Xiaofeng Qian

ATF3‐Mediated NRF2/HO‐1 Signaling Regulates TLR4 Innate Immune Responses in Mouse Liver Ischemia/Reperfusion Injury

Activating transcription factor 3 (ATF3) is a stress‐induced transcription factor that has been shown to repress inflammatory gene expression in multiple cell types and diseases. However, little is known about the roles and mechanisms of ATF3 in liver ischemia/reperfusion injury (IRI). In warm and cold liver IRI models, we showed that ATF3 deficiency significantly increased ischemia/reperfusion (IR)‐stressed liver injury, as evidenced by increased serum alanine aminotransferase levels, histological liver damage, and hepatocellular apoptosis. These may correlate with inhibition of the intrahepatic nuclear factor erythroid‐derived 2‐related factor 2/heme oxygenase‐1 (NRF2/HO‐1) signaling pathway leading to enhancing Toll‐like receptor 4/nuclear factor kappa beta (TLR4/NF‐κB) activation, pro‐inflammatory programs and macrophage/neutrophil trafficking, while simultaneously repressing anti‐apoptotic molecules in ischemic liver. Interestingly, activation of NRF2/HO‐1 signaling using an NRF2 activator, oltipraz (M2), during hepatic IRI‐rescued ATF3 anti‐inflammatory functions in ATF3‐deficient mice. For in vitro studies, ATF3 ablation in lipopolysaccharide (LPS)‐stimulated bone marrow‐derived macrophages (BMMs) depressed levels of NRF2/HO‐1 and PI3K/AKT, resulting in enhanced TLR4/NF‐κB activation. Pretreatment of LPS‐stimulated BMMs with M2 increased NRF2/HO‐1 expression, promoted PI3K/AKT, which in turn suppressed TLR4/NF‐κB‐mediated proinflammatory mediators. Thus, our results first demonstrate ATF3‐mediated NRF2/HO‐1 signaling in the regulation of TLR4‐driven inflammatory responses in IR‐stressed livers. Our findings provide a rationale for a novel therapeutic strategy for managing IR‐induced liver injury.

Antifibrotic Effect of Hepatocyte Growth Factor-Expressing Mesenchymal Stem Cells in Small-for-Size Liver Transplant Rats

Ischemia-reperfusion and chronic injuries associated with small-for-size liver transplantation (SFSLT) impair the regeneration of liver graft and induce liver fibrosis. Mesenchymal stem cells (MSCs) can prevent the development of liver fibrosis, and hepatocyte growth factor (HGF) can also attenuate liver cirrhosis. Our previous studies have demonstrated that higher occurrence of liver fibrosis existed in rats post-SFSLT, and that implantation of HGF/MSCs, the human HGF (hHGF)-expressing MSCs, can improve liver regeneration, reduce mortality of rats, as well as have the potent antifibrotic effect in this SFSLT model. In the present study, we implanted HGF/MSCs into liver grafts via the portal vein and investigated their role in antifibrosis effect, using a 30% SFSLT rat model. Fibrosis indexes, including laminin (LN), hyaluronic acid (HA) levels in serum and hydroxyproline (Hyp) content in the liver grafts, the expression of transforming growth factor-beta1 (TGF-beta(1)), rat HGF (rHGF), alpha-smooth muscle actin (alpha-SMA) in hepatic stellate cells (HSCs), alanine aminotransferase (ALT), total bilirubin (BIL), and albumin (ALB) levels in serum, in rats in different treatment groups were assessed at different time points. We found that HGF/MSCs significantly inhibited the formation of liver fibrosis in rats undergoing SFSLT, while MSCs and HGF had synergistic effects in the process. The antifibrosis effect of HGF/MSCs may have contributed in modulating the activation and apoptosis of HSCs, elevating the rHGF expression level, and decreasing the TGF-beta(1) secretion of activated HSCs. These studies suggest that HGF/MSCs may be a novel therapeutic option for the treatment of liver fibrosis after SFSLT.

Outcomes of Living-Related Liver Transplantation for Wilson's Disease : A Single-Center Experience in China

Background. Although orthotopic liver transplantation provides a therapeutic option for patients with Wilson’s disease (WD) presenting fulminant liver failure or drug resistance, it is still unclear whether the living-related liver transplantation (LRLT) can result in long-term therapeutic effect on WD. Methods. Here, we report a retrospective analysis of LRLT for 36 cases of WD patients. The indications for LRLT were fulminant hepatic failure in two patients and chronic advanced liver disease in 32 patients including 13 patients with Wilsonian neurologic manifestations. Two patients presented with severe Wilsonian neurologic manifestations even though their liver functions were stable. Results. Results revealed that the survival of posttransplant patients or grafts at 1, 3, and 5 years was 91.7%, 83.3%, 75%, or 86.1%, 77.8%, 75%, respectively. Pretransplant intensive care unit-bound and model for end-stage liver disease score were indicated as independent factors predictive of patient survival. Patients with neurologic abnormalities showed significant improvement after liver transplant. Conclusion. Our results indicate LRLT is an excellent therapeutic modality for WD patients with end-stage liver disease. Better pretransplant conditions appeared to be advantageous in gaining better survival outcomes of patients undergoing LRLT.

All-trans retinoic acid preconditioning protects against liver ischemia/reperfusion injury by inhibiting the nuclear factor kappa B signaling pathway

BACKGROUND Inflammatory response plays a pathogenic role in liver ischemia/reperfusion (I/R) injury. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A with anti-inflammatory effects. However, there are few reports on the anti-inflammatory effects of ATRA on liver I/R injury. The purpose of this study was to investigate the effects of ATRA on liver I/R injury and related mechanisms. METHODS A total of 54 male Sprague-Dawley rats were randomly divided into three groups (18 rats each), namely, sham, I/R, and I/R+ATRA groups. ATRA was intraperitoneally administered at a dose of 15mg/kg/d 14d before ischemia surgery. The segmental (70%) hepatic ischemia model was used by clamping the portal vein, hepatic artery, and bile duct of the left and median for 1h. The rats were sacrificed 3, 6, and 24h after reperfusion, and blood and liver tissue samples were obtained. Liver injury was evaluated by biochemical and histopathologic examinations. Myeloperoxidase activity was spectrophotometrically measured. The expression of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Liver nuclear factor kappa B (NF-κB) was detected by immunohistochemistry. The expression of NF-κB p65 and inhibitor NF-κB-α (IκBα) was determined by Western blot analysis. RESULTS The serum alanine aminotransferase level, Suzuki scores of hepatic histology, and hepatic myeloperoxidase activity, as indices of hepatic injury, were increased after reperfusion. The increase was attenuated by preadministration with ATRA. Compared with the I/R group, ATRA treatment increased IκBα expression and suppressed NF-κB p65 expression. Subsequently, the levels of tumor necrosis factor-α and interleukin-6 after liver I/R were effectively downregulated. CONCLUSIONS ATRA administration can significantly attenuate I/R injury in rat liver. The protective mechanism is related to its anti-inflammatory function of inhibiting NF-κB activation.

IL-17A-producing NK cells were implicated in liver injury induced by ischemia and reperfusion

NK cells play a critical role in several types of liver injury. The aim of this study was to evaluate the role of NK cells in liver ischemia reperfusion injury (IRI) and the underlying mechanism. Male Rag1-/- mice and wild type mice were subjected to partial hepatic IRI. Anti-NK1.1 (300 μg/mouse, ip) was used to deplete NK cells. Liver injury was evaluated by level of serum alanine aminotransferase (ALT). Hepatic inflammatory cytokines, neutrophils and CXCL-2 expression were measured following ischemia and reperfusion. Additionally, NK cells were cultured with or without IL-6, IL-21, IL-23 and IL-10 for 24h, then IL-17A level in the supernatants was analyzed by ELISA. Production of IL-17A was increased in NK cells after reperfusion. Various cytokines such as, IL-6, IL-21 and IL-23, which also elevated after IRI, can promote IL-17A production and up-regulate the phosphorylation of STAT3 in NK cells, while the increase was repressed in the presence of IL-10. Depletion of NK cells decreased IL-17A level in Rag1-/- mice ischemic lobes. Meanwhile, hepatic infiltration of neutrophils and CXCL-2 level were reduced and liver injury was ameliorated. Neutralization of IL-17A was used to confirm the role of this cytokine produced by NK cells in Rag1-/- mice. In conclusion, at initial stage of liver IRI, NK cells increase IL-17A production and promote liver injury.

Lipopolysaccharide preconditioning protects hepatocytes from ischemia/reperfusion injury (IRI) through inhibiting ATF4-CHOP pathway in mice.

Background Low-dose lipopolysaccharide (LPS) preconditioning-induced liver protection has been demonstrated during ischemia-reperfusion injury (IRI) in several organs but has not been sufficiently elucidated underlying causal mechanism. This study investigated the role of low-dose LPS preconditioning on ATF4-CHOP pathway as well as the effects of the pathway on tissue injury and inflammation in a mouse model of liver partial-warm IRI. Methods LPS (100 µg/kg/d) was injected intraperitoneally two days before ischemia. Hepatic injury was evaluated based on serum alanine aminotransferase levels, histopathology, and caspase-3 activity. The ATF4-CHOP pathway and its related apoptotic molecules were investigated after reperfusion. The role of LPS preconditioning on apoptosis and ATF4-CHOP pathway was examined in vitro. Moreover, the effects of the ATF4-CHOP pathway on apoptosis, Caspase-12, and Caspase-3 were determined with ATF4 small interfering RNA (siRNA). Inflammatory cytokine expression was also checked after reperfusion. Inflammatory cytokines and related signaling pathways were analyzed in vitro in macrophages treated by LPS preconditioning or ATF4 siRNA. Results LPS preconditioning significantly attenuated liver injury after IRI. As demonstrated by in vitro experiments, LPS preconditioning significantly reduced the upregulation of the ATF4-CHOP pathway and inhibited Caspase-12 and Caspase-3 activation after IRI. Later experiments showed that ATF4 knockdown significantly suppressed CHOP, cleaved caspase-12 and caspase-3 expression, as well as inhibited hepatocellular apoptosis. In addition, in mice pretreated with LPS, TNF-α and IL-6 were inhibited after reperfusion, whereas IL-10 was upregulated. Similarly, low-dose LPS significantly inhibited TNF-α, IL-6, ATF4-CHOP pathway, NF-κB pathway, and ERK1/2 in high-dose LPS-stimulated macrophages, whereas IL-10 and cytokine signaling (SOCS)-3 suppressor were induced. Importantly, ATF4 siRNA is consistent with results of LPS preconditioning in macrophages. Conclusions This work is the first time to provide evidence for LPS preconditioning protects hepatocytes from IRI through inhibiting ATF4-CHOP pathway, which may be critical to reducing related apoptosis molecules and modulating innate inflammation.

Generation of human regulatory T cells de novo with suppressive function prevent xenogeneic graft versus host disease

Treatment with rapamycin (RAPA) favorably affects regulatory T cells (Treg) in vivo, and RAPA induces the de novo expression of FOXP3 in murine alloantigen-specific T cells. Whether RAPA acts independently or with transforming growth factor beta (TGF-β) to produce ex vivo-induced Treg generation is unknown. Naïve CD4(+) T cells isolated from peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 coated beads in the presence of IL-2 for 5 to 7 days. Ten ng/ml of TGF-β (1 to 100 ng/mL RAPA) was added to some of the cultures. The phenotypes were analyzed with flow cytometry. The conditioned cells were cocultured with CFSE-labeled T cells in different ratios for 5 days. CFSE dilution indicating T response cell proliferation was analyzed by flow cytometry. Xenogeneic graft-versus-host disease (x-GVHD) was induced by transplanting human peripheral blood mononuclear cells into RAG2(-/-) γc(-/-) mice exposed to total body irradiation, and various factors in the subjects were subsequently compared. CD4 cells induced by rapamycin and TGF-β (CD4(RAPA/TGF-β)) expressed the natural Treg phenotypes and trafficking receptors, and no significant cytotoxicity was observed. CD4(RAPA/TGF-β) was anergic and demonstrated potent suppressive activity in vitro. Although the transfer of human peripheral blood mononuclear cells into RAG2(-/-) γc(-/-) mice caused x-GVHD, the cotransfer of CD4(RAPA/TGF-β) decreased human cell engraftment and extended survival in mice. RAPA plus TGF-β induces human naïve T cells to become suppressor cells, a novel strategy for treating human autoimmune diseases and preventing allograft rejection.

Rapamycin Promotes the Expansion of CD4+ Foxp3+ Regulatory T Cells After Liver Transplantation

Rapamycin can promote the generation and homeostasis of CD4(+)Foxp3(+) regulatory T cells (Tregs) both in vitro and in vivo. The mechanisms by which rapamycin mediates this effect are poorly defined. In this study, we characterized CD4(+)Foxp3(+) Tregs in liver grafts and peripheral blood following rapamycin treatment using a syngeneic liver transplant model. Orthotopic liver transplantation was performed from Lewis (LEW) to LEW rats. In the first 2 weeks the percentage of CD4(+)Foxp3(+) Tregs was increased in the liver grafts and blood only among the rapamycin group compared with control group. Conversely, the percentage of CD4(+)Foxp3(+) Tregs in the liver graft and blood decreased in the cyclosporine group. In normal rats, rapamycin did not impact the generation of CD4(+)Foxp3(+) Tregs in the thymus. Thus, rapamycin can significantly enhance the percentages of CD4(+)Foxp3(+) Tregs in the thymus and periphery, indicating that rapamycin favors Tregs expansion and may suppress other CD4(+) T cells.

Rapamycin Regulates iTreg Function through CD39 and Runx1 Pathways

It has been shown that rapamycin is able to significantly increase the expression of FoxP3 and suppress activity in induced Treg (iTreg) cells in vivo and in vitro. CD39 is a newly determined Treg marker that relates to cell suppression. Runx1, a regulator of FoxP3, controls the expression of adenosine deaminase (ADA) gene, which is found recently in the downstream of CD39 pathway in trophoblast cells. Whether rapamycin would influence CD39 pathway and regulate the expression of Runx1 remains to be determined. The addition of rapamycin to human CD4+ naïve cells in the presence of IL-2, TGF-β promotes the expression of FoxP3. In this paper, we found that CD39 positively correlated with the FoxP3 expression in iTreg cells. Rapamycin induced iTreg cells showed a stronger CD39/Runx1 expression with the enhanced suppressive function. These data suggested that CD39 expression was involved in iTreg generation and the enhanced suppressive ability of rapamycin induced Treg was partly due to Runx1 pathway. We conclude that rapamycin favors CD39/Runx1 expression in human iTreg and provides a novel insight into the mechanisms of iTreg generation enhanced by rapamycin.

Anti-IL-17 Antibody Improves Hepatic Steatosis by Suppressing Interleukin-17-Related Fatty Acid Synthesis and Metabolism

To investigate the relationship between interleukin-17 and proteins involved in fatty acid metabolism with respect to alcoholic liver disease, male ICR mice were randomized into five groups: control, alcoholic liver disease (ALD) at 4 weeks, 8 weeks, and 12 weeks, and anti-IL-17 antibody treated ALD. A proteomic approach was adopted to investigate changes in liver proteins between control and ALD groups. The proteomic analysis was performed by two-dimensional difference gel electrophoresis. Spots of interest were subsequently subjected to nanospray ionization tandem mass spectrometry (MS/MS) for protein identification. Additionally, expression levels of selected proteins were confirmed by western blot. Transcriptional levels of some selected proteins were determined by RT-PCR. Expression levels of 95 protein spots changed significantly (ratio >1.5, P < 0.05) during the development of ALD. Sterol regulatory element-binding protein-lc (SREBP-1c), carbohydrate response element binding protein (ChREBP), enoyl-coenzyme A hydratase (ECHS1), and peroxisome proliferator-activated receptor alpha (PPAR-α) were identified by MS/MS among the proteins shown to vary the most; increased IL-17 elevated the transcription of SREBP-1c and ChREBP but suppressed ECHS1 and PPAR-α. The interleukin-17 signaling pathway is involved in ALD development; anti-IL-17 antibody improved hepatic steatosis by suppressing interleukin-17-related fatty acid metabolism.