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American Journal of Physiology - Lung Cellular and Molecular Physiology logoLink to American Journal of Physiology - Lung Cellular and Molecular Physiology
. 2021 Mar 10;320(5):L803–L818. doi: 10.1152/ajplung.00441.2020

Androgen receptor activation alleviates airway hyperresponsiveness, inflammation, and remodeling in a murine model of asthma

Rama Satyanarayana Raju Kalidhindi 1,*, Nilesh Sudhakar Ambhore 1,*, Premanand Balraj 1, Taylor Schmidt 2, M Nadeem Khan 2, Venkatachalem Sathish 1,✉
PMCID: PMC8174830  PMID: 33719566

Abstract

Epidemiological studies demonstrate an apparent sex-based difference in the prevalence of asthma, with a higher risk in boys than girls, which is reversed postpuberty, where women become more prone to asthma than men, suggesting a plausible beneficial role for male hormones, especially androgens as a regulator of pathophysiology in asthmatic lungs. Using a murine model of asthma developed with mixed allergen (MA) challenge, we report a significant change in airway hyperresponsiveness (AHR), as demonstrated by increased thickness of epithelial and airway smooth muscle layers and collagen deposition, as well as Th2/Th17-biased inflammation in the airways of non-gonadectomized (non-GDX) and gonadectomized (GDX) male mice. Here, compared with non-GDX mice, MA-induced AHR and inflammatory changes were more prominent in GDX mice. Activation of androgen receptor (AR) using 5α-dihydrotestosterone (5α-DHT, AR agonist) resulted in decreased Th2/Th17 inflammation and remodeling-associated changes, resulting in improved lung function compared with MA alone challenged mice, especially in GDX mice. These changes were not observed with Flutamide (Flut, AR antagonist). Overall, we show that AR exerts a significant and beneficial role in asthma by regulating AHR and inflammation.

Keywords: 5α-dihydrotestosterone, flutamide, lung function, sex-steroids, testosterone

INTRODUCTION

Asthma is a chronic respiratory disease and a leading cause of morbidity and mortality in the United States and worldwide (1–3). Besides, asthma affects every 1 in 13 adults and 1 in 12 children making it one of the most common chronic diseases (1). Asthma is often a result of severe inflammation, hyperreactivity, and remodeling of the airways resulting in episodic bronchoconstriction (4–11). Although age, gender, and race disparities constitute significant risk factors associated with the outcome of asthma disease severity, little is known about the role of these disparities in manifesting the differential disease phenotypes at molecular and immunological levels (9–14).

Multiple clinical and epidemiological studies suggest that asthma is more common in boys compared with girls, which is reversed postpuberty with women being more susceptible to asthma than men, suggesting the role of sex/gender and sex-steroids such as estrogen, testosterone, and progesterone in the regulation of airway physiology in asthmatic lungs (9, 13, 15–17). Apart from their characteristic function in influencing reproductive organs, sex-steroids also influence a multitude of other organs and tissues eliciting their effects via sex-steroid receptors and regulate crucial cellular functions such as proliferation, migration, extracellular matrix (ECM) dynamics, intracellular calcium ([Ca2+]i), and the local inflammatory milieu (9, 11). Multiple studies, including our own, have reported a crucial role of sex-steroids on airway physiology, especially estrogen and its receptors (estrogen receptor, ERα, and ERβ) in regulating the airway structure and function (18–22). From these studies, it is evident that estrogen differentially regulates airway physiology via diversified signaling of ERα versus ERβ (detrimental vs. beneficial) in cellular aspects such as [Ca2+]i (21), proliferation (19), ECM (18) in vitro, and airway structure and function in vivo (22, 23). However, the role of androgens in airway physiology remains unclear and is still being investigated.

The association of androgens in the prevalence of asthma is apparent considering the epidemiological and clinical data reporting sex/gender differences in the pathophysiology of lung diseases (11, 13, 17, 24–26). The prevalence and severity of asthma remains constant (low compared with females) in men postpuberty, where circulating androgens are at their highest levels (11, 25, 27). However, this trend is reversed in aged men showing increased severity of asthma symptoms with declined serum testosterone levels, which suggest a potential beneficial role of androgens in asthma (25, 27, 28). Interestingly, few studies also suggest a beneficial role for androgens in asthmatic women (29). Androgens mainly exert their effects via androgen receptor (AR) (30, 31) and few studies, including our own have reported that AR activation (30, 31) regulates the contractility of various smooth muscle types by influencing [Ca2+]i levels in vitro (32–37). Studies also report a relaxant effect of testosterone on the epithelium ex vivo in rabbit trachea (38). Our own in vitro studies using human ASM cells confirmed the expression of AR in both males and females with higher fold expression detected in males, which is upregulated during inflammation/asthma (30). Apart from this, limited in vivo studies suggest androgens play a protective role in regulating airway physiology but have mostly focused on the inflammation-associated changes with local inflammatory cell recruitment (39–43). Our recent in vivo studies focused on ER activation and its effect on airways suggest that intact male mice show reduced airway hyperresponsiveness (AHR), inflammation, and remodeling compared with intact females in a mouse model of asthma (22, 23). Although earlier studies suggest a beneficial role of androgens in asthma, comprehensive mechanistic data on the role of androgens modulating airway inflammation, AHR, and remodeling are still lacking. In this context, in our current study, we tested our hypothesis that “AR activation using 5α-dihydrotestosterone (5α-DHT) downregulates AHR, inflammation and remodeling of airways in mixed allergen (MA) induced murine model of asthma.”

In this novel study, we determined the effect of specific AR activation in regulating lung mechanics, inflammation, and airway remodeling in a murine model of asthma using male C57BL/6J mice. Besides normal male C57BL/6J mice, we also used gonadectomized (GDX) mice to control the effect of endogenously secreted androgens. We did not include female mice in the study, as AR is expressed comparatively low in females (30, 31) and to avoid any cross talk with estrogen receptor (ER) associated signaling. Overall, our study is the first to evaluate the effect of AR activation in airway physiology, providing valuable insights on the AR-mediated changes in airway inflammation, structure, and function in the context of asthma.

MATERIALS AND METHODS

Allergens, Chemicals, and Reagents

Ovalbumin was procured from Sigma. Alternaria alternata, Aspergillus fumigtus, and Dermatophagoides farinae (House dust mite) were procured from Greer laboratories. Dulbecco’s phosphate-buffered saline and protease and phosphatase inhibitor (PPI) cocktail were procured from Thermo scientific. LEGENDplex Mouse Inflammation Panel (13-plex) kit was procured from Biolegend.

Study Approval

The protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at North Dakota State University and were executed in accordance with the Guide for the Care and Use of Laboratory Animals by National Institutes of Health.

Animals

Wild-type non-GDX male mice were selected from our in-house breeding protocol and were within the age range of 8 wk prior to the experiment. GDX mice were procured from Jackson Labs, Maine at the age of 7 wk and acclimatized for 1 wk before starting the experiment. Mice were housed under constant temperature and 12-h light and dark cycles provided with food and water ad libitum. A total of 72 mice (36 non-GDX and 36 GDX) were used for this study. Each population was further divided into six groups: 1) PBS, 2), MA, 3) 5α-DHT [45 µg/mice/day (44)], 4) Flut [AR antagonist, 5 mg/mice/day (45)], 5) 5α-DHT + MA, and 6) Flut + MA consisting of 06 mice in each group.

Mixed Allergen and Treatment Exposures

Mice from both populations (non-GDX and GDX) that have been allotted to MA groups were administered with a mixture of ovalbumin, extracts from A. alternata, A. fumigatus, and D. farinae at 10 µg each intranasally in 25 µL of phosphate-buffered saline (PBS), while mice allotted to vehicle groups received 25 µL of PBS alone for 28 days on every even day (22, 23, 46–48). In addition, on every odd day mice from both populations (non-GDX and GDX) and exposures (PBS and MA) were administered with 5α-DHT or flut as a solution intranasally.

Lung Mechanics

To analyze the lung mechanics, mice were subjected to flexiVent (Scireq, Montreal, Canada) analysis at the end of 28 days and parameters such as respiratory resistance (Rrs), compliance (Crs), elastance (Ers), tissue damping (G), and tissue elastance (H) were recorded according to previously published techniques (22, 23, 48, 49). Ketamine and xylazine (100 mg/kg and 10 mg/kg ip, respectively) were used to anesthetize the mice followed by mechanically ventilating them using flexiVent system, and Rrs, Ers, Crs, G, and H were recorded at baseline (0 mg/mL methacholine [MCh]) and increasing doses of nebulized MCh (6.25, 12.5, 25.0, and 50.0 mg/mL, respectively). Max Rrs, max Crs, max Ers, max G, and max H values were used to compare between different treatment groups across the two study populations. Throughout the flexiVent analysis, mice were maintained at 37°C using a heating pad underneath the mice and heated light source placed above at a 45°, with continuous observation of the heart rate.

Sample Collection and Processing

After the final dose of MCh, mice were euthanized with an overdose of pentobarbital (100 mg/kg) and bronchoalveolar lavage fluid (BALF) was collected by injecting 500 µL of PBS with a PPI cocktail via the tracheal canula and collected back. Following this, lungs were perfused to eliminate circulating blood by injecting 10 mL of PBS into the left ventricle of the heart, one lobe of the perfused lung was collected and homogenized in 500 µL of PBS with PPI cocktail and centrifuged at 14,000 rpm for 10 min to collect the supernatant. The collected supernatant was stored at −80°C until cytokine ELISA assay. Further, the left lung of mice was stored in Carnoy’s solution (100% ethanol, chloroform, and glacial acetic acid in a ratio of 6:3:1 with added ferric chloride) followed by replacing the solution with 70% ethanol and used for histology studies (22, 23). Blood was collected by cardiac puncture after the terminal flexiVent experiment and the serum testosterone levels of both non-GDX and GDX (PBS challenged) mice were determined using a mouse Testosterone ELISA kit (R&D Systems). The obtained data were calculated by constructing a standard curve by plotting absorbance for standards. The concentration of testosterone in samples was calculated by corresponding the mean absorbance from the standard curve by considering the dilution factor.

Cytokine ELISA Multiplex Assay in Lung Homogenate

Lung tissue homogenate was processed by following a previously published technique (50). Briefly, lungs were homogenized and centrifuged followed by collecting the supernatant. The collected supernatant was analyzed for cytokines using the LEGENDplex Mouse Multianalyte Inflammation Panel (BioLegend), following the manufacturer’s instructions. A BD FACSymphony flow cytometer was used for acquiring the data followed by analysis in LEGENDplex V8.0 Data Analysis Software (BioLegend).

Total and Differential Leukocyte Count in BALF

The BALF collected from the mice was processed for total and differential leukocyte count (DLC) using previously published methods (4, 7, 22, 23, 51). Briefly, BALF samples were centrifuged at 2,000 rpm for 5 min at 4°C and the cell pellet was resuspended in 200 µL of PBS followed by performing total leukocyte count using Countess-II FL cell counter (Thermo). The remaining sample was processed using a cytospin on glass slides and the resultant air-dried smear was stained using the Differential Quick Staining kit (Modified Giemsa, EMS) following the manufacturer’s protocol. The processed slides were then observed in a digital light microscope (Leica) at ×100 magnification by oil immersion technique for differential cell count. A minimum of 200 cells were counted on each slide.

Histopathology Analysis Using H&E, PAS, and SRFG Stains

Fixed lung samples were processed for histopathological studies to determine basic morphology of the airways; epithelial, and goblet cell-associated changes and collagen deposition using hematoxylin and eosin (H&E) (Sigma); PAS (EMS); and SRFG (Chrondex, Inc.) staining, respectively, using standard techniques (4, 7, 23, 49). The slides were then scanned using Motic Easy Scan (Motic, Canada), and specific regions of interest (×10) were acquired using Motic DS Assistant Lite software (Motic, Canada). Scoring was performed on a scale of 1–5 (one being the best and five being the worst) independently by two individuals for H&E-stained sections to determine inflammatory score, PAS-stained sections for epithelial and goblet cell-associated damage, and SRFG-stained sections for collagen deposition.

Western Analyses of Lung Homogenates

Fresh lung tissues collected from mice were subjected to homogenization in sucrose buffer (10 mg/100 μL) in the presence of Halt Protease and Phosphatase inhibitor cocktail (Thermo Fisher). Total protein was calculated and ∼40 μg of protein was loaded into each well. Standard protocols were followed for Western analysis and the resultant membranes were probed for Androgen receptor (AR, Santa Cruz, Cat. No. sc-7305), Vimentin (Sigma-Aldrich, Cat. No. V5255), α-smooth muscle actin (α-SMA, Sigma-Aldrich, Cat. No. A2547), and β-actin (Applied Biological Materials, Cat. No. G043) followed by scanning in LICOR near-infrared imaging system using LICOR NIR labeled secondary antibodies and densitometric analysis was performed in Image studio software (LICOR) (19, 52). The data are represented as relative protein expression of a ratio of respective proteins with β-actin.

Statistical Analysis

All the groups consisted of a minimum of five to six mice. “n” values represent the number of animals. Statistical analysis was performed using unpaired t test and two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons using GraphPad Prism version 8.1.0 for Windows (GraphPad Software, San Diego, CA) as applicable. Data are expressed as min to max showing all individual points and median line depicting mean with statistical significance tested at a minimum of P < 0.05 level.

RESULTS

Serum Testosterone Levels and Lung Tissue AR Expression

An overview of the experimental design was depicted in Fig. 1A. To confirm the effectiveness of GDX surgery, the baseline serum testosterone levels from PBS challenged non-GDX and GDX mice were measured by using ELISA kit. The results revealed a significant (P < 0.01) reduction in serum testosterone levels in GDX mice compared with non-GDX mice (Fig. 1B). Western analysis of mouse lung homogenates showed significantly increased AR expression in MA-challenged mice from both study populations (P < 0.001 for non-GDX and GDX, Fig. 1C) compared with respective PBS challenged mice. In comparison between non-GDX versus GDX mice, no significant change was observed at both baseline and MA-challenged mice lung AR expression.

Figure 1.

Figure 1.

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Experimental design of the study, serum testosterone levels, and lung AR expression. Gonadectomy (GDX) surgery was performed at the age of 4 wk. A: mice (both non-GDX and GDX) were sensitized with mixed allergen [MA, OAAH: 10 µg each of Ovalbumin, Alternaria Alternata, Aspergillus Fumigatus, and Dermatophagoides farinae (house dust mite)] or phosphate-buffered saline (PBS, vehicle) intranasally (i.n.) on alternate days for 28 days. Beginning on day 02, mice from non-GDX and GDX study populations received PBS, 5α-dihydrotestosterone [5α-DHT, androgen receptor (AR) agonist] or Flutamide (Flut, AR antagonist) according to respective groups via intranasal administration. B: serum testosterone levels in non-GDX and GDX mice. C: AR expression in non-GDX and GDX mice subjected to PBS and MA challenge using Western analysis. Data analyzed using unpaired t test and two-way ANOVA followed by Tukey’s post hoc test as applicable. Data represented as min to max showing all points of at least five to six mice per group. **P < 0.01 versus non-GDX mice; ***P < 0.001 versus respective PBS of non-GDX/GDX mice. AR, androgen receptor; BALF, bronchoalveolar lavage fluid; DLC, differential leukocyte count; H&E, hematoxylin and eosin; PAS, periodic acid Schiff; SRFG, Sirius red and fast green.

Effect of AR Activation on Airway Resistance

We have used flexiVent FX1 module to measure airway resistance (Rrs), which represents airway constriction. Here, MA-challenged mice from both study populations (P < 0.001 for non-GDX and GDX) showed a significant increase in max Rrs following MCh challenge, with substantially higher effects observed in GDX mice. Interestingly, 5α-DHT treatment significantly ablated MA-induced increase in Rrs in both the study populations (P < 0.05 for non-GDX and P < 0.001 for GDX), with a more prominent effect observed only in the GDX study population. MA-challenged mice treated with flut on the other hand did not show any significant changes in MA-induced elevated Rrs. In PBS-challenged mice, we found slightly elevated Rrs in GDX population, compared with non-GDX population, although not significant. Mice treated with 5α-DHT alone did not show any changes in Rrs of either study populations compared with PBS alone group, whereas flut alone treated non-GDX mice showed slightly increased Rrs, albeit not significant (Fig. 2, A and B). In comparison of non-GDX versus GDX mice, we did not observe any significant changes between the respective treatment groups.

Figure 2.

Figure 2.

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Effect of AR activation on airway resistance (Rrs). Effect of AR activation on Rrs in the lungs of male (A) non-GDX (left) and GDX mice (right) in control conditions (PBS challenged) and in the presence of MA. B: max Rrs was used to compare between different treatment groups of non-GDX and GDX mice. MA challenge significantly increased Rrs in both the study populations, especially in GDX mice. This effect of MA was alleviated in mice treated with 5α-DHT, which was not observed in case of flut-treated mice. Data analyzed using two-way ANOVA followed by Tukey’s post hoc test. Data represented as min to max showing all points of at least five to six mice per group; ***P < 0.001 versus respective PBS of non-GDX/GDX; #P < 0.05, ###P < 0.001 versus respective MA of non-GDX/GDX.

Effect of AR Activation on Airway Compliance

Airway compliance (Crs) was evaluated to determine the ease of airflow into the lungs of mice exposed to MA and the effect of 5α-DHT and flut against MA-induced changes. MA-challenged mice showed significantly reduced max Crs in response to MCh challenge in both the study populations (P < 0.001 for non-GDX and P < 0.05 for GDX), with notable effect observed in GDX study population, albeit not significant when compared non-GDX versus GDX. Here, although the observed statistical significance of GDX population is lower than that of non-GDX population, GDX mice showed relatively low max Crs compared with non-GDX mice at baseline (PBS challenged), which was further worsened upon MA challenge. Further, 5α-DHT-treated mice showed significant improvement in MA-induced max Crs changes in either study populations (P < 0.01 for non-GDX and P < 0.05 for GDX), which was not observed in the case of flut-treated mice. At baseline (PBS challenged), mice treated with 5α-DHT or flut did not show any significant changes in Crs (non-GDX and GDX) compared with respective PBS-treated mice (Fig. 3, A and B).

Figure 3.

Figure 3.

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Effect of AR activation on airway compliance (Crs). Effect of AR activation on Crs in the lungs of male (A) non-GDX (left) and GDX (right) mice in control conditions (PBS challenged) and in the presence of MA. B: max Crs was used to compare between different treatment groups of non-GDX and GDX mice. MA challenge significantly decreased Crs in both the study populations, especially in GDX mice. This effect of MA on Crs was alleviated in mice treated with 5α-DHT, which was not observed in case of flut-treated mice. Data analyzed using two-way ANOVA followed by Tukey’s post hoc test. Data represented as min to max showing all points of at least five to six mice per group; *P < 0.05, ***P < 0.001 versus respective PBS of non-GDX/GDX; #P < 0.05, ##P < 0.01 versus respective MA of non-GDX/GDX.

Effect of AR Activation on Airway Elastance

Airway elastance (Ers) was evaluated to identify changes associated with elastic stiffness of the airways in respective treatment groups. MA-challenged mice from both study populations showed significantly increased max Ers in response to MCh challenge (P < 0.05 for non-GDX and P < 0.001 for GDX), with a pronounced effect observed in GDX population. This MA-induced increase in max Ers was significantly alleviated in 5α-DHT-treated mice (P < 0.05 for non-GDX and P < 0.01 for GDX), which was not observed in flut-treated mice of either study population. Furthermore, max Ers was found to be slightly elevated in PBS and MA-challenged GDX mice compared with non-GDX mice, although statistically not significant. Here, 5α-DHT or flut-treated mice did not show any changes in max Ers compared with PBS-treated mice (Fig. 4, A and B).

Figure 4.

Figure 4.

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Effect of AR activation on airway elastance (Ers). Effect of AR activation on Ers in the lungs of male (A) non-GDX (left) and GDX (right) mice in control conditions (PBS challenged) and in the presence of MA. B: max Ers was used to compare between different treatment groups of non-GDX mice and GDX mice. Ers was significantly increased in MA-challenged mice of both the study populations, with prominent effect observed in GDX mice. This effect of MA-induced Ers was alleviated in mice treated with 5α-DHT, which was not observed in case of flut-treated mice. Data analyzed using two-way ANOVA followed by Tukey’s post hoc test. Data represented as min to max showing all points of at least five to six mice per group; *P < 0.05, ***P < 0.001 versus respective PBS of non-GDX/GDX; #P < 0.05, ##P < 0.01 versus respective MA of non-GDX/GDX.

Effect of AR Activation on Tissue Damping (G)

Similar to Ers, MA-challenged mice from both study populations exhibited a significantly increased max G following MCh challenge (P < 0.01 for non-GDX and P < 0.001 for GDX), with a notable a higher fold effect observed in GDX mice. Here, mice from both study populations treated with 5α-DHT significantly reduced the MA-induced changes in max G (P < 0.05 for non-GDX and P < 0.001 for GDX). Interestingly, PBS and MA-challenged GDX mice showed a slightly elevated max G compared with non-GDX mice, but not significant. Furthermore, 5α-DHT alone-treated mice did not show any significant changes in max G, whereas non-GDX mice treated with flut alone showed slightly elevated G compared with respective PBS challenged mice, albeit not significant (Fig. 5, A and B).

Figure 5.

Figure 5.

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Effect of AR activation on tissue damping (G). Effect of AR activation on G in the lungs of male (A) non-GDX (left) and GDX (right) mice in control conditions (PBS challenged). B: max G was used to compare between different treatment groups of non-GDX and GDX mice. MA challenge significantly increased G in both the study populations, especially in GDX mice. 5α-DHT treatment significantly ablated the effect of MA, which was not observed in case of flut-treated mice. Data analyzed using two-way ANOVA followed by Tukey’s post hoc test. Data represented as min to max showing all points of at least five to six mice per group; **P < 0.01, ***P < 0.001 versus respective PBS of non-GDX/GDX; #P < 0.05, ###P < 0.001 versus respective MA of non-GDX/GDX.

Effect of AR Activation on Tissue Elastance (H)

MA-challenged mice from both study populations showed significantly increased max H following MCh challenge (P < 0.05 for non-GDX and P < 0.001 for GDX), with higher fold effect observed in GDX population. 5α-DHT-treated mice from both study populations significantly ablated MA-induced changes in max H (P < 0.05 for non-GDX and P < 0.001 for GDX). Further, we did not observe any significant changes in max H between the non-GDX versus GDX animals in the respective treatment groups. However, flut alone-treated mice of either study population showed slightly elevated max H compared with PBS challenged mice, although not significant. Mice treated with 5α-DHT alone did not show any significant changes in max H of either study population (Supplemental Fig. S1, A and B; all Supplemental material is available at https://doi.org/10.6084/m9.figshare.12971594.v3).

Effect of AR Activation on the Cytokine Milieu in the Lungs

As inflammation plays a pivotal role in regulating the pathophysiology and airway integrity in asthmatic lungs, we compared the cytokine levels as a correlate of inflammation in non-GDX versus GDX mice. For the 11-cytokines detected, the baseline differences were only noted for interferon-β, interleukin-23 and IL-27 between non-GDX and GDX mice (Fig. 6, C, H, and I). Compared with PBS treated groups, the treatment with 5α-DHT did not result in significant changes in cytokine levels in both (non-GDX vs. GDX) study populations, except IL-27, where IL-27 response was suppressed in GDX mice (Fig. 6I). Further, compared with PBS challenged mice, MA-challenged mice exhibited differential inflammatory burden, in both study populations, as evidenced by increased levels of IL-1α (GDX, P < 0.001; Fig. 6A), IFN-γ (non-GDX, P < 0.001 and GDX, P < 0.01; Fig. 6D), IL-6 (GDX, P < 0.001; Fig. 6E), IL-10 (GDX, P < 0.05; Fig. 6F), IL-17α (non-GDX, P < 0.001 and GDX, P < 0.001; Fig. 6G), IL-27 (non-GDX, P < 0.001; Fig. 6H), TNFα (non-GDX, P < 0.01 and GDX, P < 0.001; Fig. 6J), and Ccl2 (GDX, P < 0.001; Supplemental Fig. S2). Conversely, the IL-23 (non-GDX, P < 0.001; Fig. 6H) and IL-27 levels (GDX, P < 0.001, Fig. 6I) were lower in MA-challenged mice compared with PBS mice. Furthermore, 5α-DHT treatment led to a suppression of MA induced increase in cytokine levels for IL-1α (GDX, P < 0.001; Fig. 6A), IFN-β (P < 0.05 for non-GDX and GDX; Fig. 6C), IFN-γ (non-GDX, P < 0.001 and GDX, P < 0.01; Fig. 6D), IL-6 (GDX, P < 0.001; Fig. 6E), IL-17α (P < 0.001 for non-GDX and GDX; Fig. 6G), TNFα (non-GDX, P < 0.05 and GDX, P < 0.001; Fig. 6J), and Ccl2 (GDX, P < 0.001; Supplemental Fig. S2). Conversely, 5α-DHT treatment alleviated MA-induced suppression of IL-27 levels (GDX, P < 0.001; Fig. 6I). Here, flut treatment did not modulate MA-induced cytokine changes, except for increased IFN-γ in GDX group. Furthermore, MA-challenged mice from GDX population showed significant increase in IL-1α (P < 0.001; Fig. 6A), IL-6 (P < 0.001; Fig. 6E) and TNFα (P < 0.001; Fig. 6J) levels compared with MA-challenged non-GDX mice. Overall, these data demonstrate that at baseline GDX mice have elevated cytokine levels compared with non-GDX mice and that MA challenge further amplifies these changes, which is ablated in 5α-DHT-treated mice.

Figure 6.

Figure 6.

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Effect of AR activation on cytokine levels in the lungs. Effect of AR activation on IL-1α (A), IL-1β (B), IFN-β (C), IFN-γ (D), IL-6 (E), IL-10 (F), IL-17α (G), IL-23 (H), IL-27 (I), and TNFα (J) levels in lung homogenate of non-GDX and GDX male mice exposed to different treatment groups. MA challenge resulted in significantly increased levels of IL-1α, IL-6, IL-10, IL-17α, IFN-β, IFN-γ, TNFα, and decreased levels of IL-23 and IL-27 in both study populations (non-GDX and GDX) compared with respective PBS challenged mice. This effect of MA was found to be Th2/17 biased and more prominent in GDX mice compared with non-GDX mice. 5α-DHT treatment resulted in ablation of MA induced increase in Th1 and Th2/Th17 cytokines in mice from both populations (non-GDX and GDX), which was not observed in case of flut-treated mice. Data analyzed using two-way ANOVA followed by Tukey’s post hoc test. Data represented as min to max showing all points of at least four to six mice per group; *P < 0.05, **P < 0.01, ***P < 0.001 versus respective PBS of non-GDX/GDX; #P < 0.05, ##P < 0.01, ###P < 0.001 versus respective MA of non-GDX/GDX; $$P < 0.01, $$$P < 0.001 versus respective non-GDX.

Effect of AR Activation on Total Cell Count and DLC in BALF

In order to determine the overall inflammatory response as a result of altered cytokine milieu, we performed a total cell count and DLC in the BALF of mice challenged with MA with/without 5α-DHT or flut treatment. In control conditions, we did not find any changes in total cell count or DLC in 5α-DHT-treated mice of either study populations. However, mice treated with flut showed a notable increase in total cell count and DLC, albeit not significant, except in GDX mice population (P < 0.01; Fig. 7, A and B) compared with PBS alone-treated mice. MA challenge significantly increased the total cell count (P < 0.05 for non-GDX and P < 0.001 for GDX; Fig. 7A) and DLC (Neutrophils: P < 0.05 for non-GDX and P < 0.001 for GDX; Fig. 7B; Eosinophils: P < 0.001 for both non-GDX and GDX, Fig. 7C; Lymphocytes: P < 0.05 for non-GDX and P < 0.01 for GDX, Fig. 7D; and Macrophages: P < 0.05 for non-GDX and P < 0.001 for GDX, Fig. 7E) compared with PBS-treated mice in respective study populations. Here, the MA-induced increase in total cell count and DLC was substantially higher in GDX mice compared with non-GDX mice. Further, MA-challenged mice treated with 5α-DHT showed significantly ablated total cell count (P < 0.05 for non-GDX and GDX; Fig. 7A) and DLC (Neutrophils: P < 0.05 for non-GDX and GDX, Fig. 7B; Eosinophils: P < 0.01 for non-GDX and P < 0.05 for GDX, Fig. 7C; and Macrophages: P < 0.05 for both non-GDX and GDX, Fig. 7E), but no significant changes with lymphocyte count.

Figure 7.

Figure 7.

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Effect of AR activation on total and differential leukocyte count (DLC) in BALF. Effect of AR activation on total (A), neutrophils (B), eosinophils (C), lymphocytes (D), and macrophage (E) cell numbers in BALF of non-GDX and GDX mice exposed to different treatment groups. MA-challenged mice from both the study populations showed significant increase in total cell count, neutrophil, eosinophil, lymphocyte, and macrophage cell populations in BALF, with higher fold increase observed in GDX mice. This MA-induced inflammatory cell recruitment into the airways was alleviated in 5α-DHT-treated mice, which was not observed in flut-treated mice. Data analyzed using two-way ANOVA followed by Tukey’s post hoc test. Data represented as min to max showing all points of at least five to six mice per group; *P < 0.05, **P < 0.01, ***P < 0.001 versus respective PBS of non-GDX/GDX; #P < 0.05, ##P < 0.01 versus respective MA of non-GDX/GDX.

Effect of AR Activation on Airway Structure

To identify remodeling and inflammation-associated changes in the airways of mice challenged with MA and the effect of respective treatments (5α-DHT or flut) on regulating MA-induced changes, we have performed histological analysis using three different stains, hematoxylin and eosin (H&E; Fig. 8A), Sirius red, and fast green (SRFG; Fig. 8B) and periodic acid Schiff (PAS; Fig. 8C). At baseline (PBS challenge), H&E-stained sections of flut-treated mice showed a significant increase in inflammatory cell recruitment (P < 0.001 for non-GDX and P < 0.01 for GDX), whereas no changes were observed in 5α-DHT-treated mice compared with PBS alone-treated mice. In addition, H&E stained of MA-challenged mice of both study populations showed significantly increased inflammatory cell recruitment (P < 0.001 for non-GDX and GDX) compared with respective PBS challenged mice. This effect of MA was significantly alleviated in mice treated with 5α-DHT (P < 0.01 for non-GDX and GDX), while flut-treated mice did not show any significant changes compared with MA-challenged mice (Fig. 8D). Furthermore, at baseline, SRFG-stained sections showed significantly increased collagen deposition around the airways in flut-treated mice (P < 0.05 for non-GDX and P < 0.001 for GDX) compared with PBS alone-treated mice. Here, sections of MA-challenged mice showed a significantly increased collagen deposition (P < 0.001 for non-GDX and GDX) compared with PBS challenged mice, which was significantly reduced in 5α-DHT-treated GDX mice (P < 0.05). MA-challenged mice treated with flut did not show any significant improvement compared with MA alone-treated mice in either population (Fig. 8E). In addition, PAS-stained sections revealed significantly increased epithelial and goblet cell damage (P < 0.001) in flut-treated GDX mice at baseline (PBS challenged) compared with PBS-treated mice. Consistent with the above findings, MA-challenged mice showed significant epithelial and goblet cell damage in both study populations (P < 0.001 for non-GDX and GDX) compared with respective PBS challenged mice. This effect of MA was abrogated in 5α-DHT-treated mice (P < 0.05 for non-GDX and P < 0.01 for GDX), whereas flut-treated mice did not show any significant changes (Fig. 8F).

Figure 8.

Figure 8.

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Effect of AR activation on airway morphology and remodeling associated proteins. A: H&E-stained mice lung sections show increased thickness of airway epithelium and ASM layer followed by inflammatory cell recruitment in MA-challenged non-GDX and GDX male mice, with prominent changes observed in GDX mice (scale 100 μm). B: SRFG-stained lung sections showing increased collagen deposition (red) in the airways of MA-challenged non-GDX and GDX mice, with GDX mice showing higher collagen deposition (scale 100 μm). C: PAS-stained lung sections show increased epithelial thickening and goblet cell hyperplasia in MA-challenged non-GDX and GDX male mice, where GDX mice showed increased remodeling compared with normal mice (scale 40 μm). Inflammatory score (D), collagen deposition score (E), and epithelial and goblet cell damage score (F) of non-GDX and GDX mice subjected to various treatments determined by three independent persons on a scale of 0–5. Expression of Vimentin (G) and α-SMA (H) in lung tissue of non-GDX and GDX mice between various treatment groups. Here, MA-induced changes were alleviated in 5α-DHT-treated mice, whereas flutamide-treated mice did not show any significant changes. Data analyzed using two-way ANOVA followed by Tukey’s post hoc test. Data presented as representative images for at least five to six mice per group with respective scoring values in D, E, and F represented as min to max showing all points of at least five to six mice per group on a scale of 1–5 (one being the best and five being the worst). *P < 0.05, **P < 0.01, ***P < 0.001 versus respective PBS of non-GDX/GDX; #P < 0.05, ##P < 0.01, ###P < 0.001 versus respective MA of non-GDX/GDX.

Effect of AR Activation on Expression of Vimentin and α-SMA in Lung Tissue

Western analysis of lung tissue homogenates of non-GDX and GDX mice showed no significant changes in Vimentin and α-SMA expression at baseline. MA challenge resulted in significantly increased expression of Vimentin (P < 0.001 for non-GDX and GDX mice; Fig. 8G) compared with respective PBS alone challenged mice. Although both study populations from MA challenged group showed increased α-SMA expression, only non-GDX mice showed significant (P < 0.01) increase compared with respective PBS challenged groups. Interestingly, flut alone-treated non-GDX mice showed a significant (P < 0.05; Fig. 8H) increase in α-SMA expression compared with respective vehicle group. Vimentin expression was significantly downregulated in MA-challenged mice treated with 5α-DHT (P < 0.001 for non-GDX and P < 0.01 for GDX mice). Here, MA-challenged GDX mice treated with Flut showed a moderate (P < 0.05) decrease in Vimentin expression, with no notable changes observed in non-GDX population compared with respective MA-challenged mice. Similar to Vimentin expression data, MA-challenged mice treated with 5α-DHT showed decreased α-SMA expression (P < 0.05 for non-GDX and GDX mice) compared with respective MA challenged groups with no changes with Flut treatment.

DISCUSSION

The pathophysiology of asthma manifests as a result of various factors such as genetic, environmental, lifestyle associated, and more importantly endogenous physiological conditions (2, 13, 24, 25, 53–55). In the quest for identifying the underlying endogenous factors contributing to the observed gender differences in the prevalence and severity of asthma, multiple research groups including our own have explored the role of sex-steroids and their receptors in asthma, especially estrogen (13, 17, 25, 56–58) and ER’s (19, 20, 22, 23), with very little information on androgens (27, 34, 43, 59, 60) and AR (30, 35, 39, 41). Androgens, such as testosterone and 5α-DHT act via the AR (nuclear receptor subfamily 3 group C member 4, NR3C4), which is a nuclear receptor that binds to androgens in the cytoplasm followed by nuclear translocation and subsequent gene transcription (31, 61–63). The role of androgens, especially testosterone, and 5α-DHT in activating AR varies and is widely dependent on specific cell or tissue type (62–64). Apart from their well-established role as a major contributing factor in regulating primary male sexual phenotype (via testosterone) and development of secondary male characteristic features (via 5α-DHT), their activity in influencing basic cellular functions has also been extensively reported in multiple cell types and diseases (34, 63, 65–71). However, the specific role of AR activation in airway physiology is limited. Considering the clinical and epidemiological evidence suggesting intrinsic sex differences in asthma pathophysiology, with men showing lower prevalence and severity of asthma compared with women (61, 64, 72, 73) and the direct correlation of serum androgen levels with reduced asthma symptoms (27, 28), warrants for studies on understanding the broad role of androgens in the airways, especially in the context of asthma.

Multiple studies, including our own, suggest that AR is ubiquitously expressed in the lungs at baseline, especially in ASM. Furthermore, the expression of AR has been reported to be higher in males compared with females, which is further increased during inflammation or asthma (30, 31). Here, few in vitro and ex vivo studies, including our own, suggest androgens (both testosterone and 5α-DHT) have physiological effects in airway cell types in the context of calcium and contractility (30, 34, 38, 74). In addition, limited in vivo studies in murine models of asthma showed that androgens reduced inflammation and Th2 cell responses (41, 43, 59, 60, 69, 75). However, the exact role of androgens in regulating inflammation, remodeling, and their contribution to the overall airway contractility is still not explored. In our current study, we show the effect of AR-specific activation by 5α-DHT (potent AR agonist) and inhibition by flut (pharmacological AR antagonist) on lung mechanics using flexiVent, airway inflammation using DLC, and Th1/Th2/Th17 inflammatory cytokine levels. Novelty of the current study lies in: 1) use of MA to induce asthma, which is a robust and effective model that mimics the Th2-biased eosinophilic inflammation, airway remodeling, and pathogenesis of human asthma (22, 23, 48); 2) employing AR-specific agonist 5α-DHT instead of testosterone, which eliminates the possibility of metabolism of testosterone to estrogen by Cyp19, thereby resulting in altered (downregulated) AR activity; 3) localized delivery of 5α-DHT and flut in the lungs via intranasal administration to avoid systemic elimination; 4) evaluating complete lung mechanics using flexiVent for airway resistance (Rrs), compliance (Crs), elastance (Ers), tissue damping (G), and tissue elastance (H); 5) evaluating Th1 and Th2/17 cytokine profiles; and 6) corroborating functional studies with basic morphology, epithelial and goblet cell changes and collagen deposition in the airways. Further, to study the effect of specific AR activation in the absence of endogenously produced and circulating androgens, we have included a GDX (castrated) study population. As a result, the data obtained from GDX mice allowed us to identify the specific effect of 5α-DHT in a controlled endogenous hormone environment. Furthermore, to minimize any additional effects of residual androgens, we have used an additional group of flut-treated mice in both study populations. However, we assume in the absence of endogenous testosterone in the GDX mice there could be some compensatory mechanisms that may be activated, which are relevant to the changes in lung structure, function, and cytokine panels, although adding an additional group of 5α-DHT and Flut combination would have further confirmed our findings in a more concrete manner. While one critical challenge would be to attribute the observed Flut-specific effects by inhibiting 5α-DHTs, since both are pharmacological compounds and there is also a possibility of several endogenous factors (especially endogenous Tes and 5α-DHT) being involved, which is challenging to regulate. The detailed mechanistic basis behind these changes is complex and will be explored in future studies.

Considering the fact that inflammation is central to the regulation of lung pathophysiology and function (10, 25, 76), we measured inflammatory cytokines in the lungs of MA-challenged non-GDX and GDX mice, with or without 5α-DHT/flut treatment. As a result of MA challenge, we found increased levels of inflammatory cytokines such as IL-1α, IL-6, IL-10, IL-17α, IFN-β, IFN-γ, and TNFα in both study populations (non-GDX and GDX) compared with respective PBS challenged mice. The effect of MA-induced changes in cytokine levels was more prominent in GDX mice compared with non-GDX mice. Here, the observed increase in MA-induced inflammation was found to be Th2/Th17-biased changes. Our findings are by accordance with previously published reports vis-à-vis the negative association of androgens with that of a Th2 response (41, 65). In addition, the Th2 predominant pattern of inflammation observed in MA-challenged mice is consistent with previously reported findings (48, 77). Moreover, our study also showed downregulated IL-23 levels in the lungs of GDX mice at baseline and MA-challenged mice of both study populations. Given the important regulatory role of IL-23 in the development of Th17 response (78, 79), our data show an IL-23-dependent Th17 response in GDX mice and mice challenged with MA (both non-GDX and GDX), which warrants further investigation. Interestingly, GDX mice showed elevated IL-27 levels at baseline, which might be due to the triggering of an intrinsic compensatory mechanism to curb the increase in Th2/Th17 inflammation in the lungs, as IL-27 has been evidenced to inhibit Th2, Th17, and Treg differentiation and promote Th1 differentiation (80–82). Here, AR activation using 5α-DHT alleviated MA-induced increase in Th2/Th17 cytokines in mice from both populations (non-GDX and GDX), which further supports existing studies suggesting that androgens suppress Th2 inflammatory response in the lungs (41, 43, 60, 69). Further, MA-induced elevated levels of Th1 cytokines (IFN-β, IFN-γ, and TNFα) were downregulated in 5α-DHT-treated mice, suggesting a potential overall anti-inflammatory role of AR activation, which is in accordance with existing findings (41, 43, 60, 69). Moreover, 5α-DHT-treated mice showed improved IL-23 and IL-27 levels as compared with MA-challenged mice, suggesting a Th1-biased inflammatory response induced by AR activation. This Th1 biased, inflammatory phenotype in the lungs as a result of AR activation using 5α-DHT further confirmed by histology studies, which showed reduced airway remodeling possibly due to the downregulation of crucial cytokines responsible for inducing remodeling associated changes in the lungs such as IL-1α, IL-1β, and TNFα.

In order to identify the changes in inflammatory immune cell milieu in the lungs, we performed total and DLC in BAL collected from mice. Inflammatory immune cell infiltration is considered as a characteristic feature of asthma, which is presented as a response to various stimuli that contribute to the pathophysiology of asthma (83–90). Our findings indicate a substantial increase in total and DLC in the airways of mice challenged with MA in both the study populations (non-GDX and GDX). Leukocytes are an important source for many cytokines, lipid mediators, growth factors, and chemokines (91). Lymphocytes, both T and B play a pivotal role in asthma by orchestrating a coordinated inflammatory response (92, 93). Clinical and preclinical findings suggest elevated lymphocyte levels in asthmatic lungs (92, 93), which are in accordance with our findings indicating increased lymphocyte count in the BAL of MA-challenged mice. Further, our data show an increase in eosinophilic infiltration in the lungs of mice (non-GDX and GDX) challenged to MA, which corroborate existing evidence, suggesting elevated eosinophil population in asthmatic lungs (4, 7, 22, 23, 48, 53). Neutrophil infiltration into the airways is not common in mild asthmatics, whereas patients with more severe asthma show elevated neutrophils in the airways (84, 87, 94). Our study showed increased neutrophils in BAL of MA-challenged mice (both non-GDX and GDX mice), suggesting severe asthmatic phenotype as a result of MA challenge. Macrophages are considered as a mainstream source for a multitude of cytokines and chemokines and are quintessential in modulating inflammatory responses (both acute vs. chronic) (95–97). Findings from our study show increased macrophage population in MA-challenged non-GDX and GDX mice, suggesting a robust and chronic inflammation, especially in GDX population. In comparison between non-GDX and GDX mice, MA-induced effect on recruiting inflammatory cells is pronounced in GDX mice population corroborating existing findings, thereby suggesting anti-inflammatory effect of androgens, which is ablated in GDX mice (39, 43, 73). The protective effect of androgens in regulating inflammatory cell recruitment is further supported by our data, where 5α-DHT treatment ablated MA-induced immune cell infiltration in the lungs, except for lymphocytes.

Furthermore, our histological studies suggested notable changes in the airways, such as increased inflammation (immune cell recruitment), airway thickening, and collagen deposition in GDX mice compared with non-GDX mice at baseline indicating an overall protective role for androgens in the airways. This difference between the two study populations was also observed in MA-challenged mice, but at a much higher degree, indicating an endogenous role of androgens in influencing lung structure and thereby its function. Given the significant role of type 2 and 17 cytokines in airway remodeling and AHR (59, 98–101), our data exhibit a strong association between elevated Th2/Th17 cytokines and histological changes such as airway thickening (both epithelium and ASM layers) and collagen deposition as observed in GDX mice at baseline and MA-challenged mice. Here, the changes in airway structure as a result of GDX (39–41) and MA challenge (22, 23) have been reported to a certain extent in previous studies. Interestingly, 5α-DHT administration ablated MA-induced structural changes in the lungs of both non-GDX and GDX mice. Vimentin, also known as fibroblast intermediate filament, is associated with ASM phenotype change to fibroblast (23). MA-challenged non-GDX and GDX mice showed increased Vimentin expression suggests a phenotype change of ASM to fibroblast, which further support by lung mechanistic data showing increased elastance in MA-challenged mice, and this was reversed in the case of MA-challenged mice treated with 5α-DHT. Similarly, α-SMA, which is a smooth muscle marker, was found to be increased in MA-challenged mice, indicating increased ASM mass and thereby observed increased remodeling, which was reversed by specific AR activation by 5α-DHT. The exacerbated remodeling observed in MA-challenged GDX mice and the protective effect observed in 5α-DHT-treated MA-challenged mice suggests that AR activation is crucial for regulating remodeling associated changes in the lungs and deficiency of circulating androgens results in prominent changes in the airway structure, which is further amplified during asthma.

In our study, we integrated the altered inflammatory and structural changes with lung mechanics using flexiVent. Our study showed increased Rrs and decreased Crs in mice challenged with MA, especially in GDX mice, suggesting obstruction of airways as a result of MA-induced Th2/Th17 inflammatory responses and subsequent remodeling-associated changes in the lungs. Increased Ers with MA on the other hand depicts the altered elastic stiffness of the airways. Similar increase in the case of G and H overall indicates increased tissue resistance and elastic recoil of the lung with MA exposure. This effect of MA on inducing AHR is well established (22, 23, 48); however, the exacerbated changes in lung function of GDX mice at baseline and with MA challenge are demonstrated for the first time in our study and is in accordance with clinical and epidemiological studies, suggesting males (higher androgen levels) show significantly less severe asthma symptoms compared with females (13, 17, 22, 23, 25, 27, 102). Furthermore, 5α-DHT treatment clearly ablated MA-induced changes in AHR in both non-GDX and GDX mice populations, thereby strengthening the fact that AR activation is quintessential for regulating AHR along with local inflammation and remodeling in the lungs.

Overall, our findings from the current study suggest MA exposure alters normal lung physiology by inducing major changes in AR expression, inflammation, remodeling, and contractility of airways leading to AHR and asthma. The extent of MA-induced exacerbations of asthma symptoms such as AHR, Th2/Th17-biased airway inflammation, and airway remodeling is generally high in GDX population compared with non-GDX population. Further, AR activation using 5α-DHT alleviated MA-induced inflammatory responses, remodeling, and AHR in mouse lungs. Interestingly, this effect of 5α-DHT was notably higher in GDX mice challenged with MA, which might be due to increased expression of AR during asthmatic conditions (30) or increased sensitivity of AR due to deprival of androgens. Here, MA-challenged mice treated with flut did not show any significant changes in MA-induced inflammation, remodeling or AHR. However, we have observed a miniscule yet not significant improvement in inflammation, remodeling, and lung function in flut-treated MA-challenged mice, which might be due to the activation of an alternative complementary pathway due to the complete inhibition of AR activation, which warrants further exploration of this phenomenon in future studies. All these novel findings suggest a crucial and beneficial role of androgens and AR in asthma. Finally, considering the existing findings and our observations, we believe therapies targeting AR can provide promising future alternative therapies against asthma.

GRANTS

This work is supported by NIH Grants R01-HL123494, R01-HL123494-02S1, and R01-HL146705 (to V. Sathish).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.S.R.K., N.S.A., and V.S. conceived and designed research; R.S.R.K., N.S.A., P.B., and T.S. performed experiments; R.S.R.K., N.S.A., and P.B. analyzed data; R.S.R.K., N.S.A., M.N.K., and V.S. interpreted results of experiments; R.S.R.K. and N.S.A. prepared figures; R.S.R.K. and N.S.A. drafted manuscript; P.B., M.N.K., and V.S. edited and revised manuscript; R.S.R.K., N.S.A., P.B., T.S., M.N.K., and V.S. approved final version of manuscript.

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