Entamoeba muris mitigates metabolic consequences of high-fat diet in mice

Abstract

Metabolic syndrome (MetS) is a cluster of several human conditions including abdominal obesity, hypertension, dyslipidemia, and hyperglycemia, all of which are risk factors of type 2 diabetes, cardiovascular disease, and metabolic dysfunction-associated steatotic liver disease (MASLD). Dietary pattern is a well-recognized MetS risk factor, but additional changes related to the modern Western life-style may also contribute to MetS. Here we hypothesize that the disappearance of amoebas in the gut plays a role in the emergence of MetS in association with dietary changes. Four groups of C57B/6J mice fed with a high-fat diet (HFD) or a normal diet (ND) were colonized or not with Entamoeba muris, a commensal amoeba. Seventy days after inoculation, cecal microbiota, and bile acid compositions were analyzed by high-throughput sequencing of 16S rDNA and mass spectrometry, respectively. Cytokine concentrations were measured in the gut, liver, and mesenteric fat looking for low-grade inflammation. The impact of HFD on liver metabolic dysfunction was explored by Oil Red O staining, triglycerides, cholesterol concentrations, and the expression of genes involved in β-oxidation and lipogenesis. Colonization with E. muris had a beneficial impact, with a reduction in dysbiosis, lower levels of fecal secondary bile acids, and an improvement in hepatic steatosis, arguing for a protective role of commensal amoebas in MetS and more specifically HFD-associated MASLD.

Keywords: Entamoeba muris; Metabolic syndrome; amoebas; cAMP; dysbiosis; hepatic steatosis; metabolic dysfunction-associated steatotic liver disease.

Figure 1.

In vitro and in vivo characterization of Entamoeba muris. (a) Representative image and (b) cycle monitorization of cyst and trophozoite forms of Entamoeba muris cultured in vitro. (c) Representative image of the cyst form in a stool sample after Merthiolate iodine formaldehyde (MIF) staining. (d) video showing E.muris behavior is vailable at https://cri1149.fr/wp-content/uploads/2024/09/E.Muris2_.mp4. (e) Representative Entamoeba PCR identifying the presence of E. muris in three independent stool samples from negative control (-) and chronically colonized C57Bl/6J mice (+). (f) Relative expression of E. muris measured by Q-PCR in fecal and cecal samples at days 7 and 70 after infestation. Data are presented as mean ± SEM. Scale bars represent 50 μm (all studies including preliminary experiments).

Figure 2.

Colonization of Entamoeba muris is associated with a cecal dysbiosis characterized by more prevotellaceae and less Desulfovibrionaceae. At day 70, the cecal microbiota of C57Bl/6J mice fed with either normal (ND) or high-fat (HFD) diet and colonized with/out Entamoeba muris (E. muris) was analyzed. (a) Alpha-diversity is represented by the Shannon index for the four experimental groups (additional indexes are available in supplementary fig S3 and S4). (b) Principal coordinate analysis (PCoA) of the Bray Curtis matrix. Ellipses represent 95% of confidence. Statistical analyses were performed using PERMANOVA. (c) Taxonomic representation in a cladogram (left) and the linear discriminant analysis (LDA) score (right) of the of HFD mice groups in the presence (green) or absence (red) of E. muris. (d–g) relative abundance of the family Prevotellaceae and Desulfovibrionaceae in (d-e) the first experiment and (f–g) in the replication study. (h) Prevotellaceae/Desulfovibrionaceae ratio calculated for the pooled data. Dot plotted data presents mean ± SEM. Statistical analyses were performed using the one-way ANOVA test followed by a Bonferroni post hoc test. Significant differences were recorded as *p < .05, **p < .01, ***p < .001, ****p < .0001. Differences corresponding to p values lower than 0,01 are reported for LDA analyses. Data were generated during study 1 (a–e), study 2 (f–g) or both (h).

Figure 3.

Cecal dysbiosis correlates with changes in bile acid composition. Bile acid composition was studied in either the feces or the cecum collected from C57Bl/6J mice fed with either normal diet (ND) or high-fat diet (HFD) and colonized with/out Entamoeba muris (E. muris). Abundance of primary (a) and secondary (b) BA in the feces. (c) relative abundance of secondary bile acids (DCA, LCA and TDCA) based on metabolomic analyses in the cecum of mice. (d) schematic representation of BA metabolism in the liver and the intestine. (e) abundance of total unconjugated bile acids and (f) farnesoid X receptor (FXR) agonist in the feces. (g) correlation analyses between the Prevotellaceae/Desulfovibrionaceae ratio and the fecal concentrations of secondary BA (top), unconjugated BA (middle), and FXR agonist (bottom). Data are presented as mean ± SEM, n = 8 - 18. Statistical analyses were performed using Mixed-effects analysis or one-way ANOVA test, both followed by a Bonferroni post hoc test. F-tests were used to determine the significance of the correlation. Significant differences were recorded as *p < .05, **p < .01, ***p < .001, ****p < .0001 when comparing the same bile acid type between groups, and ####p < 0.0001 when comparing different bile acids in one same condition. βMCA: β-Muricholic acid, CA: cholic acid, CA-7S: cholic acid-7-sulfate, CDCA: chenodeoxycholic acid, CDCA-3S: chenodeoxycholic acid-3-sulfate, DCA: deoxycholic acid, DCA-3S: deoxycholic acid-3-sulfate, GCA: glycocholic acid, HCA: hyocholic acid, HDCA: hyodeoxycholic acid, LCA: lithocholic acid, TCA: taurocholic acid, TCDCA: tauro-chenodeoxycholic acid, TDCA: tauro-deoxycholic acid, TMCA: tauro-muricholic acid, ω-MCA: ω-Muricholic acid. Analyses were done on the pooled dataset from studies 1 and 2 except for the cecum which is based on study 2 only.

Figure 4.

Entamoeba muris decreases IL-1β concentration in the liver. The concentrations of interleukin 1β (Il-1β), interleukin 6 (Il-6), interleukin 12 (Il-12), tumor necrosis factor ⍺ (Tnf-⍺), interferon γ (Ifn-γ), transforming growth factor beta (Tgf-β), and interleukin 10 (Il-10) were measured in the cecum, colon, liver, mesenteric fat of mice C57Bl/6J mice fed with either normal diet (ND) or high-fat diet (HFD) and colonized with/out Entamoeba muris (E. muris). (a) heatmap showing the cytokines levels (rows) that were significantly different between ND and HFD groups (*p < .05, **p < .01) or between HFD and HFD with E. muris groups (#p < 0.05). (b) Il-1β concentrations in the liver of the different experimental groups. (c–d) correlation analyses between Il-1β concentration and (c) unconjugated bile acids or (d) Farnesoid X receptor (FXR) agonist amounts in the feces. F-tests were used to determine the significance of the correlation. P-values are indicated in the corresponding figures. Measures were performed in study 2 mice. Il-10 was undetectable in mesenteric fat.

Figure 5.

Entamoeba muris reduces hepatic steatosis. Study of the fatty liver disease in C57Bl/6J mice fed with normal diet (ND) or high-fat diet (HFD) and colonized with/out eEntamoeba muris (E. muris). (a) microscopic examination of hepatic steatosis after mouse livers were paraffin-embedded, sectioned, and stained with H&E and contrasted with oil red O for the visualization of lipids. Bar represents 100 μm. (b) percentages of the surface stained by oil red O as a marker of lipid accumulation. Hepatic (c) triglyceride and (d) cholesterol amounts in the liver. (e) mRNA expression levels of various genes related with the fatty acid β-oxidation and de novo lipogenesis. mRNA expression levels of various genes related with bile acid activating pathways in the liver (f) and ileum (g). (h) Correlation analysis between the relative abundance of E. muris and the concentration of cyclic adenosine monophosphate (cAMP) in the cecum. (g) heatmap showing the correlations between the different parameters modified by the presence of E. muris in HFD-fed mice by Spearman correlation. The color of each spot in the heatmap corresponds to the r value. Data are presented as mean ± SEM. Statistical analyses were performed using the Mann-wWhitney U test. Significant differences/correlations were recorded as *p < 0.05 and ****p < .0001. Data were provided by both studies 1 and 2 (a–b, i) or study 2 only (c–d, e–f).