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. 2015 Mar;29(3):1043-55.
doi: 10.1096/fj.14-259515. Epub 2014 Dec 2.

Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice

Magdalena Mazagova  1 , Lirui Wang  1 , Andrew T Anfora  1 , Max Wissmueller  1 , Scott A Lesley  1 , Yukiko Miyamoto  1 , Lars Eckmann  1 , Suraj Dhungana  1 , Wimal Pathmasiri  1 , Susan Sumner  1 , Caroline Westwater  1 , David A Brenner  1 , Bernd Schnabl  2
Affiliations

Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice

Magdalena Mazagova et al. FASEB J. 2015 Mar.

Abstract

Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ-free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin-induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif-deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild-type mice. Isolated Myd88/Trif-deficient hepatocytes were more susceptible to toxin-induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis.

Keywords: bacterial translocation; innate immune system; microbiome.

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Figures

Figure 1.
Figure 1.
Chronic liver injury results in enhanced liver fibrosis in GF mice. GF and conventional mice (Conv) were treated with TAA in the drinking water for 21 wk. As control, GF and conventional mice received drinking water as vehicle (veh) alone. A) Hepatic collagen α1(I) (Col1a1) mRNA. B, C) Collagen deposition was evaluated by Sirius red staining and quantitated by image analysis. Representative sections stained with Sirius red are shown. D, E) Western blots for hepatic SMA and Cyp2e1 proteins. F, G) Hepatic gene expression of cytokines and chemokines. *P < 0.05; **P < 0.01.
Figure 2.
Figure 2.
Increased hepatic reactive oxidative stress and liver cell death in GF mice following TAA administration. A) Liver sections from conventional (Conv) and GF, vehicle, and TAA-treated mice were stained immunohistochemically for 4-HNE. Representative sections are shown. B) Liver lysates were analyzed by immunoblotting with antibodies for Gadd153 (CHOP) and tubulin. The intensity of the bands was quantified using ImageJ, and the level of Gadd153 protein was normalized to the level of tubulin protein as loading control. C) Liver sections were stained to detect dead liver cells using an In Situ Cell Death Detection kit (Roche). Representative sections are shown. D) Forty high-power fields (HPF) per single slide were counted for positively stained cells. *P < 0.05.
Figure 3.
Figure 3.
Enhanced hepatic fibrosis in GF mice subjected to CCl4 injections. GF and conventional (Conv) mice were treated with CCl4 for a total of 12 times. As control, GF and conventional mice received oil (vehicle) injections. A) Hepatic collagen α1(I) (Col1a1) mRNA. B, C) Collagen deposition was evaluated by Sirius red staining and quantitated by image analysis. Representative sections stained with Sirius red are shown. D, E) Western blots for hepatic SMA and Cyp2e1 proteins. F, G) Hepatic gene expression of cytokines and chemokines. (H) Plasma ALT levels. *P ≤ 0.05; **P < 0.01.
Figure 4.
Figure 4.
Myd88/Trif-deficient mice are more susceptible to toxin-induced liver fibrosis. C57BL/6 and Myd88−/−/TrifLPS2/LPS2 mice were injected with oil as control or CCl4 for a total of 12 times. A) Hepatic collagen α1(I) (Col1a1) mRNA. B, C) Collagen deposition was evaluated by Sirius red staining and quantitated by image analysis. Representative sections stained with Sirius red are shown. D, E) Western blots for hepatic SMA and Cyp2e1 proteins. F, G) Hepatic gene expression of cytokines and chemokines. H) Plasma ALT levels. WT, wild-type. *P < 0.05.
Figure 5.
Figure 5.
Toxin-induced cell death is higher in cultured Myd88/Trif-deficient hepatocytes. Nonparenchymal and parenchymal liver cells were isolated from C57BL/6 and Myd88−/−/TrifLPS2/LPS2 mice. A) Primary hepatic stellate cells (HSC) were cultured for 5 d. Expression of fibrotic genes (SMA, Col1a1, and TGF-β1), proliferation marker (PCNA), and Ccl2 was analyzed by quantitative PCR. The mean of 4 different hepatic stellate cell isolations is shown. B) Primary Kupffer cells (KC) were cultured for 1 d before stimulation with LPS (100 ng/ml) or poly (I:C) (10 μg/ml) for 3 h. LPS- and poly (I:C)-induced expression of IL-1β and TNF-α was analyzed by quantitative PCR. The mean of 3 different Kupffer cell isolations is shown. C) Primary Kupffer cells isolated from GF or conventional C57BL/6 mice (Conv) were stimulated with LPS (100 ng/ml) or poly (I:C) (10 μg/ml) for 6 h. IL-1β and TNF-α was analyzed by quantitative PCR. The mean of 3 different Kupffer cell isolations is shown. D) Primary mouse hepatocytes were treated with CCl4 (0.5 mM) for 24 h. CCl4-induced cell death was assessed by measuring ALT in the supernatant and by determining cell death rate. The mean of 6 different hepatocyte isolations is shown. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6.
Figure 6.
Cyp26a1 gene expression in GF and Myd88/Trif-deficient hepatocytes. A) GF and conventional mice (Conv) were treated with TAA in the drinking water for 21 wk. As control, GF and conventional mice received drinking water as vehicle alone. Hepatic Cyp26a1 mRNA expression. B) GF and conventional (Conv) mice were treated with CCl4 for a total of 12 times. As control, GF and conventional mice received oil (vehicle) injections. Hepatic Cyp26a1 mRNA expression. C) Hepatocytes were isolated from C57BL/6 and Myd88−/−/TrifLPS2/LPS2 mice and treated with CCl4 (1 mM) for 24 h. Cyp26a1 mRNA expression. The mean of 6 different hepatocyte isolations is shown. D) Liquid chromatography–mass spectrometry/mass spectrometry was used to quantitate the levels of IPA in plasma of the GF and conventional mice administered vehicle or TAA. E) Fecal levels of Clostridium Cluster I as determined by quantitative PCR. *P < 0.05; **P < 0.01.
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