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. 2019 Jul 16;51(1):77-89.e6.
doi: 10.1016/j.immuni.2019.05.004. Epub 2019 Jun 19.

The Intestine Harbors Functionally Distinct Homeostatic Tissue-Resident and Inflammatory Th17 Cells

Sara Omenetti  1 , Claudio Bussi  1 , Amina Metidji  2 , Andrea Iseppon  1 , Sunjae Lee  3 , Mauro Tolaini  1 , Ying Li  1 , Gavin Kelly  1 , Probir Chakravarty  1 , Saeed Shoaie  3 , Maximiliano G Gutierrez  1 , Brigitta Stockinger  4
Affiliations

The Intestine Harbors Functionally Distinct Homeostatic Tissue-Resident and Inflammatory Th17 Cells

Sara Omenetti et al. Immunity. .

Abstract

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases, but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here, we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines, were characterized by a metabolism typical of quiescent or memory T cells, and did not participate in inflammatory processes. In contrast, infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines, disseminated widely into the periphery, and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Colonization with SFB and Infection with C. rodentium Induce Different Th17 Responses (A) Relative abundance of SFB in the feces of mice reconstituted with SFB+ feces on 0 (n = 10), 1 (n = 10), 2 (n = 12), and 4 (n = 6) weeks after gavage. SFB genomic 16s was quantified in the feces by qPCR analysis. Abundance of SFB was normalized to Eubacteria. (B and D) Absolute numbers of Th17 cells in the small intestine of mice colonized with SFB (B) and colon of mice infected with C. rodentium (D) at 0 (n = 6 and 5), 1 (n = 10 and 6), 2 (n = 11 and 6), and 4 (n = 10 and 9) weeks after gavage. (C) C. rodentium burden in the colon of infected mice at 0 (n = 5), 1 (n = 6), 2 (n = 6), and 4 (n = 9) weeks after gavage. In the graphs, bars show the mean ± SEM (A, B, and D) or median (C) and each symbol represents an individual mouse from two pooled independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA with Dunnett’s post-test. n.d., not detected. See also Figure S1.
Figure 2
Figure 2
SFB- and C.-rodentium-Elicited Th17 Cells Have Different Cytokine Profiles (A–D) Representative intracellular staining for IL-17A and IL-22 (A) or IFN-γ (C) and corresponding quantifications in (B) and (D) in Th17 cells from the SI of mice colonized with SFB and colon of mice infected with C. rodentium at 1 (n = 9 and 6), 2 (n = 11 and 6), and 4 (n = 14 and 9) weeks after gavage. Lamina propria cells were isolated at the indicated time point, re-stimulated with PMA and ionomycin and Brefeldin A for 2 h, and analyzed by FACS. Bars show the mean ± SEM for the indicated populations, and each symbol represents an individual mouse from two pooled independent experiments. (E) Cytokine concentration in supernatants of FACS-purified Th17 cells from the SI of mice colonized with SFB or colon of mice infected with C. rodentium. In the graphs, bars show the mean ± SEM, and each symbol represents a technical replicate. Cells were FACS-purified from one sample per group, obtained by pooling 3–6 mice. Results are representative of three independent experiments. (F) Fold-change (FC) induction of listed genes quantified by qPCR in the SI (green) and colon (orange) of mice colonized with SFB or infected with C. rodentium, 1 week after gavage. Fold change is calculated on the matching organs from untreated mice and gene expression is normalized against β-2-microglobulin. Data are from two pooled independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by two-way ANOVA with Sidak’s post-test. Please see also Figure S2.
Figure 3
Figure 3
Peripheral Th17 Cells Retain Their Antigen-Specificity in Response to SFB and C. rodentium Shown in (A) are the absolute numbers of Th17 cells in the spleen of mice colonized with SFB or infected with C. rodentium at 0 (n = 11 and 5), 1 (n = 10 and 6), 2 (n = 12 and 6), and 4 (n = 6 and 8) weeks after gavage. Symbols show the mean ± SEM. Cytokine concentration in supernatants of FACS-purified Th17 cells from the spleen and SI of mice colonized with SFB (B) or from the spleen and colon of mice infected with C. rodentium (C), cultured with DCs and SFB peptide (B) or EspA (C). Bars show the mean + SEM of technical replicates (n = 3–4). Cells were FACS-purified from one sample per group, obtained by pooling 3–6 mice. Results are representative of three (B) and two (C) independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way (A) or one-way (B and C) ANOVA with Sidak’s (A) or Tukey’s (B and C) post-test. n.d., not detected. See also Figure S3.
Figure 4
Figure 4
Homeostatic Tissue-Resident Intestinal Th17 Cells Do Not Clear C. rodentium Infection (A) Schematic drawing of the experimental protocol. Mice were orally gavaged with SFB+ feces 4 weeks before the infection with C. rodentium (at day 0). Six days before the infection with C. rodentium (day −6), mice were intra-peritoneally injected with FTY720 (3 mg/kg) or control vehicle every day. FTY720 and control vehicle administration continued after the infection every other day until the end of the experiment. (B) Survival curve of C.-rodentium-infected mice treated with vehicle or FTY720. (C) C. rodentium burdens in colon and liver. (D–J) Absolute numbers of colonic Th17 cells. Representative intracellular staining for IL-17A and IFN-γ (E) or IL-22 (H) and quantification (F and I) in colonic Th17 cells. IFN-γ (G) and IL-22 (J) protein content in colon explant cultures. Bars show median (C) or mean ± SEM (D, F, G, I, and J) and each symbol represents an individual mouse (n = 8). Results are representative of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Mantel-Cox (B), Mann-Whitney (C) or Student’s t test (D, F, G, I, and J). See also Figure S4.
Figure 5
Figure 5
Intestinal Th17 Cells Elicited by SFB and C. rodentium Have Distinct Transcriptional Profiles (A) RNA-sequencing analysis of Th17 cells from the SI and colon of C. rodentium (Citro)-infected and SFB-colonized mice, presented as heatmap of condition similarity showing Poisson distance between experimental groups. (B) Gene expression in Th17 cells from the SI and colon of C.-rodentium-infected and SFB-colonized mice, presented as normalized read counts (TPM). (C) Scatter plot of differentially expressed genes (log2-fold change) in SFB SI versus C. rodentium colon against SFB colon versus C. rodentium colon. Each dot represents a gene differentially expressed (adjusted p < 0.05) in at least one comparison. Specifically, genes in green are significantly different in the SFB SI versus C. rodentium colon comparison; genes in blue are significantly different in SFB colon versus C. rodentium colon comparison; and genes in red are significantly different in both comparisons. (D) Top 15 canonical pathways in colonic SFB- versus C. rodentium-induced Th17 cells identified by ingenuity pathway analysis (IPA) and ranked on p value. The colors indicate activation Z-score of each pathway with red corresponding to positive Z-scores, blue to negative Z-scores, and white to no activity pathway available. ∗p < 0.05, ∗∗p < 0.01 by one-way ANOVA with Tukey’s post-test. See also Figure S5.
Figure 6
Figure 6
SFB- and C.-rodentium-elicited intestinal Th17 cells display different metabolic phenotypes (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways enriched in SFB- and C.-rodentium-elicited colonic Th17 cells, on the basis of upregulated genes with adjusted p < 0.001. (B) Selection of differentially upregulated reporter metabolites involved in energy generation and redox reactions in SFB- and C.-rodentium-elicited colonic Th17 cells (adjusted p < 0.05). (C) Metabolism-related genes increased in C.-rodentium-induced colonic Th17 cells (adjusted p < 0.05) (in red) as well as the associated reactions involved in glycolysis. See also Table S1.
Figure 7
Figure 7
SFB- and C.-rodentium-Intestinal Th17 Cells Have Distinct Bioenergetic Profiles and Mitochondrial Morphology Th17 cells from the SI of SFB-colonized mice and the colon of C.-rodentium-infected mice were FACS-sorted and used for further analysis. (A) Oxygen consumption rate (OCR) at baseline and in response to oligomycin (Oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone plus antimycin (R + A). (B) Baseline extracellular acidification rate (ECAR) in relation to ECAR of SFB-elicited Th17 cells. (C) Measurement of ATP-coupled respiration. (D) OCR (measure of OXPHOS) plotted against ECAR (measure of glycolysis). Data are from 2–3 pooled independent experiments and are shown as mean ± SEM ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by Student’s t test. (E–H) Z-stack images were used to evaluate mitochondrial morphology by confocal microscopy. Mitochondria were stained with MitoTracker Red CMXRos (red), nuclei with DAPI (blue), and actin with Alexa Fluor 488 phalloidin (green). A magnification (top) and a 3D surface rendered image (bottom) of the area indicated in white are shown (E). Quantification of individual mitochondria (F), mean mitochondrial branch length (G), and mitochondrial area per cell (H) obtained using MiNa Macro and Fiji software. In the violin plot, bars show the median and dotted bars the quartiles (F–H). Data are representative of two independent experiments with at least 50 cells analyzed per condition. ∗p < 0.05 by Student’s t test. n.s., not significant. Scale bars, 1 μm and 5 μm.
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