Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation

Abstract

Intestinal colonization by bacteria of oral origin has been correlated with several negative health outcomes, including inflammatory bowel disease. However, a causal role of oral bacteria ectopically colonizing the intestine remains unclear. Using gnotobiotic techniques, we show that strains of Klebsiella spp. isolated from the salivary microbiota are strong inducers of T helper 1 (T_H1) cells when they colonize in the gut. These Klebsiella strains are resistant to multiple antibiotics, tend to colonize when the intestinal microbiota is dysbiotic, and elicit a severe gut inflammation in the context of a genetically susceptible host. Our findings suggest that the oral cavity may serve as a reservoir for potential intestinal pathobionts that can exacerbate intestinal disease.

Fig. 1. Isolation of a TH1-cell–inducing, multiple-antibiotic–resistant, and proinflammatory Klebsiella pneumoniae strain from the microbiota of human saliva

(A) Aggregated relative abundance of operational taxonomic units (OTUs) typical of human oral microbiota in the fecal microbiota of healthy individuals (n=150), patients with ulcerative colitis (UC; n=51), Crohn's disease (CD; n=7), primary sclerosing cholangitis (PSC; n=27), gastroesophageal reflux disease (GERD; n=18), and alcoholism (Alc; n=16). **P < 0.001; Wilcoxon rank-sum test (fig. S1B). (B) Representative FACS plots (left) and frequencies of IFN-γ⁺ cells (right) among colonic LP CD4⁺TCRβ⁺ T cells from ex-germ free (exGF) B6 mice inoculated with saliva samples from patients with CD. Each point (right) represents an individual mouse. (C) Pyrosequencing of 16S rRNA genes from the saliva microbiota of patients (Pt) and from the resulting fecal microbiota of exGF mice (n = 3 per group). Quality filter–passed sequences were classified into OTUs on the basis of sequence similarity (96% identity), and the relative abundance of OTUs and closest known species for each OTU are shown. OTUs corresponding to the eight isolated strains are marked in green. (D) The percentage of TH1 cells in the colonic LP of exGF B6 mice colonized with 8-mix, Fu-21f+Ve-2E1, Kp-2H7, 7-mix, or Ec-2B1. (E and F) SPF B6 mice were untreated (Cont) or continuously treated with antibiotics in drinking water, starting 4 days before oral administration of 2 × 10⁸ colony-forming units (CFU) of Kp-2H7. The relative abundance of Kp-2H7 DNA over time in fecal samples was determined by qPCR (E). The percentage of TH1 cells among colonic LP CD4⁺ cells was analyzed by flow cytometry on day 21 after Kp-2H7 administration (F). Amp, ampicillin; Tyl, tylosin; Spc, spectinomycin; MNZ, metronidazole. (G to I) Representative hematoxylin and eosin staining (G), representative scanning electron micrograph (SEM) (H), and histological colitis scores (I) of the proximal colon of Kp-2H7-, Ec-2B1-, or 6-mix-colonized WT or Il10^-/- mice. Scale bars, 200 μm (G) and 30 μm (H). Symbols represent individual mice. Error bars indicate means ± SD. **P < 0.01; ***P < 0.001; one-way analysis of variance (ANOVA) with post hoc Turkey's test. Data represent at least two independent experiments with similar results.

Fig. 2. Strain-specific induction of TH1 cells by K. pneumoniae

(A) Frequency of TH1 cells in the colon of GF mice orally administered heat-killed Kp-2H7 in drinking water for 3 weeks (Kp-2H7 dead). (B) Staining with DAPI (4′,6-diamidino-2-phenylindole; blue), EUB338 FISH probe (green), and Ulex europaeus agglutinin 1 (UEA1; red) of the proximal colon of mice monocolonized with Kp-2H7. Scale bar, 100 μm. (C) GF mice were monocolonized with the indicated K. pneumoniae strain, and colonic TH1 cells were analyzed after 3 weeks. Frequencies of IFN-γ⁺ colonic LP CD4⁺TCRβ⁺ T cells are depicted. Colored shading is as in (D). (D) Comparative analysis of whole genomes of the tested Klebsiella strains revealed 61 orthologous groups correlating with TH1-inducing activity. Detailed information about gene ID for each row, is given table S3 and fig. S13. (E to G) The percentage of TH1 cells in the colon of WT, Batf3^-/-, Myd88^-/-, Tlr4^-/-, Myd88^-/-Trif^-/-, Il18^-/-, and Il1r1^-/- mice monocolonized with Kp-2H7. (H) Differential gene expression in the colonic ECs and DCs from WT mice monocolonized with Kp-2H7 or BAA-2552 for 1 week. Heatmap colors represent the z-score normalized fragments per kilobase of exon per million fragments mapped (FPKM) values for each gene. (I) SPF WT and Ifngr1^-/- mice were treated with Amp and gavaged with Kp-2H7 as in Fig. 1E. The percentage of TH1 cells among colonic LP CD4⁺ cells is depicted. Symbols represent individual mice. Error bars indicate means ± SD. **P < 0.01; ***P < 0.001; ns, not significant (P > 0.05); one-way ANOVA with post hoc Turkey's test. Data represent at least two independent experiments with similar results.

Fig. 3

Klebsiellaaeromobilis is another TH1cell–inducing oral cavity-derived bacterial species. (A) Frequencies of IFNγ⁺ within colonic LP CD4⁺TCRβ⁺ T cells from exGF mice inoculated with saliva samples from healthy donors and UC patients. (B) Pyrosequencing of 16S rRNA genes of the saliva microbiota of healthy controls and patients with UC and of the resulting fecal microbiota of exGF mice (n = 3 to 4 mice per group). The relative abundance of OTUs and closest known species for each OTU are shown. OTUs corresponding to the isolated 13 strains and Kp-40B3 are marked in green and yellow, respectively. (C) The percentage of TH1 cells in the colonic LP of B6 mice colonized with 13-mix, Ef-11A1, Ka-11E12, or 11-mix. (D) SPF B6 mice were untreated or continuously treated with antibiotics in the drinking water starting 4 days before oral administration of 2 × 10⁸ CFU of Ka-11E12. Ka-11E12 abundance in fecal samples was determined by qPCR. (E) Representative SEM images of the colon of Ka-11E12–monocolonized WT or Il10^-/- mice. (F) Staining with DAPI (blue), EUB338 FISH probe (green), and UEA1 (red) of the colon of mice monocolonized with Ka-11E12. Scale bars, 30 μm (E) and 100 μm (F). (G) The percentage of TH1 cells in the colonic LP of B6 mice colonized with Kp-40B3. Each point in (A), (C), and (G) represents an individual mouse (thick bars, means); points in (D) represent means of a group of five or six mice. Error bars, SD. ***P < 0.001; one-way ANOVA with post hoc Tukey's test [(A) and (C)] and two-tailed unpaired Student's t test (G).

Fig. 4. Association of Klebsiella species and TH1-related genes with IBD

(A) Aggregated relative abundance of OTUs assigned to the genus Klebsiella in the samples from healthy donors and patients with the indicated diseases. *P < 0.05; **P < 0.01; Wilcoxon rank-sum test. (B to D) The reads of individual samples from PRISM and UPenn cohorts were mapped to Klebsiella species (B) and to the gene sequences correlated with TH1 induction [(C) and (D)]. Detailed information about gene ID for each row is given in table S3 and fig. S13. Bars in (B) represent mean values, with error bars indicating the range. Heatmaps in [(C) and (D)] show values of reads per kilobase per million reads (RPKM) for the TH1-related genes.