Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent

Abstract

Background: The intestinal mucus layer plays a key role in the maintenance of host-microbiota homeostasis. To document the crosstalk between the host and microbiota, we used gnotobiotic models to study the influence of two major commensal bacteria, Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, on this intestinal mucus layer. B. thetaiotaomicron is known to use polysaccharides from mucus, but its effect on goblet cells has not been addressed so far. F. prausnitzii is of particular physiological importance because it can be considered as a sensor and a marker of human health. We determined whether B. thetaiotaomicron affected goblet cell differentiation, mucin synthesis and glycosylation in the colonic epithelium. We then investigated how F. prausnitzii influenced the colonic epithelial responses to B. thetaiotaomicron.

Results: B. thetaiotaomicron, an acetate producer, increased goblet cell differentiation, expression of mucus-related genes and the ratio of sialylated to sulfated mucins in mono-associated rats. B. thetaiotaomicron, therefore, stimulates the secretory lineage, favoring mucus production. When B. thetaiotaomicron was associated with F. prausnitzii, an acetate consumer and a butyrate producer, the effects on goblet cells and mucin glycosylation were diminished. F. prausnitzii, by attenuating the effects of B. thetaiotaomicron on mucus, may help the epithelium to maintain appropriate proportions of different cell types of the secretory lineage. Using a mucus-producing cell line, we showed that acetate up-regulated KLF4, a transcription factor involved in goblet cell differentiation.

Conclusions: B. thetaiotaomicron and F. prausnitzii, which are metabolically complementary, modulate, in vivo, the intestinal mucus barrier by modifying goblet cells and mucin glycosylation. Our study reveals the importance of the balance between two main commensal bacteria in maintaining colonic epithelial homeostasis via their respective effects on mucus.

Figure 1

Time course analysis of GIT in B. thetaiotaomicron mono-associated rats. Germfree (GF) rats were inoculated with a culture of B. thetaiotaomicron (10⁷ CFU) and euthanized 2 days (Bt-2d) or 30 days (Bt-30d) after inoculation. (A) Establishment of B. thetaiotaomicron in the gastrointestinal tract (GIT) of Bt-30d rats (n = 13) was monitored weekly by enumeration of the bacterial counts in the feces. (B) Scanning electron microscopy images of B. thetaiotaomicron in the cecum of Bt-2d and Bt-30d rats; scale bars, 1 μm. (C) Measurement of cecal pH in GF (n = 12), Bt-2d (n = 13) and Bt-30d rats (n = 19). (D) Cecal concentration of short-chain fatty acids (SCFA) in GF (n = 16), Bt-2d (n = 13) and Bt-30d rats (n = 19); only results for acetate, propionate and butyrate are shown, other SCFA were not detected; results are expressed in mM. Means with different letters are significantly different (P-value <0.05).

Figure 2

Characterization of the colonic epithelial response in B. thetaiotaomicron mono-associated rats. (A) Colonic crypt depth and total number of cells per crypt were determined on sections from GF (n = 3), Bt-2d (n = 5) and Bt-30d rats (n = 7) stained with Haematoxylin-Eosin-Safran (HES). (B, C) Representative pictures and graphs showing counts of goblet cells staining with (B) alcian blue (indicated as AB) and (C) periodic acid Schiff (indicated as PAS) in GF (n = 3), Bt-2d (n = 6) and Bt-30d (n = 7) samples. Scale bars, 50 μm. (D) Representative Western blots and densitometric analyses of proteins involved in the differentiation pathway of the secretory lineage, KLF4 and Chromogranin A (ChgA), in GF (n = 7), Bt-2d (n = 6) and Bt-30d (n = 7) samples. Means with different letters are significantly different (P-value <0.05).

Figure 3

Effects of bacterial metabolites on KLF4 protein in HT29-MTX cells. Representative Western blot and densitometric analyses of KLF4 and P21 proteins in HT29-MTX cells incubated with (A) 0, 10, 20 mM acetate, (B) 0, 5, 10 mM propionate, and (C) 0, 1, 5 mM butyrate. Each graph reports means of three independent experiments with three internal repeats per experiment. Means with different letters are significantly different (P-value <0.05).

Figure 4

Behavior of B. thetaiotaomicron and F. prausnitzii in combination in vitro and in vivo. (A) Acetate, butyrate and propionate concentrations from culture media, pure cultures of B. thetaiotaomicron (Bt), F. prausnitzii (Fp) and cocultures. (B) Establishment of B. thetaiotaomicron and F. prausnitzii in the gastrointestinal tract (GIT) of Bt + Fp-30d rats (n = 16) was monitored weekly by enumeration in the feces; arrows represent the various inoculations with F. prausnitzii before successful colonization. (C) Cecal concentration of short chain fatty acids (SCFA) in the cecum of germfree (GF) (n = 12), Bt-30d rats (n = 19), and Bt + Fp-30d rats (n = 16). (D) Measurement of oxidoreduction potential (mV) in the cecal contents of GF (n = 11), Bt-2d (n = 13), Bt-30d (n = 19) and Bt + Fp-30d rats (n = 16). Means with different letters are significantly different (P-value <0.05).

Figure 5

Characterization of the colonic epithelial response in B. thetaiotaomicron and F. prausnitzii di-associated rats. (A) Measurement of colonic crypt depth, total number of cells per colonic crypt and counts of (B) alcian blue- (indicated as AB) and periodic acid Schiff- (indicated as PAS) positive cells per crypt in colonic sections of Bt-30d (n = 7) and Bt + Fp-30d rats (n = 6). (C) Representative Western blot and densitometric analyses of proteins involved in the differentiation pathway of the secretory lineage, KLF4 and Chromogranin A (ChgA) in Bt-30d (n = 5) and Bt + Fp-30d rats (n = 4); protein fold induction in Bt-30d rats was used as a reference and arbitrarily defined as 1. (D) Immunostaining for MUC2 in germfree (GF) (n = 3), Bt-2d (n = 6), Bt-30d (n = 7) and Bt + Fp-30d rats (n = 6); scale bars, 50 μm. The asterisk indicates a statistical difference compared to Bt-30d rats (P-value <0.05); n.s., not significant.

Figure 6

Effects of bacterial colonization on colonic mucin glycosylation. (A) Proportion of neutral and acidic oligosaccharides (carrying sulfate, N-acetyl neuraminic acid or N-glycolyl acid residues) in germfree (GF) rats (n = 3), Bt-2d (n = 2), Bt-30d (n = 3) and Bt + Fp 30d (n = 3) rats. (B) Ratio of sialylated to sulfated O-glycan chain in each group of rats.