CCL20-CCR6 signaling alters the metabolic reprogramming to promote the pathogenic Th17 cell differentiation

Summary

CCR6 is a G-protein-coupled receptor that binds to its ligand CCL20. Th17 and Foxp3⁺CD4⁺ T cells express CCR6, enabling their migration to inflamed tissues with unique metabolic environments. The factors that regulate their metabolic adaptation and functional roles in these tissues remain unclear. Using inflammatory bowel disease patient samples and experimental colitis models in mice, we demonstrated that the intrinsic signaling of CCL20-CCR6 in CD4⁺ T cells promotes the differentiation of inflammatory Th1-like Th17 cells (T-bet⁺RORγt⁺) during colitis. This signaling induces the rapamycin-sensitive phosphorylation of PI3K, Akt, mTORC1, and STAT3 in a CCR6-dependent manner. RNA-seq and proteomics analysis revealed that the addition of CCL20 during Th17 differentiation affects several metabolic pathways, including energy metabolism. CCL20 significantly increased glycolysis and inhibited oxidative phosphorylation, thereby driving the differentiation of pathogenic Th17 cells. Our findings suggest that alterations in CCR6-induced changes in Th17 metabolism offer an interesting therapeutic target for gut inflammation and autoimmunity.

Graphical abstract

Highlights

• •Patients with UC have higher levels of Th1-like Th17 cells in their PBMCs
• •CCL20 drives pathogenic Th1-like Th17 cell differentiation via CCR6
• •CCL20 activates PI3K-Akt-mTORC1-STAT3 pathways in CD4⁺ T cells
• •CCR6 signaling reprograms the energy metabolism to promote Th1-like Th17 cells

Biological sciences; Immunology; Components of the immune system

Introduction

Chemokine receptor CCR6 (CD196) is a G protein-coupled receptor (GPCR) expressed on several immune cells, including inflammatory effector Th17 cells and regulatory CD4⁺ T cells (Tregs).^1,2 CCL20 (also known as liver and activation-regulated chemokine, LARC; macrophage inflammatory protein-3α, MIP-3α; or Exodus-1) is the only known high-affinity ligand that binds to CCR6 and induces a chemotactic function.^1,3 Colon and intestinal epithelial cells overlying Peyer’s patches show constitutive expression of CCL20.^4,5 During intestinal inflammation, CCL20 expression increases several-fold.⁶ The expression of CCR6 and its ligand, CCL20, shows a strong positive clinical association with several autoimmune diseases, including inflammatory bowel diseases (IBDs), rheumatoid arthritis (RA), psoriasis, and multiple sclerosis.¹ In IBD, intestinal epithelial cells produce substantial amounts of CCL20, and blocking CCL20-CCR6 signaling or CCR6 deficiency reduces disease severity, suggesting that CCL20-CCR6 signaling is an important therapeutic target.¹ The role of the CCR6-CCL20 axis in the migration of Th17 and Treg cells has been extensively studied. Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD), is characterized by chronic inflammation and enterocolitis. IBD is characterized by T cell and monocyte/macrophage activation, and patients with IBD exhibit increased IL-17 levels in both mucosa and serum.⁷ However, the cause of IBD is not known but is thought to be triggered by chronic gut inflammation or intestinal dysbiosis.⁸ Th1 cells produce IFN-γ, which helps clear intracellular bacteria and viruses, whereas Th17 cells produce IL-17A, IL-17F, IL-21, and IL-22 that protect the mucosal surface from extracellular bacteria and fungi.^9,10

Th17 cells exhibit a high degree of developmental plasticity, depending on the cytokines present in the tissue microenvironment. For example, IL-12 or IL-23 can rapidly modulate Th17 cells to produce IFN-γ, which is typically associated with a Th1 response.^11,12 The developmental and functional plasticity of Th17 cells toward a Th1-like phenotype increases their pathogenicity in several autoimmune diseases, including multiple sclerosis,¹³ diabetes,¹⁴ experimental autoimmune encephalomyelitis (EAE),^11,15 arthritis,¹⁶ Crohn’s disease,¹⁷ and experimental colitis.^1,18 Th17 cells play a crucial role in the pathogenesis of IBD.¹⁹ The factors regulating the phenotypic and functional plasticity of Th17 cells in the inflamed tissue microenvironment remain poorly understood. CCR6 plays a critical role in gut inflammation and autoimmunity,^1,20,21 and deficiency of CCR6 protects against low-dose dextran sodium sulfate (DSS)-induced colitis²² or blocking CCL20 with an anti-CCL20 antibody attenuates trinitrobenzene sulfonate (TNBS)-induced colitis.²³ Most of these studies focused on the role of CCR6⁺ cells in migrating into the inflamed tissues. The role of intrinsic GPCR signaling in CCR6 in the differentiation and driving of the molecular and functional plasticity of Th17 cells has not been extensively evaluated. We demonstrated that patients with ulcerative colitis exhibit an increased pathogenic RORγt⁺T-bet⁺ Th17 phenotype among CCR6⁺CD4⁺ effector cells compared to CCR6⁻CD4⁺ T cells in the venous blood. Deficiency of CCR6 in CD4 T cells prevented the differentiation of pathogenic RORγt⁺T-bet⁺ Th17 cells in DSS-induced and naive CD4 T cell-induced colitis models in mice. Further, CCR6 intrinsic signaling in CD4⁺ T cells drives Th17 differentiation by inducing the phosphorylation of Akt, mTOR, and STAT3 in human and mouse cells.

Results

CCR6⁺ CD4⁺ T cells in patients with IBD exhibit an increased Th1-like Th17 phenotype compared to CCR6⁻CD4⁺ T cells

Patients with UC were reported to have increased CCR6⁺ immune cells and CCL20-expressing epithelial cells in the rectal mucosa.^3,6,19 To study the role of CCR6 in CD4⁺ T cell differentiation in patients with UC, we collected peripheral venous blood from 13 healthy donors and 19 patients with Grade II-IV ulcerative colitis. Analysis of PBMCs revealed a significantly increased frequency of CCR6⁺CD4⁺ T cells in patients with UC compared to healthy individuals (Figure 1A). Additionally, patients with UC had a significantly higher frequency of RORγt⁺CD4⁺ T cells (Th17 phenotype) among CCR6⁺CD4⁺ cells than healthy individuals (Figure 1B). It has been shown that effector CD4⁺ T cells that co-express the transcription factors T-bet and RORγt (also known as Th1-like Th17 cells) exhibit a significantly enhanced pathogenic phenotype compared to cells expressing either T-bet or RORγt alone.^14,24 Our results showed that an increased frequency of CD4⁺ T cells co-expressing RORγt and T-bet was observed in patients with UC as compared to CCR6⁺CD4⁺ cells in healthy individuals (Figure 1C). Furthermore, cytokine analysis of PBMCs showed significantly increased frequency of IL-17A⁺IFN-γ⁺ CD4 T cells in patients with UC compared to control individuals (Figure 1D).To confirm whether CCL20 interaction with CCR6 promotes the differentiation of the Th1-like Th17 cells, we in vitro differentiated human naive CD4 T (CD4⁺CD8⁻CD25⁻CD4RA⁺) cells from PBMCs of healthy donors into the Th17 lineage in the presence or absence of recombinant CCL20. Our data showed that CCL20 increased the differentiation of RORγt⁺CD4⁺ T cells (Figure 1E). Among these Th17 cells, a higher frequency of cells co-expressed RORγt and T-bet (Figure 1F). These results showed that patients with UC have a significantly higher frequency of circulating pathogenic Th1-like Th17 cells, and that CCL20 promotes their differentiation.

Figure 1.

Patients with ulcerative colitis show increased CCR6⁺ Th1-like Th17 cells in PBMC

(A) A representative colonoscopy image of a healthy and a grade III ulcerative colitis patient (upper left panel). Peripheral blood was obtained from patients with ulcerative colitis (six women, thirteen men; age 39 ± 9.3 years) and healthy individuals (six women, seven men; age 63 ± 8 years). Peripheral blood mononuclear cells (PBMCs) were analyzed for CCR6 expression after gating on CD4⁺ cells, as shown in the dot plots (lower panel). Data from individual healthy and patients with UC are shown (right panel).

(B) RORγt expression was analyzed after gating on CD4⁺CCR6⁺ or CD4⁺CCR6^- T cells. The dot plots shown are gated on CD4⁺ T cells.

(C) The dot plot illustrates the expression of RORγt and T-bet on CCR6⁻CD4⁺ or CCR6⁺CD4⁺ gated T cell population (left panel) and provides the corresponding statistics (right panel). Each symbol represents data from an individual.

(D) PBMCs were stimulated with PMA/ionomycin in the presence of brefeldin A for 6 h. Intracellular IL-17A and IFN-γ expression was analyzed after gating on CD4⁺ cells.

(E) Purified human CD4⁺CD8⁻CD25⁻CD45RA⁺ T cells were in vitro differentiated into non-pathogenic (IL-6 plus TGF-β) or pathogenic conditions (IL-23 plus IL-1β) in the presence or absence of recombinant human CCL20 at 37°C for 4 days. On day 4, cells were stained with the indicated molecules and analyzed using flow cytometry. RORγt expression in CD4⁺ T cells is shown after gating on CD4⁺CD25⁺ cells.

(F) Expression of RORγt and T-bet in CD4⁺CD25⁺ cells is shown. Mann-Whitney test (C).

The error bar represents ±standard error of the mean (SEM). Student’s t test (A, D, E, F). Data are representative of three independent experiments (E, F). The p-value for the comparison between the two groups is indicated in the graphs. n.s., not significant): p > 0.05.

CCR6 drives the differentiation of inflammatory Th1-like Th17 cells in experimental colitis in mice

CCL20 is constitutively expressed by gut epithelial cells.² During gut inflammation, CCL20 expression increases several-fold, recruiting CCR6⁺ immune cells into the gut mucosa.^25,26 We questioned whether increased CCL20 during gut inflammation drives the differentiation of Th17 or pathogenic Th1-like Th17 cells. To test this, acute colitis was induced in C57BL/6 mice by administering dextran sulfate sodium (DSS) in the drinking water. DSS treatment increased CCL20 mRNA (data not shown) and protein expression in the colon, and mice exhibited a weight loss of more than 25% and developed colitis within 9 days (Figure 2A). Flow cytometry analysis of cells from the spleen (SP), mesenteric lymph nodes (mLN), Peyer’s patches (PPs), and lamina propria (LP) of these mice showed that the majority of CCR6⁺CD4⁺ T cells had a significantly increased frequency of RORγt⁺ as well as IL-17A expression as compared to control mice (Figure 2B). CCR6^+/+ CD4⁺ T cells also co-produced a higher amount of IL-17A and IFN-γ in the gut-associated lymphoid tissues (GALTs) compared to the CCR6^−/− CD4⁺ T cells (Figure 2B). Immunohistological analysis of Peyer’s patch showed an increased number of RORγt⁺ cells in DSS-treated mice compared to control mice (Figure 2C). Furthermore, CCR6^−/− mice developed less severe colitis than CCR6 sufficient with DSS challenge (Figure 2D). In the DSS-treated groups, CCR6^+/+ CD4⁺ but not CCR6^−/− CD4⁺ T cells had a significantly higher frequency of co-expression of RORγt⁺T-bet⁺ in the Peyer’s patches (PP) and lamina propria (LP) (Figure 2D). To confirm whether CCR6^+/+ CD4⁺ T cells under same gut inflammatory condition are mostly affected than CCR6^−/− CD4 T cells in driving the differentiation of inflammatory Th1-like Th17 cells, we adoptively transferred CCR6^−/− splenocytes (CD45.2 congenic; 10 × 10⁶ cells/mouse) into CD45.1 congenic mice (CCR6^+/+ genotype) and recipients were given 2% DSS in the drinking water (Figure 2E). On day 12, RORγt⁺ and RORγt⁺T-bet⁺ cells were analyzed after gating on CD45.2⁺CD4⁺ or CD45.2⁻CD4⁺ T cells. Immunofluorescence staining of the colon confirmed the infiltration of CD45.2⁺ cells (CCR6^−/− cells), suggesting that CCR6^−/− cells had no problem migrating to the colon (Figure 2F). However, CCR6^+/+CD4⁺ T cells (CD45.2^- cells) in these mice showed a significantly increased frequency of RORγt⁺ cells (Figure 2G) as well as the co-expression of RORγt⁺T-bet⁺ cells in the spleen and GALT as compared to CCR6^−/−CD4⁺ T cells (CD45.2⁺ cells) under the same gut inflammatory condition (Figure 2H). CCR6^+/+CD4⁺ T cells (CD45.2^- cells) in these mice also showed a significantly increased frequency of IL-17A⁺IFN-γ⁺ producing CD4⁺ T cells compared to CCR6^−/− T cells (Figure 2I). IL-23 signaling helps maintain the pathogenic Th17 cell phenotype,^27,28 and GM-CSF expression is required for the pathogenicity of Th17 cells.^29,30 Our results showed that CCR6^+/+ CD4⁺ T cells had a higher frequency of IL-23R⁺ and GM-CSF⁺ expression than CCR6^−/− CD4⁺ T cells in the GALTs (Figure S1A). These results suggest that CCR6-CCL20 signaling during gut inflammation leads to the enhanced frequency of inflammatory Th17 and Th1-like Th17 cells in the GALT.

Figure 2.

CCR6⁺CD4⁺ T cells show an increased Th1-like Th17 phenotype during inflammatory colitis

(A and B) Acute colitis was induced in wild-type C57BL/6 mice by administering 2% DSS in their drinking water, and body weight loss was monitored. On day 10, spleen (SP), mesenteric lymph nodes (mLN), Peyer’s patches (PPs), and lamina propria (LP) cells were harvested, and cells were analyzed using flow cytometry. (A) Body weight was monitored and plotted (upper panel). CCL20 expression in colon tissue sections was analyzed by immunofluorescence staining (lower panel). Original magnification 400x, scale bars 100 μm. (B) The dot plot represents the expression of RORγt on CD4⁺CCR6^- and CD4⁺CCR6⁺ T cells (upper left), and IL-17A⁺ and IFN-γ+ expression (upper right). Mean percentages of RORγt⁺ cells (lower left), mean percentages of IL-17A⁺ cells (lower middle), and IL-17 and IFN-γ expression (lower right) on CD4⁺CCR6^- and CD4⁺CCR6⁺ T cells.

(C) Representative immunofluorescence images of PP of wild-type mice treated with or without DSS are shown (upper right). Original magnification 400x, scale bars 100 μm.

(D) CCR6^−/− or CCR6^+/+ mice were given 2% DSS in the drinking water. The body weight changes in mice were monitored (left), and RORγt⁺T-bet⁺ cells were analyzed in the indicated organs by flow cytometry (right).

(E) CCR6^−/− splenocytes (CD45.2 congenic; 10 × 10⁶ cells/mouse) were adoptively transferred into CD45.1 congenic mice (CCR6^+/+ genotype), and the mice were administered 2% DSS in their drinking water. On day 12, the mice were sacrificed.

(F) The immunofluorescence staining of CD45.2 and CD4 markers was performed on the colon tissue. Original magnification 400x, scale bars 100 μm.

(G and H) RORγt⁺ and RORγt⁺T-bet⁺ cells were analyzed after gating on CD45.2⁺CD4⁺ or CD45.2⁻CD4⁺ T cells.

(I) IL-17A⁺IFN-γ⁺ cells were analyzed after gating on CD45.2⁺CD4⁺ or CD45.2⁻CD4⁺ T cells.

(J) Naive CD4⁺ T cells (CD4⁺CD25⁻CD44^loCD45RB^hiCD62L^hiFoxp3rfp⁻ T cells; 0.5 × 10⁶ cells/mouse) from CCR6^gfp/+Foxp3^rfp/rfp mice were adoptively transferred into RAG1^−/− mice. The mean percentage in body weight change from the initial weight was plotted. (n = 4 mice/group). The error bar represents ±SD.

(K) On day 21, RORγt⁺T-bet⁺ cells were analyzed after gating on CCR6gfp⁺ and CCR6gfp⁻CD4⁺ T cells in the SP, mLN, and lamina propria (LP) (left panel). The mean percentage and MFI of RORγt⁺ cells were analyzed after gating on CD4⁺CCR6gfp⁻ T cells or CD4⁺CCR6gfp⁺ cells (right panel).

The bar represents the mean ± SEM; each symbol represents data from an individual mouse, with n = 5–6 mice/group (A–K). The p-value for the comparison between the two groups is indicated in the graphs. The data shown are representative of three independent experiments (A–K). Student’s t test was used, not significant (ns): p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

To test whether CCR6 expression is also important in chronic autoimmune colitis, purified naive CD4⁺ T cells (CD4⁺CD25⁻CD44^loCD45RB^hiCD62L^hiCCR6gfp⁻ cells) from CCR6gfp transgenic mice were adoptively transferred into RAG1^−/− mice, and the development of colitis was monitored. Our results showed that mice receiving naive CD4⁺ T cells exhibited significant weight loss and developed colitis (Figure 2J). Further, flow cytometry of the spleen, mLN, and lamina propria (LP) revealed that CCR6gfp⁺CD4⁺ cells had significantly increased the frequency of pathogenic RORγt⁺T-bet⁺CD4⁺ T cells, as compared to the CCR6gfp^-CD4⁺ cells (Figure 2K). Furthermore, CCR6gfp⁺CD4⁺ T cells also had higher mean fluorescence intensity (MFI) of RORγt in the mLN and LP, compared to CCR6gfp^-CD4⁺ cells (Figure 2K). CCR6^+/+CD4⁺ T cells showed significantly increased expression of GM-CSF, Eomes, IL-23R, and Ahr molecules compared to the CCR6^−/−CD4⁺ cells (Figure S1B). Together, these data showed that CCR6 might drive the differentiation of Th1-like Th17 phenotype in autoimmune colitis.

CCL20 induces the differentiation of Th17 cells in a CCR6-dependent manner

Th17 cells express CCR6, and the phosphorylation of Akt/mTOR/STAT3 promotes their differentiation.^12,31 We tested whether CCL20 promotes Th17 cell differentiation in a CCR6-dependent manner. To answer this question, naive CD4⁺ T cells were isolated from wild-type or CCR6^−/− mice and in vitro differentiated into the Th17 lineage under various conditions, including TGF-β plus IL-6, IL-1β, or IL-23, in the presence or absence of different doses of purified recombinant CCL20, for 4 days. Our results showed that CCL20 enhanced RORγt expression in CCR6^+/+ CD4 T cells in a CCL20-dose-dependent manner, with the highest RORγt⁺ expression observed with 100 ng/mL CCL20 (Figures S2A and S2B). Furthermore, our results demonstrated that CCL20 increased RORγt mRNA expression (data not shown) and the number of RORγt⁺T-bet⁺ cells in CCR6^+/+ CD4 T cells (Figure 3A). Th17 cells differentiated in the presence of IL-1β and IL-23 were shown to cause more severe disease pathology than Th17 cells induced by TGF-β and IL-6.³² Further, deficiency of CCR6 in CD4⁺ T cells prevented CCL20-induced RORγt⁺or RORγt⁺T-bet⁺ expressing CD4 T cells (Figure 3C), and also produced low IL-17A (Figure 3C). CCL20 did not affect the differentiation of naive CD4⁺ T cells into Th1 or Th2 cells under Th1-polarizing (Figure S2C) or Th2-polarizing culture conditions (data not shown), respectively.

Figure 3.

CCL20 promotes the differentiation of the pathogenic Th1-like Th17 cells

(A) Naive CD4⁺CD25⁻CD44⁻ T cells were in vitro differentiated into the Th17 lineage (IL-1β + IL-23 condition) in the presence or absence of CCL20. On day 4, the percentages of CD25⁺RORγt⁺ cells or RORγt⁺T-bet⁺ T cells were analyzed after gating on CD4⁺ cells (left panel). The mean percentage of RORγt⁺CD4⁺ T cells is shown (right panel). Each symbol represents data from a single experiment. n = 9–10 experiments.

(B) IL-17A secretion in the cultured supernatant was analyzed using ELISA. n = 3–4 experiments.

(C) Purified CD4⁺CD25⁻CD44⁻ T cells from C57BL/6 or CCR6^−/− mice were differentiated into the Th17 lineage. The mean percentage of RORγt⁺ or RORγt⁺T-bet⁺ cells was analyzed after gating on CD25⁺CD4⁺ T cells. Intracellular IL-17A expression was analyzed after gating on CD4⁺CD25⁺ cells. n = 3–4 experiments. The error bar represents the ±SEM, and each dots represent data from an individual experiment. Student’s t test.

(D) Purified CD4⁺CD25⁻CD44⁻ T cells were differentiated into the Th17 lineage in the presence or absence of CCL20. On day 4, the proliferation of RORγt⁺ Th17 cells was analyzed by Ki-67 expression in CD4⁺ T cells. n = 2 experiments.

(E) Ki67 expression in the various lymphoid organs was analyzed from the experiments given in Figure 2D.

(F) On day 4, cells were harvested, and the expression of the indicated molecules was analyzed using quantitative real-time PCR (qRT-PCR). Data were normalized to the Cyclophilin A gene. The error bar represents ±SD. n = 2–4 independent experiments. The p-value for the comparison between the two groups is indicated in the graphs. not significant (ns): p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

To further investigate if the CCL20-induced increased the generation of Th17 is due to higher proliferation or differentiation of Th17 cells, we differentiated the CFSE-labeled naive CD4⁺ T cells into the Th17 lineage in the presence or absence of CCL20. We monitored the dilution of the CFSE dye and the proliferation marker Ki-67 in RORγt⁺CD4⁺ T cells. We did not observe any significant changes in Ki-67 expression in cultured CD4⁺ T cells from CCR6^−/− or CCR6^+/+ mice (Figure 3D), or in vivo changes in the colitic mice on CD4 T cells in CCR6^−/− or CCR6^+/+ in the various secondary lymphoid tissues (Figure 3E). These results suggest that the CCL20-induced increase in Th17 cell frequency is due to differentiation rather than increased proliferation.

Apart from RORγt and IL-17A, other Th17-associated molecules, such as IL-21, IL-22, IL-23R, GM-CSF, and the aryl hydrocarbon receptor (Ahr), play a significant role in the pathogenesis of the disease.^32,33 Our results also showed that CCL20 increased the expression of IL-21, IL-22, IL-23R, Eomes, and Ahr in the Th17 cells (Figures 3E and S2D). These results suggest that CCL20 promotes the differentiation of Th17 and pathogenic Th1-like Th17 cells in a CCR6-dependent manner.

CCL20-CCR6 signaling alters various gene signatures associated with pathogenic Th17 cells

Our results showed that CCL20 induces the differentiation of both inflammatory Th17 cells and pathogenic Th1-like Th17 cells; however, the target molecules or pathways involved in driving the differentiation of inflammatory Th17 cells have not yet been identified. To elucidate the components and pathways induced by CCR6 signaling during Th17 cell differentiation, we examined the global transcriptome and proteome of CCL20-induced Th17 cells. RNA-seq analysis showed approximately 75% overlap in gene expression between Th17 cells cultured with or without CCL20 (Figure 4A). However, 10–13% of genes are uniquely driven in the presence of CCL20 in Th17 cells (Figure 4A). The differentially expressed genes in Th17 cells versus Th17 cells treated with CCL20 were visualized in a heatmap, illustrating the overall transcriptomic alterations in Th17 cells resulting from CCL20 signaling during differentiation (Figure 4B). Furthermore, CCL20 drove the upregulated expression of Th17-specific gene signatures, including Akt, CCR6, Stat3, Itk, Nrf1, Hif1α, IL-17a, and IL-21r, whereas PTPN22, TGF-βR3, Foxp3, CCL25, and Arrb1 genes were downregulated (Figure 4C). The expression of some of these genes was further confirmed using quantitative real-time RT-PCR (Figure S3A). A gene annotation study of molecular pathways revealed that CCL20 stimulation in Th17 cells modulates several metabolic pathways (Figure 4D) and other key biological pathways essential for cell differentiation and function (Figure 4E). Further, when we investigated the enriched pathways using gene enrichment set analysis (GESA) tools, we observed that CCL20 stimulation during Th17 differentiation enriched Th17 differentiation and JAK/STAT signaling pathways (Figure 4F). These data further supported the idea that CCL20 enhances Th17 differentiation.

Figure 4.

CCR6 signaling regulates the expression of various genes involved in the activation and differentiation of Th17 cells

CD4⁺CD25⁻CD44⁻ naive T cells were in vitro differentiated into Th17-lineage (IL-1β and IL-23 condition) cells in the presence or absence of CCL20 (100 ng/mL), and total mRNA was analyzed by RNA-seq using the Illumina Mi-Seq platform.

(A) A Venn diagram illustrating the overlapping gene signatures between the Th17 culture condition in the presence or absence of CCL20.

(B) Heatmap of the fold change (log2) for differentially expressed genes in Th17 plus CCL20 Vs. Th17 cells. Each line represents one gene, and each column represents one sample. Different colors represent the expression levels (from green to red: increased expression).

(C) Volcano plot showing various differentially regulated genes in Th17 plus CCL20 Vs. Th17 cells (a few selected genes were listed in the plot) (Log2FC for upregulated genes ≥1, downregulated genes ≤ −1, pval≤0.05).

(D–F) Functional enrichment of the differential genes (with log2FC > 1<−1) in Th17 plus CCL20 cells by the GO annotation of (D) molecular functions (E) biological process, (F) GSEA enrichment plots of gene sets in Th17 plus CCL20 cells compared with Th17 cells, showing enrichment scores of GO categories including Th17 cell differentiation, JAK-STAT signaling pathway, phosphatidylinositol, and ErbB signaling pathway. CCR6 signaling upregulates important phenotypic proteins of Th17 lineages.

(G) Whole differentially expressed proteins were analyzed using mass spectrometry. A Venn diagram showing the overlapping proteins in Th17 cells with CCL20 versus those in Th17 cells.

(H) Heatmap of the fold change (log2) for differentially expressed proteins in Th17 plus CCL20 Vs. Th17 cells. Each line represents one gene, and each column represents one sample. Different colors represent expression levels (from blue to dark red, with increased expression). Functional enrichment of the differential proteins (with log2FC > 1<−1) comparing Th17 plus CCL20 Vs. Th17 cells.

(I) The volcano plot shows the differentially expressed protein. Some of the proteins are indicated within the plot.

(J) Proteins enriched in the top 10 biological functions are shown.

(K) Western blot shows the expression of selected proteins.

Several post-transcriptional modifications can cause the transcribed mRNA to fail to translate into functional proteins.³⁴ In addition to exploring global transcriptomic alterations, we sought to examine differentially regulated proteins in Th17 cells in response to CCL20 stimulation during Th17 differentiation. Based on the enrichment score of differentially expressed proteins, the top 9 enriched molecular functions were identified using the Ingenuity pathway analysis tool, and binding activity was found to be the highest enriched molecular function (Figure S3B). Moreover, we found that 1167 proteins, accounting for 43.7% of total proteins found after applying statistical filters (greater than or equal to 2, and a high level of Protein FDR confidence in the sample – combined), were exclusively detected only in Th17 cells culture in the presence of CCL20 (Figure 4G). Our results showed that CCL20 stimulation during Th17 differentiation upregulates STAT3 (Figures 4H and 4I), a master transcription factor for Th17 cell differentiation.³⁵ Moreover, we demonstrated that the highly enriched pathways include EIF2 signaling, p70S6K signaling, and the mTOR pathway, which are part of the Th17 differentiation pathways (Figure 4J). Among the several proteins, we validated several differentially expressed proteins by Western blot (Figure 4K). Consistent with our previous bulk RNA-Seq data, our mass spectrometry data also support the notion that CCL20 stimulation enhances Th17 differentiation. Our results showed that CCR6 signaling drives the expression of several genes involved in various biological processes and transcription factors, and promotes the differentiation of inflammatory Th17 cells.

CCL20 activates the mTORC1 pathway in CD4⁺ T cells

CCR6, being a GPCR, upon binding its cognate ligand CCL20, activates and transduces its signal through the Gαi family.⁴ The phosphorylation of Akt, mTOR, and STAT3 molecules is known to promote the differentiation of Th17 cells.^31,36,37 Signaling through mTORC1 promotes the differentiation of Th1 and Th17 cells,^31,38,39 while the inhibition or activation of mTORC2 promotes Th2 lineages.³⁹ Our RNA-seq and proteomics data showed that CCL20 stimulation activates the phosphatidylinositol and JAK/STAT pathways in Th17 cells. To investigate the induction of phosphorylation of Akt, mTOR, and STAT3 molecules with CCL20, CD4 T cells were purified from CCR6^gfp/gfp (CCR6^−/−) or CCR6^gfp/+ (CCR6^+/−) mice using flow cytometry. Purified cells were stimulated with purified recombinant CCL20 at various time points and monitored for the phosphorylation of Akt, mTOR, and STAT3 by flow cytometry and Western blot analysis. Our data showed that CCL20 induces the phosphorylation of Akt (Ser473), mTOR (Ser2448), and STAT3 (Tyr705) in a CCR6-dependent manner (Figures 5A and S4A–S4C). mTORC1 signaling activation is achieved through the signaling kinases phosphatidylinositol-3-OH kinase (PI3K), Akt, and S6 kinases. mTORC1 signaling is also sensitive to rapamycin signaling.⁴⁰ Our data showed that CCR6-CCL20 signaling phosphorylates PI3K and S6 kinases (Figure 5A), and that the downstream signaling of phosphorylated STAT3 is sensitive to rapamycin treatment (Figure 5B). STAT3 and RORγt bind to the IL-17A promoter and enhancer regions, positively regulating IL-17A transcription.⁴¹ IL-17A gene regulatory elements were shown to have several RORγt-binding sites among the IL-17A enhancer elements (Figure 5C). The conserved non-coding enhancer element CNS-5 is reported to play a crucial role in IL-17A gene expression.⁴¹ To demonstrate how CCL20-induced RORγt expression and STAT3 phosphorylation affect the IL-17 transcription, a reporter assay was performed using plasmid constructs containing the IL-17A promoter and CNS-5 element. Our results showed that CCR6-CCL20 signaling increased IL-17A promoter activity (Figure 5D). Furthermore, the mutation of the RORγt-binding site abolished the increased transcription activity of the IL-17A promoter (Figure 5E). Furthermore, CCL20-induced RORγt expression in Th17 cells was reduced with rapamycin, an mTOR inhibitor (Figure 5F). Collectively, these results suggested that CCL20-CCR6 induced the phosphorylation of PI3K/Akt/mTORC1/STAT3 molecules in the CD4⁺ T cells in a CCR6-dependent manner (Figure 5G), and promoted RORγt binding at IL-17A regulatory elements and positively regulated IL-17A gene expression and Th17 cell differentiation.

Figure 5.

CCR6 intrinsic signaling induces the phosphorylation of the PI3K/Akt/mTORC1/STAT3 pathway and promotes RORγt binding on IL-17A regulatory elements

(A) CCR6⁺ and CCR6^- CD4 T cells were sorted based on the expression of eGFP and stimulated with 100 ng/mL CCL20 at different time points (0, 5, 10, 15, 30, and 60 min). They were then immunoblotted with antibodies against various kinases in the downstream signaling pathways of Akt/mTOR/STAT3.

(B) Sorted CCR6⁺ cells were rested overnight with or without rapamycin (50 ng/mL) and, the next day, stimulated with recombinant CCL20 (100 ng/mL) at two time points, 0 and 30 min. The phosphorylation status of STAT3 was analyzed by immunoblotting. Cyclophilin B and total STAT3 were used as loading controls. Blots are from 2 to 3 independent experiments.

(C) The schematic presentation of IL-17A promoter (IL-17AP), IL-17F promoter (IL-17FP), conserved non-coding sequences (CNSs), and binding of RORγt.

(D) CCR6gfp⁺ Jurkat cells were transfected with the pGL4 plasmid vector containing the mouse IL-17 promoter (2 Kbp) and CNS5 enhancer elements. Transfected cells were stimulated with purified recombinant CCL20 (100 ng/mL) or TGF-β1 plus IL-6 at 37°C for 72 h. Then, cells were lysed, and luciferase activity was measured. The data shown are normalized to the Renilla luciferase activity. Each dot represents an independent experiment. Student’s t test. p-values are shown.

(E) CCR6gfp⁺ Jurkat cells were transfected with the pGL4 plasmid containing the IL-17 promoter (2 Kb) or IL-17 promoter (2 Kb) having a mutation in the RORγt binding site. Cells were cultured as given above, and luciferase activity was measured and plotted. The data shown are representative of one of the two independent experiments. The error bar represents ±SD.

(F) Naive CD4 T cells were differentiated into the Th17 lineage condition (TGF-β + IL-6) in the presence or absence of CCL20 and with or without rapamycin (25 ng/mL) for 4 days. Expression of RORγt was analyzed after gating on CD4⁺ T cells. n = 3 experiments.

(G) The schematic CCR6-CCL20 signaling pathways are shown. The p-value for the comparison between the two groups is indicated in the graphs. not significant (ns): p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

CCR6 signaling promotes glycolytic metabolism and inhibits oxidative phosphorylation in Th17 cells

mTOR signaling is known to control cellular metabolism, protein synthesis, energy balance, and cell proliferation, differentiation, and survival.^31,38,40,42 Various CD4⁺ T cell subsets exhibit distinct metabolic characteristics. For example, Th17 cells are physiologically driven by glycolysis, whereas Treg cells are supported by lipid oxidation and oxidative phosphorylation.⁴³ Blocking glycolysis limits Th17 cells' effector functions, whereas defective glycolysis increases Treg production.^44,45 Our RNA-Seq data revealed that the functional enrichment of biological processes after exposure to CCL20 during Th17 cell differentiation (Figures 4D and 4J). Furthermore, our data showed that exogenous CCL20 addition during Th17 cell differentiation induced the expression of genes involved in glycolysis and inhibited the expression of genes involved in the TCA cycle and oxidative phosphorylation (Figure 6A). Gene enrichment analysis of the data revealed that the downregulation of oxidative phosphorylation, accompanied by CCL20, occurred during Th17 cell differentiation (Figure 6B). Since RNA-seq data indicated increased modulation of glycolysis pathways, we monitored glucose utilization using a fluorescent d-glucose analog (2-NBDG) and lactate production in the culture during Th17 cell differentiation. Our data showed that CCL20 promotes the uptake of 2-NBDG (Figure 6C) and increases lactate production (Figure 6D) in both pathogenic (IL-1β + IL-23) and non-pathogenic (IL-6 + TGF-β) conditions of Th17 cell differentiation.

Figure 6.

CCR6 signaling alters the energy metabolism in Th17 cells

(A) Mouse naive T cells (CD4⁺CD25⁻CD44^lo) were in vitro differentiated into pathogenic and non-pathogenic Th17 cells. Transcriptomic analysis of Th17 cells cultured in the presence or absence of CCL20 was performed. A heatmap of the fold change (log2) for differentially expressed genes that participate in glycolysis and oxidative phosphorylation is shown. Each line represents one gene, and each column represents one sample. Different colors represent the expression levels (from red to green: increased expression).

(B) GSEA enrichment plots of gene sets in Th17 + CCL20 cells compared with Th17 cells, and showing the enrichment scores of oxidative phosphorylation.

(C) Glucose uptake was monitored using 2-NBDG by using Flow cytometry and shown as a stacked histogram (left), and the MFI of 2-NBDG uptake (right).

(D) After four days of culture of naive CD4 T cells, lactate secretion in the cultured media was measured. The error bar represents ±SD. Experiments were performed with four technical replicates.

(E) The extracellular acidification rate (ECAR) and (F) the oxygen consumption rate (OCR) were measured. Error bar represents ± S.E.M. Data shown represents one of the three independent experiments. One-way ANOVA followed by Tukey’s test was used. ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.0001.

(G) The expression of enzymes involved in glycolysis, and (H) a few of the enzymes involved in the TCA cycle in Th17 (non-pathogenic) and Th17 + CCL20 (pathogenic) cells were monitored using Western blot. The data shown represents one of the two independent experiments.

The increase in lactate secretion in the culture supernatant indicates that Th17 cells had a greater glycolytic rate than those cultured without CCL20. Interestingly, when we compared the lactate secretions between non-pathogenic and pathogenic Th17 cells, we found that the culture supernatant of pathogenic Th17 cells contained more lactate than that of non-pathogenic Th17 cells under the same culture conditions. Using the Seahorse real-time cell metabolic analyzer, we measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) during the cell culture. Our data showed that Th17 cells cultured in the presence of CCL20 exhibited a significantly higher ECAR than those cultured in its absence (Figure 6E). The CCL20 in the pathogenic Th17 (pTh17) culture condition showed increased ECAR activity compared to the non-pathogenic Th17 (npTh17) cells (Figure 6E, lower panel). Interestingly, the mitochondrial respiration measured by OCR was significantly reduced in Th17 cells cultured in the presence of CCL20 compared to those without CCL20 (Figure 6F). To further investigate the expression of enzymes involved in glycolysis and the TCA cycle, which also correlates with their activity, we performed Western blot analysis. Our results showed that stimulation with CCL20 during Th17 cell differentiation upregulated several enzymes, including hexokinase I, hexokinase II, and pyruvate kinase M2 (PKM2), which play a significant role in the glycolytic pathway (Figure 6G). However, TCA cycle enzymes, such as citrate synthase, aconitase, and fumarase, showed downregulation in response to CCL20 during Th17 cell culture (Figure 6H). Together, our data suggest that CCR6-CCL20 alters energy metabolism, driving glycolysis and inhibiting oxidative phosphorylation in Th17 cells.

Discussion

In the present study, we showed that patients with UC had increased CCR6⁺ CD4⁺ T cells in PBMCs, and that CCR6⁺CD4⁺ cells in patients with UC had a high frequency of Th17 cells and a committed, inflammatory Th1-like Th17 phenotype compared with healthy individuals. Naive CD4 T cells from humans and mice cultured under Th17 differentiation conditions with CCL20 exhibited an increased frequency of Th17 cells, elevated levels of Th17-associated molecules, and a substantial proportion of cells displayed a Th1-like Th17 cell phenotype in a CCR6-dependent manner in both in vitro and in vivo models. CCL20 promoted the phosphorylation of Akt, mTOR, and STAT3 molecules in CCR6gfp⁺ T cells as compared to CCR6gfp⁻ T cells and increased the transcription of IL-17A-promoter activity through RORγt binding elements. These observations imply that CCR6 and CCL20 play a key role in the pathogenesis of autoimmune colitis. It has been shown that JAK-STAT signaling pathways play an important role in the pathogenicity of Th17 cells.³⁵ Similarly, phosphatidylinositol signaling controls the differentiation of Th1 and Th17 cells and is a good target.⁴⁶ The ErB family gene, together with signaling, has an influence on the Th17 cell differentiation and the pathogenicity in infection and autoimmunity.^47,48 Our RNA-seq data showed that CCL20-treated Th17 cells exhibit these prominent pathways, suggesting the importance of CCR6 signaling in the inflammatory phenotypes of Th17 cells. It has been reported that Th1-like Th17 cells not only respond to canonical Th17-dependent pathogens, such as Candida albicans, but also, like Th1 cells, to intracellular pathogens, including Mycobacterium⁴⁹ and viruses.⁵⁰ Apart from the infection model, the production of IFN-γ by Th17 cells is crucial for the development of autoimmune colitis.²⁴ The plasticity of Th1, Th17, and Tregs plays a crucial role in gut inflammation and autoimmunity.^22,51,52 These findings suggest that gut inflammation increases CCL20 production by intestinal epithelial cells, thereby shaping the immune response against various gut microorganisms. These observations indicate that CCR6-CCL20 may play a role beyond recruiting immune cells to the site of inflammation. Our data demonstrated that CCL20-CCR6 signaling influences the migration of Th17 cells and the differentiation of Th17 and pathogenic Th1-like Th17 cells, as observed in both acute and chronic colitis models in mice. Genome-wide association studies have demonstrated a strong clinical association and polymorphism in several Th17-associated molecules, including IL-23R, JAK2, STAT3, and CCR6, in patients with UC.^53,54,55 Th17 cells are known to express several chemokine receptors, such as CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR10, CXCR4, CXCR5, and CXCR6.^2,56,57,58 Among these receptors, CCR6 is known to have stable expression on all subsets of Th17 cells and drives the migration of Th17 cells in both mouse and human gut.^3,9,22 The Toll-like receptor and other inflammatory cytokines cause an increase in CCL20 in colonic epithelial cells in IBD.²⁶

In addition to CCR6-CCL20, other GPCRs have been reported to affect the differentiation and function of CD4⁺ T cells. S1P1, a chemokine receptor required for T cells to egress from lymph nodes,⁵⁹ is shown to control the development and function of Treg cells through Akt-mTOR and STAT5 signaling.⁶⁰ Pertussis toxin, a known antagonist of G_αi signaling, inhibited Treg proliferation and induced Th17 differentiation in an IL-6-dependent manner.⁶¹ It is known that several GPCRs are capable of activating more than one Gα-subunit, and CCR6 can activate both Gαi and Gαq subunits.⁶² The Th17-enhancing ability may be mainly attributed to Gαq function, but this requires further investigation. Moreover, CCR2-CCL2 interactions were also shown to be involved in T cell differentiation, along with the recruitment of cells. Gu et al. observed that CCL2-deficient mice exhibited a reduced Th2 response, characterized by lower IL-4 production in draining lymph nodes.⁶³ CCL2-deficient mice also showed enhanced resistance to Leishmania infection, indicating a shift from Th2 to Th1 responses.⁶⁴ Furthermore, it has been demonstrated that CCL5-deficient mice produce significantly lower levels of IFN-γ⁶⁵ and that adding CCL3 to activated cells promotes IFN-γ production.⁶⁶ These data suggest that chemokine receptors can influence the phenotype and function of CD4⁺ T cells, thereby controlling disease pathogenesis. Recently, we demonstrated that CCR6 signaling inhibits the differentiation and function of induced T regulatory cells (Tregs) during gut inflammation and autoimmunity.²² Clinical strategies to control IBD may require interventions that prevent the differentiation of Th17 precursor cells into inflammatory Th1-like Th17 cells. As reported earlier, Th17 cells exhibit stem cell markers, possess strong potential for self-renewal,⁶⁷ and can differentiate into Th1-like Th17 cells during gut inflammation.²⁴ These studies suggest that Th17 cells may act as a strong reservoir of non-pathogenic effector cells under homeostatic conditions but may transit to pathogenic Th1-like Th17 cells under strong inflammatory conditions. It is evident from several trials on IBD that blocking antibodies to IL-12p40 (which blocks both IL-23 and IL-12)⁶⁸ and IFN-γ show a reduction in disease burden, but not with IL-17A.⁶⁹ This study demonstrated that CCL20 stimulation activates the Akt/mTORC1/STAT3 pathway, thereby increasing the frequency of Th17 cells. Various assays, including global transcriptomic and proteomic analyses, validate our findings of increased Th17 cell frequency via this pathway.

The effector functions and pathogenicity of Th17 cells are driven by shifting their metabolic pathway toward glycolysis.⁷⁰ There are reports that CNS-infiltrated pathogenic Th17 cells from diseased mice specifically upregulate genes involved in the glycolytic pathway during neuroinflammation, compared to homeostatic intestinal Th17 cells.⁷¹ Our RNA-Seq data revealed the upregulation of glycolysis and inhibition of oxidative phosphorylation in Th17 cells, driven by the CCR6-CCL20 signaling pathway. Several studies have shown that PKM molecules modulate Th17 cell differentiation.^72,73 The PI3K-Akt-mTORC1-S6K1/2 axis is known to regulate the differentiation of Th17 cells,³¹ and mTORC1 signaling also promotes the T-bet phosphorylation to control the Th1 cell differentiation.³⁸ The deficiency of the mTORC1 complex affects energy-intensive ribosomal biosynthesis, proliferation, and the cell cycle in T cells, whereas such effects are not observed with mTORC2 deficiency.⁴² mTORC1 also plays a crucial role in antigen-specific T cell differentiation and memory responses.^74,75,76 In the antigen-, virus-, and tumor-exhausted T cells, mTORC1 activity is compromised.^77,78,79 Several studies have demonstrated that Th17 cells utilize glycolysis during inflammation to fuel their rapid expansion and pathogenicity, whereas the Treg population relies on oxidative phosphorylation as its primary energy source. For Th17 cells to differentiate and become pathogenic, aerobic glycolysis is required.^80,81 Several glycolytic enzymes, such as glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate mutase (PGAM). Inhibition or blocking of these enzymes lowered the pathogenicity of Th17 cells.⁸² It has been shown that PKM2, a crucial glycolytic enzyme, fine-tunes STAT3 activation to promote the development of Th17 cells and autoimmune inflammation.⁷³ Our data showed that PKM2 is among the highly upregulated enzymes in Th17 cells cultured in the presence of CCL20. However, further extensive studies are needed to determine how chemokine receptor CCR6 influences cellular metabolism. The pathway through which the switch between glycolysis and oxidative phosphorylation occurs must also be identified using knock-out mice. We demonstrated that CCL20-CCR6 signaling can alter the metabolic reprogramming of Th17 cells, which may contribute to the pathogenesis of the disease and also the molecular adaptation of these cells in the inflamed microenvironment.

The pathogenic Th1-like Th17 cells are known to contribute to the pathogenesis of several Th17-mediated inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and IBD, and inflamed tissues in these diseases have increased expression of CCL20.¹ Biologics or therapeutic agents that target CCR6-CCL20 signaling may offer a promising strategy for managing inflammatory diseases by disrupting the cellular and metabolic programming of pathogenic Th17 cells.

Limitations of the study

In the present study, we showed that CCL20-CCR6 signaling drives the differentiation of Th1-like Th17 cells (expressing T-bet together with RORγt), which exhibit a pathogenic phenotype compared with Th17 cells expressing RORγt alone. We demonstrate that Th1-like Th17 cells can be differentiated in vitro in the presence of CCL20 and that their frequency increases in gut inflammation in both humans and mice. However, its pathogenicity needs to be further confirmed in an in vivo model using adoptive transfer experiments. Metabolic reprogramming should also be investigated in Th1-like Th17 cells isolated from colitic mice.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Girdhari Lal (glal@nccs.res.in).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •The processed bulk RNA-seq data from this study are deposited in the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra) with accession codes: SAMN47541003, SAMN47541004, SAMN47541005, SAMN47541006).
• •The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD071790 and 10.6019/PXD071790 (https://www.ebi.ac.uk/pride/archive/project/PXD071790).
• •All additional information can be obtained from the corresponding authors upon reasonable request.

Acknowledgments

G.L. received the Swarn Jayanti Fellowship (DST/LSA-01/2017-18) from the Department of Science and Technology (DST) and the TATA Innovation Fellowship (HRD-16012/5/2024-DBT) from the Department of Biotechnology, Ministry of Science and Technology, Government of India. N.K. received a senior research fellowship (SRF) from the Council of Scientific and Industrial Research (CSIR), and H.T.M. received an INSPIRE Fellowship from the DST, Government of India. The authors acknowledge the NCCS flow cytometry, Proteomics, and microscopy core facilities.

Author contributions

H.T.M. and N.K. performed the experiments, analyzed the data, and wrote the article. A.S. and S.R. assist in acquiring and analyzing proteomics data. P.K.S., V.R.M., and S.S. collected the consent and samples from the individuals. G.L. conceived the idea, designed the experiments, interpreted data, and wrote the article.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
FITC anti-human CD4	Biolegend	Cat# 317408; RRID: AB_571951
PE anti-human CD25	Biolegend	Cat# 302606; RRID: AB_314276
Biotin anti-human CD45RA	Biolegend	Cat# 304104; RRID: AB_314408
Alexa Fluor® 488 anti-mouse CD4	Biolegend	Cat# 100423; RRID: AB_389302
APC/Cy7 anti-mouse CD4	Biolegend	Cat# 100414; RRID: AB_312699
PE/Cy5 anti-mouse CD4	Biolegend	Cat# 312695; RRID: AB_100410
APC anti-mouse CD4	Biolegend	Cat# 100516; RRID: AB_312719
Biotin anti-mouse CD4	Biolegend	Cat# 312689; RRID: AB_100404
Biotin anti-mouse/human CD44	Biolegend	Cat# 103003; RRID: AB_312954
APC/Cy7 anti-mouse/human CD44	Biolegend	Cat# 103028; RRID: AB_830785
PE anti-mouse/human CD44	Biolegend	Cat# 103008; RRID: AB_312959
PerCP-Cy5.5 Anti-Mouse CD25	Biolegend	Cat# 101912; RRID: AB_10613643
PE anti-mouse CD25	Biolegend	Cat# 101904; RRID: AB_312847
FITC anti-mouse CD25	Biolegend	Cat# 101908; RRID: AB_961212
Alexa Flour 647 anti-mouse CD25	Biolegend	Cat# 102020; RRID: AB_493458
PE anti-mouse IL-17A	Biolegend	Cat# 506904; RRID: AB_315464
Anti-Mouse/Rat IL-17A eFlour-450	eBioscience	Cat#48-7177-82; RRID: AB_11149503
PerCP/Cy5.5 anti-mouse IFN-γ	Biolegend	Cat# 505822; RRID: AB_961359
PE/Cy7 anti-mouse IFN-γ	Biolegend	Cat# 505826; RRID: AB_2295770
PE/Cy7 anti-mouse GM-CSF	Biolegend	Cat# 505412; RRID: AB_2721682
PE anti-mouse GM-CSF	Biolegend	Cat# 505406; RRID: AB_315382
PE anti-mouse ROR gamma (t)	eBioscience	Cat#12-6981-82; RRID: AB_10807092
PE/Cy7 anti-mouse/hum RORγ(t)	eBioscience	Cat#25-6981-82; RRID: AB_2784671
APC anti- mouse/human RORγ(t)	eBioscience	Cat#12-6981-82; RRID: AB_10609207
PE anti-mouse T-bet	Biolegend	Cat# 644810; RRID: AB_2200542
PE/Cy7 anti-mouse T-bet	Biolegend	Cat# 644824; RRID: AB_2561761
eFlour450 anti-mouse/human T-bet	eBioscience	Cat#48-5825-82; RRID: AB_2784727
BV 421 anti-mouse IL-23R	Biolegend	Cat# 150907; RRID: AB_2715804
PE anti-mouse IL-23R	Biolegend	Cat# 150904; RRID: AB_2572189
Phospho-Stat3 (Tyr705) Rabbit mAb	Cell Signaling Technology	Cat#9145; RRID: AB_2491009
Stat3 (D3Z2G) Rabbit mAb	Cell Signaling Technology	Cat# 12640, RRID: AB_2629499
Phospho-mTOR Rabbit mAb	Cell Signaling Technology	Cat#9964; RRID: AB_10696892
Phospho-PI3 Kinase Rabbit mAb	Cell Signaling Technology	Cat# 4228, RRID: AB_659940
PI3 Kinase p110-alpha	Cell Signaling Technology	Cat#4249; RRID: AB_2165248
Phospho-p70 S6 Kinase Rabbit mAb	Cell Signaling Technology	Cat# 9862, RRID: AB_10696885
Phospho-Akt (Ser473) Rabbit mAb	Cell Signaling Technology	Cat# 4060, RRID: AB_2315049
Phospho-Akt (Ser473) Rabbit mAb	Cell Signaling Technology	Cat# 4060, RRID: AB_2315049
TAP1 Rabbit pAb	Invitrogen	Cat# PA1-28108, RRID: AB_2200193
SHP1 Rabbit mAb	Invitrogen	Cat# MA5-31989, RRID: AB_2809283
Rabbit mAb to Cyclophilin B	Abcam	Cat# ab178397, RRID: N/A
Purified anti-mouse CD3ε mAb	BioXCell	Cat# BE0001-1, RRID: AB_1107634
Purified anti-mouse CD28 mAb	BioXCell	Cat# BE0051-1, RRID: AB_1107624
LEAF™ Purified anti-human CD28	Biolegend	Cat# 302914, RRID: AB_314316
LEAF™ Purified anti-human CD3	Biolegend	Cat# 300438, RRID: AB_11146991
IFN gamma mAb	eBioscience	Cat# 16-7311-85; RRID: AB_469253
Anti-mouse IL-4	BioXCell	Cat# BE0045; RRID: AB_1107707
mTOR Pathway Antibody Sampler Kit	Cell Signaling Technology	Cat# 9964T; RRID: AB_10696892
mTOR Substrates Antibody Sampler Kit	Cell Signaling Technology	Cat# 9862T; RRID: AB_10696885
Rabbit monoclonal anti-Ki67 antibody (SP6)	Abcam	Cat# ab16667; RRID: AB_446486
Rabbit polyclonal anti-CCR6	Abcam	Cat# ab78429; RRID: AB_2072554
PE anti-mouse CD62L (MEL-14)	eBioscience	Cat# 12-0621-83; RRID: AB_465722
Glycolysis Antibody Sampler Kit	Cell Signaling Technology	Cat# 8337T; RRID: AB_10897509
Tricarboxylic Acid Cycle Antibody Sampler Kit	Cell Signaling Technology	Cat# 47767T; RRID: AB_2890191
Csk Antibody	Cell Signaling Technology	Cat# #4965; RRID: AB_2276592
PE anti-mouse CD45RB Antibody	Biolegend	Cat# 103307; RRID: AB_313014
PE anti-mouse IFN-γ Antibody	Biolegend	Cat# 505808; RRID: AB_315401
Pacific Blue™ anti-mouse IL-17A Antibody	Biolegend	Cat# 506918; RRID: AB_893545
Brilliant Violet 421™ anti-T-bet Antibody	Biolegend	Cat# 644832; RRID: AB_10896427
Alexa Fluor® 647 anti-T-bet Antibody	Biolegend	Cat# 644804; RRID: AB_1595573
Biotin anti-mouse CD45.2 Antibody	Biolegend	Cat# 109804; RRID: AB_313440
Alexa Fluor® 488 anti-mouse CD45.2 Antibody	Biolegend	Cat# 109816; RRID: AB_492869
Alexa Fluor® 647 anti-mouse CD45.1 Antibody	Biolegend	Cat# 110720; RRID: AB_492864
Alexa Fluor® 647 anti-human CD196 (CCR6) Antibody	Biolegend	Cat# 353404; RRID: AB_10918982
Brilliant Violet 421™ anti-mouse CD196 (CCR6)	Biolegend	Cat# 129818; RRID: AB_10898320
PE anti-mouse CD196 (CCR6) Antibody	Biolegend	Cat# 129804; RRID: AB_1279137
PE/Cyanine7 anti-mouse CD196 (CCR6) Antibody	Biolegend	Cat# 129815; RRID: AB_2072798
Biotin anti-mouse CD25 Antibody	Biolegend	Cat# 102004; RRID: AB_312853
Brilliant Violet 421™ anti-mouse CD25 Antibody	Biolegend	Cat# 102043; RRID: AB_10895908
PE anti-mouse CD4 Antibody	Biolegend	Cat# 100407; RRID: AB_10587288
APC anti-human CD4 Antibody	Biolegend	Cat# 300552; RRID: AB_2820224
PE/Cyanine5 anti-human CD25 Antibody	Biolegend	Cat# 302607; RRID: AB_2910402
PE/Cyanine7 anti-human CD45RA Antibody	Biolegend	Cat# 304125; RRID: AB_10708879
PE anti-human IL-17A Antibody	Biolegend	Cat# 512305; RRID: AB_961394
APC/Cyanine7 anti-human IL-17A		Cat# 512320; RRID: AB_10613103
Pacific Blue™ anti-human IFN-γ Antibody	Biolegend	Cat# 502522; RRID: AB_893525
Pacific Blue™ anti-mouse/human CD44 Antibody	Biolegend	Cat# 103019; RRID: AB_493682
Alexa Fluor® 488 anti-mouse CD8a Antibody	Biolegend	Cat# 100723; RRID: AB_493423
PE-Cy7-Streptavidin	Biolegend	Cat# 405206
eFluor 450-Streptavidin	eBiosciences	Cat# 48-4317-82
APC/Cy7-Streptavidin	Biolegend	Cat# 405208
Alexa Fluor® 647 Streptavidin	Biolegend	Cat# 405237
Brilliant Violet 421™ Donkey anti-rabbit IgG	Biolegend	Cat# 406410; RRID: AB_10897810
Bacterial and viral strains
DH5α Competent Cells	Thermo Fisher Scientific	Cat# EC0111
Biological samples
Human Peripheral Blood healthy volunteers for in vitro experiments	National Centre for Cell Science, Pune	Protocol ID: NCCS/IEC/13032014
Peripheral blood was obtained from ulcerative colitis patients and healthy individuals	Armed Force Medical College, Pune	Protocol ID: AFMC/IEC/28102014
Chemicals, peptides, and recombinant proteins
Recombinant Human IL-6	Biolegend	Cat# 570802
Recombinant Human IL-23	Biolegend	Cat# 787902
Recombinant Human IL-1β	Biolegend	Cat# 579402
Recombinant Human TGF-β1	Biolegend	Cat# 580704
Recombinant mouse IL-6	Biolegend	Cat# 575702
Recombinant Mouse IL-1β	Biolegend	Cat# 575104
Recombinant mouse IL-23	Biolegend	Cat# 589004
Recombinant mouse IL-4	Biolegend	Cat# 574302
Recombinant Human CCL20 (MIP-3α)	Biolegend	Cat# 583804
Recombinant mouse CCL20	Biolegend	Cat# 582302
Rapamycin, an mTOR inhibitor	CST	Cat# 9904S
Ficoll-Paque Plus	GE Healthcare	Cat# 17144002
1X fixation buffer	BioLegend	Cat# 420801
10X permeabilization buffer	eBioscience	Cat# 00-8333-56
HEPES	MP Biomedicals	Cat# 194549
Collagenase D	Sigma-Aldrich	Cat# 11088866001
DNase I	Thermo Fisher Scientific	Cat# AM2224
DSS	MP Biomedicals	Cat# 0216011080
2-Mercaptoethanol for Molecular Biology	Thermo Fisher Scientific - Gibco	Cat# 21985023
RPMI 1640 Medium	Thermo Fisher Scientific	Cat# 11875093
Fetal Bovine Serum	Thermo Fisher Scientific	Cat# 26140079
DMEM	Thermo Fisher Scientific	Cat# 1199507
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-free	Thermo Fisher Scientific	Cat# AM9625
ACK Lysing Buffer	Thermo Fisher Scientific	Cat# A1049201
Trizol reagent	Invitrogen	Cat# 10296-010
iScript™ cDNA Synthesis Kit	Biored	Cat# 1708890
Superscript III Reverse transcriptase	Invitrogen	Cat# 18080044
SYBR green PCR kit	Qiagen	Cat# 204054
TransFast reagent	Promega	Cat# E2431
Rneasy Plus Mini Kit	Qiagen	Cat# 74134
TruSeq RNA Sample Preparation Kit	Illumina	Cat# RS-122-2001
Trizol reagent	Invitrogen	Cat# 10296-010
Critical commercial assays
Dual luciferase assay kit	Promega	Cat# E2920
Bradford reagent	Himedia	Cat# ML106-500ML
2-NBDG	Invitrogen	Cat# N13195
Lactate estimation kit	Spinreact, Spain	Ref: 1001330
Seahorse XF Glycolysis Stress Test Kit	Agilent	Cat# 103020-100
Agilent Seahorse XF cell mito-Stress Test Kit	Agilent	Cat# 103015-100
Deposited data
RNA Sequencing data	This paper, the NCBI-SRA database (https://www.ncbi.nlm.nih.gov/sra)	Accession code: SAMN47541003, SAMN47541004, SAMN47541005, SAMN47541006
Mass Spectrometry Proteomics data	This paper, PRIDE database (https://www.ebi.ac.uk/pride/)	Accession code: PXD071790
Experimental models: Cell lines
Jurkat E-6.1∗	NCCS, Cell Repository, Pune, India	Cell line authenticated and mycoplasma-free
BW5147.3 cells	ATCC, USA	Cat# TIB-47
Experimental models: Organisms/strains
C57BL/6J	The Jackson Laboratory	RRID: IMSR_JAX:000664
B6.129S6-Ccr6tm1(EGFP)Irw/J	The Jackson Laboratory	RRID: IMSR_JAX:013061
B6.129-Ccr6tm1Dgen/J	The Jackson Laboratory	RRID: IMSR_JAX:005793
B6.129S7-Rag1tm1Mom/J	The Jackson Laboratory	RRID: IMSR_JAX:002216
B6.SJL-PtprcaPepcb/BoyJ	The Jackson Laboratory	RRID: IMSR_JAX:002014
Oligonucleotides
oligo d(T)12-16 primers	Sigma-Aldrich
qRT-PCR primers (NCBI accession number)
Mouse Cyclophilin-A Forward Primer	Sigma-Aldrich	5'-AGGGTGGTGACTTTACACGC-3'
Mouse Cyclophilin-A Reverse Primer	Sigma-Aldrich	5'-ATCCAGCCATTCAGTCTTGG-3'
Mouse STAT3 A Forward Primer (NM_213659.3)	Sigma-Aldrich	5'-GAGAGATTGACCAGCAGTATAG-3'
Mouse STAT3 A Reverse Primer (NM_213659.3)	Sigma-Aldrich	5'-CAAGATACCTGCTCTGAAGAAA-3'
Mouse Hif1a Forward Primer (NM_001313919.1)	Sigma-Aldrich	5'-CCATTCCTCATCCGTCAAATA-3'
Mouse Hif1a Reverse Primer (NM_001313919.1)	Sigma-Aldrich	5'-AAGTTCTTCCGGCTCATAAC-3'
Mouse Akt1 Forward Primer (NM_009652.3)	Sigma-Aldrich	5'-GACCATGAACGAGTTTGAGTA-3'
Mouse Akt1 Reverse Primer (NM_009652.3)	Sigma-Aldrich	5’-TTGAGGATCTTCATGGCATAG-3'
Mouse Akt2 Forward Primer (NM_001110208.2)	Sigma-Aldrich	5'-TAGATTCTTCCTCAGCATCAAC-3'
Mouse Akt2 Reverse Primer (NM_001110208.2)	Sigma-Aldrich	5'-GGTGAACTCGTCATCAAAGTA-3'
Mouse Ahr Forward Primer (NM_013464.4)	Sigma-Aldrich	5’-AGGTGCCTGCTGGATAATTC-3’
Mouse Ahr Reverse Primer (NM_013464.4)	Sigma-Aldrich	5’-CCGTCCTTCCCTTTCTTGTT-3’
Mouse IL-23R Forward Primer (NM_144548.1)	Sigma-Aldrich	5’-TGCCATGACTCCAGGATGGAACTT-3’
Mouse IL-23R Reverse Primer (NM_144548.1)	Sigma-Aldrich	5’-GCAAACAAGCAAACAAGCACAGCC-3’
Mouse IL-22 Forward Primer (NM_016971.2)	Sigma-Aldrich	5’-GTGCCTTTCCTGACCAAACT-3’
Mouse IL-22 Reverse Primer (NM_016971.2)	Sigma-Aldrich	5’-ACTGTCTCCTTCAGCCTTCT-3’
Mouse IL-21 Forward Primer (NM_021782.2)	Sigma-Aldrich	5’-TGAAAGCCTGTGGAAGTGCAAACC-3’
Mouse IL-21 Reverse Primer (NM_021782.2)	Sigma-Aldrich	5’-AGCAGATTCATCACAGGACACCCA-3’
Mouse CCR6 cloning primers
pORF9-mCCR6 Forward Primer (NM_009835.4)	Sigma-Aldrich	5'-CTAGTA GATCTGATATCCATCATGAAT-3'
pORF9-mCCR6 Reverse Primer (NM_009835.4)	Sigma-Aldrich	5'-TTCAGTCGACCTCTCGTGTTACATG-3'
Recombinant DNA
pORF9-mCCR6 plasmid	Invivogen	Cat# pORF-mCCR6
pIRES-EGFP2 vector	Invivogen
pGL4 plasmid containing mouse 2 Kbp IL-17 promoter	Adgene	Cat# 20127
pRL-TK plasmid	Promega	Cat# E2241
Software and algorithms
Leica MMAF software	Leica	https://www.leica-microsystems.com/
Image-J software	NIH, USA	https://imagej.nih.gov/ij/
FlowJo v10.2	Tree Star	https://www.flowjo.com/
GraphPad Prism 7	GraphPad	https://www.graphpad.com
Proteome Discoverer software	ThermoScientific
Ingenuity Pathway Analysis tool 9 (Content version 48207413)	Qiagen
Web-based Gene Set Analysis Toolkit	WebGestalt	https://www.webgestalt.org/
Other
70-μm cell strainers	Corning	Cat# 431751
Amicon Ultra 3KD cut-off (MWCO) filter	Millipore	Cat# UFC500324
Seahorse XF Calibrant medium	Agilent	Cat# 10840-000
24-well XF cell culture plates	Agilent	Cat# 100777-004

Experimental model and study participant details

Cell lines

Jurkat and its derivatives, such as Jurkat CCR6-EGFP⁺ cells, were maintained in complete RPMI 1640 medium (RPMI supplemented with 10% fetal bovine serum, penicillin, and streptomycin). Mouse T cell line BW5147.3 cells and other derivatives such as pIRES2-EGFP-mCCR6⁺ were also maintained in complete Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum, penicillin, and streptomycin. All cell lines were cultured in an incubator maintained at 37 °C and 5% CO₂. All cell lines used in this study were authenticated upon receipt from the NCCS cell repository and are routinely tested for mycoplasma contamination.

Mice

Wild-type C57BL/6, CCR6^-/-, Foxp3-egfp, or RAG1^-/- (C57BL/6 background) mice were procured from Jackson Laboratory (Bar Harbor, ME) and maintained in the National Centre for Cell Science (NCCS) experimental animal facility. In heterozygous F1 offspring (CCR6gfp^+/-), cells expressing EGFP could bind to anti-CCR6 mAb, validating that surface expression of CCR6 is marked by EGFP expression.⁸³ All mice used in this study were 6-8 weeks old and female. There have been no reports of gender influence on the study's results. All the experiments were performed per the approved animal usage guidelines of the Institute’s Animal Ethics Committee (Protocol IDs: NCCS/IAEC/B-158, NCCS/IAEC/B-259, and NCCS/IAEC/B-407).

Human samples

Peripheral blood was obtained from ulcerative colitis patients (six women, thirteen men; age 39 ± 9.3 years) and healthy individuals (Six women, seven men; age 63 ± 8 years) after informed consent and per a protocol approved by Institutional Ethics Committee of the National Centre for Cell Science (NCCS), Pune (Protocol ID: NCCS/IEC/13032014) and Armed Force Medical College (AFMC), Pune (Protocol ID: AFMC/IEC/28102014). Peripheral blood mononuclear cells (PBMC) were prepared using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation.

Method details

Purification and culture of human naïve CD4⁺ T cells

Peripheral blood mononuclear cells (PBMC) from healthy donors (three men and two females; age 30-45 years) were prepared using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation. PBMCs were stained with anti-human CD4, anti-human CD25, anti-human CD8, and anti-human CD45RA antibodies. CD4⁺CD8^-CD25^-CD45RA⁺ (naïve CD4⁺ T cells) and CD4^-CD8^- cells (T cell-depleted PBMCs) were purified using flow cytometry sorting. Naïve CD4⁺ T cells were cultured as described earlier.²² Naïve CD4⁺ T cells (5 X l0⁴ cells/well) were cultured in U-bottom 96 well plates along with irradiated T cell-depleted PBMCs (5 x 10⁴ cells/well) in a final volume of 0.2 ml complete 2 RPMI medium along with purified recombinant human IL-2 (10 ng/ml) and anti-human CD3ε antibody (clone OKT3; 5 μg/ml) in presence or absence of human recombinant CCL20 (100 ng/ml) at 37°C for 4 days. Recombinant human TGF-β1 (10 ng/ml), recombinant human IL-6 (20 ng/ml), and anti-human IFN-γ were added to induce differentiation of Th17 cells. After 4 days of culture, cells were harvested and analyzed for RORγt and T-bet expression using flow cytometry.

Induction of acute colitis

Acute colitis was induced in the wild-type C57BL/6 mice (6-8 weeks old female) by giving 2% dextran sodium sulfate (DSS, w/v; MP Biomedicals, Santa Ana, CA) in the drinking water. Colitis developed within seven days of DSS treatment. Development and progression of colitis were monitored daily for weight loss, loose stool, and bloody diarrhea.

Induction of chronic autoimmune colitis

Naïve CD4⁺ T cells (CD4⁺CD25^-CD44^loCD45RB^hiCD62L^hi cells; 0.5 x 10⁶ cells/mouse) purified from naïve C57BL/6 mice were adoptively transferred into RAG1^-/- mice. Six to ten-week-old female mice were used in this study. These mice develop colitis within four weeks of receiving injections of naïve CD4⁺ T cells. Development and progression of colitis were monitored daily for weight loss, loose stool, and bloody diarrhea.

In vitro CD4⁺ T cell culture

Purified naïve CD4⁺CD25^-CD44^- cells (5 x 10⁴ cells/well) and irradiated T cell-depleted splenocytes (5 x 10⁴ 10 cells/well) were cultured in 0.2 ml complete RPMI medium in presence of recombinant IL-2 (10 ng/ml) and anti-mouse CD3ε mAb (clone 145-2C11; 5 μg/ml) in U-bottom 96 well plates at 37°C for 4 days in a 5% CO2 incubator. For in vitro cultures, we used 6-8-week-old male or female mice to isolate CD4 T cells. Recombinant TGF-β1 (10 ng/ml) and recombinant IL-6 (20 ng/ml) were added to induce differentiation of Th17; recombinant IFN-γ (10 ng/ml) and anti-mouse IL-4 mAb (10 μg/ml) were added to induce differentiation of Th1; and recombinant IL-4 (10 ng/ml) and anti-mouse IFN-γ (10 μg/ml) mAb to differentiate into Th2 lineage.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)

Cells were harvested, and total RNA was prepared using Trizol reagent (Invitrogen). cDNA was synthesized using oligo (dT)_11-16 primer and Superscript cDNA synthesis kit (BioRad). Specific gene expression was performed using the SYBR green PCR kit (Qiagen) according to the manufacturer's protocol. The cyclophilin A gene was used as a housekeeping gene. Data were acquired using a CFX96 PCR machine (BioRad). Relative mRNA expression was calculated for a specific gene as: 2 ^{(Ct of cyclophilin A-Ct of specific gene)}.

Isolation of lymphocytes from lamina propria

The small intestine was dissected, and mesentery, fat, and Peyer's patches were removed. The cleaned intestine was washed in calcium-magnesium-free (CMF) HEPES solution, cut into small pieces (∼2 mm), and incubated in HEPES solution containing 10% FBS and 5 mM EDTA at 37°C for 15 minutes. Tissues were digested in RPMI medium containing collagenase D (300 units/mL; MP Biomedicals) and DNase I (0.02 mg/mL; Invitrogen) at 37°C for 60 minutes. Digested tissues were passed through a 70 μm pore containing a cell strainer, and lamina propria (LP) lymphocytes were recovered from the filtrate. Cells were washed with FACS buffer (2% FBS in PBS) and analyzed by flow cytometry.

Flow cytometry

The single-cell suspension was prepared from tissues and surface-stained with various fluorescently labeled antibodies. For intracellular staining, cells were fixed in 1X fixation buffer (eBioscience or BioLegend) and permeabilized with 1X permeabilization buffer (eBioscience or BioLegend), then stained with specific fluorescently labeled antibodies, as per the manufacturer’s guidelines. Cells were acquired using Flow cytometry (FACS Canto, BD Bioscience), and data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

Intracellular phospho-staining

Total splenocytes from CCR6gfp^+/-Foxp3rfp^+/y mice were surface-stained for CD4. Surface-stained splenocytes (10⁶ cells) were stimulated with CCL20 (100 ng/ml) in 1 ml of incomplete RPMI medium in 24-well plates for the indicated time intervals. Stimulated cells were fixed by incubating in 500 μL of 1X Phosflow Lys/Fix buffer (BD Biosciences, San Jose, CA) for 10 minutes at 37 C. Fixed cells were permeabilized by incubating them with 1 mL of ice-cold 1X Phosflow Perm Buffer II (BD Biosciences, San Jose, CA) for 30 minutes at 4°C, followed by staining with the indicated phospho specific antibodies. Cells were acquired using flow cytometry, and data were analyzed using FlowJo software.

Generation CCR6-EGFP expressing T cell line

Mouse CCR6 gene was PCR amplified from the pORF9-mCCR6 plasmid (Invivogen, San Diego, CA) using forward primer 5’-CTA GTA GAT CTG ATA TCC ATC ATG AAT-3’ and reverse primer 5’- TTC AGT CGA CCT CTC GTG TTA CAT G-3’. CCR6 amplicon (1.1 Kb) was inserted between Bgl II and Sal I restriction sites into the pIRES-EGFP2 vector (Invivogen). BW5147.3 cells (mouse T lymphoma cell line) and Jurkat J6 (human T lymphoma cell line) were electroporated with the pIRES2-EGFP-mCCR6 construct using the Gene Pulser Xcell™ system (Bio-Rad, Germany). EGFP⁺ cells were sorted using flow cytometry (FACS Aria III, BD Bioscience) and maintained in complete RPMI media containing G418 (100 μg/ml).

Luciferase assay

CCR6-EGFP⁺ Jurkat cells (10⁶ cells) were transfected with pGL4 plasmid containing mouse 2 Kbp IL-17 promoter, RORγt binding site mutated IL-17 promoter, or IL-17 promoter with CNS-5 enhancer (Addgene, Cambridge, MA; 3 μg plasmid)⁴¹ and internal control pRL-TK plasmid (Promega, Madison, WI; 0.2 μg) using TransFast reagent (Promega, Madison, WI). Transfected cells were incubated overnight and then stimulated either with CCL20 (100 ng/ml) or with recombinant TGF-β1 (10 ng/ml) and recombinant IL-6 (20 ng/ml). After 72 hours of stimulation, the cells were lysed, and luciferase activity was measured using a dual luciferase assay kit (Promega, Madison, WI). Luminescence was recorded with the Turner BioSystems Luminometer Model TD-20/20 (Promega), and data were normalized to Renilla luciferase.

Immunoblotting

CCR6⁺CD4⁺ T and CCR6^-CD4⁺ T cells were sorted from CCR6^-/- (homozygous) mice and CCR6^+/- heterozygous (CCR6-eGFP) mice using a flow cytometry sorter (FACS ARIA, BD Biosciences). After sorting, cells were stimulated with purified recombinant CCL20 at different time points, and the phosphorylation of Akt, mTOR, and STAT3 molecules was monitored. After stimulation, the cells were washed with ice-cold PBS and lysed in 100 μl of RIPA buffer 50 mM Tris-Cl, pH 7.5, with 120 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM EDTA, 1 mM PMSF, 1% NP-40 and protease inhibitor cocktail (Roche) per 3×10⁶ cells on ice for 30 minutes. Lysates were homogenized either with a homogenizer or by pipetting up and down several times. Proteins were quantified using a Bradford reagent (Himedia). An equal amount of protein samples (20-30 μg) was used for polyacrylamide gel electrophoresis. And immunoblot with different primary antibodies after blocking with 5% skimmed milk in TBST for one hour at RT. Captured the chemiluminescence from HRP-conjugated secondary antibodies using the ChemiDoc Instrument (Invitrogen).

Bulk RNA sequencing

For RNA-seq, CD4⁺CD25^-CD44^- naïve T cells were in vitro differentiated into Th17 [Anti-CD3ε mAb (10 μg/ml) + Anti-CD28 mAb (2 μg/ml) + IL-1β (20 ng/ml) + IL-23 (20 ng/ml)] cells in the presence or absence of CCL20 (100 ng/ml). On the 4th day of culture, total RNA was isolated from these cultured cells using the Rneasy Plus Mini Kit (Qiagen). mRNA was purified using oligo (dT) beads (TruSeq RNA Sample Preparation Kit, Illumina) from 1 μg of intact total RNA. The extracted RNA purity was checked using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and RNA with a RIN value of 7.0 or higher was used for library preparation. The purified mRNA was fragmented at 90°C in the presence of divalent cations. Then, using Superscript II Reverse Transcriptase (Life Technologies), fragments were converted into cDNA using random hexamers. Second-strand cDNA was prepared on the first-strand template using RNAase and DNA polymerase. The cDNAs obtained were cleaned using Beckman Coulter SPRI beads. Then, we proceeded with sequencing library preparation using TruSeq RNA Library Prep Kits for Illumina® (NEB, USA) according to the manufacturer’s instructions, and index codes were added to attribute sequences to each sample. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s protocol. Finally, paired-end reads of 150 bp were generated using an Illumina MiSeq platform, and alignment was performed using Hisat2. Assembly was performed with StringTie, and Differential analysis was conducted with DESeq2.

Mass spectrometry analysis

Cultured Th17 cells in the presence or absence of CCL20 were harvested and washed with ice-cold PBS. Cells were lysed in 100 μL of Urea lysis buffer (7 M Urea, 2 M Thiourea, 2% CHAPS, 10 mM Tris) in the presence of nuclease-phosphatase-protease inhibitors. Cell lysates were sonicated (1 minute) and centrifuged at 20°C at 16,000xg for 20 minutes. The supernatant was transferred to a new 1.5 mL centrifuge tube and stored in 6X chilled acetone at -20 °C overnight. The next day, samples were thawed and vortexed for 1 minute, and the lysate was centrifuged (8,000g) at 4°C for 15 minutes. The supernatant was discarded, and the pellet was allowed to air-dry completely. Pellets were dissolved in 100 μL Urea rehydration buffer (7 M Urea, 2 M Thiourea, 50 mM Tris). Vortexed thoroughly and spun at 8000 g at 4°C for 3 minutes to remove air bubbles. Then, the samples were passed through an Amicon Ultra 3KD cut-off (MWCO) filter, and protein estimation was performed from the eluates. Proteins (50μg) were digested with Trypsin enzyme in 1:50 proportions [1 μg trypsin for 50 μg protein sample] by incubating the samples at 37°C in a water bath overnight. Samples were desalted using Zip-Tip desalting tips. Samples were dried using SpeedVac and dissolved in 10 μL of 0.1% formic acid. Samples were analyzed using Orbitrap mass spectrometry. Acquired data were analyzed using Proteome Discoverer software (ThermoScientific).

Glucose uptake assay using 2-NBDG

In vitro differentiated Th17 cells were stimulated with an anti-CD3: anti-CD28 activation cocktail, and cells were incubated in a glucose-free medium with 30 μM 2-NBDG (Invitrogen) at 37 °C for 30 minutes. After 30 minutes of incubation, cells were washed twice with ice-cold PBS, and the fluorescence intensity was measured using flow cytometry.

Lactate estimation from the culture supernatant

Naïve CD4⁺ T cells (CD4⁺CD25^-CD44^-) were cultured in Th17 lineage differentiation conditions in the presence or absence of CCL20 for four days. Lactate secreted in the culture supernatant was quantified using a glucose and lactate estimation kit (Spinreact, Spain), following the manufacturer’s guidelines.

Glyco-stress and Mitostress assay using SeaHorse instrument

Naïve CD4⁺ T cells (CD4⁺CD25^-CD44^-) were in vitro differentiated into Th17 cells in the presence or absence of CCL20 for four days. One day before the assay, the sensor cartridge was hydrated in 1mL of Seahorse XF Calibrant medium and incubated overnight at 37°C in a CO₂-free, humidified incubator. After four days of culture, the cells were harvested and washed with Seahorse XF DMEM medium (pH 7.4) supplemented with 2 mM glutamine. Cells (2 × 10⁵ cells per well) were seeded on 0.01% Poly L-Lysine-coated 24-well XF cell culture plates containing 500μl DMEM medium. The Glyco-stress of the above-cultured cells was measured as per protocol provided by Agilent Seahorse XF Glycolysis Stress Test Kit user’s manual using 10 mM glucose, followed by one μM Oligomycin, and 50 mM 2-DG. The extracellular acidification rate (ECAR) was measured using the Seahorse XFe24 analyzer (Agilent, CA, USA). For monitoring mitochondrial functions, Th17 cells cultured as above were harvested and transferred into Agilent XF base media (102353-100) according to the Agilent Seahorse XF cell mito-Stress Test Kit manufacturer’s guidelines. 0.5M Oligomycin, 0.75 M FCCP, and 0.5 M rotenone-antimycin A were used to measure oxygen consumption rate. Agilent's Seahorse XFe24 analyzer was used to determine the oxygen consumption rate (OCR).

Quantification and statistical analysis

All statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA). Statistically significant was defined as a p-value of less than 0.05 (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) in this study. An unpaired two-tailed Student’s t-test, One-way ANOVA followed by Tukey’s test, or Mann-Whitney test was used to compare two independent groups. Individual values were presented as bar plots with mean ± standard error of the mean (SEM). The figure legends included information on the number of replicates, donors, and mice.

Published: December 8, 2025