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. 2016 Jun;22(6):598-605.
doi: 10.1038/nm.4102. Epub 2016 May 9.

CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands

Affiliations

CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands

Bruno Lamas et al. Nat Med. 2016 Jun.

Abstract

Complex interactions between the host and the gut microbiota govern intestinal homeostasis but remain poorly understood. Here we reveal a relationship between gut microbiota and caspase recruitment domain family member 9 (CARD9), a susceptibility gene for inflammatory bowel disease (IBD) that functions in the immune response against microorganisms. CARD9 promotes recovery from colitis by promoting interleukin (IL)-22 production, and Card9(-/-) mice are more susceptible to colitis. The microbiota is altered in Card9(-/-) mice, and transfer of the microbiota from Card9(-/-) to wild-type, germ-free recipients increases their susceptibility to colitis. The microbiota from Card9(-/-) mice fails to metabolize tryptophan into metabolites that act as aryl hydrocarbon receptor (AHR) ligands. Intestinal inflammation is attenuated after inoculation of mice with three Lactobacillus strains capable of metabolizing tryptophan or by treatment with an AHR agonist. Reduced production of AHR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Our findings reveal that host genes affect the composition and function of the gut microbiota, altering the production of microbial metabolites and intestinal inflammation.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
CARD9 is involved in recovery from colitis. (a) Representative immunohistochemistry images for cross-sections of the proximal colon from WT (left) and Card9−/− (right) mice stained for Ki67 (top) or cleaved caspase-3 (bottom) at day 12. Scale bars, 200 μm. (b) Quantification of Ki67 (top) and cleaved caspase-3 (bottom) staining in the proximal colon of WT and Card9−/− mice before and at days 7 and 12 after induction of colitis. Data are mean ± s.e.m. (c) Comparative expression of genes in the colon by microarray data analysis (log2-transformed (fold change, as compared to that at day 0)) on day 7 (left) and day 12 (right) after initiation of DSS treatment. (d) Il22, Reg3g, Reg3b, and Il17A transcript expression in the colon before (day 0, n = 3 mice per group) and after (day 7, n = 5 mice per group; day 12, n = 10 mice per group) initiation of DSS treatment. (e) Proportion of IL-22+ cells in the lamina propria of the colon in WT and Card9−/− mice. (f) Quantification of subpopulations of IL-22+ cells isolated from the lamina propria of the colon of WT and Card9−/− mice before (day 0) and after (day 12) DSS treatment. Cells were gated on CD3+CD4+ (for TH22), CD3CD4NKp46+ (for NKp46+ ILCs), CD3CD4+NKp46 (for LTi), and CD3CD4NKp46. In ac, n = 3 (day 0) and n = 5 (days 7 and 12) mice per group; in e,f, n = 5 mice per group. Throughout, data are mean ± s.e.m. *P < 0.05; ***P < 0.001; by two-tailed Student’s t-test.
Figure 2
Figure 2
The fungal and bacterial microbiota are altered in Card9−/− mice. (a) Fungal levels in the fecal microbiota were quantified using 18S rRNA qRT–PCR and were normalized to those of the bacterial population. Data are mean ± s.e.m. **P < 0.01 by two-tailed Student’s t-test. (b) Principal component analysis (PCA) based on fungal ITS2 rDNA gene sequence abundance in the feces. Axes correspond to principal components 1 (x axis) and 2 (y axis). d, day; KO, Card9−/− mice. (c) Fungal diversity on the basis of the operational taxonomic unit (OTU) number in the fecal samples from WT and Card9−/− mice. Horizontal line depicts the mean. (d) Fungal-taxon-based analysis at the phylum level in the fecal microbiota. (e) PCA plot based on bacterial 16S rDNA gene sequence abundance in fecal content. Axes correspond to principal components 1 (x axis) and 2 (y axis). (f) Bacterial-taxon-based analysis at the phylum level in the feces. (g) Bacterial diversity based on the OTU number in the fecal samples. Horizontal line depicts the mean. (h) Bacterial and fungal taxa differentially enriched in WT and Card9−/− mice (generated using LeFSE analysis). The heat map on the left shows the relative abundance of taxa, and the heat map on the right shows the linear differential analysis (LDA) scores. (i) Correlation between ITS2 and 16S rDNA Shannon diversity index in the fecal samples from DSS-treated WT (top) and Card9−/− (bottom) mice. Significance determined using linear regression. **P < 0.01, two-tailed Student’s t-test (a) and one-way analysis of variance (ANOVA) and post hoc Tukey test (c,g). Throughout, n = 3 mice per group for day 0, and n = 5 mice per group for days 7 and 12.
Figure 3
Figure 3
Transfer of the microbiota from Card9−/− mice is sufficient to increase susceptibility to colitis and reduce IL-22 production. (a) Weight of DSS-exposed GF WT mice colonized with microbiota from WT (WT→GF) or Card9−/− (Card9−/−→GF) mice (n = 23 per condition). Data are mean ± s.e.m. of four experiments. (b) Representative H&E-stained images (of three experiments) of mouse proximal colon samples (left) and mouse histological scores at day 12 (right). Data are representative of three experiments. Scale bars, 200 μm. (c,d) Representative immunohistochemistry images (of three experiments) for Ki67 (top) and cleaved caspase-3 (bottom) staining in proximal colon samples from WT→GF (left) and Card9−/−→GF (right) mice (c) and quantification of Ki67 and cleaved caspase-3 staining in the proximal colon at day 12. Data are representative of three experiments. Scale bars, 200 μm. (e) Comparative analysis (left) and heat map (right) of the gene expression in the colons of WT→GF or Card9−/−→GF mice on day 12, using NanoString. (f) Transcript expression in the colon of WT→GF or Card9−/−→GF mice. (g) Secreted IL-22 (left) and IL-17A (right) amounts by MLN cells from WT→GF or Card9−/−→GF mice. (h) Secreted IL-22 (left) and IL-17A (right) amounts by colon explants from WT→GF or Card9−/−→GF mice. ND, not detected. (i) Quantification of IL-22+ cells isolated from the lamina propria of the colon of WT→GF and Card9−/−→GF mice on day 12. Cells were gated for TH22 cells, NKp46+ ILCs, LTi cells, and CD3CD4NKp46 cells (n = 5 mice per group). Throughout, data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; by two-tailed Student’s t-test (a,b,d,g,h,i) and Mann–Whitney U-test (f). In bh, n = 5 mice per group (days 0 and 7) and n = 10 mice per group (day 12).
Figure 4
Figure 4
Tryptophan metabolism is impaired in the gut microbiota of Card9−/− mice, leading to defective AHR activation and colitis recovery. (a) Tryptophan (left), kynurenine (middle), and IAA (right) concentrations in the feces of WT, GF WT, Ido1−/−, WT→GF or Card9−/−→GF mice (n = 5 mice per group). (b) Quantification of AHR activity from fecal samples of the indicated mice (n = 5 for GF WT and Ido−/− mice; n = 12 mice for all other groups). NS, not stimulated. (c) Weight of DSS-exposed mice that were treated with DMSO or Ficz. For statistical comparisons, dagger (†) indicates WT→GF DMSO versus Card9−/−→GF DMSO, and asterisk (*) indicates Card9−/−→GF DMSO versus Card9−/−→GF Ficz. (d) Representative H&E-stained images of proximal colon cross-sections from WT→GF (left) and Card9−/−→GF (right) mice at day 12 that were treated with DMSO (top) or Ficz (bottom). Scale bars, 200 μm. (e) Histological scores (top) and length (bottom) of the colons of the indicated mice at day 12. (f) Transcript expression in the colons of the indicated mice at day 12. (g) Secreted IL-22 (left) and IL-17A (right) amounts by colon explants from the indicated mice at day 12. Throughout, data are mean ± s.e.m. *P < 0.05; ** P < 0.01; ††P < 0.01; *** P < 0.001; by one-way ANOVA and post hoc Tukey test (ac) or Kruskal–Wallis test followed by a post hoc Dunn’s test (eg). In cg, n = 11 DMSO-treated WT→GF mice per experiment; n = 12 DMSO-treated Card9−/−→GF mice per experiment; n = 9 Ficz-treated WT→GF mice per experiment; n = 6 Ficz-treated Card9−/−→GF mice per experiment.
Figure 5
Figure 5
Inoculation with lactobacilli that metabolize tryptophan and produce AHR ligands reduces colitis in an AHR-dependent manner. (a) AHR activation by culture supernatants from strains isolated from feces of WT and Card9−/− mice, relative to that culture media (n = 3 replicates for each strain). (b) AHR activation by culture supernatants from L. murinus CNCM I-5020, L. reuteri CNCM I-5022, and L. taiwanensis CNCM I-5019 isolated from feces of WT mice, relative to that by culture medium alone (n = 3 replicates for each strain). (c) Experimental design of AHR antagonist treatment and Lactobacilli inoculation. Vertical arrows indicate injections of vehicle or AHR antagonist. Stars indicate intragastric gavage with PBS or bacterial suspension. D, day. (d) AHR activation by day 21 feces from WT→GF, Card9−/−→GF, or WT→GF mice treated with an AHR antagonist (WT→GF + AHR), and Card9−/−→GF mice gavaged with L. murinus CNCM I-5020, L. reuteri CNCM I-5022, and L. taiwanensis CNCM I-5019 isolated from feces of WT mice and treated with either vehicle (Card9−/−→GF + Lacto) or an AHR antagonist (Card9−/−→GF + AHR and Lacto). (e) Weight of DSS-exposed mice. For statistical comparisons, asterisk (*) indicates WT→GF versus Card9−/−→GF; dagger (†) indicates Card9−/−→GF versus Card9−/−→GF + Lacto; double dagger (‡) indicates Card9−/−→GF + Lacto versus Card9−/−→GF + AHR and Lacto. (f) Weight of DSS-exposed mice. For statistical comparisons, asterisk (*) indicates WT→GF versus Card9−/−→GF; dagger (†) indicates WT→GF versus WT→GF + AHR; double dagger (‡) indicates Card9−/−→GF versus WT→GF + AHR. (g) Representative H&E-stained images of proximal colon cross-sections at day 12 after initial DSS exposure. Scale bars, 200 μm. (h) Histological scores at day 21. (i) Il22 expression in the colon. (j) Secreted IL-22 amounts by colon explants and MLN cells. Throughout, data are mean ± s.e.m. *P < 0.05; †P < 0.05; ‡P < 0.05; **P < 0.01; ††P < 0.01; ‡‡P < 0.01; ***P < 0.001, by Mann–Whitney U-test (a), two-tailed Student’s t-test (b), or one-way ANOVA with post hoc Tukey test (df, hj). In dj, n = 5 mice per group per experiment.
Figure 6
Figure 6
Reduced tryptophan metabolism and AHR activation in the gut microbiota of individuals with IBD, and its association with the CARD9 genotype. (a) Quantification of AHR activation from the feces of healthy subjects (HS; n = 37) and individuals with IBD (n = 102) in remission. NS, not stimulated (basal AHR activity of cells) (n = 9); TCDD, feces treated with AHR agonist (n = 9). (b) Concentrations of tryptophan, kynurenine, and IAA in the feces of HS (n = 32) and patients with IBD in remission (n = 54). (c) Quantification of AHR activation by the feces of HS and individuals with IBD in remission, as classified on the basis of SNP rs10781499, with ‘A’ denoting the risk allele (n = 101). Throughout, data are mean ± s.e.m. *P < 0.05; **P < 0.001; ***P < 0.0001; by the Kruskal–Wallis test followed by a post hoc Dunn’s test (a), Mann–Whitney U-test (b) or one-way ANOVA and post hoc Tukey test (c).

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References

    1. Silva MJ, et al. The multifaceted role of commensal microbiota in homeostasis and gastrointestinal diseases. J Immunol Res. 2015;2015:321241. - PMC - PubMed
    1. Molodecky NA, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142:46–54.e42. quiz e30. - PubMed
    1. Ananthakrishnan AN. Epidemiology and risk factors for IBD. Nat Rev Gastroenterol Hepatol. 2015;12:205–217. - PubMed
    1. Lanternier F, et al. Inherited CARD9 deficiency in otherwise healthy children and adults with Candida species–induced meningoencephalitis, colitis, or both. J Allergy Clin Immunol. 2015;135:1558–68.e2. - PMC - PubMed
    1. Hsu YMSM, et al. The adaptor protein CARD9 is required for innate immune responses to intracellular pathogens. Nat Immunol. 2007;8:198–205. - PubMed

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