Conclusion: These findings underscore the role of β‐catenin to modulate DC maturation and function at the innate‐adaptive interface. Activation of β‐catenin triggered PI3K/Akt, which in turn inhibited TLR4‐driven inflammatory response in a negative feedback regulatory mechanism. By identifying the molecular pathways by which β‐catenin regulates DC function, our findings provide the rationale for novel therapeutic approaches to manage local inflammation and injury in IR‐stressed liver. (HEPATOLOGY 2013)

Rapamycin protection of livers from ischemia and reperfusion injury is dependent on both autophagy induction and mammalian target of rapamycin complex 2-Akt activation.

Background Although rapamycin (RPM) have been studied extensively in ischemia models, its functional mechanisms remains to be defined. Methods We determined how RPM impacted the pathogenesis of ischemia-reperfusion injury (IRI) in a murine liver partial warm ischemia model, with emphasis on its regulation of hepatocyte death. Results Rapamycin protected livers from IRI in the presence of fully developed liver inflammatory immune response. Rapamycin enhanced liver autophagy induction at the reperfusion stage. Dual mammalian (mechanistic) target of rapamycin (mTOR)1/2 inhibitor Torin 1, despite its ability to induced autophagy, failed to protect livers from IRI. The treatment with RPM, but not Torin 1, resulted in the enhanced activation of the mTORC2-Akt signaling pathway activation in livers after reperfusion. Inactivation of Akt by Triciribine abolished the liver protective effect of RPM. The differential cytoprotective effect of RPM and Torin 1 was confirmed in vitro in hepatocyte cultures. Rapamycin, but not Trin 1, protected hepatocytes from stress and tumor necrosis factor-&agr; induced cell death; and inhibition of autophagy by chloroquine or Akt by Triciribine abolished RPM-mediated cytoprotection. Conclusion Rapamycin protected livers from IRI by both autophagy and mTORC2-Akt activation mechanisms.

Adoptive transfer of heme oxygenase-1 (HO-1)-modified macrophages rescues the nuclear factor erythroid 2-related factor (Nrf2) antiinflammatory phenotype in liver ischemia/reperfusion injury.

Macrophages are instrumental in the pathophysiology of liver ischemia/reperfusion injury (IRI). Although Nrf2 regulates macrophage-specific heme oxygenase-1 (HO-1) antioxidant defense, it remains unknown whether HO-1 induction might rescue macrophage Nrf2-dependent antiinflammatory functions. This study explores the mechanisms by which the Nrf2-HO-1 axis regulates sterile hepatic inflammation responses after adoptive transfer of ex vivo modified HO-1 overexpressing bone marrow-derived macrophages (BMMs). Livers in Nrf2-deficient mice preconditioned with Ad-HO-1 BMMs, but not Ad-β-Gal-BMMs, ameliorated liver IRI (at 6 h of reperfusion after 90 min of warm ischemia), evidenced by improved hepatocellular function (serum alanine aminotransferase (sALT) levels) and preserved hepatic architecture (Suzuki histological score). Treatment with Ad-HO-1 BMMs decreased neutrophil accumulation, proinflammatory mediators and hepatocellular necrosis/apoptosis in ischemic livers. Moreover, Ad-HO-1 transfection of Nrf2-deficient BMMs suppressed M1 (Nos2+) while promoting the M2 (Mrc-1/Arg-1+) phenotype. Unlike in controls, Ad-HO-1 BMMs increased the expression of Notch1, Hes1, phosphorylation of Stat3 and Akt in IR-stressed Nrf2-deficient livers as well as in lipopolysaccharide (LPS)-stimulated BMMs. Thus, adoptive transfer of ex vivo generated Ad-HO-1 BMMs rescued Nrf2-dependent antiinflammatory phenotype by promoting Notch1/Hes1/Stat3 signaling and reprogramming macrophages toward the M2 phenotype. These findings provide the rationale for a novel clinically attractive strategy to manage IR liver inflammation/damage.

Nuclear factor erythroid 2-related factor 2 regulates toll-like receptor 4 innate responses in mouse liver ischemia-reperfusion injury through Akt-forkhead box protein O1 signaling network.

Background Nuclear factor erythroid 2–related factor 2 (Nrf2), a master regulator of the antioxidant host defense, maintains the cellular redox homeostasis. Methods This study was designed to investigate the role and molecular mechanisms by which Nrf2 regulates toll-like receptor (TLR)4-driven inflammation response in a mouse model of hepatic warm ischemia (90 min) and reperfusion (6 hr) injury (IRI). Results Activation of Nrf2 after preconditioning of wild-type mouse recipients with cobalt protoporphyrin ameliorated liver IRI, evidenced by improved hepatocellular function (serum alanine aminotransferase levels), and preserved tissue architecture (histology Suzuki’s score). In marked contrast, ablation of Nrf2 signaling exacerbated IR-induced liver inflammation and damage in Nrf2 knockout hosts irrespective of adjunctive cobalt protoporphyrin treatment. The Nrf2 activation reduced macrophage and neutrophil trafficking, proinflammatory cytokine programs, and hepatocellular necrosis or apoptosis while increasing antiapoptotic functions in IR-stressed livers. At the molecular level, Nrf2 activation augmented heme oxygenase-1 expression and Stat3 phosphorylation and promoted PI3K-Akt while suppressing forkhead box O (Foxo)1 signaling. In contrast, Nrf2 deficiency diminished PI3K-Akt and enhanced Foxo1 expression in the ischemic livers. In parallel in vitro studies, Nrf2 knockdown in lipopolysaccharide-stimulated bone marrow-stimulated bone marrow–derived macrophages (BMMs) decreased heme oxygenase-1 and PI3K-Akt yet increased Foxo1 transcription, leading to enhanced expression of TLR4 proinflammatory mediators. Moreover, pretreatment of bone marrow–derived macrophages with PI3K inhibitor (LY294002) activated Foxo1 signaling, which in turn enhanced TLR4-driven innate responses in vitro. Conclusion Activation of Nrf2 promoted PI3K-Akt, and inhibited Foxo1 activity in IR-triggered local inflammation response. By identifying a novel integrated Nrf2-Akt-Foxo1 signaling network in PI3K-dependent regulation of TLR4-driven innate immune activation, this study provides the rationale for refined therapeutic approaches to manage liver inflammation and IRI in transplant recipients.

The myeloid heat shock transcription factor 1/β‐catenin axis regulates NLR family, pyrin domain‐containing 3 inflammasome activation in mouse liver ischemia/reperfusion injury

Heat shock transcription factor 1 (HSF1) has been implicated in the differential regulation of cell stress and disease states. β‐catenin activation is essential for immune homeostasis. However, little is known about the role of macrophage HSF1‐β‐catenin signaling in the regulation of NLRP3 inflammasome activation during ischemia/reperfusion (I/R) injury (IRI) in the liver. This study investigated the functions and molecular mechanisms by which HSF1‐β‐catenin signaling influenced NLRP3‐mediated innate immune response in vivo and in vitro. Using a mouse model of IR‐induced liver inflammatory injury, we found that mice with a myeloid‐specific HSF1 knockout (HSF1M‐KO) displayed exacerbated liver damage based on their increased serum alanine aminotransferase levels, intrahepatic macrophage/neutrophil trafficking, and proinflammatory interleukin (IL)‐1β levels compared to the HSF1‐proficient (HSF1FL/FL) controls. Disruption of myeloid HSF1 markedly increased transcription factor X‐box‐binding protein (XBP1), NLR family, pyrin domain‐containing 3 (NLRP3), and cleaved caspase‐1 expression, which was accompanied by reduced β‐catenin activity. Knockdown of XBP1 in HSF1‐deficient livers using a XBP1 small interfering RNA ameliorated hepatocellular functions and reduced NLRP3/cleaved caspase‐1 and IL‐1β protein levels. In parallel in vitro studies, HSF1 overexpression increased β‐catenin (Ser552) phosphorylation and decreased reactive oxygen species (ROS) production in bone‐marrow‐derived macrophages. However, myeloid HSF1 ablation inhibited β‐catenin, but promoted XBP1. Furthermore, myeloid β‐catenin deletion increased XBP1 messenger RNA splicing, whereas a CRISPR/CRISPR‐associated protein 9‐mediated XBP1 knockout diminished NLRP3/caspase‐1. Conclusion: The myeloid HSF1‐β‐catenin axis controlled NLRP3 activation by modulating the XBP1 signaling pathway. HSF1 activation promoted β‐catenin, which, in turn, inhibited XBP1, leading to NLRP3 inactivation and reduced I/R‐induced liver injury. These findings demonstrated that HSF1/β‐catenin signaling is a novel regulator of innate immunity in liver inflammatory injury and implied the therapeutic potential for management of sterile liver inflammation in transplant recipients. (Hepatology 2016;64:1683‐1698).

The Dichotomy of Endoplasmic Reticulum Stress Response in Liver Ischemia-Reperfusion Injury.

Abstract Endoplasmic reticulum (ER) stress plays critical roles in the pathogenesis of liver ischemia-reperfusion injury (IRI). As ER stress triggers an adaptive cellular response, the question of what determines its functional outcome in liver IRI remains to be defined. In a murine liver partial warm ischemia model, we studied how transient (30 minutes) or prolonged (90 minutes) liver ischemia regulated local ER stress response and autophagy activities and their relationship with liver IRI. Effects of chemical chaperon 4-phenylbutyrate (4-PBA) or autophagy inhibitor 3-methyladenine (3-MA) were evaluated. Our results showed that although the activating transcription factor 6 branch of ER stress response was induced in livers by both types of ischemia, liver autophagy was activated by transient, but inhibited by prolonged, ischemia. Although 3-MA had no effects on liver IRI after prolonged ischemia, it significantly increased liver IRI after transient ischemia. The 4-PBA treatment protected livers from IRI after prolonged ischemia by restoring autophagy flux, and the adjunctive 3-MA treatment abrogated its liver protective effect. The same 4-PBA treatment, however, increased liver IRI and disrupted autophagy flux after transient ischemia. Although both types of ischemia activated 5′ adenosine monophosphate-activated protein kinase and inactivated protein kinase B (Akt), prolonged ischemia also resulted in downregulations of autophagy-related gene 3 and autophagy-related gene 5 in ischemic livers. These results indicate a functional dichotomy of ER stress response in liver IRI via its regulation of autophagy. Transient ischemia activates autophagy to protect livers from IRI, whereas prolonged ischemia inhibits autophagy to promote the development of liver IRI.

Hyperglycemia and liver ischemia reperfusion injury: a role for the advanced glycation endproduct and its receptor pathway.

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.