Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-ß1-Induced OCT4/Nanog Pathway.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-ß1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-ß1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-ß1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-ß1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-ß1-induced OCT4/Nanog-dependent pathway.

Fatty Acids Activate the Transcriptional Coactivator YAP1 to Promote Liver Fibrosis via p38 Mitogen-Activated Protein Kinase.

BACKGROUND & AIMS: Patients with simple steatosis (SS) and nonalcoholic steatohepatitis can develop progressive liver fibrosis, which is associated with liver-related mortality. The mechanisms contributing to liver fibrosis development in SS, however, are poorly understood. SS is characterized by hepatocellular free fatty acid (FFA) accumulation without lobular inflammation seen in nonalcoholic steatohepatitis. Because the Hippo signaling transcriptional coactivator YAP1 (YAP) has previously been linked with nonalcoholic fatty liver disease (NAFLD)-related fibrosis, we sought to explore how hepatocyte FFAs activate a YAP-mediated profibrogenic program. METHODS: We analyzed RNA sequencing data from a GEO DataSet (accession: GSE162694) consisting of 143 patients with NAFLD. We also performed immunohistochemical, immunofluorescence, immunoblot, and quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) in liver specimens from NAFLD subjects, from a murine dietary NAFLD model, and in FFA-treated hepatic spheroids and hepatocytes. RESULTS: YAP-target gene expression correlated with increasing fibrosis stage in NAFLD patients and was associated with fibrosis in mice fed a NAFLD-inducing diet. Hepatocyte-specific YAP deletion in the murine NAFLD model attenuated diet-induced fibrosis, suggesting a causative role of YAP in NAFLD-related fibrosis. Likewise, in hepatic spheroids composed of Huh7 hepatoma cells and primary human hepatic stellate cells, Huh7 YAP silencing reduced FFA-induced fibrogenic gene expression. Notably, inhibition of p38 mitogen-activated protein kinase could block YAP activation in FFA-treated Huh7 cells. CONCLUSIONS: These studies provide further evidence for the pathological role of YAP in NAFLD-associated fibrosis and that YAP activation in NAFLD may be driven by FFA-induced p38 MAPK activation.

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.

Microrna-130a Downregulates HCV Replication through an atg5-Dependent Autophagy Pathway.

We previously identified that miR-130a downregulates HCV replication through two independent pathways: restoration of host immune responses and regulation of pyruvate metabolism. In this study, we further sought to explore host antiviral target genes regulated by miR-130a. We performed a RT² Profiler™ PCR array to identify the host antiviral genes regulated by miR-130a. The putative binding sites between miR-130a and its downregulated genes were predicted by miRanda. miR-130a and predicted target genes were over-expressed or knocked down by siRNA or CRISPR/Cas9 gRNA. Selected gene mRNAs and their proteins, together with HCV replication in JFH1 HCV-infected Huh7.5.1 cells were monitored by qRT-PCR and Western blot. We identified 32 genes that were significantly differentially expressed more than 1.5-fold following miR-130a overexpression, 28 of which were upregulated and 4 downregulated. We found that ATG5, a target gene for miR-130a, significantly upregulated HCV replication and downregulated interferon stimulated gene expression. miR-130a downregulated ATG5 expression and its conjugation complex with ATG12. ATG5 and ATG5-ATG12 complex affected interferon stimulated gene (ISG) such as MX1 and OAS3 expression and subsequently HCV replication. We concluded that miR-130a regulates host antiviral response and HCV replication through targeting ATG5 via the ATG5-dependent autophagy pathway.

Macrophage Activation Marker Soluble CD163 Is a Dynamic Marker of Liver Fibrogenesis in Human Immunodeficiency Virus/Hepatitis C Virus Coinfection.

Background: Coinfection with human immunodeficiency virus (HIV) accelerates hepatitis C virus (HCV)-related liver fibrosis. Macrophages are triggered during both viral infections and are critical in liver inflammation/fibrogenesis. Liver fibrosis strongly associates with serum soluble CD163 (sCD163, a macrophage activation marker); comprehensive evaluation in HIV/HCV coinfection is lacking. Methods: We retrospectively analyzed sCD163 (enzyme-linked immunosorbent assay) and hepatic CD163 (immunofluorescent CD163/CD68 costaining) in patients infected with HIV/HCV, HCV, or HIV, pre- and post-antiviral therapy. Results: sCD163 was significantly higher in HIV/HCV compared to either monoinfection, and decreased following successful antiviral therapy, although did not fully normalize. In HIV/HCV, sCD163 was associated with necroinflammation, Ishak fibrosis scores, and noninvasive fibrosis scores. We observed a novel trend whereby sCD163 levels progressively increase with increasing Ishak fibrosis score, peaking at stage 4, above which levels plateaued. Periportal CD163+ macrophage frequency was also higher with increasing fibrosis score. When stratified by fibrosis stage, sCD163 levels were higher in HIV/HCV than HCV but only in individuals with mild to moderate fibrosis. Conclusions: In HIV/HCV, increasing sCD163 levels accompanied periportal CD163+ macrophage enrichment in mild to moderate fibrosis, but not in established cirrhosis, suggesting that sCD163 is a dynamic biomarker of fibrogenesis rather than accumulated fibrosis. Our findings implicate HIV-related macrophage activation in accelerated fibrosis progression in HIV/HCV coinfection.

Prolonged cenicriviroc therapy reduces hepatic fibrosis despite steatohepatitis in a diet-induced mouse model of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease projected to become the leading cause of cirrhosis and liver transplantation in the next decade. Cenicriviroc (CVC), a dual chemokine receptor 2 and 5 antagonist, prevents macrophage trafficking and is under clinical investigation for the treatment of human NASH fibrosis. We assessed the efficacy and durability of short and prolonged CVC therapy in a diet-induced mouse model of NASH, the choline deficient, L-amino acid-defined, high-fat diet (CDAHFD) model. C57BL/6 mice received 4 or 14 weeks of standard chow or the CDAHFD. CVC (10 mg/kg/day and 30 mg/kg/day for 4 weeks and 20 mg/kg/day and 30 mg/kg/day for 14 weeks) was initiated simultaneously with the CDAHFD. At 4 and 14 weeks, livers were harvested for histology and flow cytometric analyses of intrahepatic immune cells. High-dose CVC (30 mg/kg/day) therapy in CDAHFD mice for 4 or 14 weeks inhibited intrahepatic accumulation of Ly6Chigh bone marrow-derived macrophages. Prolonged CVC therapy (14 weeks) yielded no significant differences in the total intrahepatic macrophage populations among treatment groups but increased the frequency of intrahepatic anti-inflammatory macrophages in the high-dose CVC group. Despite ongoing steatohepatitis, there was significantly less fibrosis in CDAHFD mice receiving high-dose CVC for 14 weeks based on histologic and molecular markers, mirroring observations in human NASH CVC trials. CVC also directly inhibited the profibrotic gene signature of transforming growth factor-ß-stimulated primary mouse hepatic stellate cells in vitro. Conclusion: CVC is a novel therapeutic agent that is associated with reduced fibrosis despite ongoing steatohepatitis. Its ability to alter intrahepatic macrophage populations and inhibit profibrogenic genes in hepatic stellate cells in NASH livers may contribute to its observed antifibrotic effect. (Hepatology Communications 2018;2:529-545).

Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFß1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFß1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells. CONCLUSION: HIV accentuates an HCV-driven profibrogenic program in hepatocyte and hepatic stellate cell lines through ROS, NFκB, and TGFß1 up-regulation; coculture of hepatocyte and hepatic stellate cell lines significantly increased expression of CoL1A1 and TIMP1; and our novel coculture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses and may provide important insights into hepatic fibrosis. (Hepatology 2016;64:1951-1968).

Circulating Soluble CD163 is Associated with Steatohepatitis and Advanced Fibrosis in Nonalcoholic Fatty Liver Disease.

OBJECTIVES: Soluble CD163 (sCD163), a marker of Kupffer cell activation detectable in serum, correlates with inflammation and fibrosis in chronic viral hepatitis, but its role in nonalcoholic fatty liver disease is unknown. We hypothesized that sCD163 would correlate with nonalcoholic fatty liver disease activity and fibrosis. METHODS: Liver biopsies and serum were obtained from 145 obese subjects undergoing gastric bypass surgery. Subjects were divided into four groups based on fibrosis stage and nonalcoholic fatty liver disease activity score (NAS); Group 1: F0, NAS=0; Group 2: F<2, 0

Blockade of the programmed death-1 (PD1) pathway undermines potent genetic protection from type 1 diabetes.

AIMS/HYPOTHESIS: Inhibition of PD1-PDL1 signaling in NOD mice accelerates onset of type 1 diabetes implicating this pathway in suppressing the emergence of pancreatic beta cell reactive T-cells. However, the molecular mechanism by which PD1 signaling protects from type 1 diabetes is not clear. We hypothesized that differential susceptibility of Idd mouse strains to type 1 diabetes when challenged with anti PDL1 will identify genomic loci that collaborate with PD1 signaling in suppressing type 1 diabetes. METHODS: Anti PDL1 was administered to NOD and various Idd mouse strains at 10 weeks of age and onset of disease was monitored by measuring blood glucose levels. Additionally, histological evaluation of the pancreas was performed to determine degree of insulitis. Statistical analysis of the data was performed using Log-Rank and Student's t-test. RESULTS: Blockade of PDL1 rapidly precipitated type 1 diabetes in nearly all NOD Idd congenic strains tested, despite the fact that all are moderately (Idd5, Idd3 and Idd10/18) or highly (Idd3/10/18 and Idd9) protected from spontaneous type 1 diabetes by virtue of their protective Idd genes. Only the Idd3/5 strain, which is nearly 100% protected from spontaneous disease, remained normoglycemic following PDL1 blockade. CONCLUSIONS: These results indicate that multiple Idd loci collaborate with PD1 signaling. Anti PDL1 treatment undermines a large portion of the genetic protection mediated by Idd genes in the NOD model of type 1 diabetes. Basal insulitis correlated with higher susceptibility to type 1 diabetes. These findings have important implications since the PD1 pathway is a target for immunotherapy.

Leptin treatment confers clinical benefit at multiple stages of virally induced type 1 diabetes in BB rats.

The adipokine, leptin, regulates blood glucose and the insulin secretory function of beta cells, while also modulating immune cell function. We hypothesized that the dual effects of leptin may prevent or suppress the autoreactive destruction of beta cells in a virally induced rodent model of type 1 diabetes. Nearly 100% of weanling BBDR rats treated with the combination of an innate immune system activator, polyinosinic:polycytidylic acid (pIC), and Kilham rat virus (KRV) become diabetic within a predictable time frame. We utilized this model to test the efficacy of leptin in preventing diabetes onset, remitting new onset disease, and preventing autoimmune recurrence in diabetic rats transplanted with syngeneic islet grafts. High doses of leptin delivered via an adenovirus vector (AdLeptin) or alzet pump prevented diabetes in>90% of rats treated with pIC+KRV. The serum hyperleptinemia generated by this treatment was associated with decreased body weight, decreased non-fasting serum insulin levels, and lack of islet insulitis in leptin-treated rats. In new onset diabetics, hyperleptinemia prevented rapid weight loss and diabetic ketoacidosis, and temporarily restored euglycemia. Leptin treatment also prolonged the survival of syngeneic islets transplanted into diabetic BBDR rats. In diverse therapeutic settings, we found leptin treatment to have significant beneficial effects in modulating virally induced diabetes. These findings merit further evaluation of leptin as a potential adjunct therapeutic agent for treatment of human type 1 diabetes.

Haptoglobin as an early serum biomarker of virus-induced autoimmune type 1 diabetes in biobreeding diabetes resistant and LEW1.WR1 rats.

Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11-21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.

The role of innate immune pathways in type 1 diabetes pathogenesis.

PURPOSE OF REVIEW: Type 1 diabetes (T1D) is an autoimmune disease typically believed to result from malfunctions in adaptive immune response signaling which result in activation of self-reactive T cells. However, recent research has indicated components of the innate immune response as having a key role in the initiation of the autoimmune process of T1D. This review will highlight recent studies which examined the role of innate immune response signaling and the connections to T1D pathogenesis. RECENT FINDINGS: Investigations indicate that components of innate immunity, including inflammation and Toll-like receptor signaling, are involved in pancreatic islet infiltration and insulitis. Recent studies examining the role of viral infections in T1D development also implicate innate immune response signaling in disease pathogenesis. SUMMARY: Current research indicates that components of innate immune response signaling are involved in the initiation of the autoimmune process which results in the eventual destruction of beta cells during T1D pathogenesis. Continuing efforts by researchers to uncover the molecular pathways of innate immunity linked to T1D development could potentially lead to therapeutics capable of preventing and curing the autoimmune disease.

TLR agonists prevent the establishment of allogeneic hematopoietic chimerism in mice treated with costimulation blockade.

Activation of TLR4 by administration of LPS shortens the survival of skin allografts in mice treated with costimulation blockade through a CD8 T cell-dependent, MyD88-dependent, and type I IFN receptor-dependent pathway. The effect of TLR activation on the establishment of allogeneic hematopoietic chimerism in mice treated with costimulation blockade is not known. Using a costimulation blockade protocol based on a donor-specific transfusion (DST) and a short course of anti-CD154 mAb, we show that LPS administration at the time of DST matures host alloantigen-presenting dendritic cells, prevents the establishment of mixed allogeneic hematopoietic chimerism, and shortens survival of donor-specific skin allografts. LPS mediates its effects via a mechanism that involves both CD4(+) and CD8(+) T cells and results from signaling through either the MyD88 or the type I IFN receptor pathways. We also document that timing of LPS administration is critical, as injection of LPS 24 h before treatment with DST and anti-CD154 mAb does not prevent hematopoietic engraftment but administration the day after bone marrow transplantation does. We conclude that TLR4 activation prevents the induction of mixed allogeneic hematopoietic chimerism through type I IFN receptor and MyD88-dependent signaling, which leads to the up-regulation of costimulatory molecules on host APCs and the generation of alloreactive T cells. These data suggest that distinct but overlapping cellular and molecular mechanisms control the ability of TLR agonists to block tolerance induction to hematopoietic and skin allografts in mice treated with costimulation blockade.