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. 2017 Sep 26:8:1804.
doi: 10.3389/fmicb.2017.01804. eCollection 2017.

Protective Effect of Akkermansia muciniphila against Immune-Mediated Liver Injury in a Mouse Model

Wenrui Wu  1   2 , Longxian Lv  1   2 , Ding Shi  1   2 , Jianzhong Ye  1   2 , Daiqiong Fang  1   2 , Feifei Guo  1   2 , Yating Li  1   2 , Xingkang He  3 , Lanjuan Li  1   2
Affiliations

Protective Effect of Akkermansia muciniphila against Immune-Mediated Liver Injury in a Mouse Model

Wenrui Wu et al. Front Microbiol. .

Abstract

Accumulating evidence indicates that gut microbiota participates in the pathogenesis and progression of liver diseases. The severity of immune-mediated liver injury is associated with different microbial communities. Akkermansia muciniphila can regulate immunologic and metabolic functions. However, little is known about its effects on gut microbiota structure and function. This study investigated the effect of A. muciniphila on immune-mediated liver injury and potential underlying mechanisms. Twenty-two C57BL/6 mice were assigned to three groups (N = 7-8 per group) and continuously administrated A. muciniphila MucT or PBS by oral gavage for 14 days. Mouse feces were collected for gut microbiota analysis on the 15th day, and acute liver injury was induced by Concanavalin A (Con A, 15 mg/kg) injection through the tail vein. Samples (blood, liver, ileum, colon) were assessed for liver injury, systemic inflammation, and intestinal barrier function. We found that oral administration of A. muciniphila decreased serum ALT and AST and alleviated liver histopathological damage induced by Con A. Serum levels of pro-inflammatory cytokines and chemokines (IL-2, IFN-γ, IL-12p40, MCP-1, MIP-1a, MIP-1b) were substantially attenuated. A. muciniphila significantly decreased hepatocellular apoptosis; Bcl-2 expression increased, but Fas and DR5 decreased. Further investigation showed that A. muciniphila enhanced expression of Occludin and Tjp-1 and inhibited CB1 receptor, which strengthened intestinal barriers and reduced systemic LPS level. Fecal 16S rRNA sequence analysis indicated that A. muciniphila increased microbial richness and diversity. The community structure of the Akk group clustered distinctly from that of mice pretreated with PBS. Relative abundance of Firmicutes increased, and Bacteroidetes abundance decreased. Correlation analysis showed that injury-related factors (IL-12p40, IFN-γ, DR5) were negatively associated with specific genera (Ruminococcaceae_UCG_009, Lachnospiraceae_UCG_001, Akkermansia), which were enriched in mice pretreated with A. muciniphila. Our results suggested that A. muciniphila MucT had beneficial effects on immune-mediated liver injury by alleviating inflammation and hepatocellular death. These effects may be driven by the protective profile of the intestinal community induced by the bacteria. The results provide a new perspective on the immune function of gut microbiota in host diseases.

Keywords: Akkermansia muciniphila; acute hepatitis; immune regulation; liver injury; microbiota.

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Figures

FIGURE 1
FIGURE 1
Akkermansia muciniphila administration inhibited Con A-induced acute liver injury. (A) Design of the animal experiment. Mice were randomly distributed to three groups (Normal, n = 8; Control, n = 7; Akk, n = 7). Mice were pretreated with PBS or A. muciniphila continuously for 14 days by gavage. On day 15, feces from each mouse were collected, and Con A was injected. Eight hours later, mice were sacrificed. (B) Serum levels of ALT and AST (n = 7–8 per group). (C) Representative liver histology. Upper panel: Left, H&E staining, scale bar, 250 μm; Right, modified HAI scores of liver histopathology. Middle panel: Left, staining of neutrophils (Ly6G+), scale bar, 100 μm; Right, percentage of Ly6G+ cells. Lower panel: Left, staining of macrophages (F4/80+), scale bar, 100 μm; Right, percentage of F4/80+ cells. Data are shown as the mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 by post hoc ANOVA one-way statistical analysis.
FIGURE 2
FIGURE 2
Pretreatment with A. muciniphila relieved Con A-induced cytokine expression in the serum. The increased production of IFN-γ, IL-2, IL-1β, and IL-12p40 was significantly diminished, along with reduced chemokines, including KC, MCP-1, MIP-1a, and MIP-1b. However, TNF-α concentration was not evidently reduced. Data are shown as the mean ± SEM. N = 5–8 per group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by post hoc ANOVA one-way statistical analysis or Kruskal–Wallis test.
FIGURE 3
FIGURE 3
Akkermansia muciniphila suppressed Con A-induced hepatocyte apoptosis. (A) TUNEL assays of liver histology 8 h after Con A injection. There was extensive damage in group Control, but only sporadic apoptosis was observed in group Akk (scale bar, 100 μm). Percentage of apoptotic cells in the liver was significantly lower in group Akk (n = 7–8 per group). (B) Liver expression of genes related to apoptosis and inflammation (n = 6–8 per group). mRNA level of an important anti-apoptotic factor, Bcl-2, was markedly reduced in group Control, whereas it was significantly elevated in group Akk. Expression of both Fas and DR5 was diminished. IFN-γ was substantially reduced in group Akk, but expression of TNF-α was not significantly influenced. Data are shown as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 by post hoc ANOVA one-way statistical analysis.
FIGURE 4
FIGURE 4
Akkermansia muciniphila reinforced gut barrier function. (A) Representative Alcian blue images of the inner mucus layer. Scale bar, 100 μm. (B) Thickness measurement of the inner mucus layer by Alcian blue staining. Data are expressed as Min to Max. (C) mRNA expression of Tjp1, Occludin, CB1, and CB2 in the ileum. Data are shown as the mean ± SEM. ∗P < 0.05 by post hoc ANOVA one-way statistical analysis.
FIGURE 5
FIGURE 5
Alteration of the microbial community. (A) PCoA among three groups based on Weighted UniFrac distances. Each point represented a sample. N, group Normal; C, group Control; A, group Akk. (B) Comparisons of pair-wised Weighted Unifrac distances. ANOVA was conducted for multiple comparisons with the LSD method for correction. N_N, C_C, and A_A, indicate intragroup distances; A_C, N_C, and N_A, indicate intergroup distances. (C) Relative abundance of taxa at the phylum level in group Control and Akk. Significance was determined by Wilcoxon rank sum tests. (D) LEfSe cladograms represented taxa enriched in group Control (green) and group Akk (red). Rings from the inside out represented taxonomic levels from phylum to genus. Sizes of circles indicate relative abundance of the taxon. (E) Discriminative biomarkers with twofold changes in LDA scores. Data are shown as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
FIGURE 6
FIGURE 6
Correlation analysis between gut microbiota and injury-related indexes. Spearman’s rho non-parametric correlation was used, and significant relationships with P < 0.05 and r > 0.5 are shown. Yellow nodes: injury-related indexes. Green nodes: differentially distributed genera between Control and Akk groups; blue nodes: the indexes showed differences but were not significantly different between the groups. Red lines between nodes represent positive relationships, and blue ones indicate negative linkages. The thickness of the connection represents the correlation coefficient, with thicker lines indicating higher r values.
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