Yanshen Peng

Interleukin-17 Contributes to the Pathogenesis of Autoimmune Hepatitis through Inducing Hepatic Interleukin-6 Expression

T helper cells that produce IL-17 (Th17 cells) have recently been identified as the third distinct subset of effector T cells. Emerging data suggests that Th17 cells play an important role in the pathogenesis of many liver diseases by regulating innate immunity, adaptive immunity, and autoimmunity. In this study, we examine the role and mechanism of Th17 cells in the pathogenesis of autoimmune hepatitis (AIH). The serum levels of IL-17 and IL-23, as well as the frequency of IL-17+ cells in the liver, were significantly elevated in patients with AIH, compared to other chronic hepatitis and healthy controls. The hepatic expressions of IL-17, IL-23, ROR-γt, IL-6 and IL-1β in patients with AIH were also significantly increased and were associated with increased inflammation and fibrosis. IL-17 induces IL-6 expression via the MAPK signaling pathway in hepatocytes, which, in turn, may further stimulate Th17 cells and forms a positive feedback loop. In conclusion, Th17 cells are key effector T cells that regulate the pathogenesis of AIH, via induction of MAPK dependent hepatic IL-6 expression. Blocking the signaling pathway and interrupting the positive feedback loop are potential therapeutic targets for autoimmune hepatitis.

Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease

Mechanisms associated with the progression of simple steatosis to non‐alcoholic fatty liver disease (NAFLD) remain undefined. Regulatory T cells (Tregs) play a critical role in regulating inflammatory processes in non‐alcoholic steatohepatitis (NASH) and because T helper type 17 (Th17) functionally oppose Treg‐mediated responses, this study focused on characterizing the role of Th17 cells using a NAFLD mouse model. C57BL/6 mice were fed either a normal diet (ND) or high fat (HF) diet for 8 weeks. Mice in the HF group had a significantly higher frequency of liver Th17 cells compared to ND‐fed mice. Neutralization of interleukin (IL)‐17 in HF mice ameliorated lipopolysaccharide (LPS)‐induced liver injury reflected by decreased serum alanine aminotransferase (ALT) levels and reduced inflammatory cell infiltrates in the liver. In vitro, HepG2 cells cultured in the presence of free fatty acids (FFA; oleic acid and palmitic acid) for 24 h and IL‐17 developed steatosis via insulin‐signalling pathway interference. IL‐17 and FFAs synergized to induce IL‐6 production by HepG2 cells and murine primary hepatocytes which, in combination with transforming growth factor (TGF‐β), expanded Th17 cells. It is likely that a similar process occurs in NASH patients, as there were significant levels of IL‐17+ cell infiltrates in NASH patient livers. The hepatic expression of Th17 cell‐related genes [retinoid‐related orphan receptor gamma (ROR)γt, IL‐17, IL‐21 and IL‐23] was also increased significantly in NASH patients compared to healthy controls. Th17 cells and IL‐17 were associated with hepatic steatosis and proinflammatory response in NAFLD and facilitated the transition from simple steatosis to steatohepatitis. Strategies designed to alter the balance between Th17 cells and Tregs should be explored as a means of preventing progression to NASH and advanced liver diseases in NAFLD patients.

Immunomodulatory and Anti-inflammatory Properties of Artesunate in Experimental Colitis

BACKGROUND Inflammatory bowel disease is a chronic and idiopathic gastrointestinal inflammation mediated by disregulated immune responses. Artemisinin (a chemical from a traditional Chinese herbal medicine Artemisia annua L.) and its derivatives have been proven to exhibit anti-inflammatory and immunomodulatory effects in the treatment of systemic lupus erythematosus and rheumatoid arthritis with low side-effects. This study is aimed to evaluate the potential therapeutic value of artesunate for inflammatory bowel disease. METHODS Murine colitis was induced by either oral administration of dextran sulfate sodium salt (DSS) or intrarectal delivery of 2,4,6- trinitrobenzene sulfonic acid (TNBS) or oxazolone. Mice were treated with artesunate (150mg/kg/day). Peritoneal macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of artesunate. Changes in cytokines or proteins of interests were analyzed by enzyme-linked immunosorbent assay (ELISA) or SDS-PAGE/Western blot. RESULTS Artesunate significantly ameliorated DSS colitis and TNBS colitis (but not oxazolone colitis), including reduced weight loss and disease activity, as well as macroscopic and microscopic colonic injury. The expression of NF-κBp65 and p-IκB-α were reduced in artesunate treated TNBS colitis compared with untreated. The levels of IFN-γ, IL-17, and TNF-α were significantly decreased in artesunate treated TNBS colitis or DSS colitis. Furthermore, in vitro artesunate treatment significantly inhibited TNF-α production by LPS-activated macrophages. CONCLUSIONS Artesunate suppresses TNF-α expression in vitro and in vivo as well as T-helper (Th)1/Th17 responses in TNBS colitis model. Our data suggest a novel clinical application of artesunate as a potential therapy for Crohns disease.

THE IMMUNOPATHOLOGY OF LIVER GRANULOMAS IN PRIMARY BILIARY CIRRHOSIS

Liver granulomas and elevated serum IgM are commonly observed in patients with primary biliary cirrhosis (PBC) but their pathogenetic significance remains largely unknown. To address this issue we performed an extensive immunostaining and colocalization study of markers associated with dendritic cells and IgM in a large cohort of tissue samples from PBC and control livers as well as from non-hepatic granulomatous diseases. First, the classical dendritic cell CD11c marker is highly expressed and more sensitive than classical hematoxylin-eosin staining in detecting granulomas associated with PBC and other conditions. Second, PBC cases with CD11c-positive granulomas have significantly higher serum IgM levels and earlier disease stages. Third, granulomas from PBC and other diseases demonstrate markers of dendritic cell immaturity, i.e. CD11b, reduced MHC II, IL-23, CCR7 and CD83 expression, and elevated C1q expression. Lastly, B cells and IgM-positive plasma cells are largely represented around PBC granulomas along with macrophages. In conclusion, our comprehensive immunohistochemical study suggests that dendritic cells are key to the pathogenesis of granulomas, regardless of their origin. More specifically, PBC liver granulomas may result from the interaction between immature dendritic cells and IgM.

CCN1 expression in hepatocytes contributes to macrophage infiltration in nonalcoholic fatty liver disease in mice

Our objective was to investigate the potential roles of CCN1 in the inflammation and macrophage infiltration of nonalcoholic fatty liver disease (NAFLD). The regulation of hepatic CCN1 expression was investigated in vitro with murine primary hepatocytes treated with free fatty acids or lipopolysaccharide (LPS) and in vivo with high-fat (HF) diet-fed mice or ob/ob mice. CCN1 protein and a liver-specific CCN1 expression plasmid were administered to mice fed a normal diet (ND) or HF diet. Myeloid-derived macrophages and RAW264.7 cells were also treated with CCN1 in vitro to determine the chemotactic effects of CCN1 on macrophages. LPS treatment significantly increased hepatic CCN1 expression in HF diet-fed mice and ob/ob mice. LPS and FFAs induced CCN1 expression in primary murine hepatocytes in vitro through the TLR4/MyD88/AP-1 pathway. CCN1 protein and overexpression of CCN1 in the liver induced more severe hepatic inflammation and macrophage infiltrates in HF mice than in ND mice. CCN1 recruited macrophages through activation of the Mek/Erk signaling pathway in myeloid-derived macrophages and RAW264.7 cells in vitro. Endotoxin and FFA-induced CCN1 expression in hepatocytes is involved in the hepatic proinflammatory response and macrophage infiltration in murine NAFLD.

Treatment of cholestatic fibrosis by altering gene expression of Cthrc1: Implications for autoimmune and non-autoimmune liver disease.

Collagen triple helix repeat containing-1 (Cthrc1) is a documented specific inhibitor of TGF-β signaling. Based on this observation, we developed the hypothesis that knocking in/knocking out the Cthrc1 gene in murine models of cholestasis would alter the natural history of cholestatic fibrosis. To study this thesis, we studied two murine models of fibrosis, first, common bile duct ligation (CBDL) and second, feeding of 3, 5-diethoxy-carbonyl-1, 4-dihydrocollidine (DDC). In both models, we administered well-defined adenoviral vectors that expressed either Cthrc1 or, alternatively, a short hairpin RNA (shRNA)-targeting Cthrc1 either before or after establishment of fibrosis. Importantly, when Cthrc1 gene expression was enhanced, we noted a significant improvement of hepatic fibrosis, both microscopically and by analysis of fibrotic gene expression. In contrast, when Cthrc1 gene expression was deleted, there was a significant exacerbation of fibrosis. To identify the mechanism of action of these significant effects produced by knocking in/knocking out Cthrc gene expression, we thence studied the interaction of Cthrc1 gene expression using hepatic stellate cells (HSCs) and human LX-2 cells. Importantly, we demonstrate that Cthrc1 is induced by TGF-β1 via phospho-Smad3 binding to the promoter with subsequent transcription activation. In addition, we demonstrate that Cthrc1 inhibits TGF-β signaling by accelerating degradation of phospho-Smad3 through a proteosomal pathway. Importantly, the anti-fibrotic effects can be recapitulated with a truncated fragment of Cthrc1. In conclusion, our findings uncover a critical negative feedback regulatory loop in which TGF-β1 induces Cthrc1, which can attenuate fibrosis by accelerating degradation of phospho-Smad3.

Quantitation of the Rank-Rankl Axis in Primary Biliary Cholangitis.

There is substantial data that suggests an abnormality of innate immunity in patients with primary biliary cholangitis (PBC) which includes the transcription factor nuclear factor-kB (NF-kB) and well as downstream inflammatory signaling pathways. In addition, ImmunoChip analysis has identified a novel PBC-associated locus near the receptor activator of NF-kB ligand (RANKL) gene. Based on these observations, we investigated the role of the RANKL axis in the liver of patients with PBC compared to controls. We used immunohistochemistry to quantitate liver expression of RANKL, its receptor (RANK), and importantly the decoy receptor osteoprotegerin (OPG), including a total of 122 liver samples (PBC = 37, primary sclerosing cholangitis = 20, autoimmune hepatitis = 26, chronic hepatitis B = 32 and unaffected controls = 7). In addition, we studied RANKL-RANK-OPG co-localization in CD4 and CD8 T cells, B cells, dendritic cells, macrophages, NK, NKT cells, hepatocytes, and cholangiocytes. We report herein that RANK is constitutively expressed by cholangiocytes in both unaffected and diseased liver. However, cholangiocytes from PBC express significantly higher levers of RANK than either the unaffected controls or liver diseased controls. CD4, CD8 and CD19 cells with in the portal areas around bile ducts in PBC express significantly higher levels of RANKL compared to controls. Importantly, the overall hepatic RANKL level and the ratio of hepatic RANKL/OPG correlated with disease severity in PBC. In conclusion, our data indicate a role of RANK-RANKL axis in the innate immune activation in PBC and we hypothesize that the damaged cholangiocytes, which express high levels of RANK, lead to the recruitment of RANKL positive cells and ultimately the classic portal tract infiltrates.

The functional characteristics CCNI modulation of myeloid- derived suppressor cells in liver inflammation

There is increasing awareness of the immunologic roles of liver mononuclear populations, including myeloid‐derived suppressor cells (MDSCs). We took advantage of a large well‐defined cohort of 148 patients with liver inflammation and 45 healthy controls to focus on the qualitative and quantitative characteristics of MDSCs. We investigated the frequency, phenotype, and functional capacities of MDSCs by using peripheral blood MDSCs in a cohort of 55 patients with primary biliary cholangitis (PBC), 40 with autoimmune hepatitis, 39 with chronic hepatitis B, 14 with nonalcoholic fatty liver disease, and 45 healthy controls. This was followed by a liver‐targeted determination in 27 patients with PBC, 27 with autoimmune hepatitis, 20 with chronic hepatitis B, 14 with nonalcoholic fatty liver disease, and 6 controls. We then focused on mechanisms of this expansion with PBC as an example, using both ursodeoxycholic acid‐naive and treated patients. HLA‐DR−/lowCD33+CD11b+CD14+CD15− monocytic MDSCs were elevated in diseases characterized by liver inflammation compared to healthy controls. Using PBC as a focus, there was a significant correlation between levels of circulating MDSCs and disease‐related biochemical markers (alkaline phosphatase, total bilirubin). We found higher amounts of MDSCs in patients with PBC who were responsive to ursodeoxycholic acid. MDSCs from PBC were found to manifest a potent immunosuppressive function. There was a significant correlation in the accumulation of hepatic MDSCs in the inflamed lesions of PBC with histologic changes, such as fibrosis. We also found that cysteine‐rich protein 61 (CCN1), a highly expressed protein in impaired cholangiocytes and hepatocytes, contributes to MDSC expansion and MDSC inducible nitric oxide synthase‐associated immune suppression. Conclusion: CCN1 modulates expansion and a suppressive function of MDSCs. Our data highlight the potential functions of CCN1 on MDSCs and suggest therapeutic implications in inflammatory liver diseases. (Hepatology HEPATOLOGY 2018;67:232‐246).

The Immunobiology of Receptor Activator for Nuclear Factor Kappa B Ligand and Myeloid‐Derived Suppressor Cell Activation in Immunoglobulin G4–Related Sclerosing Cholangitis

The primary function of myeloid‐derived suppressor cells (MDSCs) is reflected in their immune modulatory role in several immune‐mediated diseases. In immunoglobulin G4 (IgG4)–related disease (IgG4‐RD), it has been hypothesized that there are selective regulatory defects that lead to a T helper 2 (Th2) bias immune response. Herein we have taken advantage of a large cohort of patients with IgG4‐related sclerosing cholangitis (IgG4‐SC), the most common extrapancreatic involvement of IgG4‐RD, as well as controls consisting of primary sclerosing cholangitis, autoimmune hepatitis, and healthy volunteers, to study MDSCs. We report dramatically increased levels of receptor activator for nuclear factor kappa B ligand (RANKL) expression in serum and liver from patients with IgG4‐SC compared to both liver‐disease and healthy controls. Moreover, in IgG4‐SC liver, RANKL‐secreting cells specifically colocalized with cluster of differentiation 38–positive plasma cells and MDSCs, particularly monocytic MDSCs, and express the RANKL receptor in liver. Similarly, the frequency and number of peripheral blood MDSCs were significantly increased. Importantly, serum expression levels of RANKL were inversely correlated with the serum level of gamma‐glutamyltransferase but significantly positively correlated with the frequency of MDSCs. Moreover, we confirmed that RANKL induced the expansion and activation of MDSCs through the RANKL/RANK/nuclear factor kappa B signal pathway. Of note, RANKL‐treated MDSCs suppressed T‐cell proliferation and induced Th2 differentiation. Conclusion: Our data suggest that plasma cell–derived RANKL induces the expansion and activation of MDSCs, which suppress T‐cell proliferation and contribute to the Th2‐type response characteristic of IgG4‐SC.

Endoscopic biopsy in gastrointestinal neuroendocrine neoplasms: a retrospective study.

Background Gastrointestinal neuroendocrine neoplasms (GI-NENs) are often located in the deep mucosa or submucosa, and the efficacy of endoscopic biopsy for diagnosis and treatment of GI-NENs is not fully understood. Objective The current study analyzed GI-NENs, especially those diagnosed pathologically and resected endoscopically, and focused on the biopsy and cold biopsy forceps polypectomy (CBP) to analyze their roles in diagnosing and treating GI-NENs. Methods Clinical data of all GI-NENs were reviewed from January 2006 to March 2012. Histopathology was used to diagnose GI-NENs, which were confirmed by immunohistochemistry. Results 67.96% GI-NENs were diagnosed pathologically by endoscopy. Only 26.21% were diagnosed pathologically by biopsies before treatment. The diagnostic rate was significantly higher in polypoid (76.47%) and submucosal lesions (68.75%), than in ulcerative lesions (12.00%). However, biopsies were only taken in 56.31% patients, including 51.52% of polypoid lesions, 35.56% of submucosal lesions and 100.00% of ulcerative lesions. Endoscopic resection removed 61.76% of GI-NENs, including six by CBP, 14 by snare polypectomy with electrocauterization, 28 by endoscopic mucosal resection (EMR) and 15 by endoscopic submucosal dissection (ESD). 51.52% polypoid GI-NENs had infiltrated the submucosa under microscopic examination. CBP had a significantly higher rate of remnant (33.33%) than snare polypectomy with electrocauterization, EMR and ESD (all 0.00%). Conclusions Biopsies for all polypoid and submucosal lesions will improve pre-operative diagnosis. The high rate of submucosal infiltration of polypoid GI-NENs determined that CBP was inadequate in the treatment of GI-NENs. Diminutive polypoid GI-NENs that disappeared after CBP had a high risk of remnant and should be closely followed up over the long term.