Expression of immune regulatory genes correlate with the abundance of specific Clostridiales and Verrucomicrobia species in the equine ileum and cecum

Abstract

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFβ, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

Figure 1

Phyla identified by amplicon sequencing of the V3-V4 region of the 16S rRNA gene and percentage abundance based on normalized OTU numbers in the ileum, cecum and colon of 24 horses. Phyla with abundance above 0.1% in at least one compartment are shown separately. The group “Other” includes the phyla Elusimicrobia, Fusobacteria, Planctomycetes, SR1, TM7, and WPS-2 all with abundances below 0.1%. Samples from 21 horses were used to analysis of the microbial composition in the Ileum and from 24 horses for the Cecum and Colon. Significant differences were found between ileum and the hind gut (cecum/colon), but not between the cecum and colon in an ANOVA analysis. Phyla that differed significantly are marked with *(p < 0.05).

Figure 2

Heatmap illustrating a core microbiome (taxa represented in at least 32% of all samples) characterized with amplicon sequencing of 16S rRNA gene (V3-V4 region) within three sections of horse gastrointestinal tract. *Indicates taxa with significantly shifted abundance between the three sections (ANCOM, FDR p < 0.05). No major differences in the taxa abundance were detected between the colon and cecum microbiota composition. Scale on the right indicate log abundance.