Intestinal α-Defensins Play a Minor Role in Modulating the Small Intestinal Microbiota Composition as Compared to Diet

Abstract

The intestinal microbiota is at the interface between the host and its environment and thus under constant exposure to host-derived and external modulators. While diet is considered to be an important external factor modulating microbiota composition, intestinal defensins, one of the major classes of antimicrobial peptides, have been described as key host effectors that shape the gut microbial community. However, since dietary compounds can affect defensin expression, thereby indirectly modulating the intestinal microbiota, their individual contribution to shaping gut microbiota composition remains to be defined. To disentangle the complex interaction among diet, defensins, and small-intestinal microbiota, we fed wild-type (WT) mice and mice lacking functionally active α-defensins (Mmp7^-/- mice) either a control diet or a Western-style diet (WSD) that is rich in saturated fat and simple carbohydrates but low in dietary fiber. 16S rDNA sequencing and robust statistical analyses identified that bacterial composition was strongly affected by diet while defensins had only a minor impact. These findings were independent of sample location, with consistent results between the lumen and mucosa of the jejunum and ileum, in both mouse genotypes. However, distinct microbial taxa were also modulated by α-defensins, which was supported by differential antimicrobial activity of ileal protein extracts. As the combination of WSD and defensin deficiency exacerbated glucose metabolism, we conclude that defensins only have a fine-tuning role in shaping the small-intestinal bacterial composition and might instead be important in protecting the host against the development of diet-induced metabolic dysfunction. IMPORTANCE Alterations in the gut microbial community composition are associated with many diseases, and therefore identifying factors that shape the microbial community under homeostatic and diseased conditions may contribute to the development of strategies to correct a dysbiotic microbiota. Here, we demonstrate that a Western-style diet, as an extrinsic parameter, had a stronger impact on shaping the small intestinal bacterial composition than intestinal defensins, as an intrinsic parameter. While defensins have been previously shown to modulate bacterial composition in young mice, our study supplements these findings by showing that defensins may be less important in adult mice that harbor a mature microbial community. Nevertheless, we observed that defensins did affect the abundance of distinct bacterial taxa in adult mice and protected the host from aggravated diet-induced glucose impairments. Consequently, our study uncovers a new angle on the role of intestinal defensins in the development of metabolic diseases in adult mice.

Keywords: Western diet; antimicrobial peptides; defensins; gut microbiota; metabolic disease; mucosal barrier.

FIG 1

Diet is a stronger modulator of luminal microbiota composition in the jejunum than defensins. (A) Schematic experimental setup: littermate-controlled WT or Mmp7^−/− mice were fed a chow or Western-style diet (WSD) for 8 weeks. Alpha diversity according to (B) observed ASVs and (C) Shannon index and beta diversity according to (D) Bray-Curtis dissimilarity matrix and (E) Bray-Curtis distance in the jejunum lumen. (F) Average phylum relative abundance and (G) differentially abundant phyla, (H) average genera relative abundance and (I) differentially abundant genera in the jejunum lumen. Phyla or genera with less than 1% relative abundance are represented as “other (<1%).” Statistical tests calculated with the Kruskal-Wallis Pairwise test for alpha-diversity analyses, PERMANOVA for beta-diversity analyses and pairwise PERMANOVA for Bray-Curtis distance (presented as p_adj), and two-way ANOVA with Tukey’s multiple-comparison test of phyla and genera. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, < 0.0001.

FIG 2

Diet is a stronger modulator of luminal microbiota composition than defensins in the ileum. Alpha diversity according to (A) observed ASVs and (B) Shannon index, and beta diversity according to (C) Bray-Curtis dissimilarity matrix and (D) Bray-Curtis distance at the ileum lumen. (E) Average phylum relative abundance and (F) differentially abundant phyla, (G) average genera relative abundance and (H) differentially abundant genera at the ileum lumen. Phyla or genera with less than 1% relative abundance are represented as “other (<1%).” Statistical tests calculated with Kruskal-Wallis Pairwise test for alpha-diversity analyses, PERMANOVA for beta-diversity analyses and pairwise PERMANOVA for Bray-Curtis distance (presented as p_adj), and two-way ANOVA with Tukey’s multiple-comparison test of phyla and genera. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001.

FIG 3

Diet is a stronger modulator of mucosal microbiota composition in the jejunum than defensins. Alpha diversity according to (A) observed ASVs and (B) Shannon index, and beta diversity according to (C) Bray-Curtis dissimilarity matrix and (D) Bray-Curtis distance between chow- or WSD-fed WT and Mmp7^−/− mice observed for the mucosa-associated bacteria (MAB) in the jejunum. (E) Average phylum relative abundance and (F) differentially abundant phyla, (G) average genera relative abundance and (H) differentially abundant genera at the jejunum MAB. Phyla or genera with less than 1% relative abundance are represented as “other (<1%).” Statistical tests calculated with Kruskal-Wallis Pairwise test for alpha-diversity analyses, PERMANOVA for the beta-diversity analyses and pairwise PERMANOVA for Bray-Curtis distance (presented as p_adj), and two-way ANOVA with Tukey’s multiple-comparison test of phyla and genera. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001; ****, P < 0.0001.

FIG 4

Diet is a stronger modulator of mucosal microbiota composition in the ileum than defensins. Alpha diversity according to (A) observed ASVs and (B) Shannon index and beta diversity according to (C) Bray-Curtis dissimilarity matrix and (D) Bray-Curtis distance between chow- or WSD-fed WT and Mmp7^−/− mice observed for the mucosa-associated bacteria (MAB) in the ileum. (E) Average phylum relative abundance and (F) differentially abundant phyla, (G) average genera relative abundance and (H) differentially abundant genera at the ileum MAB. Phyla or genera with less than 1% relative abundance are represented as “other (<1%).” Statistical tests calculated with Kruskal-Wallis Pairwise test for alpha-diversity analyses, PERMANOVA for beta-diversity analyses and pairwise PERMANOVA for Bray-Curtis distance (presented as p_adj), and two-way ANOVA with Tukey’s multiple-comparison test of phyla and genera. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001; ****, P < 0.0001.

FIG 5

The abundance of distinct small-intestinal bacteria is modulated by defensins. (A) Orthogonal partial least squares discriminant analysis (OPLS-DA) of bacterial genera in the jejunal lumen, at the jejunal mucosa (B), in the ileal lumen (C), and at the ileal mucosa (D) of chow-fed WT and Mmp7^−/− mice. A statistically significant difference in bacterial relative abundance shown by the red rectangles was defined when the confidence interval did not contain the null hypothesis value (0).

FIG 6

WSD feeding or defensin deficiency has a minor influence on AMP expression in the jejunum. (A) Expression of host-defense molecules Defa1, Defa21/22, P-Lysozyme (P-Lys), CRS1C, Reg3g, and Pla2a2 at the jejunum. (B) Sum of all measured AMP transcripts in the jejunum of the different mouse groups. (C) Principal-component analysis (PCA) of AMP expression at the jejunum of the different mouse groups. Statistical significance was determined by two-way ANOVA analysis with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 7

Diet is a substantial driver of AMP expression in the ileum. (A) Expression of host-defense molecules Defa1, Defa21/22, P-Lysozyme (P-Lys), CRS1C, Reg3g, and Pla2a2 at the ileum. (B) Sum of all measured AMP transcripts in the ileum of the different mouse groups. (C) Principal-component analysis (PCA) of AMP expression at the ileum of the different mouse groups. (D) Antimicrobial activity of ileal protein extract against Bifidobacterium longum, Bacteroides thetaiotaomicron, Escherichia coli, and Lactobacillus reuteri, by using a radial diffusion assay (RDA). Statistical significance was determined by two-way ANOVA analysis with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. LOI, limit of inhibition.

FIG 8

Combination of WSD and defensin deficiency exacerbates glucose metabolism. Defensin-deficient Mmp7^−/− mice and WT mice were fed a chow or WSD for 8 weeks, after which (A) bodyweight, (B) body fat, (C) fasting blood insulin concentration, (D) fasting blood glucose concentration, and (E) homeostatic model assessment for insulin resistance (HOMA-IR) were determined. Two days prior to sacrifice, an oral glucose tolerance test (OGTT) was performed to determine (F) blood glucose and (G) insulin concentration and respective area under the curve (AUC). Statistical significance was determined by two-way ANOVA analysis with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.