Altered microbial biogeography in an innate model of colitis

Abstract

Changes in the spatial organization, or biogeography, of colonic microbes have been observed in human inflammatory bowel disease (IBD) and mouse models of IBD. We have developed a mouse model of IBD that occurs spontaneously and consistently in the absence of adaptive immunity. Mice expressing tumor necrosis factor-induced protein 3 (TNFAIP3) in intestinal epithelial cells (villin-TNFAIP3) develop colitis when interbred with Recombination Activating 1-deficient mice (RAG1<sup>-/-</sup>). The colitis in villin-TNFAIP3 × RAG1<sup>-/-</sup> (TRAG) mice is prevented by antibiotics, indicating a role for microbes in this innate colitis. We therefore explored the biogeography of microbes and responses to antibiotics in TRAG colitis. Laser capture microdissection and 16S rRNA sequencing revealed altered microbial populations across the transverse axis of the colon as the inner mucus layer of TRAG, but not RAG1<sup>-/-</sup>, mice was infiltrated by microbes, which included increased abundance of the classes Gammaproteobacteria and Actinobacteria. Along the longitudinal axis differences in the efficacy of antibiotics to prevent colitis were evident. Neomycin was most effective for prevention of inflammation in the cecum, while ampicillin was most effective in the proximal and distal colon. RAG1<sup>-/-</sup>, but not TRAG, mice exhibited a structured pattern of bacterial abundance with decreased Firmicutes and Proteobacteria but increased Bacteroidetes along the proximal to distal axis of the gut. TRAG mice exhibited increased relative abundance of potential pathobionts including <i>Bifidobacterium animalis</i> along the longitudinal axis of the gut whereas others, like <i>Helicobacter hepaticus</i> were increased only in the cecum. Potential beneficial organisms including <i>Roseburia</i> were decreased in the proximal regions of the TRAG colon, while <i>Bifidobacterium pseudolongulum</i> was decreased in the TRAG distal colon. Thus, the innate immune system maintains a structured, spatially organized, gut microbiome along the transverse and longitudinal axis of the gut, and disruption of this biogeography is a feature of innate immune colitis.

Keywords: A20; IBD; TRAG; biogeography; colitis; inflammatory bowel disease; innate immunity; laser capture microdissection; microbiome.

Figure 1.

Microbes invade the mucus layer of mice expressing TNFAIP3 in intestinal epithelial cells. Colon tissues were fixed in Methyl-Carnoys, sectioned, and probed for microbes, mucus, and cells. (a) Immunolocalization of microbes (red; EUB 16S rRNA FISH), mucus (green; lectin) and colon cells (blue; DAPI) with the epithelial surface approximated by a dotted line. Microbes are present in the mucus layer of villin-TNFAIP3 (v-TNFAIP3) or villin-TNFAIP3 x RAG1^−/− (TRAG) mice, but less evident in WT or RAG1^−/− mice. (b) Mucus production in TRAG mice is not defective and normal mucus thickness is present in TRAG mice treated with antibiotic (AMP; ampicillin 0.5 g/L in drinking water, 2 weeks). Scale bar = 50uM.

Figure 2.

Gammaproteobacteria and Actinobacteria invade the inner mucus layer of TRAG mice. Laser capture dissection was used to collect microbial samples from Methyl-Carnoys fixed histological sections of the colon. Microbe populations in the mucus layers were characterized by 16S rRNA sequencing. (a) PCoA plots of microbe populations from RAG1^−/− or TRAG mucus layers (IML = inner mucus layer; OML outer mucus layer). (b) Heat maps of OTUs and Shannon diversity index from RAG1^−/− and TRAG IML and OML showing reduced abundance of OTUs in TRAG layers. (c) Relative abundance of Phyla in the IML and OML or RAG1^−/− and TRAG mice showing reduced Bacteroidetes and increased Proteobacteria and Actinobacteria especially in the IML of TRAG mice. (d) Relative abundance of microbial classes in the mucus layers of RAG1^−/− and TRAG mice reveals increased abundance of Gammaproteobacteria and Actinobacteria in the IML of TRAG mice. (e) Genera that are increased in the IML of TRAG mice include Serratia, Pseudomonas, Acinetobacter and Cardiobacterium (Proteobacteria) and Corynebacterium (Actinobacteria). TRAG: n = 6; RAG1^−/−: n = 12.

Figure 2.

(Continued).

Figure 3.

Expression of antimicrobial factors is increased in the mucosa of TRAG mice. RNA collected from mucosal scrapings of RAG1^−/− and TRAG mice was analyzed and quantified by RNAseq. (a) Increased expression of phospholipases (Phopholipase A2 groupIIA (PLA2g2A), Phopholipase A2 group16 (PLA2g16), PLAA (phospholipase A2 activating protein), PLA2g4a (phospholipase A2 group IVA)), Regenerating Family member 3 gamma (REG3g), lactoferrin (Ltf), calprotectin component S100A8, and lysozyme (Lyz2). (b) Decreased expression of kallikrein 1 (KLK1) and KLK1b5 and increased expression of the KLK inhibitor Spink6 (Serine peptidase inhibitor kazal type 6) in the mucosa of TRAG vs. RAG1^−/− mice. n = 4 **p < .01; ****p < .001.

Figure 4.

Antibiotics differentially prevent colitis in TRAG mice. Colitis scores of the (a) cecum, (b) proximal colon, or (c) distal colon of TRAG mice treated with drinking water containing vancomycin (125 mg/ml), neomycin (250 mg/ml), metronidazole (62.5 mg/ml) ampicillin (500 mg/ml) or a cocktail of all four antibiotics (AVMN). (d) representative H&E sections of TRAG cecum, proximal colon and distal colon treated with antibiotics as described. n = 8–21 *p < .05, **p < .01, ***p < .005 vs. TRAG score.

Figure 4.

(Continued).

Figure 5.

Microbiome profiles of RAG1^−/− vs. TRAG cecum, proximal colon and distal colon. (a) Principal component analysis (PCOoA) of beta diversity as measured by Bray-Curtis dissimilarity of RAG1^−/− and TRAG cecum, proximal colon and distal colon. Alpha diversity (b) Pielou’s (evenness) and (c) Faith (abundance) between groups. (d-f) Beta diversity showing distance from RAG1^−/− (d) cecum, (e) proximal colon and (f) distal colon. n = 5 *p < .05.

Figure 5.

(Continued).

Figure 6.

Relative abundance of the Phyla Firmicutes, Bacteroidetes and Proteobacteria across the longitudinal axis of the colon of RAG1^−/− or TRAG mice. n = 5 *p < .05, ****p < .001.

Figure 7.

Relative abundance of microbial populations in the cecum, proximal colon and distal colon of RAG1^−/− and TRAG mice. (a) Phylum (b) Class and (c) Order. Each bar represents a single mouse (n = 5).

Figure 7.

(Continued).

Figure 8.

Phyla level differences in relative abundance between RAG1^−/− and TRAG mice. Significant differences in relative abundance were observed for the indicated Phyla in the (a) Cecum and (b) Distal colon. Only those Phyla with statistically different relative abundance are shown. No significant differences in Phyla abundance were observed in the proximal colon. n = 5 *p < .05, ****p < .001.

Figure 9.

Order and Family level differences in relative abundance between RAG1^−/− and TRAG mice. Significant differences in relative abundance were observed for (a) the Order Clostridia only in the cecum. Differences at the Family level were observed in the (b) cecum, and (c) distal colon. Only those Orders and Families with statistically different relative abundance are shown. No significant differences in Order were observed in the proximal or distal colon and no significant differences in Family were observed in the proximal colon. n = 5 **p < .01, ****p < .001.

Figure 10.

Genus level differences in relative abundance between RAG1^−/− and TRAG mice. Significant differences in relative abundance were observed for the indicated Genera in the (a) Cecum (b) Proximal colon and (c) Distal colon. Only those Genera with statistically different relative abundance are shown. n = 5 *p < .05, **p < .01, ****p < .001.

Figure 11.

Species level differences in relative abundance between RAG1^−/− and TRAG mice. Significant differences in relative abundance were observed for the indicated Species in the (a) Cecum (b) Proximal colon and (c) Distal colon. Only those Species with statistically different relative abundance are shown. n = 5 **p < .01, ***p < .005, ****p < .001.