PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis

Abstract

T helper 9 (T_H9) cells promote allergic tissue inflammation and express the type 2 cytokines, IL-9 and IL-13, as well as the transcription factor, PPAR-γ. However, the functional role of PPAR-γ in human T_H9 cells remains unknown. Here, we demonstrate that PPAR-γ drives activation-induced glycolysis, which, in turn, promotes the expression of IL-9, but not IL-13, in an mTORC1-dependent manner. In vitro and ex vivo experiments show that the PPAR-γ-mTORC1-IL-9 pathway is active in T_H9 cells in human skin inflammation. Additionally, we find dynamic regulation of tissue glucose levels in acute allergic skin inflammation, suggesting that in situ glucose availability is linked to distinct immunological functions in vivo. Furthermore, paracrine IL-9 induces expression of the lactate transporter, MCT1, in T_H cells and promotes their aerobic glycolysis and proliferative capacity. Altogether, our findings uncover a hitherto unknown relationship between PPAR-γ-dependent glucose metabolism and pathogenic effector functions in human T_H9 cells.

Fig. 1. In vitro and in vivo primed T_H9 cells display key features of pathogenic T_H2 cells.

a Venn diagram of RNA-seq data from T_H cell subsets primed in vitro showing the number of genes significantly upregulated in T_H9 cells compared to other cell subsets (p_adj <0.05). b Venn diagram of T_H9-specific transcriptome identified in a and pT_H2-associated genes identified in eosinophilic esophagitis (EoE), allergic asthma, and allergen-specific T_H2 cells (T_H2A). c Expression of selected pT_H2-associated genes as determined in a. d–f Western blot analysis of PPAR-γ in different T_H cell subsets primed in vitro (d) and in T_H2 and T_H9 clones primed in vivo (e, f). g Changes in gene expression of selected pT_H2-associated genes. h In-sample correlations of T cell cytokines with IL9. The data are representative of independent experiments with three (a–c) or six (g, h) donors or five (f (T_H2)) or 10 (f (T_H9)) clones from two donors. Statistics: a differences between cell subsets were calculated as an adjusted log-fold change, and hypothesis testing was performed using the Benjamini–Hochberg adjusted p value (DESeq2). c One-way ANOVA, followed by a Dunnett’s test for multiple comparisons. f Two-tailed unpaired t test. h Simple linear regression. The data are presented as mean ± SD. Only p values <0.05 are shown.

Fig. 2. PPAR-γ mediates the high glycolytic activity of T_H9 cells.

a Intermediates and enzymes (red) of aerobic glycolysis. b RNA-seq of T_H9 clones incubated in presence of GW9662 for 48 h and activated by αCD3/CD2/CD28 for 12 h. c Maximal glycolytic capacity of in vitro primed T_H cells in the resting state and 24 h after activation with αCD3/CD2/CD28. d ECAR measurements of in vitro primed T_H9 cells cultured in media with glucose of different levels and GW9662 for 48 h and activated by injection of glucose and αCD3/CD2/CD28. e Maximal glycolytic capacity of in vitro primed T_H9 cells from d. f Glucose uptake by in vitro primed T_H cells measured with fluorescent 2-NBDG uptake by flow cytometry at day 7. g Glucose uptake by naive T_H cells primed under T_H9 conditions for 7 days in presence of GW9662 or transfected with PPARG and control siRNA, respectively. Efficiency of knockdown was determined by measuring PPARG levels after transfection by RT-qPCR (right). h Glucose uptake of in vivo primed effector memory T_H cells (T_EM) sorted by flow cytometry into T_H1, T_H2, and T_H9 cells according to their chemokine receptor profile. Sorted T_H cells were incubated in presence or absence of GW9662 for 48 h, and activated by αCD3/CD2/CD28 for 4 h. i Proliferation of in vitro primed T_H9 cells, activated by αCD3/CD2/CD28 for 4 days in presence or absence of GW9662 and glucose of different levels, measured with CFSE dilution by flow cytometry. The data are representative of one experiment with three clones from one donor (b) or independent experiments with three (d, g (left), i), four (c, f (T_H1), h), five (e, f (T_H2 and T_H9), g (right)) or six (g, right) donors. Statistics: b differences between treatment groups were calculated as an adjusted log-fold change, and hypothesis testing was performed using the Benjamini–Hochberg adjusted p value (DESeq2). c, f, h One-way ANOVA, followed by a Tukey’s test for multiple comparisons. g Two-tailed paired t test. e, i One-way ANOVA, followed by a Šidák’s test for multiple comparisons. The data are presented as mean ± SD. Only p values <0.05 are shown.

Fig. 3. High glycolytic activity of T_H9 cells regulates specific effector functions.

a Cytokine expression measured by flow cytometry of T_H9 cells primed in vitro in media containing glucose of different levels for 7 days. b Cytokine expression of in vivo primed T_H9 clones cultured for 72 h in media containing glucose of different levels, measured as in a. c In vitro primed T_H9 cells were activated for 4 h with αCD3/CD2/CD28, then sorted by flow cytometry based on their glucose uptake measured by 2-NBDG uptake (left). Cytokine expression in the sorted T_H cell populations was measured by RT-qPCR (right). d Cytokine expression of in vivo primed T_H9 cells cultured for 7 days in the presence of 2-DG, measured as in a. e Cytokine expression of in vitro primed T_H9 cells cultured for 48 h in media containing glucose of different levels and in presence or absence of GW9662, measured as in a. The data are representative of independent experiments with three (e), six (c), or seven (a) donors or fourteen clones from two donors (b, d). Statistics: a, c One-way ANOVA, followed by a Tukey’s test for multiple comparisons. b, e One-way ANOVA, followed by a Šidák’s test for multiple comparisons. d Two-tailed paired t test. The data are presented as mean ± SD. Only p values <0.05 are shown.

Fig. 4. mTORC1 integrates bioenergetics with effector function in T_H9 cells.

a Western blot analysis of pS6 in T_H9 cells primed in vitro. b In vitro primed T_H9 cells were cultured in glucose of different levels for 48 h, and pS6 and IL-9 were measured 18 h after activation with αCD3/CD2/CD28 by flow cytometry. The histogram shows pS6 positive cells, split into high and low pS6. Dot-plots represent IL-9⁺/IL-13⁺ clusters. c The IL-9⁺/IL-13⁺ ratio in T_H9 cells primed in vitro from b. d, e Cytokine expression of in vitro primed T_H9 cells after incubation in glucose of different levels and rapamycin for 48 h, measured as in b. f Immunofluorescence staining for CD4 and pS6 on skin samples of allergic contact dermatitis (ACD) and quantification of CD4⁺pS6⁺ cells in normal skin (NS) and ACD skin samples. Scale bars, 50 μM. g Cytokine expression of T cells isolated from ACD skin biopsies incubated with GW9662 for 48 h, measured as in b. h Immunofluorescence staining for pS6 and PPAR-γ on skin samples of ACD and quantification of PPAR-γ⁺pS6⁺ cells in NS and ACD skin samples. Scale bars, 50 μM. The data are representative of independent experiments with one (g), three (c), four (f (ACD), h (ACD)), five (f (NS), h (NS)), six (a), or nine (e) donors. Statistics: a, c, e One-way ANOVA, followed by a Šidák’s test for multiple comparisons. f, h Two-tailed unpaired t test. g Two-tailed paired t test. The data are presented as mean ± SD. Only p values <0.05 are shown.

Fig. 5. Paracrine IL-9 promotes aerobic glycolysis in IL-9R⁺ T_H cells by inducing the lactate transporter MCT1.

a IL-9R levels of in vivo primed T_H clones analyzed by flow cytometry. b IL-9R levels of PBMCs stained for their chemokine receptor profiles analyzed by flow cytometry. c Immunofluorescence staining for CD3 and IL-9R on ACD skin. Scale bars, 50 μM. d IL-9R levels of T cells isolated from ACD analyzed by flow cytometry. e–f RNA-seq of IL-9R⁺ T_H cells isolated from blood and ACD in presence of IL-9 shows (e) pathway analysis of the 250 most significant IL-9-induced genes and (f) changes in the expression of selected aerobic glycolysis genes. g Western blot analysis of MCT1 expression in IL-9R⁺ T_H clones incubated with IL-9 or IL-2 for 48 h. h SLC16A1 expression measured by RT-qPCR in IL-9R⁺ T_H clones in presence of JAK3 inhibitor (JAK3-i) ritlecitinib and IL-9 for 24 h. i RNA expression of SLC16A1 in in vitro primed T_H cells after 7 days. j, k Time course transcriptomic data shows RNA expression levels of SLC16A1 in i and correlation between IL9 and SLC16A1 expression in j. l ECAR measurements of in vivo primed IL-9R⁺ T_H clones incubated with IL-9 for 16 h. m IL-9R⁺ T_H clones incubated with the MCT1 inhibitor (MCT1-i) BAY-8002 or transfected with SLC16A1 siRNA. Extracellular (e.c.) lactate was measured with the Lactate-Glo^TM Assay (Promega) after 48 h in presence of IL-9. The data are representative of one experiment with one (c, l) donor or two (a (T_H17)), three (h), five (a (T_H1)) or twenty-two (a (T_H9)) clones from one donor or independent experiments with eight clones from one (g) or two (e, f) donors or nine clones from two donors (m (left)) or two (m (right)), three (i), five (b) six (j, k) or eleven (d) donors. Statistics: a Two-tailed unpaired t test. b, d, g, m (right) Two-tailed paired t test. e Fisher’s one-tailed test. f Differences between treatment groups were calculated as an adjusted log-fold change, and hypothesis testing was performed using the Benjamini–Hochberg adjusted p value (DESeq2). h, I One-way ANOVA, followed by a Dunnett’s test for multiple comparisons. k Simple linear regression. h, j, I, m One-way ANOVA, followed by a Tukey’s test for multiple comparisons. The data are presented as mean ± SD. Only p values <0.05 are shown.

Fig. 6. IL-9 promotes T-cell proliferation in the high-glucose environment of allergic contact dermatitis.

a Proliferation of in vivo primed IL-9R⁺ T_H clones in presence of IL-9 and different concentrations of IL-2, measured with CFSE dilution by flow cytometry after 3 days. b Proliferation of in vivo primed IL-9R⁺ T_H clones in presence of IL-9 and MCT1 inhibitor (MCT1-i) BAY-8002 measured as in a. c In vivo primed IL-9R⁺ T_H clones were cultured in media containing glucose of different levels for 7 days. Proliferation was measured in the presence of IL-9 with CFSE dilution by flow cytometry as in a. d Glucose concentrations measured with the Glucose-Glo^TM Assay (Promega) of interstitial fluids of lesional skin of positive patch test reactions to different allergens (Supplementary Table S3) 48 h post allergen application and adjacent non-lesional skin biopsies. e Schematic presentation of the main conclusions. The mTORC1-HIF-1α-IL-9 axis has previously been established by others^–. The data are representative of one experiment with four a or five c clones from one donor or independent experiments with eight clones from one donor b or independent experiments with six donors d. Statistics: a, c Two-way ANOVA, followed by a Šidák’s test for multiple comparisons. b One-way ANOVA, followed by a Tukey’s test for multiple comparisons. d Two-tailed paired t test. The data are presented as mean ± SD. Only p values <0.05 are shown.