Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling

Abstract

The gut microbiota is a unique ecosystem of microorganisms interacting with the host through several biochemical mechanisms. The endocannabinoidome (eCBome), a complex signaling system including the endocannabinoid system, approximately 50 receptors and metabolic enzymes, and more than 20 lipid mediators with important physiopathologic functions, modulates gastrointestinal tract function and may mediate host cell-microbe communications there. Germ-free (GF) mice, which lack an intestinal microbiome and so differ drastically from conventionally raised (CR) mice, offer a unique opportunity to explore the eCBome in a microbe-free model and in the presence of a reintroduced functional gut microbiome through fecal microbiota transplant (FMT). We aimed to gain direct evidence for a link between the microbiome and eCBome systems by investigating eCBome alterations in the gut in GF mice before and after FMT. Basal eCBome gene expression and lipid profiles were measured in various segments of the intestine of GF and CR mice at juvenile and adult ages using targeted quantitative PCR transcriptomics and LC-MS/MS lipidomics. GF mice exhibited age-dependent modifications in intestinal eCBome gene expression and lipid mediator levels. FMT from CR donor mice to age-matched GF male mice reversed several of these alterations, particularly in the ileum and jejunum, after only 1 week, demonstrating that the gut microbiome directly impacts the host eCBome and providing a cause-effect relationship between the presence or absence of intestinal microbes and eCBome signaling. These results open the way to new studies investigating the mechanisms through which intestinal microorganisms exploit eCBome signaling to exert some of their physiopathologic functions.

Keywords: endocannabinoids; fecal microbiota transplant; gene expression; germ-free phenotype; gut microbiome; intestine; lipidomics..

Conflict of interest statement

P.D.C. is a senior research associate at FRS-FNRS (Fonds de la Recherche Scientifique), Belgium. All other authors declare that they have no conflicts of interest with the contents of this article.

Copyright © 2020 Manca et al.

Figures

Fig. 1.

Experimental protocols. A: Six male CR and GF mice were euthanized at 4 and 13 weeks of age. B: For FMT, 12-week-old GF mice were gavaged with either intestinal contents and stools coming from one and four donor mice, respectively (FMT group; n = 6), or sterile PBS (SHAM group; n = 5). For both experiments, sections of duodenum, jejunum, ileum, cecum, and colon were isolated, and the intestinal contents were harvested.

Fig. 2.

Intestinal expression levels of the eCBome receptors Cnr1 (A), Gpr18 (B), Gpr55 (C), and Pparα (D) in 4- and 13-week-old CR and GF mice and in 13-week-old male GF mice gavaged with sterile PBS (SHAM) or fecal microbiota (FMT). The mRNA expression levels were measured by qPCR array in duodenum, jejunum, ileum, and colon for all experimental groups and are expressed relative to CR mice for each age within each tissue. Values are mean ± SEM of n = 5–10 mice. Bars marked with different letters were significantly different at the P ≤ 0.05 level. E, F: Gpr55 gene expression in 7-week-old CR, GF, and FMT Swiss Webster mice (E) and control (CR) or antibiotic-treated (Ab) C57BL/6J mice (F).

Fig. 3.

Concentrations in intestinal tissues of eCBs and select congeners in 4- and 13-week-old CR or GF mice and in 13-week-old male GF mice gavaged with sterile PBS (SHAM) or fecal microbiota (FMT). Levels of NAEs (A) and 2-acylglycerols (B) are expressed as picomoles per milligram of tissue and were determined in duodenum, jejunum, ileum, and colon. Values are the mean ± SEM of n = 5–10. Bars marked with different letters were significantly different at the P ≤ 0.05 level. 2-AG, the three enantiomers of mono-arachidonoyl­glycerol (added together because presumably coming from the isomerization of 2-arachidonoylglycerol); 2-EPG, the three enantiomers of mono-eicosapentaenoyl-glycerol (added together because presumably coming from the isomerization of 2-eicosapentaenoyl-glycerol); 2-DHG, the three enantiomers of mono-docosahexaenoyl-glycerol (added together because presumably coming from the isomerization of 2-docosahexaenoyl -glycerol).

Fig. 4.

Concentrations of AA expressed as picomoles per milligram of tissue (A) and mRNA expression levels of Pla2g5 (B) and Ptges (C) in 4- and 13-week-old male CR or GF mice and in 13-week-old male GF mice gavaged with sterile PBS (SHAM) or fecal microbiota (FMT). Values are the mean ± SEM of n = 5–10. Bars marked with different letters were significantly different at the P ≤ 0.05 level.

Fig. 5.

Expression levels of select intestinal eCBome-related anabolic (A, C) and catabolic (B, D) enzymes for N-aminoacylethanolamines and 2-acylglycerols in 4- and 13-week-old male CR and GF mice and in 13-week-old male GF mice gavaged with sterile PBS (SHAM) or fecal microbiota (FMT). The mRNA expression levels of all the genes represented were measured by qPCR array in the duodenum, jejunum, ileum, and colon for all experimental groups. Values are the mean ± SEM of n = 5–10. Bars marked with different letters were significantly different at the P ≤ 0.05 level.

Fig. 6.

Composition of the intestinal microbiome of 13-week-old male GF mice after FMT and in CR mice. A: Average bacterial family composition of different intestinal segments (jejunum, ileum, colon, and cecum) in CR and FMT mice and in the intestinal contents and feces pool (FMT pool) used for microbiota transfer. Family names (right) are presented from most (top) to least (bottom) abundant. B: Comparison of colon and cecum microbiota composition of FMT mice and CR mice. Barplots show the coefficient associated bacterial families after PERMANOVA. Positive values indicate a higher prevalence in CR mice and negative indicate a higher prevalence in FMT mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.