Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability - PubMed (original) (raw)

doi: 10.1016/j.jcmgh.2015.01.001.

John J Garber 1, Hamed Khalili 1, Maneesh Dave 2, Shyam Sundhar Bale 3, Rohit Jindal 3, Daniel L Motola 1, Sanjana Luther 1, Stefan Bohr 3, Soung Won Jeoung 1, Vikram Deshpande 1, Gurminder Singh 4, Jerrold R Turner 4, Martin L Yarmush 3, Raymond T Chung 1, Suraj J Patel 3

Affiliations

Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability

Jay Luther et al. Cell Mol Gastroenterol Hepatol. 2015 Mar.

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 (TNF_α_) induction. In vitro studies reveal that MCD medium induces hepatic injury and TNF_α_ production yet has no direct effect on intestinal epithelial cells. Although these data suggest a role for hepatic TNF_α_ in altering intestinal permeability, we found that mice genetically resistant to TNF_α_-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 TNF_α_ and MLCK.

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

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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.

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