Nonalcoholic fatty liver disease with cirrhosis increases familial risk for advanced fibrosis

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Abstract

BACKGROUND. The risk of advanced fibrosis in first-degree relatives of patients with nonalcoholic fatty liver disease and cirrhosis (NAFLD-cirrhosis) is unknown and needs to be systematically quantified. We aimed to prospectively assess the risk of advanced fibrosis in first-degree relatives of probands with NAFLD-cirrhosis. METHODS. This is a cross-sectional analysis of a prospective cohort of 26 probands with NAFLD-cirrhosis and 39 first-degree relatives. The control population included 69 community-dwelling twin, sib-sib, or parent-offspring pairs (n = 138), comprising 69 individuals randomly ascertained to be without evidence of NAFLD and 69 of their first-degree relatives. The primary outcome was presence of advanced fibrosis (stage 3 or 4 fibrosis). NAFLD was assessed clinically and quantified by MRI proton density fat fraction (MRI-PDFF). Advanced fibrosis was diagnosed by liver stiffness greater than 3.63 kPa using magnetic resonance elastography (MRE). RESULTS. The prevalence of advanced fibrosis in first-degree relatives of probands with NAFLD-cirrhosis was significantly higher than that in the control population (17.9% vs. 1.4%, P = 0.0032). Compared with controls, the odds of advanced fibrosis among the first-degree relatives of probands with NAFLD-cirrhosis were odds ratio 14.9 (95% CI, 1.8–126.0, P = 0.0133). Even after multivariable adjustment by age, sex, Hispanic ethnicity, BMI, and diabetes status, the risk of advanced fibrosis remained both statistically and clinically significant (multivariable-adjusted odds ratio 12.5; 95% CI, 1.1–146.1, P = 0.0438). CONCLUSION. Using a well-phenotyped familial cohort, we demonstrated that first-degree relatives of probands with NAFLD-cirrhosis have a 12 times higher risk of advanced fibrosis. Advanced fibrosis screening may be considered in first-degree relatives of NAFLD-cirrhosis patients. TRIAL REGISTRATION. UCSD IRB: 140084. FUNDING. National Institute of Diabetes and Digestive and Kidney Diseases and National Institute of Environmental Health Sciences, NIH.

Authors

Cyrielle Caussy, Meera Soni, Jeffrey Cui, Ricki Bettencourt, Nicholas Schork, Chi-Hua Chen, Mahdi Al Ikhwan, Shirin Bassirian, Sandra Cepin, Monica P. Gonzalez, Michel Mendler, Yuko Kono, Irine Vodkin, Kristin Mekeel, Jeffrey Haldorson, Alan Hemming, Barbara Andrews, Joanie Salotti, Lisa Richards, David A. Brenner, Claude B. Sirlin, Rohit Loomba, the Familial NAFLD Cirrhosis Research Consortium

Long-lived keratin 15⁺ esophageal progenitor cells contribute to homeostasis and regeneration

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Abstract

The esophageal lumen is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (Krt15). Genetic in vivo lineage tracing revealed that the Krt15 promoter marks a long-lived basal cell population able to self-renew, proliferate, and generate differentiated cells, consistent with a progenitor/stem cell population. Transcriptional profiling demonstrated that Krt15⁺ basal cells are molecularly distinct from Krt15^– basal cells. Depletion of Krt15-derived cells resulted in decreased proliferation, thereby leading to atrophy of the esophageal epithelium. Further, Krt15⁺ cells were radioresistant and contributed to esophageal epithelial regeneration following radiation-induced injury. These results establish the presence of a long-lived and indispensable Krt15⁺ progenitor cell population that provides additional perspective on esophageal epithelial biology and the widely prevalent diseases that afflict this epithelium.

Authors

Véronique Giroux, Ashley A. Lento, Mirazul Islam, Jason R. Pitarresi, Akriti Kharbanda, Kathryn E. Hamilton, Kelly A. Whelan, Apple Long, Ben Rhoades, Qiaosi Tang, Hiroshi Nakagawa, Christopher J. Lengner, Adam J. Bass, E. Paul Wileyto, Andres J. Klein-Szanto, Timothy C. Wang, Anil K. Rustgi

Folate cycle enzyme MTHFD1L confers metabolic advantages in hepatocellular carcinoma

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Abstract

Cancer cells preferentially utilize glucose and glutamine, which provide macromolecules and antioxidants that sustain rapid cell division. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of these nutrients. The folate cycle, through transfer of a carbon unit between tetrahydrofolate and its derivatives in the cytoplasmic and mitochondrial compartments, produces other metabolites that are essential for cell growth, including nucleotides, methionine, and the antioxidant NADPH. Here, using hepatocellular carcinoma (HCC) as a cancer model, we have observed a reduction in growth rate upon withdrawal of folate. We found that an enzyme in the folate cycle, methylenetetrahydrofolate dehydrogenase 1–like (MTHFD1L), plays an essential role in support of cancer growth. We determined that MTHFD1L is transcriptionally activated by NRF2, a master regulator of redox homeostasis. Our observations further suggest that MTHFD1L contributes to the production and accumulation of NADPH to levels that are sufficient to combat oxidative stress in cancer cells. The elevation of oxidative stress through MTHFD1L knockdown or the use of methotrexate, an antifolate drug, sensitizes cancer cells to sorafenib, a targeted therapy for HCC. Taken together, our study identifies MTHFD1L in the folate cycle as an important metabolic pathway in cancer cells with the potential for therapeutic targeting.

Authors

Derek Lee, Iris Ming-Jing Xu, David Kung-Chun Chiu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Lynna Lan Li, Cheuk-Ting Law, Felice Ho-Ching Tsang, Larry Lai Wei, Cerise Yuen-Ki Chan, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong

Mesothelin/mucin 16 signaling in activated portal fibroblasts regulates cholestatic liver fibrosis

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Abstract

Cholestatic liver fibrosis is caused by obstruction of the biliary tract and is associated with early activation of portal fibroblasts (PFs) that express Thy-1, fibulin 2, and the recently identified marker mesothelin (MSLN). Here, we have demonstrated that activated PFs (aPFs) and myofibroblasts play a critical role in the pathogenesis of liver fibrosis induced by bile duct ligation (BDL). Conditional ablation of MSLN⁺ aPFs in BDL-injured mice attenuated liver fibrosis by approximately 50%. Similar results were observed in MSLN-deficient mice (Msln^–/– mice) or mice deficient in the MSLN ligand mucin 16 (Muc16^–/– mice). In vitro analysis revealed that MSLN regulates TGF-β1–inducible activation of WT PFs by disrupting the formation of an inhibitory Thy-1–TGFβRI complex. MSLN also facilitated the FGF-mediated proliferation of WT aPFs. Therapeutic administration of anti-MSLN–blocking Abs attenuated BDL-induced fibrosis in WT mice. Liver specimens from patients with cholestatic liver fibrosis had increased numbers of MSLN⁺ aPFs/myofibroblasts, suggesting that MSLN may be a potential target for antifibrotic therapy.

Authors

Yukinori Koyama, Ping Wang, Shuang Liang, Keiko Iwaisako, Xiao Liu, Jun Xu, Mingjun Zhang, Mengxi Sun, Min Cong, Daniel Karin, Kojiro Taura, Chris Benner, Sven Heinz, Tapan Bera, David A. Brenner, Tatiana Kisseleva

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation

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Abstract

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the Apc^Min mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling–induced proliferation, particularly during regeneration and adenoma formation.

Authors

Daniel O. Kechele, R. Eric Blue, Bailey Zwarycz, Scott T. Espenschied, Amanda T. Mah, Marni B. Siegel, Charles M. Perou, Shengli Ding, Scott T. Magness, P. Kay Lund, Kathleen M. Caron

Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis

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Abstract

Congenital tufting enteropathy (CTE) is a severe autosomal recessive human diarrheal disorder with characteristic intestinal epithelial dysplasia. CTE can be caused by mutations in genes encoding EpCAM, a putative adhesion molecule, and HAI-2, a cell surface protease inhibitor. A similar phenotype occurs in mice whose intestinal epithelial cells (IECs) fail to express the tight junction–associated protein claudin-7. EpCAM stabilizes claudin-7 in IECs, and HAI-2 regulates the cell surface serine protease matriptase, a known modifier of intestinal epithelial physiology. Therefore, we hypothesized that HAI-2, matriptase, EpCAM, and claudin-7 were functionally linked. Herein we have demonstrated that active matriptase cleaves EpCAM after Arg80 and that loss of HAI-2 in IECs led to unrestrained matriptase activity and efficient cleavage of EpCAM. Cleavage of EpCAM decreased its ability to associate with claudin-7 and targeted it for internalization and lysosomal degradation in conjunction with claudin-7. CTE-associated HAI-2 mutant proteins exhibited reduced ability to inhibit matriptase and also failed to efficiently stabilize claudin-7 in IECs. These results identify EpCAM as a substrate of matriptase and link HAI-2, matriptase, EpCAM, and claudin-7 in a functionally important pathway that causes disease when it is dysregulated.

Authors

Chuan-Jin Wu, Xu Feng, Michael Lu, Sohshi Morimura, Mark C. Udey

Antibiotic effects on gut microbiota and metabolism are host dependent

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Abstract

Interactions of diet, gut microbiota, and host genetics play important roles in the development of obesity and insulin resistance. Here, we have investigated the molecular links between gut microbiota, insulin resistance, and glucose metabolism in 3 inbred mouse strains with differing susceptibilities to metabolic syndrome using diet and antibiotic treatment. Antibiotic treatment altered intestinal microbiota, decreased tissue inflammation, improved insulin signaling in basal and stimulated states, and improved glucose metabolism in obesity- and diabetes-prone C57BL/6J mice on a high-fat diet (HFD). Many of these changes were reproduced by the transfer of gut microbiota from antibiotic-treated donors to germ-free or germ-depleted mice. These physiological changes closely correlated with changes in serum bile acids and levels of the antiinflammatory bile acid receptor Takeda G protein–coupled receptor 5 (TGR5) and were partially recapitulated by treatment with a TGR5 agonist. In contrast, antibiotic treatment of HFD-fed, obesity-resistant 129S1 and obesity-prone 129S6 mice did not improve metabolism, despite changes in microbiota and bile acids. These mice also failed to show a reduction in inflammatory gene expression in response to the TGR5 agonist. Thus, changes in bile acid and inflammatory signaling, insulin resistance, and glucose metabolism driven by an HFD can be modified by antibiotic-induced changes in gut microbiota; however, these effects depend on important interactions with the host’s genetic background and inflammatory potential.

Authors

Shiho Fujisaka, Siegfried Ussar, Clary Clish, Suzanne Devkota, Jonathan M. Dreyfuss, Masaji Sakaguchi, Marion Soto, Masahiro Konishi, Samir Softic, Emrah Altindis, Ning Li, Georg Gerber, Lynn Bry, C. Ronald Kahn

Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice

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Abstract

Radiotherapy causes dose-limiting toxicity and long-term complications in rapidly renewing tissues, including the gastrointestinal tract. Currently, there is no FDA-approved agent for the prevention or treatment of radiation-induced intestinal injury. In this study, we have shown that PD 0332991 (PD), an FDA-approved selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), prevents radiation-induced lethal intestinal injury in mice. Treating mice with PD or a structurally distinct CDK4/6 inhibitor prior to radiation blocked proliferation and crypt apoptosis and improved crypt regeneration. PD treatment also enhanced LGR5⁺ stem cell survival and regeneration after radiation. PD was an on-target inhibitor of RB phosphorylation and blocked G₁/S transition in the intestinal crypts. PD treatment strongly but reversibly inhibited radiation-induced p53 activation, which blocked p53-upregulated modulator of apoptosis–dependent (PUMA-dependent) apoptosis without affecting p21-dependent suppression of DNA damage accumulation, with a repair bias toward nonhomologous end joining. Further, deletion of PUMA synergized with PD treatment for even greater intestinal radioprotection. Our results demonstrate that the cell cycle critically regulates the DNA damage response and survival of intestinal stem cells and support the concept that pharmacological quiescence is a potentially highly effective and selective strategy for intestinal radioprotection.

Authors

Liang Wei, Brian J. Leibowitz, Xinwei Wang, Michael Epperly, Joel Greenberger, Lin Zhang, Jian Yu

EGFR regulates macrophage activation and function in bacterial infection

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Abstract

EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

Authors

Dana M. Hardbower, Kshipra Singh, Mohammad Asim, Thomas G. Verriere, Danyvid Olivares-Villagómez, Daniel P. Barry, Margaret M. Allaman, M. Kay Washington, Richard M. Peek Jr., M. Blanca Piazuelo, Keith T. Wilson

Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function

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Abstract

Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT₄ agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT₄–mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation.

Authors

Kara Gross Margolis, Zhishan Li, Korey Stevanovic, Virginia Saurman, Narek Israelyan, George M. Anderson, Isaac Snyder, Jeremy Veenstra-VanderWeele, Randy D. Blakely, Michael D. Gershon