Fig. 5.

Mucosal colonization of B. fragilis requires suppression of host Th17 responses. (A) A population of B. fragilis is associated with the colonic mucosa. Colons from germ-free or B. fragilis mono-associated mice were fixed, stained with chicken anti-B. fragilis (green) and nuclear counterstained with DAPI (white) and imaged by whole mount confocal microscopy. Images are similar to five different z-stack images per colon, and representative of 5 mice. (B) Colon sections or luminal contents from B. fragilis mono-associated mice were homogenized and serially diluted to obtain live bacterial counts. CFUs (colony forming units) per gram of tissue were determined after microbiologic plating. Each symbol represents an individual animal. ***p value <0.001. (C) qRT-PCR analysis was performed using Bacteriodes-specific primers on RNA extracted from colon tissue or luminal contents. GF: germ-free; B.frag: B. fragilis. Error bars represent SD from individual mice in the same experiment and are representative of two independent trials. (D) qRT-PCR analysis for B. fragilis was performed on RNA extracted from colon homogenates from indicated animals. The bar furthest to the right shows colonization of B. fragilisΔPSA in animals orally treated with purified PSA. GF: germ-free; B.frag: B. fragilis; ΔPSA: B. fragilisΔPSA. Data shown for 4 animals per group, and are representative of 2 independent trials. **p value < 0.01. (E) B. fragilis is able to colonize the mucosa in the presence of other Th17 inducing organisms. Germ-free animals were colonized with SFB alone, or co-colonized with B. fragilis & SFB or B. fragilisΔPSA & SFB. RNA was extracted from colon homogenates and primers specific for Bacteroides were used to determine the relative abundance of tissue associated B. fragilis. Data shown for 4 animals per group, and are representative of 2 independent trials. *p value <0.05. (F) TLR2 deletion on CD4+ T cells leads to decreased B. fragilis mucosal colonization. Germ-free RAG−/− animals were reconstituted with TLR2−/− or WT CD4+ T cells and colonized with either WT B. fragilis or B. fragilisΔPSA. Colons were prepared and analyzed as in panel (D). **p value < 0.01. (G) Ablation of Tregs leads to decreased B. fragilis mucosal colonization. Germ-free RAG−/− animals were reconstituted with Foxp3-DTR bone and colonized. Two months post-reconstitution animals were treated with PBS (+Tregs) or with DT (-Tregs), and colons prepared as described in panel (D). **p value < 0.01. (H and I) Neutralization of IL-17A increases B. fragilis colonization. Germ-free animals were colonized with B. fragilisΔPSA and either treated with an antibody that neutralizes IL-17A (α-IL17A) or an isotype control. Colon homogenates were analyzed by live bacterial plating (H) or qRT-PCR (I) as described above. Each symbol in (H) represents an individual animal. Error bars in (I) show SD from triplicate samples and are representative of two independent trials. *p value <0.05.