Androgen and Androgen Receptor as Enhancers of M2 Macrophage Polarization in Allergic Lung Inflammation

Abstract

Allergic asthma is a disease initiated by a breach of the lung mucosal barrier and an inappropriate Th2 inflammatory immune response that results in M2 polarization of alveolar macrophages (AM). The number of M2 macrophages in the airway correlates with asthma severity in humans. Sex differences in asthma suggest that sex hormones modify lung inflammation and macrophage polarization. Asthmatic women have more M2 macrophages than asthmatic men and androgens have been used as an experimental asthma treatment. In this study, we demonstrate that although androgen (dihydrotestosterone) reconstitution of castrated mice reduced lung inflammation in a mouse model of allergic lung inflammation, it enhanced M2 polarization of AM. This indicates a cell-specific role for androgens. Dihydrotestosterone also enhanced IL-4-stimulated M2 macrophage polarization in vitro. Using mice lacking androgen receptor (AR) in monocytes/macrophages (AR^floxLysMCre), we found that male but not female mice exhibited less eosinophil recruitment and lung inflammation due to impaired M2 polarization. There was a reduction in eosinophil-recruiting chemokines and IL-5 in AR-deficient AM. These data reveal an unexpected and novel role for androgen/AR in promoting M2 macrophage polarization. Our findings are also important for understanding pathology in diseases promoted by M2 macrophages and androgens, such as asthma, eosinophilic esophagitis, and prostate cancer, and for designing new approaches to treatment.

Figure 1.. Androgen reconstitution of castrated C57BL/6 wild-type mice with DHT-releasing pellets decreases allergic lung inflammation.

C57BL/6 male mice were castrated at 3 weeks of age. At 7 weeks, allergic lung inflammation was induced with the OVA protocol. (A) Schematic of the timeline for OVA-induced allergic lung inflammation and pellet implantation. IP, intraperitoneal; OVA, ovalbumin; PBS, phosphate-buffered saline; Neb, nebulization. On day 16, mice were anesthetized and BALF was collected. The left lobe from each mouse lung was inflated with 10% formalin and paraffin-embedded for sectioning. (B) Percent change in mouse body weight on day 7 after OVA or PBS. (C) Immunohistochemical analysis of lung tissue from DHT- and placebo-implanted mice. Mucus production was measured by Periodic acid–Schiff (PAS) staining. Cell infiltration was analyzed by hematoxylin and eosin (H&E) staining. Images were acquired with a 20X objective. Periodic acid–Schiff⁺ quantification is plotted as PAS⁺ cells / 1000 pixels. Hematoxylin and eosin graphic represents cell infiltration scored on a scale of 0 to 3 (0 = no inflammation, 1 = light infiltration in only a few areas, 2 = moderate cell infiltration around <50% of the airways and vessels, and 3 = dense cell infiltration around >50% of the airways and vessels. (D) ELISA quantification of total IgE in serum from the different implanted groups. (E) Gating strategy for BALF cells analyzed by FACS. Total cells (left panel), live cells (middle panel), and eosinophils (LIVE/DEAD^-CD11c^-Siglec F⁺) and AM (LIVE/DEAD^-CD11c⁺Siglec F⁺) (right panel). (F) Number of total live cells in BALF from DHT- and placebo-implanted mice. Each data point represents one mouse. (G) Quantification of eosinophils (left panel) and AM (right panel) in BALF. (H) YM1 quantification in BALF by ELISA. (I) Analysis of intracellular YM1 in AM by FACS. The change in mean fluorescence intensity (MFI) of YM1 (MFI target – MFI isotype) is shown, with female data pooled from our other experiments here and previously. Results are representative of three different experiments. n = 9–12 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.. DHT enhances M2 gene expression, YM1 and FIZZ1 production in BMM.

Bone marrow from female and male C57BL/6 mice was cultured for 10 days with M-CSF (40 ng/mL) to obtain BMM. (A) Validation of the specificity of anti-AR antibody. AM from female and male mice were cultured for 24 h in the presence or absence of the AR-degrading drug, ASC-J9 (10 μM), left panel. AR expression in BMM from C57BL/6 female and male mice, middle panel. Comparison of AR expression in BMM and AM, right panel. (B) BMM were cultured for 10 days with 40 ng/mL of GM-CSF or M-CSF in 6-well plates, cells were collected and viability determined. F4/80⁺ cells were selected in the live population. Dot plots of CD11c⁺Siglec F⁺ cells from F4/80⁺ cells cultured with GM-CSF or M-CSF. (C) Analysis of double-positive CD11c⁺Siglec F⁺ macrophages with GM-CSF or M-CSF (D) Analysis of AR⁺ cells from F4/80⁺ cells cultured with GM-CSF or M-CSF. (E) BMM were pretreated overnight with the indicated concentrations of DHT and then stimulated with IL-4 (1 ng/mL) for 48 h. Expression of the indicated M2 genes was determined by qPCR using the 2^-ΔΔCT method, and compared to the amount of mRNA in the male IL-4 sample (= 100%). (F) Expression of YM1 and FIZZ1 protein was determined by Western blot in the same BMM supernatants after 48 h of IL-4 stimulation. Equal volumes of supernatant were loaded in each lane. Densitometry of the specific bands was normalized to the amount of protein in male IL-4 (= 100%). Results are representative of four independent in vitro experiments. †p < 0.05, ††p < 0.01, ††p < 0.001 for the comparison between DHT pretreatment and IL-4 stimulation alone; *p < 0.05; **p < 0.01, ***p < 0.001 for the comparison between females and males in that specific treatment.

Figure 3.. Bronchoalveolar cell recruitment is impaired in AR^floxLysMCre male mice in the OVA model of allergic lung inflammation.

(A) Genotyping of AR^flox and AR^floxLysMCre male and female mice. Zfy, AR (AR [855 bp], AR floxed gene [952 bp] and the excision product [404 bp]), and LysM (LysM and LysMCre) genes were analyzed by electrophoresis on a 3% agarose gel with ethidium bromide (left panel). AR measurement by intracellular FACS stain of AM from AR^flox and AR^floxLysMCre female (middle panel) and male (right panel) mice. (B) Total live cells (left panel), eosinophils (middle panel), and AM (right panel) recovered from the BALF of AR^flox and AR^floxLysMCre female and male mice on day 16 of the OVA protocol. (C) Immunohistochemical analysis of lung tissue from AR^flox and AR^floxLysMCre male mice. Mucus production was measured by Periodic acid–Schiff (PAS) staining. Cell infiltration was analyzed by hematoxylin and eosin (H&E) staining. Images were acquired with a 20X objective. PAS⁺ quantification is plotted as PAS⁺ cells / 1000 pixels. The H&E graph represents cell infiltration scored on a scale of 0–3, as in Figure 1H. Collagen deposition was analyzed by Masson trichrome stain. Concentration of (D) testosterone and (E) total IgE in serum were determined by ELISA. (F) Proliferation of AM by BrdU incorporation is shown as percent of BrdU⁺ cells in the total alveolar macrophage population. Results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.. Absence of AR results in decreased expression of chemokines and cytokines in AM of male mice.

BALF and AM were obtained from AR^flox and AR^floxLysMCre male mice on day 16 of the allergic lung inflammation model. (A) CCL24, CCL5, and IL-5 were measured in the BALF by ELISA. (B–E) BALF cells were seeded for 2 h in αMEM, and non-adherent cells were vigorously washed away. Relative gene expression in AM was analyzed by qPCR using the 2^-ΔΔCT method and compared to the amount of mRNA in the AR^flox male PBS sample (=1). (B) Ccl2 gene expression. (C) Ccl3, Ccl5, Ccl24, and Ccl11 gene expression. (D) Ccl17 and Ccl22 gene expression. (E) Tgfb and Il10 gene expression. Results are representative of two independent experiments. *p < 0.05, **p <0.01, ***p <0.001.

Figure 5.. Impaired M2 polarization of AR-deficient alveolar macrophages.

BALF and AM were obtained from AR^flox and AR^floxLysMCre male mice after induction of allergic lung inflammation with OVA. (A) The amount of YM1 and FIZZ1 protein in the BALF was determined by Western blot. Densitometry of the specific bands was normalized to the amount in the AR^flox OVA group (= 100%). (B) MMP-12 and MMP-9 were measured in BALF by ELISA. (C) AM from BALF were isolated as described in Figure 4A and analyzed for expression of the indicated M2 genes by qPCR with the 2^-ΔΔCT method. Amount of mRNA was compared to that in the AR^flox PBS sample (= 1). (D) BMM cultured from AR^flox and AR^floxLysMCre male mice as described in Figure 2B were pretreated overnight with DHT at the concentrations shown and then stimulated with IL-4 (1 ng/mL) for 48 h. Relative gene expression was determined by qPCR with the 2^-ΔΔCT method and compared to the amount of mRNA in the IL-4 sample (= 100%) for each genotype (n = 3 independent experiments). Results are representative of three independent experiments. *p < 0.05, **p <0.01, ***p <0.001.