Intestinal Microbiota Protects against MCD Diet-Induced Steatohepatitis

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in western countries, with a continuously rising incidence. Gut-liver communication and microbiota composition have been identified as critical drivers of the NAFLD progression. Hence, it has been shown that microbiota depletion can ameliorate high-fat diet or western-diet induced experimental Non-alcoholic steatohepatitis (NASH). However, its functional implications in the methionine-choline dietary model, remain incompletely understood. Here, we investigated the physiological relevance of gut microbiota in methionine-choline deficient (MCD) diet induced NASH. Experimental liver disease was induced by 8 weeks of MCD feeding in wild-type (WT) mice, either with or without commensal microbiota depletion, by continuous broad-spectrum antibiotic (AB) treatment. MCD diet induced steatohepatitis was accompanied by a reduced gut microbiota diversity, indicating intestinal dysbiosis. MCD treatment prompted macroscopic shortening of the intestine, as well as intestinal villi in histology. However, gut microbiota composition of MCD-treated mice, neither resembled human NASH, nor did it augment the intestinal barrier integrity or intestinal inflammation. In the MCD model, AB treatment resulted in increased steatohepatitis activity, compared to microbiota proficient control mice. This phenotype was driven by pronounced neutrophil infiltration, while AB treatment only slightly increased monocyte-derived macrophages (MoMF) abundance. Our data demonstrated the differential role of gut microbiota, during steatohepatitis development. In the context of MCD induced steatohepatitis, commensal microbiota was found to be hepatoprotective.

Keywords: Gut-liver-Axis; MCD; NASH; microbiota.

Figure 1

Antibiotic treatment (ABx) aggravates non-alcoholic steatohepatitis (NASH) in the murine methionine-choline deficient (MCD) model. (A) Representative liver histology (hematotoxylin and eosin staining) showing livers of the wild-type (WT) mice with (+ABx) and without (–ABx) treatment on the normal chow diet (NCD) and after MCD treatment. (B) Increased “Inflammation” score in the ABx treated mice. (C) ABx treated mice had a higher histopathological non-alcoholic fatty liver disease (NAFLD) activity score (NAS). (D) ABx resulted in significantly increased Liver-to-Body-Weight ratios. Data are expressed as the mean ± SD from 2–5 mice per group and were considered significant if * p < 0.05, ** p < 0.01.

Figure 2

Antibiotic treatment increased the hepatic fat accumulation in the MCD-fed mice. (A) Representative Oil Red O stainings demonstrated increased the hepatic lipid accumulation upon an antibiotic treatment. (B) Steatosis score was higher in the ABx-treated mice, compared to the MCD-fed control mice. (C) Colorimetric hepatic triglyceride assay confirmed significantly increased hepatic triglycerides (TG) levels in +ABx group. Data are expressed as the mean ± SD from 2–5 mice per group and were considered significant if ** p < 0.01.

Figure 3

Microbiota depletion augments the inflammatory response during the MCD-induced steatohepatitis. (A) Representative immunofluorescence staining against CD11b, showing an increased infiltration of the CD11b+ cells in the Abx group. (B) Flow cytometry (FACS) shows increased infiltration of the neutrophils (CD11b+ Ly6G+ living leukocytes) after antibiotic treatment. (C) Monocyte-derived macrophages (MoMFs) (defined as CD11b^hi F4/80+ living leukocytes) abundance is lower in the ABx group, compared to the MCD-fed control mice. (D) Pro-inflammatory mRNA expression of the Mcp, Tnf, and Il1beta. GAPDH was used as a housekeeping gene. (E) ABx treatment prompted pronounced mRNA expression of pathogen recognition receptors (PRRs), including Tlr2, Tlr4, Tlr9, Nlrp3, and Caspase-1. GAPDH was used as a housekeeping gene. Data are expressed as the mean ± SD from 2–5 mice per group and were considered significant if * p < 0.05, ** p < 0.01, *** p < 0.001. **** p < 0.0001.

Figure 4

Antibiotic treatment fuels excessive liver fibrosis in experimentally-induced MCD-NASH. (A) Representative Sirius red staining of the liver sections showing the collagen fibers in red. (B) Quantification of the Sirius Red positive area, using the ImageJ software (at least 5 areas in 100× magnification per mouse). (C) Serum liver function tests. Data are expressed as the mean ± SD from 2–5 mice per group and were considered significant if * p < 0.05, ** p < 0.01.

Figure 5

MCD diet impacts intestinal homeostasis and microbiota composition. (A) MCD diets leads to shortening of small and large intestines. (B) Representative histology of the paraffin-fixed duodenum sections. (C) Clustered heatmap analysis of the microbiota composition of the normal chow or the MCD-fed mice. (D) “Observed species” and “Chao1” alpha diversity metrics were reduced, upon MCD feeding. (E) Occludin protein levels in the ileum tissue lysates. (F) Tnf, Il1beta, and Mcp1 mRNA expression, determined by the qRT-PCR in the ileum and the colon samples. * p < 0.05, ** p < 0.01, *** p < 0.001.