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Dietary tryptophan deficiency promotes gut RORγt+ Treg cells at the expense of Gata3+ Treg cells and alters commensal microbiota metabolism - PubMed

  • ️Sun Jan 01 2023

Dietary tryptophan deficiency promotes gut RORγt+ Treg cells at the expense of Gata3+ Treg cells and alters commensal microbiota metabolism

Lucille C Rankin et al. Cell Rep. 2023.

Abstract

Micronutrient deficiency is a major cause of disease throughout the world. Yet, how perturbations influence the immune-microbiome interface remains poorly understood. Here, we report that loss of dietary tryptophan (Trp) reshapes intestinal microbial communities, including the depletion of probiotic L. reuteri, drives transcriptional changes to immune response genes in the intestinal ileum, and reshapes the regulatory T cell (Treg) compartment. Dietary Trp deficiency promotes expansion of RORγt+ Treg cells and the loss of Gata3+ Tregs in a microbiota-dependent manner. In the absence of dietary Trp, provision of the AhR ligand indole-3-carbinol is sufficient to restore the Treg compartment. Together, these data show that dietary Trp deficiency perturbs the interaction between the host and its bacterial symbionts to regulate Treg homeostasis via the deprivation of bacterially derived Trp metabolites. Our findings highlight an essential role for immune-microbiome crosstalk as a key homeostatic regulator during nutrient deficiency.

Keywords: CP: Immunology; CP: Metabolism; aryl hydrocarbon receptor; gut inflammation; intestinal Tregs; microbiota; mucosal immunity; nutrient deficiency; tryptophan metabolism.

Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. A Trp-deficient diet changes the ileal microbiota and fecal metabolome

SPF mice were fed Bakers Amino Acid diet (CD) for 1–2 weeks, then placed on Bakers Amino Acid diet without Trp (TDD) for 3 weeks. (A) Ileal contents and/or feces were collected followed by 16S rRNA sequencing and CE-TOFMS metabolite analysis. (B) A Bray-Curtis PCoA plot representing the differences in the beta diversity of bacterial communities in the ileum of SPF mice fed CD or TDD. (C) Relative abundance of main bacterial genus detected within the ileum of SPF mice fed CD or TDD at 3 weeks. (D) Forest plot that shows Log2FC of the most differentially abundant taxa within the ileum of SPF mice fed CD compared with those fed TDD. Data are representative of two experiments (n = 4–5 mice per group/experiment); error bars represent SD. (E) Unbiased metabolite analysis was carried out in the stool of animals fed CD or TDD using CE-TOFMS (E–H) and principal-component analysis of detected metabolites is shown in (E). (F) Differentially abundant known putative metabolites were detected and shown as a heatmap in (F). (G) Heatmaps of selected metabolites identified in the stool of SPF mice fed CD or TDD that are involved in tryptophan metabolism (upper) and nicotinamide metabolism (lower). (F) is a schematic representation of pathways of Trp metabolism. p values in (F) and (G) were computed by Welch’s t test. (*<0.05, **<0.01, ***<0.001). Heatmaps were generated using standardized value of relative area in detected peaks. Data are from one independent experiment (n = 3–4 mice/group). CD, control diet; CE-TOFMS, capillary electrophoresis time-of-flight mass spectrometry; TDD, Trp-deficient diet. See also (Figure S1).

Figure 2.
Figure 2.. Dietary Trp deficiency profoundly alters ileal gene expression networks in a microbiota-dependent manner

(A–C) SPF and antibiotics (ABX)-treated mice were fed CD or TDD for 3 weeks and transcriptional analysis was performed on the terminal ileum (A). Shown in (B) is a principal-component analysis (PCA) plot of gene expression within the ileum of SPF or ABX-treated mice fed a CD or TDD. Lists of differentially expressed genes were generated between groups (q < 0.05) and shown in (C) as a Venn diagram representing the intersection of differentially expressed genes in SPF mice fed CD versus TDD and ABX-treated mice fed CD versus TDD (black is total number of differentially expressed genes, blue is downregulated genes, and red is upregulated genes). GO term enrichment analysis of biological process was carried out on differentially expressed genes between CD versus TDD. (D) Heatmap of downregulated genes (left) and upregulated genes (right) within the GO term immune system process CD versus TDD. (E) Statistically significant GO term enrichment analysis of biological process carried out using the list of differentially regulated genes in SPF mice only. Respective dot size and numbers represent relative and actual p values. Data are from one experiment, n = 4–5 mice per group. ABX, pre-treated with antibiotics (ampicillin, gentamycin, vancomycin, metronidazole, and neomycin) to deplete microbiota; CD, control diet; SPF, specific pathogen free; TDD, Trp-deficient diet. See also Figure S2.

Figure 3.
Figure 3.. Dietary Trp deficiency drives expansion of intestinal RORγt+ Treg cells and loss of Gata3+ Treg cells

(A) SPF mice were fed CD for 2 weeks, and switched to TDD for 1, 2, or 3 weeks. (B) Representative FACs plots and frequencies of Treg populations (gated on TCRb+NK1.1−CD4+Foxp3+ cells) in the indicated organs after 1, 2, and 3 weeks on a CD or TDD diet. (C and D) Quantification of mean (±min/max) frequencies of RORγt+ (C) and Gata3+ (D) Treg cells isolated from the siLPL and mLN of mice fed control (black) or Trp-deficient diets (blue) for the indicated timepoints. Data are representative of at least two independent experiments (n = 3–5 mice/group). (E) A representative FACs plot of CD44 versus CD62L expression within Tregs isolated from the mLN at the indicated timepoints after TDD feeding. (F and G) Representative FACs plots (left) and frequencies (right) of Ki67-positive cells within RORγt+FoxP3+ T cells (F) and Gata3+FoxP3+ T cells (G) at the indicated timepoints. Data are representative of at least two independent experiments where n = 3–5 mice per group. Weeks 2 and 3 in (F) and (G) are pooled from two independent experiments. (H–J) Numbers and function of ILC populations isolated from CD- and TDD-fed mice. Frequency of ILC3 (RORγt+ LinNeg) (left) and ILC2 (Gata3+ LinNeg) (right) within CD45+ si-LP lymphocytes are shown in (H). (I) is frequency of IL22+ ILC3 isolated from the si-LP after 3 weeks on CD or TDD and re-stimulated with PMA/ionomycin and IL-23 for 3 h. Representative plots of Ki67+ ILC2 (top) and Ki67+ ILC3 (bottom) isolated from the siLPL of CD- and TDD-fed mice are shown in (I). Frequencies are enumerated on the right. Data are representative of at least three independent experiments, where n = 3–5 mice per group. Statistical analysis was performed using two-tailed Student’s t test. Floating bars are min to max, and line is at mean. SPF, specific pathogen free; CD, control diet; mLN, mesenteric lymph node; siLPL, small intestinal lamina propria; TDD, Trp-deficient diet. See also Figure S3.

Figure 4.
Figure 4.. Microbiota mediates the Treg cell changes induced by Trp-deficient diet

(A) Schematic for experimental setup. (B) Representative plots and frequencies of Treg populations (gated on TCRb+NK1.1−CD4+Foxp3+cells) isolated from SPF (no ABX) or ABX-treated mice fed a CD or TDD as in (A). (C–E) Frequencies (C and D) and numbers (E) of RORγt+ Treg cells and Gata3+ Treg cells from the indicated organs treated as in (A). Data in (C and D) are pooled from three of four independent experiments (n = 4–5 mice per group). Statistical analysis was performed using two-tailed Student’s t test. Floating bars show min to max, and line is at mean. ABX, pre-treated with antibiotics (ampicillin, gentamycin, vancomycin, metronidazole, and neomycin) to deplete microbiota; CD, control diet; mLN, mesenteric lymph node; siLPL, small intestinal lamina propria; SPF, specific pathogen free; TDD, Trp-deficient diet. See also Figure S4.

Figure 5.
Figure 5.. Trp-deficient diet-induced RORγt+ Treg cells remain expanded after enteric bacterial infection and result in increased susceptibility to disease

(A) SPF mice were fed CD or TDD for 1 week and gavaged with 2 × 109 CFU of C. rodentium. (B) Bacterial load in feces, liver, and spleen at day 10 post C. rodentium infection on the indicated diets. (C) Data show representative FACs plots and frequencies of Treg populations (gated on TCRb+NK1.0031−CD4+Foxp3+cells) isolated from indicated organs and treated as (A). (D) Frequencies of RORγt+ (upper) and Gata3+ (lower) Treg cells isolated from the siLPL, cLPL, and mLN of mice treated as in (A). (E) Representative FACs plots of CD4 vs Ki67 (upper) and quantification of the frequencies (lower) of Ki67+ cells within RORγt+FoxP3+ Treg cells isolated from the siLPL, cLPL, and mLN at day 10 post C. rodentium infection on the indicated diets. (F) Data are frequencies of RORγt+ conventional T cells (gated on TCRb+NK1.1–CD4+Foxp3− cells) (upper) and frequencies of interleukin (IL)-17-producing cells (gated on TCRb+NK1.1−CD4+Foxp3−) and re-stimulated with PMA/ionomycin together with IL-23 for 3 h (lower). (G) Representative plots and frequencies of IL-22-producing cells within LinNeg, CD127+ Rorγt+ ILC3 stimulated as in (F). (H) Data show frequency of LinNeg, CD127+ Rorγt+ ILC3 among CD45+ lymphocytes isolated from the si-LP and cLP of CD- or TDD-fed mice at day 10 post C. rodentium infection. (I) Representative plots show frequencies of Ki67+ cells within ILC3 isolated from the si-LP and cLP of CD- and TDD-fed mice on day 10 post C. rodentium infection. Data are representative of two to three independent experiments where n = 4–5 mice per group. Statistical analysis was performed using two-tailed Student’s t test. Floating bars show min to max, and line is at mean. CD, control diet; CFU, colony-forming units; cLPL, colon lamina propria; mLN, mesenteric lymph node; siLPL, small intestinal lamina propria; SPF, specific pathogen free; TDD, Trp-deficient diet.

Figure 6.
Figure 6.. Supplementation with I3C on a Trp-deficient background is sufficient to normalize Treg cell populations

(A) SPF mice were fed CD, TDD, CD + 200 ppm I3C or TDD diet + 200 ppm I3Cfor 3 weeks. (B) Representative plots and frequencies of Treg populations (gated onTCRb+NK1.1−CD4+Foxp3+cells) in the si-LP isolated from animals treated as indicated. (C and D) Frequencies of RORγt+ Treg cells (C) and Gata3+ Treg cells (D) within Foxp3+ Tregs isolated from the indicated organs of animals fed the indicated diets. Data are representative of two independent experiments (n = 4 mice per group). (E) Relative AhR expression (to hPRT) within TCRβ+CD4+Foxp3–GFP+ Treg cells sorted from the si-LP and spleen of FoxP3-GFP reporter mice. Statistical analysis was performed using two-tailed Student’s t test. Floating bars show min to max, and line is at mean. CD, control diet; I3C, indole-3-carbinol; mLN, mesenteric lymph node; siLPL, small intestinal lamina propria; TDD, Trp-deficient diet. See also Figure S5.

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