Microbiota-derived indoles alleviate intestinal inflammation and modulate microbiome by microbial cross-feeding

Abstract

Background: The host-microbiota interaction plays a crucial role in maintaining homeostasis and disease susceptibility, and microbial tryptophan metabolites are potent modulators of host physiology. However, whether and how these metabolites mediate host-microbiota interactions, particularly in terms of inter-microbial communication, remains unclear.

Results: Here, we have demonstrated that indole-3-lactic acid (ILA) is a key molecule produced by Lactobacillus in protecting against intestinal inflammation and correcting microbial dysbiosis. Specifically, Lactobacillus metabolizes tryptophan into ILA, thereby augmenting the expression of key bacterial enzymes implicated in tryptophan metabolism, leading to the synthesis of other indole derivatives including indole-3-propionic acid (IPA) and indole-3-acetic acid (IAA). Notably, ILA, IPA, and IAA possess the ability to mitigate intestinal inflammation and modulate the gut microbiota in both DSS-induced and IL-10^-/- spontaneous colitis models. ILA increases the abundance of tryptophan-metabolizing bacteria (e.g., Clostridium), as well as the mRNA expression of acyl-CoA dehydrogenase and indolelactate dehydrogenase in vivo and in vitro, resulting in an augmented production of IPA and IAA. Furthermore, a mutant strain of Lactobacillus fails to protect against inflammation and producing other derivatives. ILA-mediated microbial cross-feeding was microbiota-dependent and specifically enhanced indole derivatives production under conditions of dysbiosis induced by Citrobacter rodentium or DSS, but not of microbiota disruption with antibiotics.

Conclusion: Taken together, we highlight mechanisms by which microbiome-host crosstalk cooperatively control intestinal homoeostasis through microbiota-derived indoles mediating the inter-microbial communication. These findings may contribute to the development of microbiota-derived metabolites or targeted "postbiotic" as potential interventions for the treatment or prevention of dysbiosis-driven diseases. Video Abstract.

Keywords: Lactobacillus; Indole derivatives; Intestinal inflammation; Microbial tryptophan metabolites.

Fig. 1

L. reuteri alters the intestinal microbiota composition and microbial tryptophan metabolite. A Differential enrichment of the mouse colonic microbiota in response to L. reuteri supplementation on day 28 (see the Figure S1A legend for the experimental scheme). The taxonomic classifications of the bacteria are shown in the left panel. Blanks are unassigned taxa. The 36 differentially enriched bacterial taxa are ranked by estimating the mean decrease in accuracy based on random forest analysis. The right panel shows the LEfSe analysis of the 36 bacterial taxa. B Serum concentrations of metabolites in tryptophan metabolism among three groups of mice (n = 10). C Volcano plot showing differential gene expression in the colon of DSS-treated mice with or without L. reuteri supplementation. Each red dot indicates a significantly upregulated gene, while a blue dot represents a significantly downregulated gene, with each gray dot showing a gene with no significant difference. D Pairwise comparisons of tryptophan metabolites, with a color gradient denoting Spearman’s correlation coefficient. The correlation between the bacterial/gene expression profiles and each metabolite using partial Mantel tests. The edge width corresponds to Mantel’s R statistic for the corresponding distance correlations, and the edge color denotes statistical significance. E Real-time PCR assay showing the expression of Pxr and its target genes (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 2

IPA and IAA ameliorate DSS-induced colitis. A Experimental scheme of the mouse trial (n = 5). Six-week-old C57BL/6 mice were administered with a cocktail of antibiotics for 2 weeks, followed by oral administration of 40 mg/kg IPA and/or IAA daily with or without 3% DSS in drinking water for another 7 days. The severity of colitis was assessed by determining the changes in body weight (B) and colon length (C). D Serum FITC-dextran levels among different groups of mice (n = 4). E Goblet cell changes in the colon of mice. The left panel shows the goblet cells per crypt of the colon, and the right panel shows representative images of alcian blue staining for goblet cells within the inner mucus layer of colonic sections. F Levels of IL-6 and IL-1β in the colonic tissue using ELISA (n = 4). G The levels of occludin and E-cadherin proteins determined by Western blotting (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

Fig. 3

L. reuteri alters the expression of major enzymes involved in microbial metabolism of tryptophan. Six-week-old C57BL/6 mice were orally gavaged with 200 µl of PBS with or without L. reuteri I5007 (10⁹ CFU/mL) daily for 3 weeks, followed by a week of 3% DSS administration in drinking water to induce colitis (See Figure S1A for experimental scheme). Metatranscriptomics was performed with the colonic microbiota collected on day 28. A Major metabolic pathways and enzymes involved in microbial tryptophan metabolism. B PCoA plot of the Bray–Curtis distance showing the differences in the colonic microbiota function. C The differences in mRNA expression levels of major enzymes involved in microbial tryptophan metabolism among three groups of mice. Statistics was performed using DESeq2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

Fig. 4

Indole derivatives alleviate intestinal inflammation in IL-10^−/− mice. Six-week-old IL-10^−/− (KO) mice were orally gavaged daily with or without ILA (20 mg/kg), IPA (40 mg/kg), or IAA (40 mg/kg) for 4 weeks (n = 4). Congenic wild-type (WT) mice were also gavaged daily for 4 weeks as negative controls. A Colonic mRNA expression levels of proinflammatory cytokines (IL-6 and IL-1β) (n = 4) by RT-qPCR. B The levels of TNF-α and LPS in the colon (n = 4) using ELISA. C PCoA plot of the Bray–Curtis distance of the colonic microbiota among different groups of mice. D Heatmap showing the relative abundance of different bacterial genera. E Co-occurrence network analysis of bacterial genera among different groups of mice. Edges representing significant SparCC correlations indicate |r|> 0.6 and p < 0.05. Each light blue line represents a significant negative correlation, while each light red line represents a significant positive correlation. The size of the points represents the degree of the node. The thickness of the line is proportional to the degree of correlation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

Fig. 5

ILA promotes the microbial production of IPA and IAA in vitro. The colonic microbiota was cultured under anaerobic conditions with or without an addition of de Man Rogosa Sharpe (MRS) medium, cell-free supernatant (CFS) of L. reuteri I5007, or ILA for 12 h at 37 °C. A The concentrations of IPA and IAA in the anaerobic culture supernatant of the colonic microbiota (n = 4). B PCoA plot of the Bray–Curtis distance among different treatment groups (n = 4). C Relative abundance of three major enzymes involved in microbial tryptophan metabolism as predicted by PICRUSt2. D Network analysis of differentially enriched bacterial genera and enzymes. Edges representing significant Spearman’s correlations indicate |r|> 0.7 and p < 0.05. The thickness of each line is proportional to the magnitude of the correlation. The pie chart shows the relative abundance (%) of bacterial genera or enzymes among different groups. ArAT, aromatic amino acid aminotransferase; ID, indolepyruvate decarboxylases. Relative abundances of acyl-CoA dehydrogenase (ACD) (E) and indoleacetamide hydrolase (IAAH) (F) predicted by PICRUSt2 among DSS-treated mice administered with or without ILA (see the Figure S7 legend for experimental details). G The C. sporogenes was cultured with or without an addition of Tryptophan, L. reuteri I5007, ArAT-deficient mutant L. reuteri I5007(△L.R), CFS of L. reuteri I5007 or △L.R, ILA for 24 h at 37 °C (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

Fig. 6

ILA promotes the microbial synthesis of IPA and IAA. A Experimental scheme to examine the role of cross-feeding (n = 4). The intestinal microbiota was depleted by 2 weeks of antibiotic administration in the drinking water, followed by oral gavage of ArAT-deficient mutant (△L.R) or heat-killed L. reuteri for another 2 weeks with or without oral administration of 40 mg/kg ILA in the second week. Colitis was induced by adding 3% DSS in drinking water in the fifth week. Colitis severity was assessed by determining the changes in body weight (B) and colon length (C). The concentrations of IAA (D) and IPA (E) were measured in the colon contents. F Experimental scheme to evaluate the role of the intestinal microbiota on ILA-mediated synthesis of IPA and IAA. Mice were administered with or without a cocktail of antibiotics for 2 days, followed by induction of dysbiosis for a week (n = 7 or 8). Three different dysbiosis models were employed including 3% DSS in drinking water, antibiotic cocktail in drinking water, and oral daily challenged with 10⁸ CFU/ml C. rodentium. G Richness and Shannon Index of the colonic microbiota among different groups of mice on day 9. H PCoA plot of the Bray–Curtis distance. Comparisons of the concentrations of IAA (I) and IPA (J) in the colon contents among groups are shown (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant