Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability

Jay Luther, John J Garber, Hamed Khalili, Maneesh Dave, Shyam Sundhar Bale, Rohit Jindal, Daniel L Motola, Sanjana Luther, Stefan Bohr, Soung Won Jeoung, Vikram Deshpande, Gurminder Singh, Jerrold R Turner, Martin L Yarmush, Raymond T Chung, Suraj J Patel, Jay Luther, John J Garber, Hamed Khalili, Maneesh Dave, Shyam Sundhar Bale, Rohit Jindal, Daniel L Motola, Sanjana Luther, Stefan Bohr, Soung Won Jeoung, Vikram Deshpande, Gurminder Singh, Jerrold R Turner, Martin L Yarmush, Raymond T Chung, Suraj J Patel

Abstract

Background & aims: Emerging data suggest that changes in intestinal permeability and increased gut microbial translocation contribute to the inflammatory pathway involved in nonalcoholic steatohepatitis (NASH) development. Numerous studies have investigated the association between increased intestinal permeability and NASH. Our meta-analysis of this association investigates the underlying mechanism.

Methods: A meta-analysis was performed to compare the rates of increased intestinal permeability in patients with NASH and healthy controls. To further address the underlying mechanism of action, we studied changes in intestinal permeability in a diet-induced (methionine-and-choline-deficient; MCD) murine model of NASH. In vitro studies were also performed to investigate the effect of MCD culture medium at the cellular level on hepatocytes, Kupffer cells, and intestinal epithelial cells.

Results: Nonalcoholic fatty liver disease (NAFLD) patients, and in particular those with NASH, are more likely to have increased intestinal permeability compared with healthy controls. We correlate this clinical observation with in vivo data showing mice fed an MCD diet develop intestinal permeability changes after an initial phase of liver injury and tumor necrosis factor-α (TNFα) induction. In vitro studies reveal that MCD medium induces hepatic injury and TNFα production yet has no direct effect on intestinal epithelial cells. Although these data suggest a role for hepatic TNFα in altering intestinal permeability, we found that mice genetically resistant to TNFα-myosin light chain kinase (MLCK)-induced intestinal permeability changes fed an MCD diet still develop increased permeability and liver injury.

Conclusions: Our clinical and experimental results strengthen the association between intestinal permeability increases and NASH and also suggest that an early phase of hepatic injury and inflammation contributes to altered intestinal permeability in a fashion independent of TNFα and MLCK.

Keywords: Meta-Analysis; Myosin Light Chain Kinase; Steatosis; Tight Junctions.

Figures

Supplementary Figure 1
Supplementary Figure 1
Flow diagram for meta-analysis.
Figure 1
Figure 1
Meta-analysis of increased intestinal permeability rates in nonalcoholic fatty liver disease (NAFLD) patients versus healthy controls. (A) Forest plot of increased intestinal permeability in patients with NAFLD as compared to healthy controls using a fixed-effects model. (B) Exclusion sensitivity plot of increased intestinal permeability in NAFLD patients versus healthy controls. CI, confidence interval; OR, odds ratio.
Figure 2
Figure 2
Meta-analysis of increased intestinal permeability rates in nonalcoholic steatohepatitis (NASH) patients versus healthy controls. (A) Forest plot of increased intestinal permeability in patients with NASH as compared with healthy controls using a fixed-effects model. (B) Exclusion sensitivity plot of increased intestinal permeability in NASH patients versus healthy controls. CI, confidence interval; OR, odds ratio.
Figure 3
Figure 3
Temporal characterization of liver injury and intestinal permeability changes in a murine dietary nonalcoholic steatohepatitis (NASH) model. C57BL/6 mice (N = 5 mice/group) were fed a diet deficient in methionine and choline (MCD) and were sacrificed at multiple time points up to 21 days. We found evidence for significant MCD-induced liver injury as early as day 6 based on both (A) serum alanine aminotransferase (ALT) and (B, C) H&E liver histology (original magnification: 20×) that progressed to a peak value at day 21. Specifically, liver histologic examination revealed a progressively increasing number of inflammatory foci and steatosis throughout the experiment. In parallel, hepatic mRNA expression and systemic levels of tumor necrosis factor-α (TNF-α) were elevated at an early phase of the diet (D, E).Temporal evaluation of intestinal permeability changes, based on (F) fluorescein isothiocyanate (FITC) dextran serum measurements, and tight junction architecture based on (G) immunofluorescence staining for zona-occludens-1 (ZO-1), revealed evidence for a significant increase in intestinal permeability and disruption of normal tight junction architecture (loss of chicken-wire appearance of ZO-1: arrows) at day 10. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 4
Figure 4
In vitro assessment of MCD-induced changes to the liver and intestine. Rat hepatocytes (H35) were grown in coculture with primary rat Kupffer cells (KC) at a ratio of 2:1. Cells were exposed to standard medium (SM) or methionine-and-choline-deficient (MCD) medium for 24 hours, after which the supernatant was harvested for further analysis. (A) Coculturing of hepatocytes and KCs in the presence of MCD caused the most significant elevation in hepatocyte injury, based on supernatant levels of alanine aminotransferase (ALT). (B) Further, MCD-exposed hepatocytes and KCs produced significantly more tumor necrosis factor-α (TNF-α) compared to cells grown in standard medium. (C) Intestinal epithelial cells (Caco2 cells) were grown to confluence and allowed to form strong tight junctions, after which they were exposed to either MCD medium or SM. We found no difference in tight junction function between cells grown in MCD medium versus SM, suggesting MCD medium is not directly toxic to these cells. TEER, transepithelial electrical resistance.
Figure 5
Figure 5
In vivo assessment of tumor necrosis factor-α (TNF-α) on intestinal permeability in nonalcoholic steatohepatitis (NASH) pathogenesis. TNF-α-induced increases in intestinal permeability are mediated through myosin light chain kinase (MLCK); therefore, genetic deletion of MLCK renders mice impervious to intestinal permeability changes caused by TNF-α. We tested whether mice deficient in the long isoform of MLCK would be protected against MCD-induced liver injury and intestinal permeability changes. MLCK-knockout (KO) and wild-type (WT) mice were fed the MCD diet for 24 days and then euthanized (N = 5 mice per group). There were no differences in (A) serum levels of ALT or (B) in H&E liver histology (original magnification: 20×) between MLCK-KO and WT mice. (C) Furthermore, we were unable to detect a difference in intestinal permeability changes between the MCD-fed MLCK-KO and WT mice.

References

    1. Szczepaniak L.S., Nurenberg P., Leonard D. Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population. Am J Physiol Endocrinol Metab. 2005;288:E462–E468.
    1. Dixon J.B., Bhathal P.S., O’Brien P.E. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology. 2001;121:91–100.
    1. Gabele E., Dostert K., Hofmann C. DSS induced colitis increases portal LPS levels and enhances hepatic inflammation and fibrogenesis in experimental NASH. J Hepatol. 2011;55:1391–1399.
    1. Henao-Mejia J., Elinav E., Jin C. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature. 2012;482:179–185.
    1. Pendyala S., Neff L.M., Suarez-Farinas M., Holt P.R. Diet-induced weight loss reduces colorectal inflammation: implications for colorectal carcinogenesis. Am J Clin Nutr. 2011;93:234–242.
    1. Zhu L., Baker S.S., Gill C. Characterization of gut microbiome in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH. Hepatology. 2013;57:601–609.
    1. DerSimonian R., Laird N. Meta-analysis in clinical trials. Control Clin Trials. 1986;7:177–188.
    1. Higgins J.P., Thompson S.G., Deeks J.J. Measuring inconsistency in meta-analysis. BMJ. 2003;327:557–560.
    1. Wells GA, Shea B, O’Connell D, et al. The Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses. Ottawa: Ottawa Health Research Institute, nd. Available at: .
    1. Sterne J.A., Gavaghan D., Egger M. Publication and related bias in meta-analysis: power of statistical tests and prevalence in the literature. J Clin Epidemiol. 2000;53:1119–1129.
    1. Kleiner D.E., Brunt E.M., Van Natta M. Design and validation of a histological scoring system for nonalocholic fatty liver disease. Hepatology. 2005;41:1313–1321.
    1. Napolitano L.M., Koruda M.J., Meyer A.A., Baker C.C. The impact of femur fracture with associated soft tissue injury on immune function and intestinal permeability. Shock. 1996;5:202–507.
    1. Clayburgh D.R., Barrett T.A., Tang Y. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell-activation-induced diarrhea in vivo. J Clin Invest. 2005;115:2702–2715.
    1. King K.R., Wang S., Irimia D. A high-throughput microfluidic real-time gene expression living cell array. Lab Chip. 2007;7:77–85.
    1. McMullen M., Pritchard M., Nagy L. Isolation of Kupffer cells from rates fed chronic ethanol. In: Nagy L.E., editor. Alcohol: methods and protocols. Humana Press; Totowa, NJ: 2008. pp. 199–212.
    1. Sahai A., Pan X., Paul R. Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium. Am J Physiol Gastrointest Liver Physiol. 2006;291:G55–G62.
    1. Wigg A.J., Roberts-Thomson I.C., Dymock R.B. The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor α in the pathogenesis of non-alcoholic steatohepatitis. Gut. 2001;48:206–211.
    1. Farhadi A., Gundlapalli S., Shaikh M. Susceptibility to gut leakiness: a possible mechanism for endotoxaemia in non-alcoholic steatohepatitis. Liver Int. 2008;28:1026–1033.
    1. Miele L., Valenza V., La Torre G. Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology. 2009;49:1877–1887.
    1. Volynets V., Kuper M.A., Strahl S. Nutrition, intestinal permeability, and blood ethanol levels are altered in patients with nonalcoholic fatty liver disease. Dig Dis Sci. 2012;57:1932–1941.
    1. Giorgio V., Miele L., Principessa L. Intestinal permeability is increased in children with non-alcoholic fatty liver disease, and correlates with liver disease severity. Dig Liver Dis. 2014;46:556–560.
    1. van Wijck K., Bessems B.A., van Eijk H.M. Polyethylene glycol versus dual sugar assay for gastrointestinal permeability analysis: is it time to choose? Clin Exp Gastroenterol. 2012;5:139–150.
    1. Miura K., Kodama Y., Inokucki S. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice. Gastroenterology. 2010;139:323–334.
    1. Mas E., Danjoux M., Garcia V. IL-6 deficiency attenuates murine diet-induced non-alcoholic steatohepatitis. PLoS ONE. 2009;4:e7929.
    1. Marchiando A.M., Shen L., Graham W.V. Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo. J Cell Biol. 2010;189:111–126.
    1. Su L., Nalle S.C., Shen L. TNFR2 activates MLCK-dependent tight junction dysregulation to cause apoptosis-mediated barrier loss and experimental colitis. Gastroenterology. 2013;145:407–415.
    1. Bardella M.T., Valenti L., Pagliari C. Searching for coeliac disease in patients with non-alcoholic fatty liver disease. Dig Liver Dis. 2004;36:333–336.
    1. McGowan C.E., Jones P., Long M.D., Barritt A.S., 4th Changing shape of disease: nonalcoholic fatty liver disease in Crohn’s disease—a case series and review of the literature. Inflamm Bowel Dis. 2012;18:49–54.
    1. Reddy S.K., Zhan M., Alexander H.R., El-Kamary S.S. Nonalcoholic fatty liver disease is associated with benign gastrointestinal disorders. World J Gastroenterol. 2013;19:8301–8311.
    1. Ma Y.Y., Li L., Yu C.H. Effects of probiotics on nonalcoholic fatty liver disease: a meta-analysis. World J Gastroenterol. 2013;19:6911–6918.
    1. Du Plessis J., Vanheel H., Janssen C.E. Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function. J Hepatol. 2013;58:1125–1132.
    1. Seki E., Schanbl B. Role of innate immunity and the microbiota in liver fibrosis: crosstalk between the liver and gut. J Physiol. 2012;590:447–458.
    1. Crespo J., Cayon A., Fernandez-Gil P. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology. 2001;34:1158–1163.
    1. Alaaeddine N., Sidaoui J., Hilal G. TNF-α messenger ribonucleic acid (mRNA) in patients with nonalcoholic steatohepatitis. Eur Cytokine Netw. 2012;23:107–111.
    1. Bahccioglu I.H., Yalniz M., Ataseven H. Levels of serum hyaluronic acid, TNF-alpha and IL-8 in patients with nonalcoholic steatohepatitis. Hepatogastroenterology. 2005;52:1549–1553.
    1. Zolotarevsky Y., Hecht G., Koutsouris A. A membrane-permeant peptide that inhibits mlc kinase restores barrier function in in vitro models of intestinal disease. Gastroenterology. 2002;123:163–172.
    1. Wang F., Graham W.V., Wang Y. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005;166:409–419.
    1. Al-Sadi R., Guo S., Ye D. Mechanism of IL-1β modulation of intestinal epithelial barrier involves p38 kinase and activating transcription factor-2 activation. J Immunol. 2013;190:6596–6606.
    1. Lee J.K., Bettencourt R., Brenner D. Association between serum interleukin-6 concentrations and mortality in older adults: the Rancho Bernardo study. PLoS ONE. 2012;7:e34218.
    1. Suzuki T., Yoshinaga N., Tanabe S. Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium. J Biol Chem. 2011;286:31263–31271.
    1. Csak T., Pillai A., Ganz M. Both bone marrow-derived and non-bone marrow-derived cells contribute to AIM2 and NLRP3 inflammasome activation in a MyD88-dependent manner in dietary steatohepatitis. Liver Int. 2014;34:1402–1413.
    1. Pena A.D., Leclercq I., Field J. NF-κB activation, rather than TNF, mediates hepatic inflammation in a murine dietary model of steatohepatitis. Gastroenterology. 2005;129:1663–1674.
    1. Tomita K., Tamiya G., Ando S. Tumour necrosis factor signalling through activation of Kupffer cells plays an essential role in liver fibrosis of nonalcoholic steatohepatitis in mice. Gut. 2006;55:415–424.
    1. Koca S.S., Bahcecioglu I.H., Poyrazoglu O.K. The treatment with antibody to TNF-alpha reduces the inflammation, necrosis, and fibrosis in the non-alcoholic steatohepatitis induced by the methionine-and choline-deficient diet. Inflammation. 2008;31:91–98.
    1. Pinto Lde F., Compri C.M., Fornari J.V. The immunosuppressant drug, thalidomide, improves hepatic alterations induced by a high-fat diet in mice. Liver Int. 2010;30:603–610.
    1. Campanati A., Ganzetti G., Di Sario A. The effect of etanercept on hepatic fibrosis risk in patients with non-alcoholic fatty liver disease, metabolic syndrome, and psoriasis. J Gastroenterol. 2013;48:839–846.
    1. Chen P., Starkel P., Turner J.R. Dysbiosis-induced intestinal inflammation activates TNFRI and mediates alcoholic liver disease in mice. Hepatology. 2014 Published online.
    1. Peled Y., Watz C., Gilat T. Measurement of intestinal permeability using 51Cr-EDTA. Am J Gastroenterol. 1985;80:770–773.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅