Metabolites produced by the intestinal microbiota are potentially important physiological modulators. Here we present a metabolomics strategy that models microbiota metabolism as a reaction network and utilizes pathway analysis to facilitate identification and characterization of microbiota metabolites. Of the 2,409 reactions in the model, B53% do not occur in the host, and thus represent functions dependent on the microbiota. The largest group of such reactions involves amino-acid metabolism. Focusing on aromatic amino acids, we predict metabolic products that can be derived from these sources, while discriminating between microbiota-and host-dependent derivatives. We confirm the presence of 26 out of 49 predicted metabolites, and quantify their levels in the caecum of control and germ-free mice using two independent mass spectrometry methods. We further investigate the bioactivity of the confirmed metabolites, and identify two microbiota-generated metabolites (5-hydroxy-L-tryptophan and salicylate) as activators of the aryl hydrocarbon receptor.
Nutritional modulation of the immune system is an often exploited but poorly characterized process. In chickens and other food production animals, dietary enhancement of the immune response is an attractive alternative to antimicrobial use. A yeast cell wall component, beta-1,3/1,6-glucan, augments the response to disease in poultry and other species; however, the mechanism of action is not clear. Ascorbic acid and corticosterone are better characterized immunomodulators. In chickens, the spleen acts both as reservoir and activation site for leukocytes and, therefore, splenic gene expression reflects systemic immune function. To determine effects of genetic line and dietary immunomodulators, chickens of outbred broiler and inbred Leghorn and Fayoumi lines were fed either a basal diet or an experimental diet containing beta-glucans, ascorbic acid, or corticosterone from 56 to 77 d of age. Spleens were harvested, mRNA was isolated, and expression of interleukin (IL)-4, IL-6, IL-18, macrophage inflammatory protein-1beta, interferon-gamma, and phosphoinositide 3-kinase p110gamma transcripts was measured by quantitative reverse transcription PCR. Effects of diet, genetic line, sex, and diet x genetic line interaction on weight gain and gene expression were analyzed. At 1, 2, and 3 wk after starting the diet treatments, birds fed the corticosterone diet had gained less weight compared with birds fed the other diets (P < 0.001). Sex affected expression of IL-18 (P = 0.010), with higher levels in males. There was a significant interaction between genetic line and diet on expression of IL-4, IL-6, and IL-18 (P = 0.021, 0.006, and 0.026, respectively). Broiler line gene expression did not change in response to the experimental diet. Splenic expression of IL-6 was higher in Leghorns fed the basal or ascorbic acid diets, rather than the beta-glucan or corticosterone diets, whereas the opposite relationship was observed in the Fayoumi line. Expression of IL-4 and IL-18 responded to diet only within the Fayoumi line. The differential splenic expression of birds from diverse genetic lines in response to nutritional immunomodulation emphasizes the need for further study of this process.
and Implications Chickens from broiler, Leghorn, and Fayoumi lines were fed diets with ingredients to affect immune function: β-glucans, ascorbic acid, or corticosterone. Spleens were tested for expression of genes involved in immune response: interleukin-4 (IL-4), IL-6, IL-18, and macrophage inflammatory protein-1β (MIP-1β). Birds from the broiler line did not show any change in splenic gene expression associated with the dietary immunomodulators, perhaps due to the stringent selection of these birds for growth. The corticosterone diet was associated with increased expression of IL-4, indicative of an immune response relying primarily on humoral defenses. The Leghorn and Fayoumi lines showed opposing changes in expression of IL-4, IL-6, and IL-18 in response to the ascorbic acid and β-glucans enhanced diets, suggesting that processing of these immunomodulators and/or immune signaling in these lines are different. Our findings emphasize the need to further evaluate the effects of dietary immunomodulators before applying them in commercial settings.
The gut mucosa normally exhibits tolerance towards the commensal microbiota by active suppression of inflammation. When homeostasis is disrupted, inflammatory bowel diseases may develop, during which microbiota dysbiosis may also occur. Resulting chronic inflammation induces loss of intestinal epithelial integrity, gastrointestinal (GI) distress, and colon cancer risk. We hypothesize that under normal conditions, the microbiota produce beneficial compounds that promote homeostasis. Previously, we reported that indole exerts immunomodulatory and anti-inflammatory effects on intestinal epithelial cells (IECs). We propose that indole, an abundant, freely diffusible, and strictly microbiota-derived molecule in the GI tract, conditions IECs and immune cells for optimal gut functions. Here, we report that indole potently modulates dendritic cell (DC) responses, which are central regulators of gut homeostasis. Specifically, indole-conditioned DCs produce decreased pro-inflammatory cytokines after microbial activation, and indole strongly synergizes with TGF-β for this effect. In addition, indole regulates the expression of the mucosal DC markers CCR9, B220, and aldh1a2. Furthermore, indole-treatment rescues host inflammation in a murine model of colitis. Given its availability and immunomodulatory properties, we suggest indole is a member of a novel class of microbiota compounds that regulate gut immune cells and may be a potential treatment of inflammatory bowel disease.
The presence of a normal microbiota and immune regulation in the gut limits the onset of several inflammatory disorders. However, the specific microbiota mechanisms or chemical effectors that promote homeostasis are largely unknown. Previously, we reported that indole, a microbiota-derived (not host derived) tryptophan-metabolite, attenuates indicators of inflammation in intestinal epithelial cells. In addition, we show that indole treatment rescues mice from colitis in vivo. Based on our results, we hypothesized that indole influences mucosal CD4 T-cell function. Because Foxp3+ regulatory T cells (Tregs) and pro-inflammatory Th17 cells are key players in the balance of gut homeostasis, we tested the impact of indole on Treg and Th17 development. Here, we reveal that indole regulates Treg/Th17 lineage fate by dramatically augmenting TGF-β-induced Treg expansion, function, and stability. Interestingly, indole also promoted Treg Foxp3 expression independent of TGF-β or IL-2 suggesting a novel mechanism for the induction of Foxp3 expression in CD4 T cells. In a reciprocal fashion, indole inhibited Th17 development via STAT3 and RORgt pathways and decreased IL-17 production. These data reveal a novel mechanistic paradigm on how the microbiota influence mucosal immunobiology and establish the metabolite indole as a potent regulator of Treg and Th17 cell balance. Moreover, our results suggest microbiota-derived metabolites offer a rich pool of potent immunomodulatory compounds.
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