Blocking integrin α4β7-mediated CD4 T cell recruitment to the intestine and liver protects mice from western diet-induced non-alcoholic steatohepatitis - PubMed (original) (raw)

. 2020 Nov;73(5):1013-1022.

doi: 10.1016/j.jhep.2020.05.047. Epub 2020 Jun 12.

Yunshan Liu 2, Smita S Iyer 3, Silvia Liu 4, Biki Gupta 1, Chirayu Desai 5, Pradeep Kumar 2, Tekla Smith 2, Aatur D Singhi 6, Asma Nusrat 7, Charles A Parkos 7, Satdarshan P Monga 8, Mark J Czaja 2, Frank A Anania 9, Reben Raeman 10

Affiliations

Blocking integrin α4β7-mediated CD4 T cell recruitment to the intestine and liver protects mice from western diet-induced non-alcoholic steatohepatitis

Ravi P Rai et al. J Hepatol. 2020 Nov.

Abstract

Background & aims: The heterodimeric integrin receptor α4β7 regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of α4β7-mediated recruitment of CD4 T cells to the intestine and liver in NASH.

Methods: Male littermate F11r+/+ (control) and junctional adhesion molecule A knockout F11r-/- mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR.

Results: WD-fed knockout mice developed NASH and had increased hepatic and intestinal α4β7+ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in α4β7+ CD4 T cells was associated with markedly higher expression of the α4β7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. α4β7 blockade in WD-fed knockout mice significantly decreased α4β7+ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α4 and β7 integrins.

Conclusions: These findings establish α4β7/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment.

Lay summary: Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor α4β7-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of α4β7 antibody in the treatment of human NASH.

Keywords: Epithelial barrier; Gut permeability; Inflammation; Microbiota; Non-alcoholic fatty liver disease.

Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Conflict of interest statement

Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details.

Figures

Figure 1.

Figure 1.. Western diet increases integrin α4β7+ CD4 T cells in the peripheral blood.

(A) Gating strategy for the isolation of CD4 T cells. Plots progressively gated on lymphocytes, singlets, live, CD3+, CD8+ T cells. (B) Representative flow plots show percent of CD4 T cells, and scatter plots show total number and percentage of CD4 T cells in peripheral blood of F11r +/+ and F11r −/− mice fed a normal diet (ND) or a western diet (WD) for eight-weeks (n = 5 – 7 mice per group). (C) Representative flow plots show percent of α4β7+ CD4 T cells, and scatter plots show total number and percentage of α4β7+ CD4 T cells in peripheral blood (n = 5 – 7 mice per group). Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between F11r +/+ and F11r −/− mice fed an identical diet. Hashtags indicate significant differences (p < 0.05) between ND or WD-fed F11r +/+ and F11r −/− mice.

Figure 2.

Figure 2.. Western diet increases α4β7+ CD4 T cell recruitment to the intestine.

(A-B) Scatter plots show total number and percentage of (A) CD4 T cells (n = 5 −12 mice per group) and (B) α4β7+ CD4 T cells (n = 5 – 8 mice per group) in Peyer’s patches (PP) of F11r +/+ and F11r −/− mice fed a normal diet (ND) or western diet (WD) for eight-weeks. (C-D) Scatter plots show total number and percentage of (C) CD4 T cells and (D) α4β7+ CD4 T cells in the colonic lamina propria (LP; n = 4 – 6 mice per group). Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between F11r +/+ and F11r −/− mice fed an identical diet. Hashtags indicate significant differences (p < 0.05) between ND or WD-fed F11r +/+ and F11r −/− mice. (E) Representative confocal images of CD4 (green) and α4β7 (red) immunofluorescence in the colonic mucosa of F11r −/− mice fed a WD for eight weeks (n = 5 mice per group). Nuclei are stained blue. Scale bar 100 μm.

Figure 3.

Figure 3.. Western diet promotes microbiota-dependent colonic MAdCAM-1 expression.

(A-B) Representative confocal images of MAdCAM-1 (red) immunofluorescence in the colonic mucosa of F11r +/+ and F11r −/− mice fed a western diet (WD) or WD plus antibiotics for eight-weeks (n = 5 mice per group). Nuclei are stained blue. White arrowheads, MAdCAM-1 expression. Scale bar 100 μm. (C) Quantification of MAdCAM-1 expression in the colon of F11r +/+ and F11r −/− mice fed a WD or WD plus antibiotics for eight weeks (n = 5 mice per group). Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between F11r +/+ and F11r −/− mice fed a WD. Hashtags indicate significant differences (p < 0.05) between WD or WD plus antibiotics fed F11r −/− mice. (D-E) Cecum mucosa-associated microbiota from F11r +/+ and F11r −/− mice fed a ND or WD for eight-weeks were analyzed using 16S rRNA sequencing followed by phylogenetic analysis and a comparison of the microbial community structure using the unweighted UniFrac algorithm (n = 4 – 5 mice per group). (D) Jackknifed principal coordinate analysis (PCoA) of the un-weighted UniFrac distance matrix of the mucosa-associated microbiota. The ovals represent clustering by treatment groups. (E) Relative abundance of mucosa-associated bacterial phyla in F11r +/+ and F11r −/− mice. Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between F11r +/+ and F11r −/− mice fed an identical diet. Hashtags indicate significant differences (p < 0.05) between the ND or WD-fed F11r +/+ and F11r −/− mice.

Figure 4.

Figure 4.. Western diet-induced increase in hepatic MAdCAM-1 expression is associated with increased recruitment of α4β7+ CD4 T cells to the liver.

(A) Representative confocal images of MAdCAM-1 (red) immunofluorescence in the liver of F11r +/+ and F11r −/− mice fed a normal (ND) or western diet (WD), or WD plus antibiotics for eight-weeks (n = 5 mice per group). Nuclei are stained blue. White arrowheads, MAdCAM-1 expression. Scale bar 100 μm. (B) Quantification of MAdCAM-1 expression in the liver of F11r +/+ and F11r −/− mice fed a WD or WD plus antibiotics for eight weeks (n = 5 mice per group). (C) Representative confocal images of MAdCAM-1 (red) and αSMA (green) immunofluorescence in the liver of F11r −/− mice fed a WD for eight weeks (n = 5 mice per group). Nuclei are stained blue. Scale bar 100 μm. (D-E) Scatter plots show total number and percentage of (D) α4β7+ CD4 T cells (n = 5 – 8 mice per group) and (E) CD4 T cells (n = 5 – 11 mice per group) in the liver of F11r +/+ and F11r −/− mice fed a ND or WD for 8 weeks. (F) Representative confocal images of CD4 (green) and α4β7 (red) immunofluorescence in the liver of F11r −/− mice fed a WD for eight weeks (n = 5 mice per group). Nuclei are stained blue. Scale bar 100 μm. (G-H) Expression of (E) integrin α4, Itga4 and (F) integrin β7, Itgb7 in the liver of F11r −/− mice fed a WD for eight weeks (n = 5 mice per group). Data are presented as means ± SEM. Asterisks indicate significant differences (p < 0.05) between F11r +/+ and F11r −/− mice fed an identical diet. Hashtags indicate significant differences (p < 0.05) between ND or WD-fed control (F11r +/+) and F11r −/− mice.

Figure 5.

Figure 5.. α4β7 blockade decreases α4β7+ CD4 T cell recruitment to the intestine.

(A-B) Scatter plots show total number and percentage of (A) α4β7+ CD4 T cells and (B) CD4 T cells in the Peyer’s patches (n = 4 – 6 mice per group). (C-D) Scatter plots show total number and percentage of (C) α4β7+ CD4 T cells and (D) CD4 T cells in the colonic lamina propria (LP) (n = 3 – 4 mice per group). (E-I) Expression of (E) Madcam1, (F) integrin α4, Itga4, (G) integrin β7, Itga7, (H) occludin and (I) Zo1 in the colonic mucosa (n = 5 mice per group). F11r −/− mice fed a western diet (WD) for eight-weeks were treated with IgG or α4β7 mAb for four weeks starting at week four after the initiation of WD. Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between IgG and α4β7 mAb treatment.

Figure 6.

Figure 6.. α4β7 blockade attenuates hepatic inflammation and fibrosis.

(A-B) Scatter plots show total number and percentage of (A) α4β7+ CD4 T cells and (B) CD4 T cells in the liver (n = 7 – 8 mice per group). F11r −/− mice fed a WD for eight weeks were treated with IgG or α4β7 mAb for four weeks starting at week four after initiation of the WD (n = 10 mice per group). (C-E) Expression of (C) Madcam1, (D) integrin α4, Itga4 and (E) integrin β7, Itgb7 in the liver (n = 5 mice per group). (F) Representative photomicrographs of Hematoxylin and Eosin (H&E) stained liver tissue sections. Black arrowheads, immune cells. (G-H) Serum ALT and AST levels. (I-K) Expression of key molecules associated with hepatic inflammation (n = 10 mice per group). (L) Representative photomicrographs of Sirius Red-stained liver tissue sections. Blue arrowheads, collagen deposition. (M-P) Expression of hepatic stellate cell activation markers and markers of fibrosis in the liver (n = 10 mice per group). Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between IgG and α4β7 mAb treatment.

Figure 7.

Figure 7.. α4β7 blocking improves metabolic syndrome.

Changes in (A) body, and (B) liver and (C) visceral fat weight reported as percent of body weight. F11r −/− mice fed a WD for eight-weeks were treated with IgG or α4β7 mAb for four weeks starting at week four following initiation of the WD (n = 10 mice per group). (D) Glucose and (E) insulin tolerance after four weeks of IgG or α4β7 mAb treatment (n = 5 mice per group). (F) Confocal microscopic images of BODIPY (lipids) stained liver tissue sections. Scale bar 20 μm. (G) Volcano plot showing differentially regulated genes following α4β7 mAb treatment. (H) Heatmap for the differentially expressed genes. (I) Ingenuity Pathway Analysis of most significantly enriched signaling pathways. Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between IgG and α4β7 mAb treatment.

Figure 8.

Figure 8.. MAdCAM-1 expression is higher in the liver of NASH patients and correlates with increased integrin a4 and b7 expression in the liver.

(A) Representative photomicrographs of Hematoxylin and Eosin (H&E) stained human liver tissue biopsies obtained from NASH patients (n = 15) and subjects without NASH (controls; n = 8). Scale bar 100 μm. (B-C) Serum AST and ALT levels. (D) Representative confocal images of MAdCAM-1 (green) immunofluorescence in the liver of NASH patients and subjects without NASH. Nuclei are stained blue. Scale bar 100 μm. (E-H) Expression of (E) MADCAM1, (F) ACTA2, (G) integrin α4, ITGA4 and (H) integrin β7, ITGB7 in the liver (n = 5). Data are presented as mean ± SEM. Asterisks indicate significant differences (p < 0.05) between controls and NASH patients.

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