Dissecting the respective roles of microbiota and host genetics in the susceptibility of Card9-/- mice to colitis

Abstract

Background: The etiology of inflammatory bowel disease (IBD) is unclear but involves both genetics and environmental factors, including the gut microbiota. Indeed, exacerbated activation of the gastrointestinal immune system toward the gut microbiota occurs in genetically susceptible hosts and under the influence of the environment. For instance, a majority of IBD susceptibility loci lie within genes involved in immune responses, such as caspase recruitment domain member 9 (Card9). However, the relative impacts of genotype versus microbiota on colitis susceptibility in the context of CARD9 deficiency remain unknown.

Results: Card9 gene directly contributes to recovery from dextran sodium sulfate (DSS)-induced colitis by inducing the colonic expression of the cytokine IL-22 and the antimicrobial peptides Reg3β and Reg3γ independently of the microbiota. On the other hand, Card9 is required for regulating the microbiota capacity to produce AhR ligands, which leads to the production of IL-22 in the colon, promoting recovery after colitis. In addition, cross-fostering experiments showed that 5 weeks after weaning, the microbiota transmitted from the nursing mother before weaning had a stronger impact on the tryptophan metabolism of the pups than the pups' own genotype.

Conclusions: These results show the role of CARD9 and its effector IL-22 in mediating recovery from DSS-induced colitis in both microbiota-independent and microbiota-dependent manners. Card9 genotype modulates the microbiota metabolic capacity to produce AhR ligands, but this effect can be overridden by the implantation of a WT or "healthy" microbiota before weaning. It highlights the importance of the weaning reaction occurring between the immune system and microbiota for host metabolism and immune functions throughout life. A better understanding of the impact of genetics on microbiota metabolism is key to developing efficient therapeutic strategies for patients suffering from complex inflammatory disorders. Video Abstract.

Keywords: Lactobacillus; AhR ligands; CARD9; Genetics; Gut microbiota; IL-22; Metabolism; Trp metabolism.

Fig. 1

Card9 contributes to colitis recovery and controls intestinal immune response independently of the gut microbiota. A Weight (left) and Disease Activity Index (DAI, right) score of DSS-exposed GF WT and GF Card9^−/− mice. B Representative H&E-stained images of colon cross sections from DSS-exposed GF WT (upper panel) and GF Card9^−/− (lower panel) mice at day 7 (left) and day 12 (right). Scale bars, 200 μm. C Histological score of colon sections at days 7 and 12. One representative experiment out of two. D Gene Ontology analysis of microarray data showing downregulation of the expression of genes involved in host defense (GO:0006952), immune response (GO:0006955), and inflammatory response (GO:0006954) (top signature, DAVID annotation) in the colon of GF Card9^−/− versus GF WT mice at day 7 of colitis. E Il-22, Reg3β, and Reg3γ and F Il-1β and TNF-α expression by qRT-PCR in total colon tissue of DSS-exposed GF WT and GF Card9.^−/− mice at days 0, 7, and 12, normalized to Gapdh. Data points represent individual mice. Data are mean ± SEM of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, as determined by as determined by two-way analysis of variance (ANOVA) with Sidak’s posttest (A, C) and Mann–Whitney test (E, F)

Fig. 2

Card9^−/− mice susceptibility to colitis is not overridden by colonization with a WT microbiota. A Schematic representation of the DSS-induced colitis experiment preceded by 3 weeks of oral administration (gavage) of GF WT and GF Card9^−/− mice with the microbiota of WT mice (WT ➔ GF WT and Card9^−/− ➔ GF WT) or Card9^−/− mice (WT ➔ GF Card9^−/− and Card9^−/− ➔ GF Card9^−/−). DSS 2% in drinking water for 7 days and then water for 5 days. B Weight of DSS-exposed GF Card9^−/− or GF WT mice colonized with the microbiota of WT mice (WT ➔ GF Card9^−/− and WT ➔ GF WT) or Card9^−/− mice (Card9^−/− ➔ GF WT and Card9^−/− ➔ GF Card9^−/−). C Representative H&E-stained images of colon cross sections from DSS-exposed GF Card9^−/− colonized with the microbiota of WT mice (WT ➔ GF Card9^−/−, left) or Card9^−/− mice (Card9^−/− ➔ GF Card9^−/−, right) at day 12. Scale bars, 500 μm. D Histological score of colon sections at day 12. E Il-22, Reg3β, and Reg3γ expression by qRT-PCR in total colon tissue at day 12, normalized to Gapdh. IL-17, IL-6, IL-10, and IL-22 concentration measured by ELISA in total colon tissue (F) or mesenteric lymph nodes (MLN, G) of WT ➔ GF Card9^−/− and Card9^−/− ➔ GF Card9^−/.⁻ mice at day 12. Data points represent individual mice. Data are mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, as determined by two-way analysis of variance (ANOVA) with Sidak’s posttest (B) and Mann–Whitney test (D, E, F, G)

Fig. 3

WT microbiota shaped by Card9 gene deletion exhibits altered composition and AhR activity. A Alpha-diversity analysis (left panel: observed ASVs, right panel: Shannon index) of the WT inoculum and the fecal microbiota of WT ➔ GF WT and WT ➔ GF Card9^−/− mice at day 7 and 21. B Beta-diversity analysis (PCoA) using Jaccard index (binary) and PERMANOVA. C Pairwise distance (Bray–Curtis divergence) of the fecal microbiota of WT ➔ GF WT and WT ➔ GF Card9^−/− mice at day 7 versus 21. D AhR activity (shown as “fold change”) measured in feces of WT ➔ GF WT and WT ➔ GF Card9^−/.⁻ mice at days 6 and 21 after colonization. Data are mean ± SEM. *P < 0.05 and **P < 0.01, as determined by Wilcoxon test of pairwise (C) and unpaired t-test (D)

Fig. 4

Card9 regulates Lactobacillus strains capacity to produce AhR ligands. A Schematic representation of the gavage of GF WT or Card9^−/− mice five different bacterial strains and analyses performed. B Concentration of bacterial strains in feces reported as CFU/g of feces of GF WT and GF Card9^−/− mice after gavage with three Lactobacillus strains known to produce AhR ligands, L. murinus CNCM I-5020, L. reuteri CNCM I-5022, and L. taiwanensis CNCM I-5019 (left panel), Bacteroides thetaiotaomicron VPI-5482 (middle panel), or Escherichia coli MG1655 (right panel). C qRT-PCR detection of each of the three gavaged Lactobacillus strains (L. murinus CNCM I-5020, L. reuteri CNCM I-5022, and L. taiwanensis CNCM I-5019) in colon of GF WT and GF Card9^−/− mice. D Induction of AhR activity (shown as “fold change”) induced by Lactobacillus strains colonization in GF WT and GF Card9^−/− mice at days 11 and 23 after gavage. E Percentage of IL-22⁺ cells among CD4⁺ αβ T cells (left) and NKp46⁺ ILCs in the colon lamina propria of GF WT and GF Card9^−/− mice, at days 0 and 24 after gavage with the three Lactobacillus strains. Data points represent individual mice. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by Mann–Whitney test

Fig. 5

Inherited Card9^−/− microbiota controls Trp metabolism independently of the host genotype. A Schematic representation of the cross-fostering experiment with conventional WT and Card9^−/− mice showing the adoption of half of the offspring by a nursing mother of different phenotypes, leaving half of each original litter with their birth mother (WT mothers with half WT and half Card9^−/− pups; Card9^−/− mothers with half WT and half Card9^−/− pups). Pups were weaned at week 4 and kept in separate cages according to their genotype and nursing mother until week 9, when targeted metabolomics focused on Trp metabolism on feces and serum of the pups was performed, i.e., 5 weeks after weaning. B Beta-diversity analysis (PCoA) of the fecal microbiota of mothers and pups, separated according to the nursing mothers genotype (left) or the pups genotype (right), at week 9 of age, i.e., 5 weeks after weaning, using Jaccard index (binary) and PERMANOVA (based on 16 s sequencing). C Principal coordinates analysis showing metabolites composition of the feces (upper panel) and serum (lower panel) of the pups, separated according to the nursing mothers genotype (left) or the pups genotype (right), at week 9 of age. D Trp or E indoles concentration measured in feces (top) or serum (bottom) of the pups separated according to either the microbiota inherited from the nursing mother genotype (left) or the pups genotype (right) at week 9 of age. F Correlation between AhR activity induction (shown as fold change) and functionality of Card9 gene (in the nursing mother genotype and/or the pups genotype) at week 9 of age. Significance was determined by using Spearman linear. Data points represent individual mice. *P < 0.05, **P < 0.01, as determined by Mann–Whitney test. Trp, tryptophan