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Science 332 (6032): 974-977

Copyright © 2011 by the American Association for the Advancement of Science

The Toll-Like Receptor 2 Pathway Establishes Colonization by a Commensal of the Human Microbiota

June L. Round1,*, S. Melanie Lee1, Jennifer Li1, Gloria Tran1, Bana Jabri2, Talal A. Chatila3, and Sarkis K. Mazmanian1,*

1 Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
2 Department of Pathology, Department of Pediatrics, Department of Medicine, University of Chicago, Chicago, IL 60637, USA.
3 Division of Immunology, Allergy and Rheumatology, Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.


Figure 1
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Fig. 1. PSA actively suppresses TH17 cell development during B. fragilis colonization through Foxp3+ Tregs. (A) Colonic lamina propria lymphocytes (LPLs) were harvested and stained with antibodies against CD4 and IL-17A and analyzed by flow cytometry. Numbers indicate the percentage of CD4+IL-17A+ (TH17) cells. Conventional mice are specific pathogen free. (B) Compiled data from three independent experiments as in (A). CV, conventional; GF, germ-free; B.frag, B. fragilis; {Delta}PSA, B. fragilis{Delta}PSA. ***P < 0.001, two-way analysis of variance. (C and D) CD4+ T cells were isolated from the mesenteric lymph nodes of the indicated animals. RNA was collected and used as a template to determine the relative levels of IL-17A (C) and ROR{gamma}t (D) transcript. Error bars represent SDs from triplicate samples. The data are representative of three independent experiments. (E) B. fragilis{Delta}PSA mono-associated mice were treated with either phosphate-buffered saline (PBS) or PSA, and the LPLs were isolated and the percentage of CD4+IL-17A–producing cells was determined by flow cytometry. Each symbol represents an individual animal (n = 3 to 4 mice per group). ***P < 0.001. (F and G) Germ-free Rag1–/– animals were reconstituted with bone marrow from Foxp3-DTR mice and then mono-associated with B. fragilis. Animals were treated with either PBS (–DT) or diphtheria toxin (+DT) as described (16). Colonic LPLs were harvested after Treg ablation and restimulated with phorbol 12-myristate 13-acetate (PMA)–ionomycin and brefeldin A. Cells were stained with antibodies to CD4, Foxp3, and IL-17A and analyzed by flow cytometry. (Right) Symbols represent T cell proportions from individual mice within a single experiment (n = 3 to 4 mice per group) and are representative of two independent trials. ***P <0.001.

 

Figure 2
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Fig. 2. PSA signals through TLR2 on CD4+ T cells to suppress TH17 cell responses. (A) Bone marrow–derived dendritic cells from wild-type (WT) or Tlr2–/– mice were incubated with splenic CD4+ T cells. Amounts of secreted IL-10 were determined by enzyme-linked immunosorbent assay (ELISA). Error bars represent SDs from two independent assays performed in quadruplicate. ***P < 0.001; NS, not significant. (B) CD4+ T cells were isolated from WT mice, and the indicated knockout mice and cells were stimulated as in (A). MyD is Myd88–/–. IL-10 was assayed by ELISA. **P < 0.01. Error bars represent SDs for quadruplicate samples and are representative of two independent trials. (C) CD4+Foxp3+ Tregs were purified from Foxp3EGFP mice (26) and Tlr2–/– X Foxp3EGFP mice and stimulated with plate-bound antibodies against CD3 and recombinant TGF-β, with PSA or with the indicated TLR ligands. Equal numbers of live cultured Tregs were then incubated with CFSE (carboxyfluorescein diacetate succinimidyl ester)–pulsed responder cells (CD4+Foxp3). Percent suppression is determined by the ratio of proliferating responder cells in each condition relative to proliferation in the absence of added Tregs. Error bars are SDs from a single experiment performed in duplicate and are representative of two independent trials. (D and E) Germ-free Rag1–/– animals were reconstituted with CD4+ T cells from WT or Tlr2–/– mice and then mono-associated with either WT B. fragilis or B. fragilis{Delta}PSA. Colonic LPLs were isolated and analyzed for TH17 cell proportions by flow cytometry. Plots are gated on CD4+ cells. (D) Each symbol represents an individual animal (n = 3 to 4 mice per group), and data are representative of two independent trials. **P < 0.01; ***P < 0.001.

 

Figure 3
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Fig. 3. Mucosal colonization of B. fragilis requires suppression of host TH17 responses. (A) Colons from germ-free or B. fragilis mono-associated mice were fixed, stained with chicken antibodies against B. fragilis (green) and nuclear-counterstained with 4',6-diamidino-2-phenylindole (DAPI) (white) and imaged by whole-mount confocal microscopy. Images are similar to five different z-stack images per colon and representative of five 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 (n = 3 to 4 animals per group) and is representative of three independent trials. ***P < 0.001. (C) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis was performed with Bacteriodes-specific primers on RNA extracted from colon tissue or luminal contents. GF, germ-free; BF, B. fragilis. Error bars represent SDs 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. fragilis{Delta}PSA in animals orally treated with purified PSA. GF, germ-free; B.frag, B. fragilis; {Delta}PSA, B. fragilis{Delta}PSA. Data are shown for four animals per group and are representative of two independent trials. **P < 0.01. (E) Germ-free Rag1–/– animals were reconstituted with Tlr2–/– or WT CD4+ T cells and colonized with either WT B. fragilis or B. fragilis{Delta}PSA. Colons were prepared and analyzed as in (D). **P < 0.01. (F) Germ-free Rag1–/– animals were reconstituted with Foxp3-DTR bone marrow and colonized with B. fragilis. Two months after reconstitution animals were treated with either PBS (–DT) or with diphtheria toxin (+DT), and colons were prepared as described in (D). **P < 0.01. (G and H) Neutralization of IL-17A increases B. fragilis colonization. Germ-free animals were colonized with B. fragilis{Delta}PSA and treated with either an antibody that neutralizes IL-17A (α-IL-17A) or an isotype control (Iso). Colon homogenates were analyzed by live bacterial plating (G) or qRT-PCR (H) as described in (B) and (C). Each symbol in (G) represents an individual animal. Error bars in (H) show the SDs from individual animals and are compiled data from two independent trials with three or four animals per group. *P < 0.05.

 


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