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Interleukin-33 Induces a Potent Cholangiocyte Proliferation via a Novel Paracrine Circuit

Jun Li, Pranavkumar Shivakumar, Stephanie Walters, Tatsuki Mizuochi, Reena Mourya, Kazuhiko Bessho, Jorge A. Bezerra
Division of Gastroenterology, Hepatology and Nutrition and the Pediatric Liver Core Center, Cincinnati Children's Hospital Medical Center, Cincinnati, OH

Background and Aims: Cholangiocyte proliferation is critical for tissue repair and carcinogenesis. We reported previously that the hepatic expression of ST2, the receptor for interleukin-33 (IL33, a Th2 cytokine) increases in patients with biliary atresia, the most common obstructive cholangiopathy in children. Here, we aimed to investigate the role of the IL33/ST2 axis in bile duct repair using complementary human and murine experiments. Methods and Results: We quantified serum IL33 in 81 children with biliary atresia and found it to be higher (93.7±52.6 pg/mL) than the undetectable levels of agematched controls. In a mouse model of biliary atresia, IL33 expression correlated with increased numbers of cholangiocytes in livers and extrahepatic bile ducts (EHBDs), and both IL33 and ST2 were expressed in cholangiocytes of EHBDs of neonatal and adult mice. To determine if IL33 induces mitosis of cholangiocytes, we injected 0.1 μg IL33 daily to adult mice. IL33 induced marked proliferation, with a peak of 76-fold increase in EHBD at day 1(BrdU+ cholangiocytes: 37.9±3.4% vs 0.5±1% in controls, P<0.0001) and ongoing proliferation at days 4 (13.1 ±2.5%) and 7 (5.7±1.2%), evidenced histologically as epithelial hyperplasia. IL33 induced an expansion of the hepatic population of lineage-negative mononuclear cells that expressed ST2 with surface staining for Sca1 +CkitlowICoS+CD127+CD25+CD44+ (known as nuocytes), which overexpressed IL4, IL5, and IL13 upon treatment with PMA-ionomycin in culture. To determine the relevance of nuocytes and IL13 in the proliferative response, we injected IL33 into Rag2-/Il2rgc-/- and Roro% mice, which lack nuocytes, and found decreased proliferation compared to wild-type (WT) in both mouse lines (2.6±0.6% vs WT=26.3±4.3%, P=0.005; 6.3±0.8% vs WT=22.5±3.9%, P=0.02, respectively), but remained above baseline (P<0.01). Adoptive transfer of nuocytes restored the IL33-induced cholangiocyte proliferation. Similarly, proliferation was suppressed in Il1-/- mice (1.3±0.3%), while injections of IL13 induced cholangiocyte proliferation in a dose-dependent manner. By RNAseq, IL33 resulted in the activation of the E2f1, Hif1a, Prrx2, and Myb pathways. Last, injection of IL33 to neonatal mice with atresia suppressed epithelial injury, promoted cholangiocyte hyperplasia, and decreased duct obstruction. Conclusions: IL33 induces proliferation of cholangiocytes directly and by the release of IL13 from nuocytes in a paracrine fashion. The activation of inflammation and cell proliferation pathways and epithelial hyperplasia in a disease model point to a potential role IL33 in tissue repair and oncogenesis.

Disclosures:

Jorge A. Bezerra - Grant/Research Support: Molecular Genetics Laboratory,

The following people have nothing to disclose: Jun Li, Pranavkumar Shivakumar, Stephanie Walters, īatsuki Mizuochi, Reena Mourya, Kazuhiko Bessho

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Signaling via the osteopontin and high-mobility group box-1 axis drives the fibrogenic response to liver injury

Elena Arriazu1, Xiaodong Ge1, Tung Ming Leung1, Aritz Lopategi1, Yongke Lu1, Naoto Kitamura1, Raquel Urtasun1, Neil D. Theise2, Natalia Nieto1
1Medicine/liver diseases, Mount Sinai School of Medicine, New York, NY; 2Division of Digestive Diseases, Beth Israel Medical Center, New York, NY

Background: Activation of hepatic stellate cells (HSC) and increased fibrillar collagen-I synthesis are critical events in the pathogenesis of liver fibrosis. Recent studies from our laboratory demonstrated that osteopontin (OPN), a secreted cytokine up-regulated during liver fibrosis, conditions the dynamics of extracellular matrix deposition and remodelling. Additional work, identified high-mobility group box-1 (HMGB1) as an early proinflammatory cytokine responsive to sterile and nonsterile injury that increases in plasma and in liver from fibrotic patients; yet, factors that drive HMGB1 cytosolic localization and/or promote HMGB1 export are still largely unknown. So far, a link among OPN, a damage-associated molecular patter such as HMGB1 and scarring has not been established. Hence, we hypothesized that OPN acting as a trigger could target HMGB1, which as a proinflammatory mediator could regulate collagen-I expression in HSC contributing to the pathogenesis of liver fibrosis. Methods: Human liver biopsies from long-term HCV were analyzed for oPN and HMGB1 expression. WT, Hmgb1-/+, opn-/- and transgenic mice expressing oPN in hepatocytes (OpnHEP Tg) were injected CCl4 for 1 month to induce liver fibrosis. In vitro studies were performed with primary mouse HSC from WT and opn-/- mice and with embryonic skin fibroblasts from WT and Hmgb1-/- mice. Primary HSC were co-cultured with hepatocytes, a major source of OPN and HMGB1 or were treated with recombinant oPN or HMGB1.Results: Human liver biopsies from long-term HCV progressors showed co-induction of oPN and HMGB1 expression correlating with fibrosis stage. CCl4-treated 〇pnHEP Tg mice showed increased HMGB1 and collagen-I expression compared to WT mice but the increase in these two proteins was blunted in opn-/- mice. Moreover, Hmgb1+/- mice were significantly protected from CCl4-induced fibrosis compared to WT mice. In vitro studies identified OPN as upstream of HMGB1 in regulating collagen-I expression since 〇pn deletion lowered HMGB1 and collagen-I and Hmgb1 ablation decreased collagen-I without altering 0PN expression. Hepatocytes, which produce large amounts of OPN and condition HMGB1 compartmentalization and secretion, enhanced collagen-I synthesis by HSC in co-culture. Likewise, treatment with roPN increased HMGB1 and collagen I deposition by HSC. Last, challenge with rHMGB1 increased collagen I synthesis in HSC by binding the receptor for advanced glycation-end products and activating the PI3K-pAkt signalling pathway. Conclusion: These results unveil the mechanism whereby OPN induction of HMGB1 secretion could amplify the profibrogenic potential of HSC hence contributing to liver fibrosis.

Disclosures:

The following people have nothing to disclose: Elena Arriazu, Xiaodong Ge, Tung Ming Leung, Aritz Lopategi, Yongke Lu, Naoto Kitamura, Raquel Urtasun, Neil D. Theise, Natalia Nieto

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Cell Fate Tracking Reveals Hepatocytes as the Primary Cellular Source for Hepatocellular Carcinoma

Regina Español Suñer2, Xueru Mu1, Christine Sempoux2, Dianne H. Dapito1, Frederic Lemaigre2, Isabelle A. Leclercq2, Robert Schwabe1
1Medicine, Columbia University, New York, NY; 2Institut de Recherche Expérimentale eftClinique, Université catholique de Louvain, Brussels, Belgium

BACKGROUND: Hepatocellular carcinoma (HCC), in particular in patients with unfavorable prognosis, often displays a progenitor signature, suggesting an origin from liver progenitor cells (Lee et al, Nature Medicine 2006). However, it is also conceivable that HCC is derived from hepatocytes that dedifferentiate into progenitor-like cells. No cell fate tracking studies have been performed to test these hypotheses. AIM: To determine the cellular source for HCC by genetic cell fate tracking approaches. METHODS: Liver progenitor cells were tracked in tamoxifen-inducible Osteopontin-Cre (OPN-CreERT2) mice via YFP Cre reporter. Hepatocytes were tracked by an adeno-associated virus serotype 8, expressing Cre under control of the hepatocyte-specific TBG promoter (AAV8-TBG-Cre), using mTomato/mGFP Cre reporter mice. HCC was induced by diethylnitrosamine (DEN), DEN+carbon tetrachloride (CCl4), or PTEN deletion. RESULTS: Although oPN-CreERT2marked 69.1% of liver progenitor cells and cholangiocytes, no HCCs induced by DEN were YFP-positive, thus excluding a contribution of progenitors to HCC formation. Interestingly, some HCCs contained YFP-, CK19+ cells, suggesting dedifferentiation of hepatocytes into progenitor-like cells. In contrast, >80% of tumors from AAV8-TBG-Cre injected mice induced by DEN- or DEN+CCl4- were mGFP+, demonstrating their hepatocyte origin. Notably, AAV8-TBG-Cre marked >95% of hepatocytes but no CK+ progenitors, bile ducts, endothelial or stellate cells, confirming specificity of this cell fate tracking approach. Accordingly, mGFP+ HCCs contained numerous mGFP- .mTomato+ CD31+endothelial cells. Deletion of PTEN by AAV8TBG-Cre resulted in development of HCCs, similar to those induced in Alb-Cre transgenic mice (that delete in hepatocytes as well as in progenitors), again confirming hepatocytes as source for HCCs. CONCLUSION: Hepatocytes, but not liver progenitor cells are the primary source for HCC in genotoxic and genetic HCC models.

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Disclosures:

The following people have nothing to disclose: Regina Español Suñer, Xueru Mu, Christine Sempoux, Dianne H. Dapito, Frederic Lemaigre, Isabelle A. Leclercq, Robert Schwabe

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The mast cell stabilizer, cromolyn sodium, reduces bile duct ligated-induced biliary hyperplasia: a novel role for the in vivo paracrine influence of mast cells on biliary proliferation

Laura Hargrove2, Lindsey Kennedy2, Taylor Francis3, Kyle M. Hodges2, Allyson B. Graf1, Yoshiyuki Ueno4, John F. Greene2, Heather L. Francis1, 2
1Central Texas Veterans Health Care System, Temple, TX; 2Scott and White Memorial Hospital, Temple, TX; 3Texas A&M Heath Science Center, Temple, TX; 4Yamagata University, Yamagata, Japan

Background: Cholangiopathies are characterized by dysregulation of the balance between biliary growth/damage. After bile duct ligation (BDL) (i) histamine (HA) levels increase; (ii) hepatic mast cells infiltrate; and (iii) cholangiocyte histidine decarboxylase (HDC) levels are elevated. Inhibition of biliary HDC expression decreases cholangiocyte growth and the expression of VEGF; and in vitro, co-culture with mast cells increases biliary proliferation and VEGF expression. Aim: to evaluate the effects of mast cell stabilization on biliary proliferation. Methods: Male sham and BDL rats were treated by IP implanted osmotic pumps with saline or cromolyn sodium (inhibits mast cell histamine release, 24 mg/kg BW/day) for 1 wk. Serum, liver blocks and cholangiocytes were collected. HDC expression was evaluated by immunohistochemistry (IHC) in liver sections and qPCR in cholangiocytes. Intrahepatic bile duct mass (IBDM) was evaluated by IHC for CK-19.Hepatic mast cell number was evaluated by toluidine blue and correlated to IBDM. Proliferation was evaluated by PCNA in liver sections and immunoblots in cholangiocytes. Expression of VEGF and the mast cell stem factor receptor, c-kit and Pro-Caspase 3 and Bax was measured by qPCR in cholangiocytes. HA levels were measured in serum and cholangiocyte supernatants by EIA. Liver morphology was measured by H&E and Sirius Red staining. In vitro, mast cells (MC) were treated with 0.1% BSA (basal), compound 48/80 (MC activator, 10 μM) and cromolyn (10 μM) for up to 30 minutes prior to assessing HA levels and tryptase expression. Supernatants from MCs were added to cholangiocyte cultures before measuring (i) proliferation by MTT assays; (ii) VEGF gene expression by qPCR; and (iii) HA release by EIA. Results: In vivo, cromolyn treatment decreased BDL-induced: (i) IBDM and hepatic mast cell number; (ii) cholangiocyte HDC, VEGF and c-kit expression; and (iii) cholangiocyte proliferation and HA levels. Cromolyn treatment increased cholangiocyte apoptosis compared to BDL, but did not significantly alter liver morphology. In vitro, compound 48/80 activated MCs, whereas cromolyn decreased HA release and tryptase expression. Cromolyn-treated MC supernatants decreased biliary proliferation, VEGF expression and HA release compared to cholangiocytes treated with supernatant from untreated MCs. Summary/Conclusion: These studies provide novel evidence that (i) cholangiocyte proliferation is regulated by mast cells and (ii) mast cell histamine is critical to biliary proliferation. Manipulation of hepatic mast cell mediators may provoke a shift in the current treatment paradigm in patients suffering from cholangiopathies.

Disclosures:

Yoshiyuki Ueno - Advisory Committees or Review Panels: Jansen

The following people have nothing to disclose: Laura Hargrove, Lindsey Kennedy, Taylor Francis, Kyle M. Hodges, Allyson B. Graf, John F. Greene, Heather L. Francis

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Cholangiocyte senescence via N-Ras activation is a characteristic of primary sclerosing cholangitis

James H. Tabibian, Steven O'Hara, Patrick L. Splinter, Christy E. Trussoni, Nicholas F. LaRusso
Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN

Introduction: Primary sclerosing cholangitis (PSC) is an incurable cholangiopathy of unknown etiology. Given recent evidence that cellular senescence is important in the pathogenesis of other diseases, we tested the hypothesis that cholangiocyte senescence plays a role in PSC. Methods: After obtaining IRB approval, we assessed markers of senescence and senescence associated secretory phenotype (SASP) in cholangiocytes in PSC, PBC, HCV, and normal livers by in situ hybridization (FISH) and immunofluorescence microscopy (IFM). We tested if endogenous (eg bile acids) and exogenous (eg LPS) bile constituents induce senescence and SASP in cultured human cholangiocytes. We determined in co-culture if senescent cholangiocytes induce senescence in bystander cholangiocytes. We explored signaling pathways potentially involved in cholangiocyte senescence and SASP. Results: In vivo, FISH showed greater p16 mRNA expression in PSC cholangiocytes vs. PBC, HCV, and normal (3-20 fold, all p<0.01). IFM showed a greater proportion (47%) of yH2Ax-positive cholangiocytes in PSC (2-6-fold increase, all p<0.01). By IFM, expression of inflammatory SASP proteins (IL6/IL8, CCL2, and PAI-1)was also higher in PSC cholangiocytes compared to all other conditions (3-20 fold, p<0.001). In vitro, several endogenous (eg oxysterols) and exogenous (eg LPS) biliary constituents increased cholangiocyte senescence (p<0.01) and expression of IL6/8 and CCL2 mRNA (5, 5 and 6- fold, respectively, all p<0.05) and protein (8, 8, and 5- fold, respectively, all p<0.05) vs. control; others (eg deoxycholate) had no effect. Experimentally-induced senescent cholangiocytes induced senescence in bystander cholangiocytes (3-fold, p<0.05 vs. control). IFM revealed activated N-Ras, a known inducer of cellular senescence, was increased 4-fold (p<0.05) in cholangiocytes in PSC vs. normal livers. An N-Ras activation assay showed that LPS-induced senescent cultured cholangiocytes exhibited 6-fold increased activated N-Ras compared to control cells (p<0.01). Pharmacologic inhibition of N-Ras decreased LPS-induced cholangiocyte senescence in vitro by 60% (p<0.01 vs. control). Consistent with our in vivo human data, cholangiocytes in livers of the MDR2-/- PSC mouse model showed increased markers of senescence (3-fold, p<0.05). Conclusions: Our data suggest that cholangiocyte senescence is an important pathogenic mechanism in PSC. Moreover, we identified a novel signaling pathway through which biliary constituents induce this cellular state (N-Ras activation). Pharmacologic inhibition of this pathway and the senescent cholangiocyte phenotype may represent a new potential therapeutic strategy for PSC.

Disclosures:

The following people have nothing to disclose: James H. Tabibian, Steven O΄Hara, Patrick L. Splinter, Christy E. Trussoni, Nicholas F. LaRusso

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Innate immune signaling couples endoplasmic reticulum stress to hepatocyte apoptosis via STING and mitochondrial IRF3 interactions

Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo
Medicine, UMASS Medical School, Worcester, MA

Background: Endoplasmic reticulum (ER) stress resulting from accumulation of unfolded proteins in the ER is a major determinant of liver diseases. The mechanisms by which ER stress induces hepatocyte death are not known. Stimulator of Interferon Genes (STING), is a member of the ER-associated protein family and also involved in antiviral immune signaling that activates Interferon Regulatory Factor (IRF)−3 and Type I interferon (IFN) responses. Aim: To investigate mechanisms by which ER stress induces hepatocyte damage. Methods: ER stress was assessed in acute or chronic liver injury induced by alcohol or carbon-tetrachloride (CCl4) in wild-type (WT), IRF3-, STING-, TRAM-, TRIF- or Type I IFN receptor (IFNAR1)-deficient mice. Clodronate was used for Kupffer cell (KC) depletion, and thapsigargin or Fas ligand were used to study ER stress or hepatocyte apoptosis, respectively. Protein interactions were evaluated using immunoprecipitation. Results: Administration of alcohol in WT mice or treatment of primary hepatocytes with thapsigargin induced ER stress and apoptosis, and resulted in activation of IRF3 and induction of Type-I IFNs. While deficiency in IRF3 prevented hepatocyte apoptosis, there was no protection in mice lacking the IRF3 canonical activators, TRAM or TRIF, or deficient in Type-I IFN receptors. We found that the pathogenic role of IRF3 was independent of KCs and inflammation suggesting that IRF3 contributed to hepatocyte death and that non-canonical IRF3 activation and ER stress were involved. We observed that IRF3 phosphorylation required the ER-associated adaptor, STING, and found that following ER stress, STING associated with IRF3 in the ER. In turn, activated IRF3 associated with the pro-apoptotic proteins, caspase-8 and Bax, and triggered the mitochondrial apoptotic pathway. Deficiency of STING completely prevented IRF3 activation and hepatocyte damage; further, deficiency of IRF3 provided full protection from hepatocyte apoptosis in vitro and in vivo. Deficiency of STING or IRF3 also protected from CCl4-induced liver injury and fibrosis suggesting that the ER stress-STING-IRF3 pathway represents a common mechanism in acute (hepatocyte death) and chronic (fibrosis) liver pathology. Conclusions: Our novel findings demonstrate that ER stress mediates liver damage involving innate immune signaling pathways and show that ER stress activates IRF3 via the ER adaptor STING, and phosphorylated IRF3 triggers hepatocyte apoptosis. These discoveries indicate that innate immunity modulates outcomes of metabolic stress in the liver and may open a new direction in liver pathobiology.

Disclosures:

The following people have nothing to disclose: Jan Petrasek, Arvin Iracheta-Vellve, Timea Csak, Karen Kodys, Evelyn A. Kurt-Jones, Katherine A. Fitzgerald, Gyongyi Szabo