Modulatory Influence of Segmented Filamentous Bacteria on Transcriptomic Response of Gnotobiotic Mice Exposed to TCDD

Abstract

Environmental toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR), are known to induce host toxicity and structural shifts in the gut microbiota. Key bacterial populations with similar or opposing functional responses to AhR ligand exposure may potentially help regulate expression of genes associated with immune dysfunction. To examine this question and the mechanisms for AhR ligand-induced bacterial shifts, C57BL/6 gnotobiotic mice were colonized with and without segmented filamentous bacteria (SFB) - an immune activator. Mice were also colonized with polysaccharide A producing Bacteroides fragilis - an immune suppressor to serve as a commensal background. Following colonization, mice were administered TCDD (30 μg/kg) every 4 days for 28 days by oral gavage. Quantified with the nCounter^® mouse immunology panel, opposing responses in ileal gene expression (e.g., genes associated with T-cell differentiation via the class II major histocompatibility complex) as a result of TCDD dosing and SFB colonization were observed. Genes that responded to TCDD in the presence of SFB did not show a significant response in the absence of SFB, and vice versa. Regulatory T-cells examined in the mesenteric lymph-nodes, spleen, and blood were also less impacted by TCDD in mice colonized with SFB. TCDD-induced shifts in abundance of SFB and B. fragilis compared with previous studies in mice with a traditional gut microbiome. With regard to the mouse model colonized with individual populations, results indicate that TCDD-induced host response was significantly modulated by the presence of SFB in the gut microbiome, providing insight into therapeutic potential between AhR ligands and key commensals.

Keywords: B. fragilis; TCDD; gnotobiotic mice; gut dysbiosis; host microbe response; regulatory T-cells; segmented filamentous bacteria.

FIGURE 1

nCounter^® mouse immunology panel analysis of genes in ileal tissue that were significantly influenced in response to TCDD in mice (A) with SFB, and (B) without SFB. Line color indicates gene regulation due to TCDD (blue). For comparative analysis of genes significantly influenced by TCDD, fold change expression due to SFB and B. fragilis colonization is also shown regardless of significance. For comparative analysis of SFB colonization (red), the vehicle dosed group colonized with B. fragilis was compared with the vehicle dosed group co-colonized with SFB and B. fragilis. For comparative analysis of B. fragilis colonization (gray), the vehicle dosed group colonized with B. fragilis was compared with the uncolonized vehicle dosed group.

FIGURE 2

nCounter^® mouse immunology panel analysis and functional response. (A) Genes in ileal tissue that responded to SFB colonization. Line color and direction indicate upregulated (red) and downregulated (blue) genes. Abbreviations include mice that were mono-colonized with SFB (SFB), mono-colonized with B. fragilis (B) and co-colonized (SFB+B). (B,C) Functional clusters identified using the DAVID using nCounter^® mouse immunology panel analysis with gene expression of ileal tissue showing (B) clusters of genes that responded to colonization with SFB, and (C) clusters of genes that responded to TCDD in presence and absence of SFB. Functional clusters with scores =1.3 were included as significantly enriched.

FIGURE 3

Percent CD4⁺ T_reg measured in (A) spleen, (B) whole blood, and (C) mesenteric lymph nodes in C57BL/6 female mice after TCDD (30 μg/kg) or vehicle (sesame oil) treatment by oral gavage once every 4 days for 28 days. Gray bars are TCDD dosed and white bars are vehicle dosed mice. Values represent mean percent and error bars represent standard error in presence/absence of SFB. P-values are shown between vehicle- (Veh) and TCDD-treated groups.

FIGURE 4

Bacteroides fragilis and SFB abundance and selected functional gene expression based on isolated RNA. (A) Absolute abundance of B. fragilis expression measured with specific rplB gene primers in cecum content from respective groups. (B) Absolute abundance of SFB expression using species-specific 16S rRNA gene primers from ileum of respective groups. (C) PSA biosynthesizing wcfQ gene normalized expression, measured with cecum content from respective groups. (D) SFB functional genes putative to interaction with host immunity (hemolysin A producing gene) normalized expression, measured with ileal RNA from SFB mono-colonized mice. SFB functional genes in co-colonized group are not included as the genes were expressed below detection limit in two or more biological replicates. Bars represent mean normalized abundance. Open dots indicate mono-colonized groups, closed dots indicate co-colonized groups. Abbreviations include vehicle dosed (Veh) and TCDD treated (TCDD).