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. 2015 Oct 8;163(2):367-80.
doi: 10.1016/j.cell.2015.08.058. Epub 2015 Sep 24.

Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells

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Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells

Koji Atarashi et al. Cell. .
Free PMC article


Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.


Figure 1
Figure 1. EC Adhesion and Th17 Induction by SFB in Mice and Rats
B6 or IQI mice (A–D) and F344 rats (E–H) were monocolonized with M-SFB or R-SFB for 3 weeks. (A and E) qPCR analysis for SFB DNA in feces, SI luminal contents, and mucosal tissue specimens. (B and F) SEM images of epithelial surfaces in the SI and colon. (C, D, G, and H) Th17 cell frequencies in SI and colon LP. Representative dot plots gated on total lymphocytes (C and G) and summarized data gated on CD4+ T cells (D and H) are shown. Error bars represent SD. See also Figure S1.
Figure 2
Figure 2. Effects of SFB Adhesion on Antigen-Specific Th17 Cells, IgA+ Cells, and ILC3s
(A and B) Cytokine responses of SI LP CD4+ T cells from M-SFB and R-SFB co-colonized B6 mice to the indicated ex vivo stimulation. A/C, autoclaved cecal contents; P/I, PMA/ionomycin. (C) Fecal IgA levels of indicated monocolonized B6 mice and F344 rats (n = 5). (D) The frequencies of ILC3s among CD90+ cells (left dot plots) and IL22+ cells among ILC3s (right histograms and bar graphs) in SI LP cells of the indicated mice. Error bars represent SD. See also Figure S2.
Figure 3
Figure 3. Adhesion-Mediated EC Activation
(A) Heatmap showing the relative abundance for gene transcripts upregulated in SI ECs of M-SFB-mono versus GF and R-SFB-mono IQI mice. Each column represents a single mouse. (B) qPCR for the selected genes relative to Gapdh in SI ECs from the indicated mice. (C) Immunostaining of SIs from the corresponding mice for SAA1 (red) and DAPI (blue). Scale bar, 100 μm. (D and E) The percentage of RORγt+IL-17+ cells after culture of splenic naive CD4 T cells in the presence of various combinations of splenic CD11c+ cells, IL-6, TGF-β, SAA1, and/or anti-IL-1R1 antibody. (F) Il1b expression in splenic CD11c+ cells from B6 SPF mice stimulated with recombinant SAA1. (G) SI LP Th17 cell frequencies of IQI M-SFB-mono mice either untreated (−) or treated with NAC. (H) RNA-seq and DNase-seq data from SI ECs of GF and conventional (CV) mice at the region surrounding the Saa1 locus. H3K27ac ChIP-seq data for SI and various other tissues obtained from (Shen et al., 2012). ChIP-seq datasets for TFs assayed in various cell and tissue types. SI EC regulatory regions are highlighted in gray. (I) The relative abundance for TF genes that are >2-fold different between M-SFB-mono mice versus GF or R-SFB-mono mice. (J) SAA1 and C/EBPδ genes expression in aMoS7 cells cultured with cecal content from GF mice or a Percoll-enriched M-SFB or R-SFB either in contact or separated by a Transwell membrane (TW). Error bars represent SD. See also Figure S3.
Figure 4
Figure 4. Influences of Mouse Genetic Background
(A and B) The percentages of Th17, IL-22+ ILC3, and CD138+IgA+ cells in SI LP of the indicated mice. (C) SEM images of SI epithelial surface of the indicated BALB/c mice. (D and E) IL-1β protein production (D) and mRNA expression (E) from SI LP CD11c+ and CD11c cells from the indicated mice. (F–I) Saa1/2 mRNA expression in SI ECs from the indicated mice. (J and K) The percentage of RORγt+IL-17+ cells among SI LP CD4+ T cells in IL-1β-injected BALB/c mice (J) or Il1r1−/− B6 mice (K). Error bars represent SD. See also Figure S4.
Figure 5
Figure 5. EC Adhesion-Mediated Th17 Induction by C. rodentium
IQI mice were monocolonized with wild-type (WT) or Δeae mutant of C. rodentium for 5 days. (A) DNA of C. rodentium in colonic luminal contents and mucosal tissues. (B) SEM images of colonic villi of the indicated mice. (C) C. rodentium localization visualized by O antigen antisera (green) and DAPI (blue) staining. Scale bar, 100 μm. (D) The percentage of RORγt+IL-17+ colonic CD4+ T cells. (E) Fecal IgA levels of the indicated mice (n = 4). (F) The relative abundance of gene transcripts that were commonly upregulated in M-SFB-mono mice versus R-SFB-mono mice and WT C. rodentium-mono mice versus Δeae C. rodentium-mono mice. (G) Duox2 and Duoxa2 mRNA expression in colonic ECs of the indicated mice. (H) Th17 cell frequencies of WT C. rodentium-mono IQI mice either untreated (−) or treated with NAC. Error bars represent SD. See also Figure S5.
Figure 6
Figure 6. EC Adhesion-Mediated Th17 Induction by Extracellular Pathogens
(A) SEM images of colonic villi from IQI GF mice monocolonized with Δstx1Δstx2 or Δstx1Δstx2Δeae EHEC O157:H7 for 3 weeks. (B) O antigen antisera (green) and DAPI (blue) staining for EHEC O157 visualization. Scale bar, 50 μm. (C–E) Th17 and Th1 cell proportions in the colonic LP CD4+ T cells from IQI mice inoculated with EHEC O157:H7 (C), C. albicans (D), or L. monocytogenes (E). (F) SEM images of the colon of IQI GF mice and mice infected orally with L. monocytogenes. Error bars represent SD. See also Figure S6.
Figure 7
Figure 7. Th17 Induction and EC Adhesion by 20 Bacterial Strains Derived from the Human Intestine
(A) Th17 cell frequencies in IQI GF mice colonized with stool from patients with UC or healthy adults. (B) Th17 cell frequencies in UC5-1 or H23-colonized IQI mice either untreated (cont) or treated with the indicated antibiotics. (C) Cecal microbiota compositions of each mouse (n = 4 or 5 per group). OTUs positively correlated with the frequency of Th17 cells are marked in red and those negatively correlated marked in blue. OTUs corresponding to the isolated 20 strains are marked in green. ID, isolated strain ID. See also Table S1. (D–F) The percentage of Th17 and Th1 cells in the colonic LP of B6 mice (D and E) and F344 rats (F) colonized with the mixture of 20 strains (20st.). (G) qPCR for Nos2 and Duoxa2 in colonic EC (upper panels) and FACS for LP IgA+ cells and IL-22+ ILC3s (lower panels) of B6 mice colonized with the 20 strains. (H and I) SEM images and FISH staining with EUB338 of the proximal colon of mice and rats colonized with the 20 Th17-inducing strains versus the 17 Treg-inducing strains. Scale bar, 100 μm. Error bars represent SD. See also Figure S7.

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