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 Suzukis 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.

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.

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.

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.

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-α 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.

UNLABELLED: 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).

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 (HSF1^M-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 (HSF1^FL/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).

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.

Notch signaling plays important roles in the regulation of immune cell functioning during the inflammatory response. Activation of the innate immune signaling receptor NLRP3 promotes inflammation in injured tissue. However, it remains unknown whether Jagged1 (JAG1)-mediated myeloid Notch1 signaling regulates NLRP3 function in acute liver injury. Here, we report that myeloid Notch1 signaling regulates the NLRP3-driven inflammatory response in ischemia/reperfusion (IR)-induced liver injury. In a mouse model of liver IR injury, Notch1-proficient (Notch1^FL/FL) mice receiving recombinant JAG1 showed a reduction in IR-induced liver injury and increased Notch intracellular domain (NICD) and heat shock transcription factor 1 (HSF1) expression, whereas myeloid-specific Notch1 knockout (Notch1^M-KO) aggravated hepatocellular damage even with concomitant JAG1 treatment. Compared to JAG1-treated Notch1^FL/FL controls, Notch1^M-KO mice showed diminished HSF1 and Snail activity but augmented NLRP3/caspase-1 activity in ischemic liver. The disruption of HSF1 reduced Snail activation and enhanced NLRP3 activation, while the adoptive transfer of HSF1-expressing macrophages to Notch1^M-KO mice augmented Snail activation and mitigated IR-triggered liver inflammation. Moreover, the knockdown of Snail in JAG1-treated Notch1^FL/FL livers worsened hepatocellular functioning, reduced TRX1 expression and increased TXNIP/NLRP3 expression. Ablation of myeloid Notch1 or Snail increased ASK1 activation and hepatocellular apoptosis, whereas the activation of Snail increased TRX1 expression and reduced TXNIP, NLRP3/caspase-1, and ROS production. Our findings demonstrated that JAG1-mediated myeloid Notch1 signaling promotes HSF1 and Snail activation, which in turn inhibits NLRP3 function and hepatocellular apoptosis leading to the alleviation of IR-induced liver injury. Hence, the Notch1/HSF1/Snail signaling axis represents a novel regulator of and a potential therapeutic target for liver inflammatory injury.