The Role of Reactive Oxygen Species and the Microbiome in Toxicant Induced Liver Fibrosis

Research Project 4


Project Narative

Obesity and non-alcoholic fatty liver (NAFL) are prevalent in many impoverished communities, contributes to health disparities, and increases the risk in these individuals developing toxicant-associated steatohepatitis (TASH) if exposed to environmental toxicants such as those found at NPL-Superfund sites. Since Hispanic and Native Americans are genetically predisposed to develop NAFL as a result of polymorphisms in the PNPLA3 gene, we propose to investigate the contribution of reactive oxygen species towards promoting TASH when the PNPLA3 gene with human polymorphic alleles are combined in mice expressing the inducible human CYP2E1 gene. The contribution of intestinal microbiota on increased susceptibility of toxicants generating TASH will be pursued, and the mechanisms of these responses will be examined in “human livers in the dish” (exVive3D™ Liver) following exposure to Superfund toxicants to examine if this novel culture system can be exploited to investigate TASH.

Summary of Project

Environmental exposures to industrial chemicals, such as carbon tetrachloride (CCl4) causes toxicant-induced steatohepatitis (TASH) characterized by fatty liver, inflammation, and fibrosis. Although susceptibility to occupational and environmental TASH may be modified by genetic predisposition (SNPs) and nutritional factors, our central hypothesis are that obesity and non-alcoholic fatty liver (NAFL) increase the severity of TASH by (1) Inducing the activity of cytochrome P450 2E1, (2) Increasing the generation of reactive oxygen species (ROS) by activating NADPH oxidases (NOXs), and (3) Inducing dysbiosis of the gut microbiome and increased intestinal permeability. The goal is to determine the mechanism by which obesity and CCl4 synergistically facilitate progression of TASH to liver cirrhosis, and develop new approaches to identifying toxicants that induce TASH. The following specific aims have been developed: (AIM1) To study the effect of “fast food diet” (FFD)-induced obesity and NAFL on CCl4-induced TASH. We propose to use improved diet-induced and genetic mouse models, including mice expressing human CYP2E1 gene and knock-in PNPLA3 mice (expressing the human SNP associated with NAFL). Since hCYP2E1 mice are more susceptible to toxicants, we anticipate that production of Nox1 and Nox4, release of pro-inflammatory cytokines by activated Kupffer cells, and development of liver fibrosis will closely recapitulate the pathology observed in patients with NASH/TASH. The specific role of NOX 1 and 4 in this TASH model will be assessed by pharmacological inhibition. (AIM2) To investigate the role of dysbiosis in transgenic mice subjected to fecal microbiota transplantation (FMT) from patients with NASH or matched normal controls from a well phenotyped cohort. We propose that the microbiome from NASH patients will render mice more sensitive to CCl4, while the transfer of “healthy” microbiota will decrease liver fibrosis in these mice. We will then do a therapeutic intervention by FMT of normal flora into a mouse with liver fibrosis to assess reversal of TASH. (AIM3) To translate our findings in mice to humans, we will utilize a “human liver in a dish” (exVive3D™ Livers, Organovo), a 3D culture composed of 4 primary hepatic cell types that maintains architectural and functional features of the human liver for greater than 40 days. These cultures will be subjected to Superfund toxicants and then assessed for TASH. The effects of potential drugs, such as Nox inhibitors, will be assessed in these 3D culture models of TASH. Overall, we will develop a high through-put system for ex vivo drug screening by measuring hepatotoxicity of Superfund toxicants and effectiveness of therapeutic interventions.

Publication

PubMed Central ID: 

Shalapour S., Lin X.J., Bastian I.N., Brain J., Burt A.D., Aksenov A.A., Vrbanac A.F., Li W., Perkins A., Matsutani T., Zhong Z., Dhar D., Navas-Molina J.A., Xu J., Loomba R., Downes M., Yu R.T., Evans R.M., Dorrestein P.C., Knight R., Benner C., Anstee Q.M., Karin M. (2017). Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity. Nature. 551, 340-345. doi: 10.1038/nature24302.

Hartmann P., Hochrath K., Horvath A., Chen P., Seebauer C.T., Llorente C., Wang L., Alnouti Y., Fouts D.E., Stärkel P., Loomba R., Coulter S., Liddle C., Yu R.T., Ling L., Rossi S.J., DePaoli A.M., Downes M., Evans R.M., Brenner D.A., Schnabl B. (2017) Modulation of the intestinal bile acid-FXR-FGF15 axis improves alcoholic liver disease in mice. Hepatology. doi: 10.1002/hep.29676.

PubMed Central ID: 

Xu J., Ma H.Y., Liang S., Sun M., Karin G., Koyama Y., Hu R., Quehenberger O., Davidson N.O., Dennis E.A., Kisseleva T., Brenner D.A. (2017) The role of human cytochrome P450 2E1 in liver inflammation and fibrosis. Hepatol Commun. 1(10):1043-1057. doi: 10.1002/hep4.1115. eCollection 2017 Dec.

PubMed Central ID: 

Song IJ, Yang YM, Inokuchi-Shimizu S, Roh YS, Yang L, Seki E. (2018) The contribution of toll-like receptor signaling to the development of liver fibrosis and cancer in hepatocyte-specific TAK1-deleted mice. Int J Cancer. 142(1):81-91. doi: 10.1002/ijc.31029. Epub 2017 Sep 23.

PubMed Central ID: 

Caussy C., Soni M., Cui J., Bettencourt R., Schork N., Chen C.H., Ikhwan M.A., Bassirian S., Cepin S., Gonzalez M.P., Mendler M., Kono Y., Vodkin I., Mekeel K., Haldorson J., Hemming A., Andrews B., Salotti J., Richards L., Brenner D.A., Sirlin C.B., Loomba R., Familial NAFLD Cirrhosis Research Consortium. (2017) Nonalcoholic fatty liver disease with cirrhosis increases familial risk for advanced fibrosis. J Clin Invest. 127(7):2697-2704. doi: 10.1172/JCI93465. Epub 2017 Jun 19.

PubMed Central ID: 

Vollmann E.H., Cao L., Amatucci A., Reynolds T., Hamann S., Dalkilic-Liddle I., Cameron T.O., Hossbach M., Kauffman K.J., Mir F.F., Anderson D.G., Novobrantseva T., Koteliansky V., Kisseleva T., Brenner D., Duffield J., Burkly L.C. (2017) Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing. Mol Ther Nucleic Acids. 7:314-323. doi: 10.1016/j.omtn.2017.04.014. Epub 2017 Apr 20.

PubMed Central ID: 

Reinders M.E., Wardi G., Bettencourt R., Bouland D., Bazick J., Mendler M., Vodkin I., Kalmaz D., Savides T., Brenner D., Sell R.E., Loomba R. (2017) Increased Risk of Death, in the Hospital and Outside the Intensive Care Unit, for Patients With Cirrhosis After Cardiac Arrest. Clin Gastroenterol Hepatol. 15(11):1808-1810. doi: 10.1016/j.cgh.2017.05.044. Epub 2017 Jun 7.

PubMed Central ID: 

Kim R.G., Loomba R., Prokop L.J., Singh S. (2017) Statin Use and Risk of Cirrhosis and Related Complications in Patients With Chronic Liver Diseases: A Systematic Review and Meta-analysis. Clin Gastroenterol Hepatol. 15(10):1521-1530.e8. doi: 10.1016/j.cgh.2017.04.039. Epub 2017 May 4.

PubMed Central ID: 

Loomba R., Seguritan V., Li W., Long T., Klitgord N., Bhatt A., Dulai P.S., Caussy C., Bettencourt R., Highlander S.K., Jones M.B., Sirlin C.B., Schnabl B., Brinkac L., Schork N., Chen C.H., Brenner D.A., Biggs W., Yooseph S., Venter J.C., Nelson K.E. (2017) Gut Microbiome-Based Metagenomic Signature for Non-invasive Detection of Advanced Fibrosis in Human Nonalcoholic Fatty Liver Disease. Cell Metab. 25(5):1054-1062.e5. doi: 10.1016/j.cmet.2017.04.001.

PubMed Central ID: 

Koyama Y., Wang P., Liang S., Iwaisako K., Liu X., Xu J., Zhang M., Sun M., Cong M., Karin D., Taura K., Benner C., Heinz S., Bera T., Brenner D.A., Kisseleva T. (2017) Mesothelin/mucin 16 signaling in activated portal fibroblasts regulates cholestatic liver fibrosis. J Clin Invest. 127(4):1254-1270. doi: 10.1172/JCI88845. Epub 2017 Mar 13.

PubMed Central ID: 

Dulai P.S., Singh S., Patel J., Soni M., Prokop L.J., Younossi Z., Sebastiani G., Ekstedt M., Hagstrom H., Nasr P., Stal P., Wong V.W., Kechagias S., Hultcrantz R., Loomba R. (2017) Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta-analysis. Hepatology. 65(5):1557-1565. doi: 10.1002/hep.29085. Epub 2017 Mar 31.

PubMed Central ID: 

Lin S.C., Heba E., Bettencourt R., Lin G.Y., Valasek M.A., Lunde O., Hamilton G., Sirlin C.B., Loomba R. (2017) Assessment of treatment response in non-alcoholic steatohepatitis using advanced magnetic resonance imaging. Aliment Pharmacol Ther. 45(6):844-854. doi: 10.1111/apt.13951. Epub 2017 Jan 24.

PubMed Central ID: 

Koyama Y., Brenner D.A. (2017) Liver inflammation and fibrosis. J Clin Invest. 127:55-64. doi: 10.1172/JCI88881.

PubMed Central ID: 

Park C.C., Nguyen P., Hernandez C., Bettencourt R., Ramirez K., Fortney L., Hooker J., Sy E., Savides M.T., Alquiraish M.H., Valasek M.A., Rizo E., Richards L., Brenner D., Sirlin C.B., Loomba R. (2017) Magnetic Resonance Elastography vs Transient Elastography in Detection of Fibrosis and Noninvasive Measurement of Steatosis in Patients With Biopsy-Proven Nonalcoholic Fatty Liver Disease. Gastroenterology. 152:598-607. doi: 10.1053/j.gastro.2016.10.026.

PubMed Central ID: 

Koyama Y., Xu J., Liu X., Brenner D.A. (2016) New Developments on the Treatment of Liver Fibrosis. Dig Dis. 34:589-96. doi: 10.1159/000445269.

PubMed Central ID: 

Wang P., Koyama Y., Liu X., Xu J., Ma H.Y., Liang S., Kim I.H., Brenner D.A., Kisseleva T. (2016) Promising Therapy Candidates for Liver Fibrosis. Front Physiol. 7:47. doi: 10.3389/fphys.2016.00047.

PubMed Central ID: 

Liang S., Kisseleva T., Brenner D.A. (2017) The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts. Front Physiol. 7:17. doi: 10.3389/fphys.2016.00017. eCollection 2016.

PubMed Central ID: 

Brenner, D. A., Paik, Y. H., Schnabl, B. (2015) Role of Gut Microbiota in Liver Disease. J Clin Gastroenterol. 49 Suppl 1:S25-7. doi: 10.1097/MCG.0000000000000391.

PubMed Central ID: 

Lan, T., Kisseleva, T., Brenner, D.A. (2015) Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation. PLoS One. 10(7):e0129743. doi: 10.1371/journal.pone.0129743.

PubMed Central ID: 

Koyama, Y., Brenner, D. A. (2015) New therapies for hepatic fibrosis. Clin Res Hepatol Gastroenterol. 39 Suppl 1, S75-9. doi: 10.1016/j.clinre.2015.06.011.

PubMed Central ID: 

Seki, E., Brenner, D. A. (2015) Recent advancement of molecular mechanisms of liver fibrosis. J Hepatobiliary Pancreat Sci. 22(7):512-8. doi: 10.1002/jhbp.245

PubMed Central ID: 

Kim, I. H., Kisseleva, T., Brenner, D. A. (2015) Aging and liver disease. Curr Opin Gastroenterol. 31(3):184-91. doi: 10.1097/MOG.0000000000000176.

PubMed Central ID: 

Fang, S., Suh, J. M., Reilly, S. M., Yu, E., Osborn, O., Lackey, D., Yoshihara, E., Perino, A., Jacinto, S., Lukasheva, Y., Atkins, A. R., Khvat, A., Schnabl, B., Yu, R. T., Brenner, D. A., Coulter, S., Liddle, C., Schoonjans, K., Olefsky, J. M., Saltiel, A. R., Downes, M., Evans, R. M. (2015) Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance. Nat Med. 21(2):159-65. doi: 10.1038/nm.3760.

PubMed Central ID: 

Mazagova, M., Wang, L., Anfora, A. T., Wissmueller, M., Lesley, S. A., Miyamoto, Y., Eckmann, L., Dhungana, S., Pathmasiri, W., Sumner, S., Westwater, C., Brenner, D. A., Schnabl, B. (2015) Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice. FASEB J. 29(3):1043-55. doi: 10.1096/fj.14-259515.

PubMed Central ID: 

Lopez-Sanchez, I., Dunkel, Y., Roh, Y. S., Mittal, Y., De Minicis, S., Muranyi, A., Singh, S., Shanmugam, K., Aroonsakool, N., Murray, F., Ho, S. B., Seki, E., Brenner, D. A., Ghosh, P. (2014) GIV/Girdin is a central hub for profibrogenic signalling networks during liver fibrosis. Nat Commun. 5:4451. doi: 10.1038/ncomms5451.

PubMed Central ID: 

Inokuchi-Shimizu, S., Park, E. J., Roh, Y. S., Yang, L., Zhang, B., Song, J., Liang, S., Pimienta, M., Taniguchi, K., Wu, X., Asahina, K., Lagakos, W., Mackey, M. R., Akira, S., Ellisman, M. H., Sears, D. D., Olefsky, J. M., Karin, M., Brenner, D. A., Seki, E. (2014) ETAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis. J Clin Invest. 124(8), 3566-78. doi: 10.1172/JCI74068.

PubMed Central ID: 

Liu, C., Chen, X., Yang, L., Kisseleva, T., Brenner, D. A., Seki, E. (2014) Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells. J Biol Chem. 289(10):7082-91. doi: 10.1074/jbc.M113.543769.

PubMed Central ID: 

Schnabl, B., Brenner, D. A. (2014) Interactions between the intestinal microbiome and liver diseases. Gastroenterology. 146(6):1513-24. doi: 10.1053/j.gastro.2014.01.020.

PubMed Central ID: 

Paik, Y. H., Kim, J., Aoyama, T., De Minicis, S., Bataller, R., Brenner, D. A. (2014) Role of NADPH Oxidases in Liver Fibrosis. Antioxid Redox Signal. 20(17):2854-72. doi: 10.1089/ars.2013.5619.

PubMed Central ID: 

Madsen, D. H., Leonard, D., Masedunskas, A., Moyer, A., Jürgensen, H. J., Peters, D. E., Amornphimoltham, P., Selvaraj, A., Yamada, S. S., Brenner, D. A., Burgdorf, S., Engelholm, L. H., Behrendt, N., Holmbeck, K., Weigert, R., Bugge, T. H. (2013) M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway J Cell Biol. 202(6):951-66. doi: 10.1083/jcb.201301081.

PubMed Central ID: 

Seki, E., Brenner, D. A., Karin, M. (2012) A liver full of JNK: signaling in regulation of cell function and disease pathogenesis, and clinical approaches. Gastroenterology. 143(2), 307-20. doi: 10.1053/j.gastro.2012.06.004.

PubMed Central ID: 

Österreicher, C.H., Penz-Österreicher, M., Grivennikov, S. I., Guma, M., Koltsova, E. K., Datz, C., Sasik, R., Hardiman, G., Karin, M., Brenner, D. A., (2011) Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver. Proc Natl Acad Sci USA. 108(1), 308-13. doi: 10.1073/pnas.1017547108.

Main Contact Information


Project Leaders
  • Dr. David A. Brenner
    Dean, UCSD School of Medicine; Vice Chancellor for Health Sciences

  • Dr. Rohit Loomba
    Professor, Division of Gastroenterology, Department of Medicine, UCSD School of Medicine
Superfund Project Members
  • Tatiana Kisseleva, Researcher
  • Jacopo Baglieri, Post-Doctoral Scholar
  • Shuang Liang, Post-Doctoral Scholar
  • Karin Diggle, Lab Manager

Contact

UCSD Superfund Research Center
University of California, San Diego
Pharmacology Department
9500 Gilman Drive, Mail Code 0722
La Jolla, CA 92093-0722