Androgens Alleviate Allergic Airway Inflammation by Suppressing Cytokine Production in Th2 Cells Free

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Sex difference in immunity is well recognized. Females evoke stronger immune responses than males. Probably because of this, females have an increased prevalence of autoimmune diseases and allergy compared with males. However, numbers of patients with asthma exhibit a distinct tendency compared with other allergic diseases. In childhood, more boys suffer from asthma than girls, whereas numbers of female patients increase and reverse after puberty (1, 2). Therefore, it is considered that sex steroid hormones such as androgens and estrogens may cause a sex difference in asthma. It is reported that androgen receptor (AR), estrogen receptor (ER)α, and ERβ are expressed in immune cells (3, 4). In addition, macrophage-specific AR-deficient mice exhibit impaired M2 macrophage differentiation in allergic lung inflammation (5). In contrast, macrophage-specific ERα-deficient mice promote M2 macrophage differentiation (6). Similarly, AR-deficient mice enhance homeostasis of type 2 innate lymphoid cells (ILC2s) in the house dust mite (HDM)–induced asthma model (7). In addition, AR-deficient mice exhibit increased Th17 cells and enhanced lung inflammation in the HDM-induced asthma model (8). Notably, regulatory T (Treg) cell–specific AR-deficient mice show reduced airway inflammation in the Alternaria extract–induced asthma model (9). Thus, it is supposed that androgens suppress and estrogens enhance asthma. However, because sex hormones have pleiotropic effects on diverse cells in the body as well as immune cells, molecular mechanisms of how sex hormones affect the pathology of asthma remain unclear.

Asthma is chronic airway inflammation caused by an excessive response of Th2 cells against environmental Ags. Naive T cells activated by Ags differentiate into Th2 cells, which produce type 2 cytokines such as IL-4, IL-5, and IL-13. IL-4 helps B cells to differentiate into IgE-producing plasma cells, and IgE triggers an immediate allergic reaction after binding to the high-affinity IgE Fc receptor on mast cells. In parallel, IL-4 promotes the differentiation of Th2 cells (10). In contrast, IL-5 induces differentiation, recruitment, and activation of eosinophils (11). IL-13 elevates mucus production of goblet cells in bronchial epithelium. These pathways lead to airway inflammation and dyspnea by airway narrowing (12–14). After acute inflammation, some Th2 cells differentiate into memory Th2 cells and are maintained for a long time. When Ags are rechallenged, memory Th2 cells are rapidly activated and produce type 2 cytokines, exacerbating allergic airway inflammation. In the chronic phase, even weak stimuli such as dryness and temperature change cause airway narrowing because of airway hypersensitivity (15). However, how sex steroid hormones suppress Th2 cells is still unknown.

The dual specificity phosphatase (DUSP) family suppresses the MAPK and AP-1 pathways and reduces production of inflammatory cytokines. There are several DUSP family members with effects on T cells (16). DUSP-2 is highly expressed in T cell–rich tissues such as thymus, spleen, and lymph nodes. In addition, DUSP-2 expression is dramatically elevated in naive CD4 T cells after TCR stimulation (17). Glucocorticoids are commonly used in the treatment of asthma (18), and glucocorticoid receptor (GR) signaling induces expression of DUSP-1, which inactivates ERK1/2 and AP-1 by dephosphorylation (19). In addition, DUSP-10 reportedly suppresses type 2 cytokine production in memory Th2 cells during allergic airway inflammation by dephosphorylating p38 (20). Because GR, AR, and ER are similar in structure and share some consensus sequences in their target gene response elements, they regulate a subset of common genes (21, 22). Thus, steroid hormones other than glucocorticoids might also suppress cytokine expression via the DUSP–p38 pathway. However, whether sex steroid hormones suppress cytokine expression of Th2 cells via DUSP is still unknown.

In this study, to address whether sex steroid hormones have any effects on T cells in allergic airway inflammation, we analyzed T cell–specific AR- and ERα/β-deficient mice. We found that androgens, but not estrogens, alleviate allergic airway inflammation by inhibiting differentiation and effector function of Th2 cells. AR signaling in T cells suppresses expression of Th2 cytokines by inducing DUSP-2 expression. Thus, this study might explain, at least partly, the decrease in prevalence of asthma in males after adolescence.

C57BL/6 mice (CD45.2) were purchased from Japan SLC. B6.CD45.1 congenic mice were maintained in our laboratory. CD4-Cre transgenic (Tg) (23), AR^flox/y (24), ERα^flox/flox (25), ERβ^flox/flox (26), OT-II TCR Tg (27), and Rag2^−/− mice on a C57BL/6 background were used. CD4-Cre Tg⁺ AR^flox/y and CD4-Cre Tg⁺ ERα^flox/flox ERβ^flox/flox mice were used as T cell–specific AR and ER conditional knockout mice, respectively. AR^flox/y and ERα^flox/flox ERβ^flox/flox mice were used as controls. Eight- to 12-wk-old mice were analyzed. All mice were maintained under specific pathogen-free conditions in the Experimental Research Center for Infectious Diseases in the Institute for Life and Medical Sciences, Kyoto University. All mouse protocols were approved by the Animal Experimentation Committee of the Institute for Life and Medical Sciences, Kyoto University.

Lymphocytes were isolated from lung as described previously (28). Briefly, lung was cut into small fragments and digested for 40 min at 37°C with 1 mg/ml collagenase D (Roche) and 100 µg/ml DNase (Worthington Biochemical). Digests were filtered through a 40-µm cell strainer and centrifuged at 1200 rpm for 7 min. The cell pellet was suspended in 30% Percoll and centrifuged at 620 × g for 20 min to deplete epithelial cells. RBCs were lysed by ACK (ammonium, chloride, potassium) buffer. Lymph node cells were obtained from submandibular, axillary, brachial, inguinal, mesenteric, and pelvic lymph nodes. Naive CD4 T cells were isolated from lymph nodes using a MagniSort mouse CD4 naive T cell enrichment kit (Thermo Fisher Scientific).

The following fluorescent dye– or biotin-conjugated Abs against the indicated proteins were used: TCRβ (H57-597), CD4 (GK1.5), Foxp3 (3G3), Gr-1 (RB6-8C5), CD11b (M1/70), Siglec-F (E50-2440), CD11c (N418), Ki67 (SolA15), IFN-γ (XMG1.2), IL-4 (11B11), IL-13 (eBio13A), IL-17A (TC11-18H10.1), IL-5 (TRFK5), IL-10 (JES5-16E3), CD45.1 (A20), CD45.2 (104), mouse IgG1κ isotype control (MOPC-21), ERK1/2 pT202/pY204 (20A), and p38 MAPK pT180/pY182 (36/p38). Fluorescent dye– or biotin-conjugated Abs were purchased from Thermo Fisher Scientific, BD Biosciences, BioLegend, and Tonbo Biosciences. Stained cells were analyzed on FACSVerse flow cytometer (BD Biosciences) using FlowJo software (BD Biosciences). Debris and dead cells were excluded from analysis by forward and side scatter and propidium iodide gating.

For intracellular staining of cytokines, T cells were stimulated with 50 ng/ml PMA and 2 μg/ml ionomycin for 4 h in the presence of 10 μg/ml brefeldin A. Then, the cells were stained for surface Ags, fixed, permeabilized, and stained with the relevant anti-cytokine Abs using IC (intracellular) fixation buffer (eBioscience). For intracellular staining of Ki67, T cells were stained for surface Ags, fixed, permeabilized, and stained with anti-Ki67 Ab using a Foxp3 staining buffer set (eBioscience). For intracellular staining of p-p38 and p-ERK1/2, the cells were fixed using a Foxp3 staining buffer set, permeabilized in ice-cold methanol, and stained with the relevant Abs.

For Th2 differentiation, naive CD4 T cells were cultured with 10 μg/ml plate-bound anti-CD3 Ab (145-2C11, Bio X Cell) and 8 μg/ml soluble anti-CD28 Ab (37.51, Bio X Cell), 20 ng/ml human IL-2 (BioLegend), 10 ng/ml mouse IL-4 (BioLegend), 5 ng/ml anti–IFN-γ Ab in RPMI 1640 medium containing 10% FBS, 50 μM 2-ME, and 10 mM HEPES (pH 7.4).

In some experiments, the cells after 4 d of the Th2 culture were stimulated with 10 nM dihydrotestosterone (DHT) (Sigma-Aldrich) or DMSO as a vehicle control for 48 h. The cells were then stimulated with PMA and ionomycin for 4 h in the presence of brefeldin A, fixed, stained with Abs, and analyzed by flow cytometry.

Mice were sensitized i.p. with 10 μg of HDM (ITEA) and Imject Alum (Thermo Scientific) at days 0 and 14 and challenged intranasally (i.n.) with 10 μg of HDM in 20 μl of PBS at days 21, 22, and 23. The mice were dissected at day 26, and bronchoalveolar lavage fluid (BALF) was recovered with 800 μl of PBS.

The concentrations of IL-4, IL-5, and IL-13 in BALF were assessed by a IL-4 mouse uncoated ELISA kit (Invitrogen), IL-5 mouse uncoated ELISA kit (Invitrogen), and mouse IL-13 ELISA Ready-SET-Go! (eBioscience), respectively. IgE in serum was measured by an IgE mouse uncoated ELISA kit (Invitrogen).

Lungs were dissected after perfusion with 50 ml of PBS. Tissues were fixed in 10% formalin for 3 h, stored in 70% ethanol, and embedded in paraffin. Sections with a thickness of 5 μm were stained with H&E. Images were acquired on a KEYENCE Biozero BZ-8000 microscope. Images of 32–80 bronchiolar regions were examined per each mouse per each examiner, and six mice per each group were examined. Histological severity of inflammation was scored by two independent examiners. Cell infiltration was scored on a scale of 0–3 according to a previous report (5).

Mice were anesthetized with i.p. injection of pentobarbital sodium (100 μg/kg), tracheostomized, and cannulated with an 18G steel needle in the supine position. Then, the mice were connected to flexiVent (SCIREQ, Montreal, QC, Canada) and ventilated with a tidal volume of 8 ml/kg at a frequency of 150 breaths/min. Thereafter, airway resistance was determined using SnapShot perturbation at baseline and after administration of nebulized methacholine by AeroNeb (Aerogen, Galway, Ireland) (8, 29).

Naive CD4 T cells from CD4-Cre AR^flox/y and AR^flox/y, or CD4-Cre AR^flox/y OT-II Tg⁺ Rag2^−/− and AR^flox/y OT-II Tg⁺ Rag2^−/− male mice (CD45.2) were cultured in the Th2 differentiation condition for 6 d. Cultured Th2 cells (3 × 10⁶ cells) were i.v. transferred into wild-type (WT) male mice (CD45.1). The mice were i.n. challenged with 40 μg of OVA in 20 μl of PBS at days 36, 38, 42, and 44. The mice were analyzed at day 34 or 47.

Naive CD4 T cells of CD4-Cre AR^flox/y and AR^flox/y male mice were cultured in the Th2 condition and stimulated with DHT as described above. Th2 cells were lysed with Buffer LTR (Qiagen), and RNA was purified with an RNeasy micro kit (Qiagen). Double-stranded cDNA was synthesized using a SMART-seq HT kit for sequencing (Takara), and sequencing libraries were constructed with a Nextera XT DNA library preparation kit (Illumina) according to the manufacturers’ instructions. The libraries were sequenced with 150-bp paired-end reads on an Illumina HiSeq X Ten sequencer (Illumina). To exclude low-quality sequences, raw sequenced reads were filtered by Trimmomatic (version 0.33) (30). The filtered sequences were aligned to the mouse reference genome (mm10) with HISAT2 (version 2.1.0) (31), and the aligned reads were used for the transcript quantification by using featureCounts (version 1.6.5) (32). edgeR (version 3.28.1) (33) was used to normalize and identify differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed with the ShinyGO Web analysis tool (34). Next-generation sequencing data generated in this study were deposited in the Gene Expression Omnibus under accession number GSE200840 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE200840).

Real-time RT-PCR was carried out on a StepOnePlus real-time PCR system (Thermo Fisher Scientific) as described previously (35). PCR results were normalized to corresponding levels of Hprt mRNA in cDNA from whole thymocytes from WT mice. Primer sequence are as follows: Dusp2, 5′-GAAGATAACCAGATGGTGGAGATAA-3′ and 5′-CCCCACTATTCTTCACCGAGT-3′; Dusp1, 5′-GTGCCTGACAGTGCAGAATC-3′ and 5′-CACTGCCCAGGTACAGGAAG-3′; Dusp10, 5′-GGGCTACGCTTATTGATGAAAC-3′ and 5′-CCTGTCGTCTAAAGGAGATGGA-3′; and Hprt, 5′-GTTGGATACAGGCCAGACTTTGTTG-3′ and 5′-GATTCAACTTGCGCTCATCTTAGGC-3′.

The mouse Dusp2 promoter plus a 5′ untranslated region (UTR) (852 bp) were subcloned by PCR into pGL4.10[Luc2] plasmid (Promega). Mouse AR cDNA was subcloned by PCR into pcDNA3 plasmid. Primer sequences are as follows: Dusp2 promotor, 5′-TCGGTACCTGGGAGAGCCCAGCATCGTCAA-3′ and 5′-AGAAGCTTAAAGCCTCCTGCTTCCGGATGT-3′; and AR cDNA, 5′-CAGGATCCAAGCTCAAGGATGG-3′ and 5′-TAAGCTCGAGGTTTCCAAATCTTCA-3′ (underlined nucleotides indicate restriction enzyme recognition sites for subcloning). Jurkat cells (4 × 10⁵) were transiently transfected with 100 ng of pGL4.74[hRluc/TK] internal control plasmid (Promega), 200 ng of AR/pcDNA3 expression vector, and 200 ng of Dusp2 promoter/pGL4.10[Luc2] reporter vector using ViaFect transfection reagent (Promega). A reporter assay was performed 24 h after transfection. Cell lysate was then subjected to the Dual-Luciferase reporter assay system (Promega), and luciferase activity was measured using a luminometer (ARVO X3; PerkinElmer). Firefly luciferase activity was normalized to Renilla luciferase activity. In each experiment, samples were analyzed in duplicate, and each experiment was repeated at least three times.

Naive CD4 T cells (5 × 10⁶) were incubated for 2 h in RPMI 1640 medium containing 10% FBS, 50 μM 2-ME, and 10 nM DHT. A chromatin immunoprecipitation (ChIP) assay was done as described previously (36). Purified chromatin was immunoprecipitated with 10 μg of anti-AR Ab (441, Santa Cruz Biotechnology) or isotype control IgG1κ. Purified ChIP and input DNA were measured by real-time PCR. DNA from thymocytes served as a calibration control. ChIP DNA levels were normalized to those of input DNA. Primer sequences were as follows: Dusp2 promoter ChIP, 5′-TAAGCCCGGGCTCGACGAAG-3′ and 5′-AGCCTCCTGCTTCCGGATGT-3′.

Mouse Dusp2 cDNA was subcloned by PCR into MSCV MIGR1 retrovirus vector (37). Primer sequences are as follows: Dusp2 virus, 5′-GCAGGATCCCAGGAGGCTTTGCGATGCC-3′ and 5′-GTCTAGAATTCGGCTGGGCTTTGCCTCA-3′ (underlined sequences are restriction enzyme recognition sites used for subcloning).

Naive CD4 T cells were cultured in the Th2 differentiation condition for 4 d as describe above. MSCV-Dusp2 vector or empty vector was transfected into PLAT-E packaging cells (a gift of Dr. Toshio Kitamura, University of Tokyo) by the TransIT-X2 dynamic delivery system (Takara). Two days after transfection, supernatant was collected and centrifuged at 8000 × g for 16 h at 4°C. The pellet was resuspended in 500 μl of RPMI 1640 medium containing 10% FBS and 20 ng/ml human IL-2. At day 4 of the Th2 culture, CD4 T cells were infected with the concentrated retrovirus in the presence of 100 μg/ml RetroNectin T100 (Takara). After 48 h, the medium was removed and exchanged to RPMI 1640 medium containing 10% FBS and 20 ng/ml human IL-2. At 3 d postinfection, EGFP⁺ cells were analyzed for cytokine expression by flow cytometry. Frequencies of EGFP⁺ cells were 4.4–10.7% for empty retrovirus vector and 5.4–10.2% for Dusp2 retrovirus vector.

Each experiment was carried out at least three independent times. All data are presented as means ± SEM. Comparisons between two samples were performed using the unpaired two-tailed Student t test. Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA).

It is reported that female BALB/c mice exhibit severer OVA-induced allergic airway inflammation than do male BALB/c mice (38). To test whether a sex difference is observed in allergic airway inflammation induced with HDM, we injected WT C57BL/6J mice i.p. with HDM and alum on days 0 and 14, challenged i.n. the mice with HDM on days 21, 22, and 23, and analyzed the mice by flow cytometry on day 26 (Fig. 1A). Cell subpopulations in BALF and lung parenchyma were identified as eosinophils (Gr-1⁻CD11c^intSiglec-F⁺), macrophages (Gr-1⁻CD11c^highSiglec-F⁺), neutrophils (Gr-1⁺CD11b⁺), and CD4 T cells (TCRβ⁺CD4⁺ or TCRβ⁺CD4⁺CD8⁻) (Fig. 1B). The total number of BALF cells was significantly elevated in female mice compared with male mice (Fig. 1C). Numbers of eosinophils and CD4 T cells were significantly elevated in female mice, whereas numbers of macrophages and neutrophils were unchanged. In lung parenchyma, numbers of total cells and CD4 T cells were elevated in female mice, whereas the number of Treg cells (TCRβ⁺CD4⁺Foxp3⁺) was unchanged (Fig. 1D). Next, we measured serum IgE by ELISA. The serum level of IgE was higher in female mice than in male mice (Fig. 1E). Taken together, these results indicate that female mice develop severer allergic airway inflammation than do male mice.

Because CD4 T cells, but not Treg cells, were elevated in the lungs of female mice compared with male mice (Fig. 1D), we next investigated whether sex steroid hormones have a role in T cells during allergic airway inflammation. To test this hypothesis, we employed T cell–specific AR-deficient (CD4-Cre AR^flox/y) mice. First, to exclude the possibility that T cell–specific AR deficiency has some effects on development and maintenance of T cells, we analyzed AR-deficient male mice in the steady state. There was no difference in numbers of naive and effector CD4 T cells, Treg cells, and CD8 T cells in lung, lymph nodes, and spleen between control and T cell–specific AR-deficient male mice (Supplemental Fig. 1). Additionally, the frequency of Ki67⁺ CD4 T cells was also unchanged, suggesting that proliferation of CD4 T cells may be unaffected by AR deficiency.

Next, we addressed whether T cell–specific AR deficiency has some impact on allergic airway inflammation. T cell–specific AR-deficient male mice were sensitized with HDM and alum, challenged intranasally with HDM, and analyzed by flow cytometry (Fig. 1A). The total number of BALF cells was significantly elevated in T cell–specific AR-deficient male mice compared with control male mice (Fig. 2A). Especially, cell numbers of eosinophils, neutrophils, and CD4 T cells were significantly elevated in the BALF of AR-deficient male mice, whereas the number of macrophages was reduced. Similarly, numbers of total cells and eosinophils were elevated in the BALF of T cell–specific AR-deficient female mice, whereas neutrophils and CD4 T cells only showed a tendency to increase (Supplemental Fig. 2A). In lung parenchyma, the numbers of total cells, CD4 T cells, and Treg cells were elevated in T cell–specific AR-deficient male mice (Fig. 2B). In contrast, T cell–specific AR-deficient female mice did not show any difference in cell numbers of each population in lung parenchyma (Supplemental Fig. 2B). Because the numbers of lung CD4 T cells were elevated in T cell–specific AR-deficient male mice, we next analyzed the proliferation marker Ki67. Frequencies of Ki67⁺ cells in CD4 T cells increased in T cell–specific AR-deficient male mice compared with control male mice (Fig. 2C, 2D). Therefore, androgens might suppress proliferation of CD4 T cells in allergic airway inflammation.

In asthma, type 2 cytokines such as IL-4, IL-5, and IL-13 enhance allergic inflammation. Thus, we next examined production of type 2 cytokines in BALF of AR-deficient male mice. Levels of IL-4, IL-5, and IL-13 were elevated in BALF of AR-deficient male mice compared with control male mice (Fig. 2E). Furthermore, frequencies of type 2 cytokine-producing CD4 T cells, as well as IL-17–producing CD4 T cells, were significantly elevated in lung parenchyma of T cell–specific AR-deficient male mice (Fig. 2F, 2G). Consistent with the elevated Th2 cytokine production, the serum level of IgE was elevated in AR-deficient male mice (Fig. 2H). In contrast, frequencies of type 2 cytokine-producing CD4 T cells were unchanged in lung parenchyma of T cell–specific AR-deficient female mice compared with control female mice (Supplemental Fig. 2C).

To test whether the allergic airway inflammation causes any functional insufficiency in lung, we next measured airway hyperresponsiveness (AHR) with methacholine. Mice were administered methacholine under ventilation, and airway resistance was measured. Methacholine-induced AHR was significantly elevated in T cell–specific AR-deficient male mice compared with control male mice (Fig. 2I). The data suggest that androgens suppress airway hyperresponsiveness in male mice via T cells. Next, we analyzed pathological changes in lungs of T cell–specific AR-deficient male mice. Histological analysis revealed that T cell–specific AR-deficient male mice exhibited more cell infiltration into the peribronchiolar region of the lung (Fig. 2J, 2K). Taken together, these results suggest that T cell–specific AR deficiency exacerbates allergic airway inflammation in male mice.

Next, to test whether estrogens have a role in T cells during allergic airway inflammation, we analyzed T cell–specific ER-deficient (CD4-Cre ERα^flox/flox ERβ^flox/flox) mice. Mice were sensitized with HDM and alum, challenged i.n. with HDM, and analyzed by flow cytometry (Fig. 1A). Total cell number and numbers of eosinophils, macrophages, neutrophils, and CD4 T cells were unchanged in BALF of T cell–specific ER-deficient female mice compared with control female mice (Fig. 3A). Similarly, total cell number and numbers of CD4 T cells and Treg cells were unchanged in lung parenchyma of ER-deficient female mice (Fig. 3B). Furthermore, frequencies of type 2 cytokine-producing CD4 T cells were unchanged in lung parenchyma of ER-deficient female mice (Fig. 3C). Thus, these results suggest that T cells are hardly affected by estrogens in allergic airway inflammation.

Because the production of type 2 cytokines was elevated in T cell–specific AR-deficient male mice during allergic airway inflammation (Fig. 2E–G), we hypothesized that androgens might suppress differentiation and cytokine production of Th2 cells. To test this hypothesis, we cultured naive CD4 T cells from AR-deficient male mice in the Th2 cell differentiation condition with DHT or vehicle control (Fig. 4A). At day 6 of culture, the cells were stimulated with PMA and ionomycin and analyzed by flow cytometry. Of note, frequencies of IL-4⁺ and IL-13⁺ cells were elevated more in AR-deficient cells without DHT than in control cells (Fig. 4B, 4C). Furthermore, frequencies of IL-4⁺ and IL-13⁺ cells were downregulated in control cells, but not in AR-deficient cells, with DHT stimulation. Because GATA3 is the master transcription factor of Th2 cells (39), we next analyzed GATA3 expression. CD4 T cells differentiated in the Th2 condition exhibited GATA3 expression as a single peak, suggesting that most of the cells were under the differentiation into Th2 cells (Fig. 4D). The level of GATA3 expression was unchanged in AR-deficient Th2 cells compared with control cells (Fig. 4E). Consistently, stimulation with DHT did not alter GATA3 expression in control Th2 cells, nor in AR-deficient Th2 cells. Collectively, these results suggest that androgens appear to suppress cytokine production of Th2 cells, but not differentiation from naive CD4 T cells.

Because AR signaling enhances the suppressive function of Treg cells during allergic airway inflammation (9), it is important to analyze the effects of androgens on memory Th2 cells. To investigate whether androgens affect the maintenance and/or response of memory Th2 cells, we carried out adoptive transfer of memory Th2 cells and induced airway inflammation (Fig. 5A). First, naive CD4 T cells of CD4-Cre AR^flox/y and control mice (CD45.2) were cultured in the Th2 condition for 6 d and transferred i.v. into WT male mice (CD45.1). We detected memory Th2 cells as CD45.2⁺TCRβ⁺CD4⁺ T cells, which were maintained for 4 wk. Before challenging the mice, we checked colonization of the transferred Th2 cells (CD45.2⁺TCRβ⁺CD4⁺) on day 34. The numbers of AR-deficient Th2 cells were unchanged in spleen and lung compared with control Th2 cells (Fig. 5B). Moreover, frequencies of IL-4⁺, IL-13⁺, and IFN-γ⁺ cells in AR-deficient Th2 cells in lung were similar to those in control Th2 cells (Fig. 5C). These results suggest that maintenance of memory Th2 cells is not impaired by AR deficiency.

Next, to investigate whether androgens affect the response of memory Th2 cells, naive CD4 T cells of CD4-Cre AR^flox/y Rag2^−/− OT-II Tg⁺ male mice (CD45.2) were cultured in the Th2 condition and transferred into WT male mice (CD45.1). On days 35, 38, 42, and 44, we challenged the mice with OVA and analyzed them by flow cytometry (Fig. 5D). In the mice transferred with AR-deficient OT-II T cells, the total numbers of BALF cells were elevated compared with the mice transferred with control OT-II T cells (Fig. 5E, 5F). The numbers of eosinophils increased, whereas the numbers of macrophages, neutrophils, and CD4 T cells were unchanged. In lung parenchyma, the numbers of transferred AR-deficient OT-II T cells were elevated in lung compared with control OT-II T cells (Fig. 5G). Consistently, Ki67 was expressed at higher levels in the transferred AR-deficient OT-II Th2 cells compared with control Th2 cells (Fig. 5H). Furthermore, frequency of IL-5⁺ cells in the transferred AR-deficient OT-II T cells was slightly elevated in lung parenchyma compared with control OT-II T cells, whereas frequencies of IL-4⁺, IL-13⁺, and IL-10⁺ cells were unchanged (Fig. 5I). In addition, concentrations of Th2 cytokines in BALF and serum IgE level were unchanged (Fig. 5J, 5K). Together, these results suggest that androgens might suppress proliferation and IL-5 production of memory Th2 cells.

To investigate the molecular mechanism suppressing proliferation and type 2 cytokine production of Th2 cells by androgens, we performed RNA-seq analysis of AR-deficient Th2 cells. Naive CD4 T cells of CD4-Cre AR^flox/y and AR^flox/y male mice were cultured in the Th2 condition and stimulated with DHT. In AR-deficient Th2 cells, 651 genes were upregulated and 311 genes were downregulated (Fig. 6A). Notably, KEGG pathway analysis showed that the pathways related with cell cycle, metabolism (metabolic pathways), Th1 and Th2 cell differentiation, and Th17 cell differentiation were enriched in DEGs (Fig. 6B). Cell cycle–related genes such as Cdk1 (CDK1), Ccna1 (cyclin A1), and Ccnd3 (cyclin D3) were upregulated in AR-deficient Th2 cells. These results suggested that cell proliferation is accelerated in AR-deficient Th2 cells and that androgens inhibit the expansion of Th2 cells. As for the pathways related with Th1 and Th2 cell differentiation and Th17 cell differentiation, a heatmap of individual genes is shown (Fig. 6C). Gata3 (GATA3), Ppp3ca (calcineurin Aα), and Rbpj (RBP-J) were upregulated in AR-deficient Th2 cells. Gata3 is the master transcription factor of Th2 cells (39). Ppp3ca enhances the transcription of the IL-2 gene in T cells (40). Rbpj transmits the Notch signal and promotes survival of memory CD4 T cells (41). These results suggested that T cell activation, and thereby differentiation into Th2 cells as well, is enhanced in AR-deficient Th2 cells and that androgens suppress the differentiation into Th2 cells. In the TCR signaling–related pathway, Mapk3 (ERK1/2) and Mapk11 (p38) were upregulated in AR-deficient Th2 cells (Fig. 6C). To investigate whether AR eventually has some effects on activation of ERK1/2 and p38, we assessed their phosphorylation in control and AR-deficient Th2 cells by flow cytometry. Control Th2 cells slightly downregulated phosphorylated p38 by DHT stimulation, whereas AR-deficient Th2 cells did not, suggesting that androgens suppress the phosphorylation of p38 (Fig. 6D, 6E). In contrast, DHT stimulation reduced phosphorylated ERK1/2 similarly in control and AR-deficient Th2 cells, suggesting that DHT might have some effects independent of AR. These results suggest that AR signaling induces Dusp2 expression, which may suppress the phosphorylation of p38 and the production of type 2 cytokines.

Because the DUSP family dephosphorylates MAPK in T cells (16) and Dusp2 was downregulated in AR-deficient Th2 cells (Fig. 6C), we hypothesized that DUSP-2 might negatively regulate p38 signaling in Th2 cells. First, to assess DUSP-2 expression in Th2 cells, we performed quantitative RT-PCR analysis of Dusp2 mRNA in AR-deficient and control Th2 cells stimulated with DHT or vehicle. Dusp2 transcripts were elevated by DHT stimulation in control Th2 cells but not in AR-deficient Th2 cells (Fig. 7A). Because DUSP-1 and DUSP-10 are expressed in T cells (17), we also measured transcripts of Dusp1 and Dusp10. Transcripts of Dusp1 and Dusp10 were unchanged between control and AR-deficient Th2 cells by DHT stimulation (Supplemental Fig. 3A). Furthermore, we compared transcripts of Dusp2, Dusp1, and Dusp10 in naive CD4 T cells isolated from male and female WT mice. The level of Dusp2 transcripts was slightly higher in male T cells than in female T cells, whereas the expression of Dusp1 and Dusp10 was unchanged (Supplemental Fig. 3B). These results suggest that androgens induce Dusp2 expression in Th2 cells.

To identify the mechanism of Dusp2 induction by androgens, we searched androgen response elements (AREs) in the Dusp2 gene locus. We identified an atypical ARE in the 5′ UTR of the Dusp2 gene (Fig. 7B). To test whether AR binds to the ARE, we performed a ChIP assay with naive CD4 T cells of T cell–specific AR-deficient and control male mice stimulated with DHT. Specific AR binding to the 5′ UTR was detected in control, but not AR-deficient, CD4 T cells (Fig. 7C). Next, to test whether the AR transactivates the Dusp2 promoter through the ARE, we performed a luciferase reporter assay with Jurkat cells by reporter plasmids containing WT and mutated ARE (Fig. 7D). The luciferase activity was unchanged between WT and mutant vectors without DHT stimulation, whereas the activity was elevated with WT but not mutant vector by DHT stimulation (Fig. 7E). In addition, the promoter activity was enhanced with WT but not mutant reporter in a dose-dependent manner of the AR expression vector (Fig. 7F). Thus, these results suggest that the Dusp2 promoter activity is enhanced by AR binding to the ARE.

Finally, to test whether DUSP-2 has the potential to suppress the production of type 2 cytokines and proliferation, we overexpressed DUSP-2 in Th2 cells by retrovirus vector. The frequency of IL-4⁺ Th2 cells in EGFP⁺ cells was slightly reduced by enforced expression of Dusp2 (Fig. 7G). Furthermore, the frequency of Ki67⁺ Th2 cells was reduced by DUSP-2 overexpression (Fig. 7H, 7I). Thus, these results suggest that DUSP-2 suppresses IL-4 expression and the proliferation of Th2 cells.

It is well recognized that there is sex bias in the prevalence of asthma. It is reported that androgens suppress asthma (42), whereas estrogens enhance asthma (43). Our study confirmed that female mice develop severer asthma than do male mice in allergic airway inflammation (Fig. 1). We also demonstrated that androgen but not estrogen signaling in T cells is important for the sex bias in asthma (Figs. 2, 3). Previous studies reported the roles of androgens in the pathogenesis of asthma either by castration, systemic AR-deficient mice, or macrophage-specific AR-deficient mice. To our knowledge, this study is the first to demonstrate the direct effects of androgens on T cells in allergic airway inflammation by using T cell–specific AR-deficient mice. We found that androgens, but not estrogens, suppress cytokine production of Th2 cells. However, because the prevalence of asthma in females gradually increases after puberty, it is possible that estrogens also have some positive effects on the development of asthma. Pulmonary function generally deteriorates at the late follicular phase of the menstrual cycle, when estrogen levels become the highest (43). Similarly, the use of oral contraceptives, which contain estrogens, reportedly exacerbates the symptoms of asthma (43). Notably, ERβ is highly expressed in the lung and ovary, and ERβ-deficient mice exhibit pulmonary dysfunction (44). Thus, it might be that androgens and estrogens have different mechanisms of action in asthma. Androgens seem to regulate T cells, whereas estrogens might control stromal cells. It will be a future question whether estrogens modulate asthma by T cell–independent mechanisms.

It is reported that AR signaling enhances suppressive function of Treg cells in a cell-intrinsic manner (9). Treg cell–specific AR-deficient mice exhibit enhanced allergic airway inflammation by the Alternaria extract–induced asthma model. Thus, it is certain that the suppressive function of Treg cells is impaired in CD4-Cre AR^flox/y mice, which will exacerbate allergic airway inflammation. We showed that cytokine production is impaired in AR-deficient Th2 cells compared with control Th2 cells (Fig. 4). Furthermore, AR-deficient OT-II Th2 cells transferred into WT mice aggravated allergic airway inflammation (Fig. 5). Thus, these results demonstrate that AR signaling in Th2 cells reduces cytokine production of Th2 cells, which contributes to mitigate allergic airway inflammation. Because the frequency of IL-17–producing CD4 T cells was elevated in CD4-Cre AR^flox/y mice in airway inflammation (Fig. 2G), and AR-deficient mice exhibit increased Th17 cells and enhanced lung inflammation (8), AR signaling might also reduce IL-17 production in Th17 cells. Thus, it is possible that androgens might have a wide range of effects on Th2, Th17, and Treg cells, which in total enhances allergic airway inflammation.

In this study, we employed the priming protocol by i.p. injection of allergen and alum. This sensitization method elicits Th2 cell–mediated allergic airway inflammation with elevated IgE and infiltration of eosinophils in the lung (5, 6). However, it is rather a controversial model for asthma to use the adjuvant for priming the mice. In recent studies, repetitive i.n. administration of allergen also induces ILC2- or Th2 cell–mediated allergic airway inflammations (7, 8). Although both protocols can induce typical allergic airway inflammation mediated by Th2 cells, there is a need to evaluate the impact of AR in alum-free models in the future.

It is reported that androgens negatively regulate the development and maintenance of ILC2s at steady state and that AR signaling in ILC2s alleviates HDM-induced allergic airway inflammation (7). These results suggested that androgens suppress the type 2 inflammation by ILC2s. Thus, taking previous studies and our studies into account, androgens appear to suppress both innate and adaptive type 2 immune responses. Because ILC2s and Th2 cells have common features in development (e.g., GATA3 dependency) and function (e.g., type 2 cytokine expression), it is possible that a similar mechanism of suppression by androgens might operate in ILC2s and Th2 cells. It will be an interesting question in the future whether androgens also suppress effector function of ILC2s via the DUSP–p38 pathway in allergic airway inflammation.

The DUSP family members dephosphorylate JNK, p38, and ERK and inhibit their functions. Deficiency in DUSP members is associated with some autoimmune diseases (16). DUSP-2 also dephosphorylates STAT3 and thereby inhibits differentiation of Th17 cells (17) and expression of proinflammatory factors in macrophages (45). DUSP-10 alleviates allergic airway inflammation by dephosphorylating p38 in pathogenic memory Th2 cells (20). In our RNA sequencing analysis, the expression of DUSP-2 was reduced in AR-deficient Th2 cells (Fig. 6C). In addition, the expression of DUSP-2, but not DUSP-1 and DUSP-10, was elevated in Th2 cells by stimulation with DHT (Fig. 7A). Furthermore, DUSP-2 is highly expressed in Th2 cells, and its expression becomes higher after TCR stimulation (17). Thus, androgens might suppress asthma via the DUSP-2/p38 axis.

GATA3 is the master transcription factor of Th2 cells and promotes production of type 2 cytokines (39). After Ag stimulation of CD4 T cells, GATA3 is phosphorylated by p38 and transported from the cytoplasm into the nucleus by importin-α (46). Thus, the nuclear transportation of GATA3 depends on the phosphorylation of serine residues in p38. Glucocorticoids bind to the GR and suppress the expression of inflammatory genes by translocating to the nucleus (47, 48). Indeed, GR binds to importin-α and inhibits translocation of GATA3 into the nucleus. In addition, GR increases expression of DUSP-1, which dephosphorylates p38 (19). Then, p38 suppresses phosphorylation of GATA3. We found that androgens upregulate DUSP-2 expression in Th2 cells (Fig. 7A) and reduce the phosphorylation of p38 in Th2 cells (Fig. 6D). In addition, overexpression of DUSP-2 reduced IL-4 production in Th2 cells (Fig. 7F). Because DUSP-1 and DUSP-2 have similar structures, the dephosphorylation of p38 by DUSP-2 might also suppress the nuclear transport and function of GATA3.

In this study, we proposed the mechanism of how androgens suppress type 2 cytokine expression in T cells by the DUSP-2–p38 pathway. This may lead to develop more effective therapy of asthma instead of using androgens. It is reported that administration of androgens alleviates asthma symptoms in mice (49). In a clinical trial, administration of dehydroepiandrosterone-3-sulfate, a type of androgen, mitigates asthma symptoms. However, the safety of long-term androgen administration has not been proved (50). Thus, the DUSP-2–p38 axis in Th2 cells might become a new therapeutic target for asthma in future.

We thank Drs. C.B. Wilson and T. Honjo for CD4-Cre Tg mice, Dr. N. Minato for OT-I TCR Tg mice, Drs. F.W. Alt and M. Ito for Rag2^−/− mice, Satsuki Kitano and Hitoshi Miyachi for manipulation of mouse embryos, Dr. Fumiko Toyoshima for encouragement, and members of the K. Ikuta laboratory for discussions.

The authors have no financial conflicts of interest.