The macrophage STING-YAP axis controls hepatic steatosis by promoting the autophagic degradation of lipid droplets.

BACKGROUND AND AIMS: The hallmark of NAFLD or hepatic steatosis is characterized by lipid droplet (LD) accumulation in hepatocytes. Autophagy may have profound effects on lipid metabolism and innate immune response. However, how innate immune activation may regulate the autophagic degradation of intracellular LDs remains elusive. APPROACH AND RESULTS: A mouse model of a high-fat diet-induced NASH was used in the myeloid-specific stimulator of interferon genes (STING) knockout or STING/yes-associated protein (YAP) double knockout mice. Liver injury, lipid accumulation, lipid droplet proteins, autophagic genes, chromatin immunoprecipitation coupled with massively parallel sequencing, and RNA-Seq were assessed in vivo and in vitro . We found that high-fat diet-induced oxidative stress activates STING and YAP pathways in hepatic macrophages. The acrophage STING deficiency (myeloid-specific STING knockout) enhances nuclear YAP activity, reduces lipid accumulation, and increases autophagy-related proteins ATG5, ATG7, and light chain 3B but diminishes LD protein perilipin 2 expression. However, disruption of STING and YAP (myeloid STING and YAP double knockout) increases serum alanine aminotransferase and triglyceride levels and reduces ß-fatty acid oxidation gene expression but augments perilipin 2 levels, exacerbating high-fat diet-induced lipid deposition. Chromatin immunoprecipitation coupled with massively parallel sequencing reveals that macrophage YAP targets transmembrane protein 205 and activates AMP-activated protein kinase α, which interacts with hepatocyte mitofusin 2 and induces protein disulfide isomerase activation. Protein disulfide isomerase activates hypoxia-inducible factor-1α signaling, increases autophagosome colocalization with LDs, and promotes the degradation of perilipin 2 by interacting with chaperone-mediated autophagy chaperone HSC70. CONCLUSIONS: The macrophage STING-YAP axis controls hepatic steatosis by reprogramming lipid metabolism in a transmembrane protein 205/mitofusin 2/protein disulfide isomerase-dependent pathway. These findings highlight the regulatory mechanism of the macrophage STING-driven YAP activity on lipid control.

Macrophage RIPK3 triggers inflammation and cell death via the XBP1-Foxo1 axis in liver ischaemia-reperfusion injury.

Background & Aims: Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) is a central player in triggering necroptotic cell death. However, whether macrophage RIPK3 may regulate NOD1-dependent inflammation and calcineurin/transient receptor potential cation channel subfamily M member 7 (TRPM7)-induced hepatocyte death in oxidative stress-induced liver inflammatory injury remains elusive. Methods: A mouse model of hepatic ischaemia-reperfusion (IR) injury, the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific RIPK3 knockout (RIPK3M-KO) and RIPK3-proficient (RIPK3FL/FL) mice. Results: RIPK3M-KO diminished IR stress-induced liver damage with reduced serum alanine aminotransferase/aspartate aminotransferase levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the RIPK3FL/FL controls. IR stress activated RIPK3, inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α), x-box binding protein 1 (XBP1), nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), NF-κB, forkhead box O1 (Foxo1), calcineurin A, and TRPM7 in ischaemic livers. Conversely, RIPK3M-KO depressed IRE1α, XBP1, NOD1, calcineurin A, and TRPM7 activation with reduced serum tumour necrosis factor α (TNF-α) levels. Moreover, Foxo1M-KO alleviated IR-induced liver injury with reduced NOD1 and TRPM7 expression. Interestingly, chromatin immunoprecipitation coupled with massively parallel sequencing revealed that macrophage Foxo1 colocalised with XBP1 and activated its target gene Zc3h15 (zinc finger CCCH domain-containing protein 15). Activating macrophage XBP1 enhanced Zc3h15, NOD1, and NF-κB activity. However, disruption of macrophage Zc3h15 inhibited NOD1 and hepatocyte calcineurin/TRPM7 activation, with reduced reactive oxygen species production and lactate dehydrogenase release after macrophage/hepatocyte coculture. Furthermore, adoptive transfer of Zc3h15-expressing macrophages in RIPK3M-KO mice augmented IR-triggered liver inflammation and cell death. Conclusions: Macrophage RIPK3 activates the IRE1α-XBP1 pathway and Foxo1 signalling in IR-stress livers. The XBP1-Foxo1 interaction is essential for modulating target gene Zc3h15 function, which is crucial for the control of NOD1 and calcineurin-mediated TRPM7 activation. XBP1 functions as a transcriptional coactivator of Foxo1 in regulating NOD1-driven liver inflammation and calcineurin/TRPM7-induced cell death. Our findings underscore a novel role of macrophage RIPK3 in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases. Impact and implications: Macrophage RIPK3 promotes NOD1-dependent inflammation and calcineurin/TRPM7-induced cell death cascade by triggering the XBP1-Foxo1 axis and its target gene Zc3h15, which is crucial for activating NOD1 and calcineurin/TRPM7 function, implying the potential therapeutic targets in stress-induced liver inflammatory injury.

Behavioural activity pattern, genetic factors, and the risk of nonalcoholic fatty liver disease: A prospective study in the UK Biobank.

BACKGROUND & AIMS: Physical activity, sedentary behaviour, and genetic variants have been associated with the nonalcoholic fatty liver disease (NAFLD). However, whether and how the degree of healthy activity patterns may modify the impact of genetic susceptibility on NAFLD remains unknown. METHODS: Behaviour activity factors were determined according to total physical activity (TPA) and sedentary time. The polygenic risk score (PRS) was calculated by variants in PNPLA3, TM6SF2, MBOAT7, and GCKR. Cox regression was used to analyse the associations of genetic and behaviour activity factors with incident NAFLD in the UK Biobank (N = 338 087). RESULTS: During a median follow-up of 12.4 years, 3201 incident NAFLD cases were ascertained. Analyses of TPA and sedentary time simultaneously showed a dose-response association with the risk of NAFLD (ptrend < .001). The association of behaviour activity patterns with NAFLD varied by genetic variants. Of the subjects with high genetic risk, we observed a null protective effect of moderate or high TPA on NAFLD risk, while sitting less than three hours a day significantly decreased the risk of NAFLD (p = 3.50 × 10-4 ). The high genetic risk of NAFLD can also be offset by the combination of moderate physical activity and shorter sedentary time. Moreover, the high genetic risk group has the greatest reduction of 10-year absolute risk (6.95 per 1000 person-years) if reaching both healthy activities. CONCLUSIONS: Moderate-to-high physical activity and favourable sedentary behaviour may be lifestyle modifications in preventing NAFLD, which could offset the harmful effect of predisposing genetic factors.

Novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death.

Background & Aims: The stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress. Methods: A mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice. Results: The TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-ß (IFN-ß) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis. Conclusions: Macrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases. Lay summary: Liver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

The cGAS-STING Pathway: Novel Perspectives in Liver Diseases.

Liver diseases represent a major global health burden accounting for approximately 2 million deaths per year worldwide. The liver functions as a primary immune organ that is largely enriched with various innate immune cells, including macrophages, dendritic cells, neutrophils, NK cells, and NKT cells. Activation of these cells orchestrates the innate immune response and initiates liver inflammation in response to the danger signal from pathogens or injured cells and tissues. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a crucial signaling cascade of the innate immune system activated by cytosol DNA. Recognizing DNA as an immune-stimulatory molecule is an evolutionarily preserved mechanism in initiating rapid innate immune responses against microbial pathogens. The cGAS is a cytosolic DNA sensor eliciting robust immunity via the production of cyclic GMP-AMPs that bind and activate STING. Although the cGAS-STING pathway has been previously considered to have essential roles in innate immunity and host defense, recent advances have extended the role of the cGAS-STING pathway to liver diseases. Emerging evidence indicates that overactivation of cGAS-STING may contribute to the development of liver disorders, implying that the cGAS-STING pathway is a promising therapeutic target. Here, we review and discuss the role of the cGAS-STING DNA-sensing signaling pathway in a variety of liver diseases, including viral hepatitis, nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), primary hepatocellular cancer (HCC), and hepatic ischemia-reperfusion injury (IRI), with highlights on currently available therapeutic options.

CD47-Mediated Hedgehog/SMO/GLI1 Signaling Promotes Mesenchymal Stem Cell Immunomodulation in Mouse Liver Inflammation.

BACKGROUND AND AIMS: The cluster of differentiation 47 (CD47)-signal regulatory protein alpha (SIRPα) signaling pathway plays important roles in immune homeostasis and tissue inflammatory response. Activation of the Hedgehog/smoothened (SMO)/GLI family zinc finger 1 (Gli1) pathway regulates cell growth, differentiation, and immune function. However, it remains unknown whether and how the CD47-SIRPα interaction may regulate Hedgehog/SMO/Gli1 signaling in mesenchymal stem cell (MSC)-mediated immune regulation during sterile inflammatory liver injury. APPROACH AND RESULTS: In a mouse model of ischemia/reperfusion (IR)-induced sterile inflammatory liver injury, we found that adoptive transfer of MSCs increased CD47 expression and ameliorated liver IR injury. However, deletion of CD47 in MSCs exacerbated IR-induced liver damage, with increased serum ALT levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators. MSC treatment augmented SIRPα, Hedgehog/SMO/Gli1, and Notch1 intracellular domain (NICD), whereas CD47-deficient MSC treatment reduced these gene expressions in IR-stressed livers. Moreover, disruption of myeloid SMO or Notch1 increased IR-triggered liver inflammation with diminished Gli1 and NICD, but enhanced NIMA related kinase 7 (NEK7) and NLR family pyrin domain containing 3 (NLRP3) activation in MSC-transferred mice. Using a MSC/macrophage co-culture system, we found that MSC CD47 and macrophage SIRPα expression were increased after LPS stimulation. The CD47-SIRPα interaction increased macrophage Gli1 and NICD nuclear translocation, whereby NICD interacted with Gli1 and regulated its target gene Dvl2 (dishevelled segment polarity protein 2), which in turn inhibited NEK7/NLRP3 activity. CONCLUSIONS: The CD47-SIRPα signaling activates the Hedgehog/SMO/Gli1 pathway, which controls NEK7/NLRP3 activity through a direct interaction between Gli1 and NICD. NICD is a coactivator of Gli1, and the target gene Dvl2 regulated by the NICD-Gli1 complex is crucial for the modulation of NLRP3-driven inflammatory response in MSC-mediated immune regulation. Our findings provide potential therapeutic targets in MSC-mediated immunotherapy of sterile inflammatory liver injury.

Functional crosstalk between myeloid Foxo1-ß-catenin axis and Hedgehog/Gli1 signaling in oxidative stress response.

Foxo1 transcription factor is an evolutionarily conserved regulator of cell metabolism, oxidative stress, inflammation, and apoptosis. Activation of Hedgehog/Gli signaling is known to regulate cell growth, differentiation, and immune function. However, the molecular mechanisms by which interactive cell signaling networks restrain oxidative stress response and necroptosis are still poorly understood. Here, we report that myeloid-specific Foxo1 knockout (Foxo1M-KO) mice were resistant to oxidative stress-induced hepatocellular damage with reduced macrophage/neutrophil infiltration, and proinflammatory mediators in liver ischemia/reperfusion injury (IRI). Foxo1M-KO enhanced ß-catenin-mediated Gli1/Snail activity, and reduced receptor-interacting protein kinase 3 (RIPK3) and NIMA-related kinase 7 (NEK7)/NLRP3 expression in IR-stressed livers. Disruption of Gli1 in Foxo1M-KO livers deteriorated liver function, diminished Snail, and augmented RIPK3 and NEK7/NLRP3. Mechanistically, macrophage Foxo1 and ß-catenin colocalized in the nucleus, whereby the Foxo1 competed with T-cell factor (TCF) for interaction with ß-catenin under inflammatory conditions. Disruption of the Foxo1-ß-catenin axis by Foxo1 deletion enhanced ß-catenin/TCF binding, activated Gli1/Snail signaling, leading to inhibited RIPK3 and NEK7/NLRP3. Furthermore, macrophage Gli1 or Snail knockout activated RIPK3 and increased hepatocyte necroptosis, while macrophage RIPK3 ablation diminished NEK7/NLRP3-driven inflammatory response. Our findings underscore a novel molecular mechanism of the myeloid Foxo1-ß-catenin axis in regulating Hedgehog/Gli1 function that is key in oxidative stress-induced liver inflammation and necroptosis.

Tissue Inhibitor of Metalloproteinase 3 Deficiency Disrupts the Hepatocyte E-Cadherin/ß-Catenin Complex and Induces Cell Death in Liver Ischemia/Reperfusion Injury.

Tissue inhibitor of metalloproteinase (TIMP) 3 is a naturally occurring inhibitor of a broad range of proteases, with key roles in extracellular matrix turnover and in the pathogenesis of various diseases. In this study, we investigated the response of mice lacking TIMP3 (TIMP3-/-) to hepatic ischemia/reperfusion injury (IRI). We report here that TIMP3-/- mice showed an enhanced inflammatory response, exacerbated organ damage, and further impaired liver function after IRI when compared with their wild-type littermates. Loss of TIMP3 led to the cleavage and shedding of E-cadherin during hepatic IRI; the full-length 120-kDa E-cadherin and the ratio of 38-kDa C-terminal fragment/120-kDa E-cadherin were decreased and increased, respectively, in TIMP3-/- livers after IRI. Moreover, GI254023X, a potent inhibitor of a disintegrin and metalloprotease (ADAM) 10, was capable of partially rescuing the expression of E-cadherin in the TIMP3-null hepatocytes. The proteolysis of E-cadherin in the TIMP3-/- livers was also linked to the loss of ß-catenin from the hepatocyte membranes and to an increased susceptibility to apoptosis after liver IRI. In a similar fashion, depression of the E-cadherin/ß-catenin complex mediated by TIMP3 deletion and knockdown of ß-catenin by small interfering RNA were both capable of inducing caspase activation in isolated hepatocytes subjected to H2 O2 oxidative stress. Hence, these results support a protective role for TIMP3 expression in sheltering the hepatocyte E-cadherin/ß-catenin complex from proteolytic processing and inhibiting apoptosis after hepatic IRI.

Jagged1-mediated myeloid Notch1 signaling activates HSF1/Snail and controls NLRP3 inflammasome activation in liver inflammatory injury.

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 (Notch1FL/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 (Notch1M-KO) aggravated hepatocellular damage even with concomitant JAG1 treatment. Compared to JAG1-treated Notch1FL/FL controls, Notch1M-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 Notch1M-KO mice augmented Snail activation and mitigated IR-triggered liver inflammation. Moreover, the knockdown of Snail in JAG1-treated Notch1FL/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.

The Modulation of Regulatory T Cells via HMGB1/PTEN/ß-Catenin Axis in LPS Induced Acute Lung Injury.

Sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) remains the leading complication for mortality caused by bacterial infection. The regulatory T (Treg) cells appear to be an important modulator in resolving lung injury. Despite the extensive studies, little is known about the role of macrophage HMGB1/PTEN/ß-catenin signaling in Treg development during ALI. Objectives: This study was designed to determine the roles and molecular mechanisms of HMGB1/PTEN/ß-catenin signaling in mediating CD4+CD25+Foxp3+ Treg development in sepsis-induced lung injury in mice. Setting: University laboratory research of First Affiliated Hospital of Anhui Medical University. Subjects: PTEN/ß-catenin Loxp and myeloid-specific knockout mice. Interventions: Groups of PTENloxp/ß-cateninloxp and myeloid-specific PTEN/ß-catenin knockout (PTENM-KO/ß-cateninM-KO) mice were treated with LPS or recombinant HMGB1 (rHMGB1) to induce ALI. The effects of HMGB1-PTEN axis were further analyzed by in vitro co-cultures. Measures and Main Results: In a mouse model of ALI, blocking HMGB1 or myeloid-specific PTEN knockout (PTENM-KO) increased animal survival/body weight, reduced lung damage, increased TGF-ß production, inhibited the expression of RORγt and IL-17, while promoting ß-catenin signaling and increasing CD4+CD25+Foxp3+ Tregs in LPS- or rHMGB-induced lung injury. Notably, myeloid-specific ß-catenin ablation (ß-cateninM-KO) resulted in reduced animal survival and increased lung injury, accompanied by reduced CD4+CD25+Foxp3+ Tregs in rHMGB-induced ALI. Furthermore, disruption of macrophage HMGB1/PTEN or activation of ß-catenin significantly increased CD4+CD25+Foxp3+ Tregs in vitro. Conclusions: HMGB1/PTEN/ß-catenin signaling is a novel pathway that regulates Treg development and provides a potential therapeutic target in sepsis-induced lung injury.

Hippo Signaling Controls NLR Family Pyrin Domain Containing 3 Activation and Governs Immunoregulation of Mesenchymal Stem Cells in Mouse Liver Injury.

The Hippo pathway, an evolutionarily conserved protein kinase cascade, tightly regulates cell growth and survival. Activation of yes-associated protein (YAP), a downstream effector of the Hippo pathway, has been shown to modulate tissue inflammation. However, it remains unknown as to whether and how the Hippo-YAP signaling may control NLR family pyrin domain containing 3 (NLRP3) activation in mesenchymal stem cell (MSC)-mediated immune regulation during liver inflammation. In a mouse model of ischemia/reperfusion (IR)-induced liver sterile inflammatory injury, we found that adoptive transfer of MSCs reduced hepatocellular damage, shifted macrophage polarization from M1 to M2 phenotype, and diminished inflammatory mediators. MSC treatment reduced mammalian Ste20-like kinase 1/2 and large tumor suppressor 1 phosphorylation but augmented YAP and ß-catenin expression with increased prostaglandin E2 production in ischemic livers. However, disruption of myeloid YAP or ß-catenin in MSC-transferred mice exacerbated IR-triggered liver inflammation, enhanced NLRP3/caspase-1 activity, and reduced M2 macrophage phenotype. Using MSC/macrophage coculture system, we found that MSCs increased macrophage YAP and ß-catenin nuclear translocation. Importantly, YAP and ß-catenin colocalize in the nucleus while YAP interacts with ß-catenin and regulates its target gene X-box binding protein 1 (XBP1), leading to reduced NLRP3/caspase-1 activity after coculture. Moreover, macrophage YAP or ß-catenin deficiency augmented XBP1/NLRP3 while XBP1 deletion diminished NLRP3/caspase-1 activity. Increasing NLRP3 expression reduced M2 macrophage arginase1 but augmented M1 macrophage inducible nitric oxide synthase expression accompanied by increased interleukin-1ß release. Conclusion: MSCs promote macrophage Hippo pathway, which in turn controls NLRP3 activation through a direct interaction between YAP and ß-catenin and regulates XBP1-mediated NLRP3 activation, leading to reprograming macrophage polarization toward an anti-inflammatory M2 phenotype. Moreover, YAP functions as a transcriptional coactivator of ß-catenin in MSC-mediated immune regulation. Our findings suggest a therapeutic target in MSC-mediated immunotherapy of liver sterile inflammatory injury.

Recipient HO-1 inducibility is essential for posttransplant hepatic HO-1 expression and graft protection: From bench-to-bedside.

By documenting potent antioxidative and anti-inflammatory functions, preclinical studies encourage heme oxygenase-1 (HO-1)-inducing regimens in clinical orthotopic liver transplantation (OLT). We aimed to determine the importance of recipient-derived HO-1 in murine and human OLTs. Hepatic biopsies from 51 OLT patients were screened for HO-1 expression (Western blots) prior to put-in (basal) and post reperfusion (stressed) and correlated with the hepatocellular function. In parallel, livers from HO-1 proficient mice (WT; C57/BL6), subjected to ex vivo cold storage (18 hour), were transplanted to syngeneic myeloid HO-1 deficient (mHO-1 KO) or FLOX (control) hosts, and sampled postreperfusion (6 hour). In human OLT, posttransplant but not pretransplant HO-1 expression correlated negatively with ALT levels (P = .0178). High posttransplant but not pretransplant HO-1 expression trended with improved OLT survival. Compared with controls, livers transplanted into mHO-1 KO recipient mice had decreased HO-1 levels, exacerbated hepatic damage/frequency of TUNEL+ cells, increased mRNA levels coding for TNFα/CXCL1/CXCL2/CXCL10, higher frequency of Ly6G+/4HN+ neutrophils; and enhanced MPO activity. Peritoneal neutrophils from mHO-1 KO mice exhibited higher CellRox+ ratio and increased TNFα/CXCL1/CXCL2/CXCL10 expression. By demonstrating the importance of posttransplant recipient HO-1 phenotype in hepatic macrophage/neutrophil regulation and function, this translational study identifies recipient HO-1 inducibility as a novel biomarker of ischemic stress resistance in OLT.

Loss of ATF3 exacerbates liver damage through the activation of mTOR/p70S6K/ HIF-1α signaling pathway in liver inflammatory injury.

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that plays important roles in regulating immune and metabolic homeostasis. Activation of the mechanistic target of rapamycin (mTOR) and hypoxia-inducible factor (HIF) transcription factors are crucial for the regulation of immune cell function. Here, we investigated the mechanism by which the ATF3/mTOR/HIF-1 axis regulates immune responses in a liver ischemia/reperfusion injury (IRI) model. Deletion of ATF3 exacerbated liver damage, as evidenced by increased levels of serum ALT, intrahepatic macrophage/neutrophil trafficking, hepatocellular apoptosis, and the upregulation of pro-inflammatory mediators. ATF3 deficiency promoted mTOR and p70S6K phosphorylation, activated high mobility group box 1 (HMGB1) and TLR4, inhibited prolyl-hydroxylase 1 (PHD1), and increased HIF-1α activity, leading to Foxp3 downregulation and RORγt and IL-17A upregulation in IRI livers. Blocking mTOR or p70S6K in ATF3 knockout (KO) mice or bone marrow-derived macrophages (BMMs) downregulated HMGB1, TLR4, and HIF-1α and upregulated PHD1, increasing Foxp3 and decreasing IL-17A levels in vitro. Silencing of HIF-1α in ATF3 KO mice ameliorated IRI-induced liver damage in parallel with the downregulation of IL-17A in ATF3-deficient mice. These findings demonstrated that ATF3 deficiency activated mTOR/p70S6K/HIF-1α signaling, which was crucial for the modulation of TLR4-driven inflammatory responses and T cell development. The present study provides potential therapeutic targets for the treatment of liver IRI followed by liver transplantation.

Serelaxin induces Notch1 signaling and alleviates hepatocellular damage in orthotopic liver transplantation.

Liver ischemia-reperfusion injury (IRI) represents a risk factor for early graft dysfunction and an obstacle to expanding donor pool in orthotopic liver transplantation (OLT). We have reported on the crucial role of macrophage Notch1 signaling in mouse warm hepatic IRI model. However, its clinical relevance or therapeutic potential remain unknown. Here, we used Serelaxin (SER), to verify Notch1 induction and putative hepatoprotective function in ischemia-reperfusion-stressed OLT. C57BL/6 mouse livers subjected to extended (18-hour) cold storage were transplanted to syngeneic recipients. SER treatment at reperfusion ameliorated IRI, improved post-OLT survival, decreased neutrophil/macrophage infiltration, and suppressed proinflammatory cytokine programs, while simultaneously increasing Notch intracellular domain (NICD) and hairy and enhancer of split 1 (Hes1) target genes. In bone marrow-derived macrophage cultures, SER suppressed proinflammatory while enhancing antiinflammatory gene expression concomitantly with increased NICD and Hes1. Hepatic biopsies from 21 adult primary liver transplant patients (2 hours postreperfusion) were divided into low-NICD (n = 11) and high-NICD (n = 10) expression groups (western blots). Consistent with our murine findings, human livers characterized by high NICD were relatively IRI resistant, as shown by serum alanine aminotransferase (ALT) levels at day 1 post-OLT. Our study documents the efficacy of SER-Notch1 signaling in mouse OLT and highlights the protective function of Notch1 in liver transplant patients.

Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench-to-bedside.

Hepatic ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and acute/chronic rejection as well as a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although glucocorticoid receptor (GR) signaling may enhance cytoprotective programs, clinical use of glucocorticoid is limited because of adverse effects, whereas clinical relevance of GR-facilitated cytoprotection in OLT remains unknown. We aimed to evaluate the significance of hepatic GR in clinical OLT and verify the impact of recombinant human relaxin (rhRLX), which may function as a GR agonist in a tissue/disease-specific manner. Fifty-one OLT patients were recruited under an institutional research board (IRB) protocol. Liver biopsies were collected after cold storage (presurgery) and 2 hours postreperfusion (before abdominal closure), followed by western blotting-assisted hepatic analyses. Forty-three percent of OLTs failed to increase GR perioperatively under surgical stress. Post-/pre-GR ratios at postoperative day 1 correlated negatively with serum aspartate aminotransferase (AST)/cleaved caspase-3 and positively with B-cell lymphoma-extra large (Bcl-xL)/B-cell lymphoma 2 (Bcl-2) levels. In a murine OLT model with extended (18-hour) cold storage, treatment with rhRLX ameliorated ischemia-reperfusion (IR) damage and improved survival while up-regulating hepatocyte GR and Bcl-xL/Bcl-2 expression in OLT. rhRLX-induced GR suppressed hepatocyte high-mobility group box 1 (HMGB1) translocation/release, accompanied by decreased Toll-like receptor 4 (TLR4)/receptor for advanced glycation end products (RAGE), suppressed interleukin 1 beta (IL1ß), chemokine (C-C motif) ligand 2 (CCL2), C-X-C motif chemokine (CXCL)10, tumor necrosis factor alpha (TNFα), CXCL1, and CXCL2 levels, and attenuated neutrophil/macrophage accumulation in OLT. Inhibition of GR in hepatocyte culture and in OLT diminished rhRLX-mediated cytoprotection. CONCLUSION: This translational study underscores the role of rhRLX-GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258-273).

Myeloid Notch1 deficiency activates the RhoA/ROCK pathway and aggravates hepatocellular damage in mouse ischemic livers.

Notch signaling plays an emerging role in the regulation of immune cell development and function during inflammatory response. Activation of the ras homolog gene family member A/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates ras homolog gene family member A/ROCK-mediated immune responses in liver ischemia and reperfusion (IR) injury. This study investigated intracellular signaling pathways regulated by Notch receptors in the IR-stressed liver and in vitro. In a mouse model of IR-induced liver inflammatory injury, we found that mice with myeloid-specific Notch1 knockout showed aggravated hepatocellular damage, with increased serum alanine aminotransferase levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and proinflammatory mediators compared to Notch1-proficient controls. Unlike in the controls, myeloid Notch1 ablation diminished hairy and enhancer of split-1 (Hes1) and augmented c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), JNK, ROCK1, and phosphatase and tensin homolog (PTEN) activation in ischemic livers. Disruption of JSAP1 in myeloid-specific Notch1 knockout livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and toll-like receptor 4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed toll-like receptor 4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain promoted Hes1 and inhibited JSAP1 in lipopolysaccharide-stimulated bone marrow-derived macrophages. Hes1 deletion enhanced JSAP1/JNK activation, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated JSAP1 knockout diminished ROCK1/PTEN and toll-like receptor 4 signaling. CONCLUSION: Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).

Heme oxygenase-1 regulates sirtuin-1-autophagy pathway in liver transplantation: From mouse to human.

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.