Androgen signaling restricts glutaminolysis to drive sex-specific Th17 metabolism in allergic airway inflammation

Abstract

Female individuals have an increased prevalence of many Th17 cell-mediated diseases, including asthma. Androgen signaling decreases Th17 cell-mediated airway inflammation, and Th17 cells rely on glutaminolysis. However, it remains unclear whether androgen receptor (AR) signaling modifies glutamine metabolism to suppress Th17 cell-mediated airway inflammation. We show that Th17 cells from male humans and mice had decreased glutaminolysis compared with female individuals, and that AR signaling attenuated Th17 cell mitochondrial respiration and glutaminolysis in mice. Using allergen-induced airway inflammation mouse models, we determined that females had a selective reliance upon glutaminolysis for Th17-mediated airway inflammation, and that AR signaling attenuated glutamine uptake in CD4+ T cells by reducing expression of glutamine transporters. In patients with asthma, circulating Th17 cells from men had minimal reliance upon glutamine uptake compared to Th17 cells from women. AR signaling thus attenuates glutaminolysis, demonstrating sex-specific metabolic regulation of Th17 cells with implications for Th17 or glutaminolysis targeted therapeutics.

Keywords: Asthma; Immunology; Pulmonology; Sex hormones; T cells.

Figure 1. CyTOF of lung-draining lymph nodes of deceased donor patients reveals sex differences in T cells and metabolic protein expression.

CyTOF was conducted on human lung draining lymph nodes from deidentified female and male deceased donors. (A) UMAP visualization of cell surface markers in all samples. (B–D) Median expression of metabolic markers on CD4⁺ T cells, Th1, Th2, Th17, and Treg cells. Gating strategies and population definitions are shown in Figure S1. Data are expressed as mean ± SEM: n = 14 females and n = 17 males *P < 0.05, **P < 0.01; 2-tailed Mann-Whitney U test. See Supplemental Figures 1 and 2 and Supplemental Tables 1 and 2.

Figure 2. AR signaling in CD4⁺ T cells reduces Th17-driven neutrophilic inflammation during airway inflammation.

(A) Model of HDM allergen challenge. Female and male Ar^fl and Cd4^Cre+ Ar ^fl mice were challenged intranasally with 40 μg of HDM 3 times per week for 3 weeks. BAL fluid and lungs were harvested 24 hours after the last challenge. (B) Quantification of eosinophils and neutrophils in the BAL fluid from mice after HDM challenge. (C) IL-13 and IL-17A protein expression in BAL fluid after HDM challenge. (D) Airway hyperresponsiveness as measured by FlexiVent with increasing doses of methacholine was also conducted on HDM-challenged female and male Ar^fl and Cd4^Cre+ Ar^fl mice 48 hours after last challenge (Data show mean ± SEM, n = 4–6 mice per group). (E) Representative flow diagrams of IL-13⁺ Th2 cells and IL-17A⁺ Th17 cells. (F) Quantification of lung Th2 and Th17 cells after HDM challenge (G and H) Expression of GLUD1, Glut1, HIF1-α, and pS6 in lung Th2 cells (G) and Th17 cells (H) after HDM challenge. (B, C, and E–H) Data are expressed as mean ± SEM: n = 7–9 mice per group combined from 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ANOVA with Tukey’s post hoc test. See Supplemental Figures 3, 4, and 5.

Figure 3. AR signaling modifies mitochondrial metabolism but not glycolysis in Th17 cells.

(A) Seahorse MitoStress Test on differentiated Th17 cells from WT female, WT male, and Ar^Tfm male mice to measure mitochondrial respiration using oxygen consumption rate (OCR) (n = 5 mice per group from 2 independent experiments). (B) Quantified measures of basal respiration, maximal respiration, and spare respiratory capacity from panel A. (C) Seahorse GlycoStress test on Th17 cells from WT female, WT male, and Ar^Tfm male mice to measure glycolysis using extracellular acidification rate (ECAR) (n = 5–6 mice per group from 2 independent experiments). (D) Quantified measures of basal glycolysis, glycolytic capacity, and glycolytic reserve from panel C. (E and F) Expression of MitoSOX Red, a mitochondrial superoxide marker, and Mitotracker green (MTG), a stain to measure mitochondrial mass, in differentiated Th17 cells from WT female, WT male, and Ar^Tfm male mice (n = 3–4 mice per group, representative of 2 independent experiments). Data show mean ± SEM, *P < 0.05, **P < 0.01; ANOVA with Tukey’s post hoc test. See Supplemental Figure 6 and 7.

Figure 4. AR signaling reduces glutamine metabolism in Th17 cells.

(A) Pathway analysis on targeted metabolomics from differentiated Th17 cells from WT male and Ar^Tfm male mice (n = 5–6 mice per group from 2 independent experiments, statistical analysis by MetaboAnalyst Global test, larger circle indicates larger impact, darker shade indicates increased significance). (B) Intracellular glutamine and glutamate levels from differentiated Th17 cells from WT male and Ar^Tfm male mice measured by mass spectrometry (Data show mean ± SEM, n = 5–6 mice per group). (C) Seahorse Substrate Oxidation Assay using CB-839, an inhibitor of glutaminase, on differentiated Th17 cells from WT male and Ar^Tfm male mice to measure dependance of mitochondrial respiration of Th17 cells on glutamine metabolism. (D) Quantified ΔΔOCR from C. ΔOCR was calculated by determining the difference in measured OCR after DMSO or CB-839 injection. ΔΔOCR was calculated as follows: ΔOCR_{CB-839 basal} – ΔOCR_{DMSO basal}. See Supplemental Figure 5A (E) Quantified from C and calculated as OCR_{CB-839 Max} – OCR_{DMSO Max}. *P < 0.05, unpaired 2-tailed t test. See Supplemental Figure 5 and Supplemental Table 3. (F and G) HDM-induced airway inflammation in female and sham or gonadectomized (GNX) male Gls^fl/fl and Cd4^Cre+ Gls^fl/fl mice were sensitized and challenged with HDM as described in Figure 2A. A day following the last challenge, BAL fluid and lungs were harvested. (F) Eosinophils and neutrophils in BAL fluid and (G) Lung Th2 and Th17 cells quantified by flow cytometry. Data show mean ± SEM, n = 3–4 mice per group, *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ANOVA with Tukey’s post hoc test. See Supplemental Figure 8 and 9 and Supplemental Table 3.

Figure 5. AR signaling reduces glutamine uptake in T cells.

(A) Model of targeted CRISPR screen using a glutamine library on differentiated Th17 cells from male and female OT-II Cas9 mice in an OVA-induced lung inflammation model. (B) Change in gRNA abundance in lung Th17 cells from male mice (y-axis) and GNX male mice (x-axis) with table showing statistics (Data show mean ± SEM, n = 4–5 mice, statistical analysis by MAGeCK affected represented by color of dot as shown in legend). (C) Model of F18-Glutamine PET studies in HDM-induced lung inflammation as shown in Figure 2A. (D) Quantification of ¹⁸F-Glutamine concentration in whole lung by PET/CT imaging (n = 4–6 mice per group). (E) Quantification of ¹⁸F-Glutamine concentration in lung CD4⁺ T cells by magnetic separation and γ counting, normalized to viable cells (Data show mean ± SEM, n = 4–6 mice per group). ***P < 0.001, unpaired 2-tailed t test. See Supplemental Figure 10 and 11.

Figure 6. AR signaling reduces glutamine uptake and TCA cycle intermediates.

Th17 cells were differentiated from WT male and Ar^Tfm male mice and pulsed with ¹³C₅-glutamine for 4 hours. ¹³C-labelled metabolites were measured by liquid chromatography-tandem mass spectrometry and normalized to total protein within the sample. Colored bars indicate the number of carbons labelled on each isotopologue for each metabolite. Data show mean ± SEM, n = 3–5 samples per group, *P < 0.05, Welch’s 2-tailed t test.

Figure 7. AR signaling alters H3K27 trimethylation in Th17 cells.

CUT & RUN analysis for H3K27me3 was conducted on Th17 cells differentiated from WT male, WT female, and Ar^Tfm male mice (n = 3 per group). (A) Heatmaps of H3K27me3 to IgG control ratio in ± 10 kb regions around promoters of UCSC known genes in Th17 cells measured by CUT&RUN. (B and C) Analysis of differentially methylated areas in Th17 cells from male and Ar^Tfm male mice (panel B) or Th17 cells from males and females (panel C). (D) GREAT analysis to find differences in gene ontology pathways in Th17 cells from WT male versus Ar^Tfm male mice. (E) GREAT analysis to find differences in H3K27me3 tagged genomic regions in Th17 cells from WT male versus Ar^Tfm male mice. (D and E) Pathways and genes shown had a P < 0.05. (F) qPCR analysis of Slc1a5 in Th17 cells. Data show mean ± SEM, n = 3–5 samples, ** P < 0.01, ANOVA with Tukey’s post hoc analysis. See Supplemental Figure 12.

Figure 8. Males with severe asthma have decreased dependence on glutamine uptake in circulating CD4⁺ T cell subsets compared with females with severe asthma.

(A) Model of SCENITH protocol on frozen PBMCs restimulated overnight with anti-CD3/CD28/CD2. (B–D) Glucose dependence, mitochondrial dependence, and glutamine uptake dependence in males and females measured using SCENITH inhibitors (2-deoxyglucose, oligomycin, and V9302, respectively) in (B) CD4⁺ TEMs (defined as Live, CD3⁺, CD4⁺, CD8^–, CD45RA^–, and CCR7^–), (C) CCR4⁺ CD4⁺ TEMs, and (D) TEM 17 cells (defined as B and CCR4⁺, CCR6⁺). Data show mean ± SEM, *P < 0.05, **P < 0.01, or P value as shown; Mann-Whitney U test. See Supplemental Figure 13 and Supplemental Table 4.