Loss of Junctional Adhesion Molecule A Promotes Severe Steatohepatitis in Mice on a Diet High in Saturated Fat, Fructose, and Cholesterol

doi:10.1053/j.gastro.2016.06.022

Gastroenterology

Volume 151, Issue 4, October 2016, Pages 733-746.e12

https://doi.org/10.1053/j.gastro.2016.06.022 Get rights and content

Background & Aims

There is evidence from clinical studies that compromised intestinal epithelial permeability contributes to the development of nonalcoholic steatohepatitis (NASH), but the exact mechanisms are not clear. Mice with disruption of the gene (F11r) encoding junctional adhesion molecule A (JAM-A) have defects in intestinal epithelial permeability. We used these mice to study how disruption of the intestinal epithelial barrier contributes to NASH.

Methods

Male C57BL/6 (control) or F11r^-/- mice were fed a normal diet or a diet high in saturated fat, fructose, and cholesterol (HFCD) for 8 weeks. Liver and intestinal tissues were collected and analyzed by histology, quantitative reverse-transcription polymerase chain reaction, and flow cytometry. Intestinal epithelial permeability was assessed in mice by measuring permeability to fluorescently labeled dextran. The intestinal microbiota were analyzed using 16S ribosomal RNA sequencing. We also analyzed biopsy specimens from proximal colons of 30 patients with nonalcoholic fatty liver disease (NAFLD) and 19 subjects without NAFLD (controls) undergoing surveillance colonoscopy.

Results

F11r^-/- mice fed a HFCD, but not a normal diet, developed histologic and pathologic features of severe NASH including steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis, whereas control mice fed a HFCD developed only modest steatosis. Interestingly, there were no differences in body weight, ratio of liver weight:body weight, or glucose homeostasis between control and F11r^-/- mice fed a HFCD. In these mice, liver injury was associated with significant increases in mucosal inflammation, tight junction disruption, and intestinal epithelial permeability to bacterial endotoxins, compared with control mice or F11r^-/- mice fed a normal diet. The HFCD led to a significant increase in inflammatory microbial taxa in F11r^-/- mice, compared with control mice. Administration of oral antibiotics or sequestration of bacterial endotoxins with sevelamer hydrochloride reduced mucosal inflammation and restored normal liver histology in F11r^-/- mice fed a HFCD. Protein and transcript levels of JAM-A were significantly lower in the intestinal mucosa of patients with NAFLD than without NAFLD; decreased expression of JAM-A correlated with increased mucosal inflammation.

Conclusions

Mice with defects in intestinal epithelial permeability develop more severe steatohepatitis after a HFCD than control mice, and colon tissues from patients with NAFLD have lower levels of JAM-A and higher levels of inflammation than subjects without NAFLD. These findings indicate that intestinal epithelial barrier function and microbial dysbiosis contribute to the development of NASH. Restoration of intestinal barrier integrity and manipulation of gut microbiota might be developed as therapeutic strategies for patients with NASH.

Section snippets

Mice

C57BL/6 (control) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and F11r^-/- mice were originally a gift from Dr Thomas N. Sato (Cornell University, New York, NY). F11r^-/- mice were generated as previously described¹⁴ and were backcrossed to C57BL/6 mice for 7 generations. The intestine-specific JAM-A knockout mice, Villin^CreF11r^FL/FL, were obtained by crossing Villin^Cre mice with F11r^FL/FL mice. C57BL/6, F11r^-/-, F11r^FL/FL, and Villin^CreF11r^FL/FL mice were bred and maintained

F11r^-/- Mice Fed a HFCD Develop Severe Histologic Features of NASH Including Markers of Hepatic Fibrosis

To induce NAFLD, F11r^-/- and control littermates were fed a HFCD for 8 weeks, F11r^-/- and control mice fed a normal diet served as controls. While consumption of HFCD resulted in modest NAFL-related histologic findings in control mice, F11r^-/- mice developed severe histologic features of NASH including ballooning degeneration of hepatocytes, inflammatory cell infiltration, and extensive pericentral, periportal, and sinusoidal fibrosis (Figure 1A, B, and F, and Supplementary Figure 1A and B). As

Discussion

In the current study, we provide experimental evidence that intestinal epithelial barrier function plays a major role in NAFLD progression. The HFCD-fed F11r^-/- model overcomes key limitations that have hindered reliable induction of NASH along with typical features of the metabolic syndrome (MetS) in murine models even after long-term feeding. To date, investigators have struggled to reliably replicate MetS and typical features of human NASH in murine liver, including substantial evidence for

Acknowledgments

The authors acknowledge the contribution of graphic artist Deepali Gupta ([email protected]) for the generation of the summary figure 7.

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: Conflicts of interest The authors disclose no conflicts.

: Funding This work was supported by National Institutes of Health grant DK062092, VA grant I01BX001746, and funds from Emory University School of Medicine (F.A.A.), and the Emory University Integrated Cellular Imaging Microscopy Core of the Emory and Children's Pediatric Research Center.

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Original ResearchFull Report: Basic and Translational—LiverLoss of Junctional Adhesion Molecule A Promotes Severe Steatohepatitis in Mice on a Diet High in Saturated Fat, Fructose, and Cholesterol

Background & Aims

Methods

Results

Conclusions

Section snippets

Mice

F11r-/- Mice Fed a HFCD Develop Severe Histologic Features of NASH Including Markers of Hepatic Fibrosis

Discussion

Acknowledgments

Clin Liver Dis

Gastroenterology

Hepatology

Mucosal Immunol

Immunity

Gastroenterology

J Nutr Biochem

Best Pract Res Clin Gastroenterol

Cell Host Microbe

Kidney Int

Cell Metab

J Hepatol

Trends Microbiol

Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease

Hepatology

The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis

Gut

Small intestinal bacterial overgrowth in nonalcoholic steatohepatitis: association with toll-like receptor 4 expression and plasma levels of interleukin 8

Dig Dis Sci

Metabolic endotoxemia initiates obesity and insulin resistance

Diabetes

Etiopathogenesis of nonalcoholic steatohepatitis

Semin Liver Dis

JAM-A regulates permeability and inflammation in the intestine in vivo

J Exp Med

JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function

Mol Biol Cell

Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice

J Clin Invest

Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition

Am J Physiol Gastrointest Liver Physiol

The impact of femur fracture with associated soft tissue injury on immune function and intestinal permeability

Shock

Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice

Sci Rep

Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample

Proc Natl Acad Sci U S A

Original Research
Full Report: Basic and Translational—Liver
Loss of Junctional Adhesion Molecule A Promotes Severe Steatohepatitis in Mice on a Diet High in Saturated Fat, Fructose, and Cholesterol

F11r^-/- Mice Fed a HFCD Develop Severe Histologic Features of NASH Including Markers of Hepatic Fibrosis