Age-related immune-modulating properties of seminal fluid that control the severity of asthma are gender specific

Ovary-independent eosinophilic asthma development in adult female mice

Given that women who suffer from asthma are already sensitized to certain antigens, for clinical purposes it is important to determine whether the ovaries contribute to disease development and severity in antigen-sensitized animals. We therefore performed bilateral ovariectomy in young (2-month-old) adult female mice 1 week after OVA sensitization and housed them for at least 3 weeks until endogenous ovarian hormone production ceased (Figure S1A). Upon OVA inhalation, sensitized female mice with intact ovaries displayed extensive eosinophilic airway inflammation (Figure S1B, red). Although previous studies have reported that ovariectomy before antigen sensitization effectively reduces allergic responses [14,16,18], we failed to observe any change in the degree of eosinophilic airway inflammation in ovariectomized mice (Figure S1A B, purple). Histological analysis also showed strong infiltration of immune cells under the epithelial layer of the airways, as well as epithelial thickening and subepithelial fibrosis in the lungs of both intact and ovariectomized asthmatic female mice (Figure S1C, upper). Furthermore, hyperplasia of PAS-positive mucus-producing cells—a characteristic pathological alteration in asthmatic patients—was observed in the epithelial lining of the airways of both intact and ovariectomized mice (Figure S1C, lower), suggesting that ovary-derived female factors are not essential in the pathogenesis of adult female asthma once antigen sensitization has been established.

Controlling eosinophilic female asthma with murine seminal fluid

Next, we explored whether factors derived from male reproductive organs contribute to asthma pathogenesis and can be utilized to control female asthma (Figure 1A). Of the male accessory sex glands, we focused on the seminal vesicles and epididymis because of their major roles in semen production and maturation. Systemic exposure to murine epididymal fluid (EpF) via intraperitoneal injection successfully reduced OVA-induced eosinophilic inflammation in the lungs of 2-month-old sensitized female mice exposed to EpF from 2-month-old and 10-month-old male mice (Figure 1B, **P < 0.01 and *P < 0.05 versus OVA asthma group). Since middle-aged male seminal fluid exerted more potent anti-inflammatory activity than young adult male fluid, we used murine seminal fluid from 10-month-old male mice for further experiments. Seminal vesicle fluid (SVF) from middle-aged mice effectively suppressed eosinophilic airway inflammation in OVA-challenged asthmatic female mice (Figure 1C, left, *P < 0.05 versus OVA asthma group). Consistent with this finding, we observed a significant decrease in the levels of the Th2-related pro-inflammatory cytokine IL-13 in BALF and of OVA-specific IgE in the sera of asthmatic female mice exposed to SVF (Figure 1C, center and right, **P < 0.01 and *P < 0.05 versus OVA asthma group). Furthermore, mucus-producing cell hyperplasia and airway inflammation in asthmatic female mice were attenuated on exposure to SVF or EpF (Figure 1D). Taken together, our data indicate that murine seminal fluid from middle-aged animals suppresses antigen-induced pathological alterations in adult female mice that have been sensitized to antigen, suggesting that female asthma can be controlled by systemic exposure to seminal fluid.

Figure 1. Murine seminal fluid ameliorates asthmatic features in adult female mice. (A) Schematic representation of experimental design for murine seminal fluid (SF) exposure. Young adult female mice sensitized with ovalbumin (OVA) were given murine SF intraperitoneally 30 min before OVA challenge. (B) Age-related functional alteration in murine SF in asthmatic female mice. Numbers of eosinophils (Eos) in bronchoalveolar lavage fluid (BALF) of asthmatic female mice exposed to epididymal fluid (EpF) from 2-month-old (2M) or 10-month-old (10M) male mice are shown. White box: control group (n = 3); colored boxes: asthma groups (n = 6–12). Data are presented as means ± SEM. **P < 0.01 and *P < 0.05 versus OVA asthma group. (C) Changes in Th2-cell-driven allergic responses in asthmatic female mice exposed to 10M-seminal vesicle fluid (SVF) or 10M-EpF. Eosinophil number, IL-13 section, and OVA-specific IgE antibody production are shown. White box: control group (n = 3); colored boxes: asthma groups (n = 5 each). Data are presented as means ± SEM. **P < 0.01 and *P < 0.05 versus OVA asthma group. (D) Representative images of airway inflammation and mucus-producing cell hyperplasia in lungs from asthmatic female mice exposed to 10M-SVF or 10M-EpF. Hematoxylin and eosin (HE, upper) and periodic acid-Schiff (PAS, lower) staining reveals immune cell infiltration and mucus-producing cell hyperplasia, respectively. AW: airway.

Functional conservation of human seminal fluid

To examine whether human seminal fluid (hSF) also exerts an anti-asthma effect, hSF was collected from healthy middle-aged volunteers (34 to 54 years of age). Daily exposure to hSF did not induce immune activation or histological alteration in the lungs of OVA-sensitized young adult female mice (data not shown). In addition, hSF-exposed sensitized female mice had normal reproductive cycles, as verified by vaginal smear (data not shown), indicating that hSF does not impair the ovarian function of adult female mice. hSF exposure prior to OVA challenge successfully suppressed eosinophilic airway inflammation in sensitized young adult female mice (Figure 2A, left, **P < 0.01 versus OVA asthma group). We also observed significant decreases in IL-13 secretion and OVA-specific IgE production in hSF-exposed asthmatic female mice (Figure 2A, center and right, **P < 0.01 and *P < 0.05 versus OVA asthma group). Since vaginal exposure to hSF was sufficient to improve the Th2-mediated allergic reaction (Figure S2), insemination through sexual intercourse may provide a systemic benefit to adult females with asthma.

Figure 2. Human seminal fluid improves pathological changes in asthmatic female mice. (A) Changes in Th2-cell-driven allergic responses in asthmatic female mice exposed to human seminal fluid (hSF). White box: control group (n = 3); colored boxes: asthma groups (n = 7 each). Data are presented as means ± SEM. **P < 0.01 and *P < 0.05 versus ovalbumin (OVA) asthma group. (B) Representative images of PAS staining of lungs from asthmatic female mice exposed to hSF. AW: airway. (C) Assessment of airway hyper-responsiveness in asthmatic female mice exposed to hSF. The response to methacholine at each dose was quantified as the average of the peak measurements of airway resistance (Rrs). control group (n = 3, black); OVA asthma groups (n = 5, red); hSF/OVA group (n = 5, blue). Data are presented as means ± SEM. **P < 0.01 and *P < 0.05 versus OVA asthma group. (D) Transcriptional repression of osteopontin and IL-17A in lungs from asthmatic female mice exposed to hSF. White box: control group (n = 3); colored boxes: asthma groups (n = 7 each). Data are presented as means ± SEM. *P < 0.05 versus OVA asthma group. (E) Transcriptional repression of osteopontin by hSF in antigen-stimulated bone-marrow-derived dendritic cells (BM-DCs) of 2-month-old female mice. White box: control group (n = 8); colored boxes: OVA-stimulated groups (n = 8 each). Data are presented as means ± SEM. *P < 0.05 versus OVA group. BM: bone marrow, GM-CSF: granulocyte macrophage colony-stimulating factor.

Declines in pulmonary function in patients with asthma are mostly due to structural alterations in the lung tissue. Therefore, we next examined whether hSF could prevent pathological and functional alterations in the lungs. Histological analysis showed that hSF exposure reduced hyperplasia of PAS-positive mucus-producing cells in the airways of asthmatic female mice (Figure 2B). Furthermore, airway hyper-responsiveness to methacholine—a hallmark clinical manifestation of asthma—was significantly decreased by hSF exposure in asthmatic female mice (Figure 2C, **P < 0.01 and *P < 0.05 versus OVA asthma group), indicating that mammalian seminal fluid restores the lung compliance of asthmatic females.

Osteopontin is a pro-inflammatory cytokine involved in asthma pathogenesis, and its level in the serum or sputum of asthma patients is positively correlated with disease severity [43–46]. Accordingly, we tested whether hSF influences osteopontin expression in asthmatic adult female mice. Real-time PCR revealed a significant increase in osteopontin mRNA transcripts in the lungs of asthmatic female mice, but this increase was completely repressed by hSF exposure prior to OVA challenge (Figure 2D, left, *P < 0.05 versus OVA asthma group). Similarly, we found that hSF repressed the transcriptional induction of IL-17A in the lungs of asthmatic female mice (Figure 2D, right, *P < 0.05 versus OVA asthma group). Given the pivotal role of dendritic-cell-derived osteopontin in Th17 differentiation [32,33], together with the transcriptional repression of both the osteopontin and IL-17A genes by hSF, we surmised that hSF decreases osteopontin gene expression in antigen-stimulated dendritic cells. Bone marrow cells collected from young adult female mice were cultured in the presence of GM-CSF to obtain bone-marrow-derived dendritic cells (BM-DCs); this was followed by OVA stimulation with or without hSF. Transcriptional induction of the osteopontin gene in OVA-stimulated BM-DCs was completely repressed in the presence of hSF (Figure 2E, *P < 0.05 versus OVA group). Collectively, these results suggest that mammalian seminal fluid ameliorates the antigen-stimulated immunological reaction, at least in part, through the suppression of dendritic cell activity.

Possible negative impact of seminal fluid in asthmatic male mice

Lastly, we asked whether hSF would exert a similar beneficial function in asthmatic male mice (Figure 3A). In the absence of exogenous seminal fluid, the allergic immune reaction of young (2-month-old) adult male mice was lower than that of young adult females (Figure 3B, compared with Figure 2A): 22% reduction in eosinophilic airway inflammation (P < 0.1 versus asthmatic female mice), 45% reduction in IL-13 secretion (P < 0.1 versus asthmatic female mice), and 75% reduction in OVA-IgE production (P < 0.01 versus asthmatic female mice). Histological analysis showed sporadic staining of PAS-positive mucus-producing cells in the airways of asthmatic male mice (Figure 3C). However, all asthmatic features tested, except for OVA-IgE production, remained unchanged upon hSF exposure. Consistent with these findings, we found reduced transcription of the osteopontin and IL-17A genes in asthmatic male mice compared with asthmatic female mice in the absence of exogenous seminal fluid (Figure 3D, red, compared with Figure 2D, red). Unexpectedly, however, we observed enhance transcriptional induction of these molecules in the lungs of asthmatic male mice exposed to hSF (Figure 3D, blue). This observation was reproduced in our in vitro assay system, in which OVA-induced osteopontin transcription was enhanced in BM-DCs derived from 2-month-old male mice and exposed to hSF throughout their differentiation process to mimic the endocrine environment of mature males in vitro (Figure 3E). These data strongly support the concept that age-related changes in seminal fluid shift the immune environment in males from anti-inflammatory to pro-inflammatory. To address this point, we produced asthmatic mature male mice by sensitizing 2-month-old mice with OVA and performing an OVA challenge when they reached 10 months of age (Figure 4A). Consistent with the data shown in Figure 3D, we observed a significant increase in the antigen-induced transcription of osteopontin and IL-17A in the lungs of asthmatic mature male mice (Figure 4D), but no signs of exacerbation of the asthmatic response were detected (Figure 4B, 4C).

Figure 3. Human seminal fluid does not improve pathological changes in asthmatic male mice. (A) Schematic representation of experimental design for human seminal fluid (hSF) exposure. Young adult male mice sensitized with ovalbumin (OVA) were given hSF intraperitoneally 30 min before OVA challenge. (B) Changes in Th2-cell-driven allergic responses in asthmatic male mice exposed to hSF. White box: control group (n = 3); colored boxes: asthma groups (n = 5–7). Data are presented as means ± SEM. *P < 0.05 versus OVA asthma group. (C) Representative images of PAS staining of lungs of asthmatic male mice exposed to hSF. AW: airway. (D) Transcriptional induction of osteopontin and IL-17A in lungs of asthmatic male mice exposed to hSF. White box: control group (n = 3); colored boxes: asthma groups (n = 5 - 7). Data are presented as means ± SEM. *P < 0.05 versus OVA asthma group. (E) Transcriptional induction of osteopontin by hSF in antigen-stimulated BM-DCs of 2-month-old male mice. White box: control group (n = 3); colored boxes: OVA-stimulated groups (n = 6 each). Data are presented as means ± SEM. *P < 0.05 versus OVA group.

Figure 4. Enhanced transcription of osteopontin and IL-17A in lungs of asthmatic mature male mice. (A) Schematic representation of experimental design for asthma induction in mature male mice. Sensitized young adult male mice were challenged with ovalbumin (OVA) at 10 months of age. (B) Changes in Th2-cell-driven allergic responses in asthmatic mature male mice. White box: control group (n = 3); colored boxes: asthma groups (n = 5 - 7). (C) Representative images of PAS staining of lungs from asthmatic mature male mice. AW: airway. (D) Enhanced transcription of osteopontin and IL-17A in lungs of asthmatic mature male mice. White box: control group (n = 3); colored boxes: asthma groups (n = 5–7). Data are presented as means ± SEM. **P < 0.01 and *P < 0.05 versus young OVA asthma group.

Animals

Wild-type C57BL/6Ncr mice (6 weeks of age) were purchased from the Sankyo Lab Service Corporation, Inc. (Tokyo, Japan). Mice were housed in a specific pathogen-free environment in groups of 4 or 5 per cage and allowed access to food and water ad libitum. Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Musashino University (H25-13011, H26-14011, H27-15011, H28-16005, H29-17007, and 05-A-2018 for animal ethic number) and were performed in accordance with Guide for the Care and Use of Laboratory Animals, 8^th edition, NIH (https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf).

Antigen sensitization and challenge

All animals were sensitized intraperitoneally with 2 μg of ovalbumin (OVA; A5503, Sigma-Aldrich, MO, USA) in conjunction with aluminum adjuvant in 0.5 mL of phosphate-buffered saline (PBS) on day 0 and day 14 [58]. One week after the second sensitization, the mice were challenged with either aerosolized saline or OVA (20 mg/mL saline) intranasally for 10 min on each of 5 days in a closed chamber by using a MicroMist nebulizer (DeVilbiss, PA, USA). In some experiments, sensitized mice were left until they reached 10 months of age. The mature sensitized mice were then challenged with OVA as described above to study the influence of sexual maturation and reproductive aging on the pathogenesis of asthma in adults.

Ovariectomy in adult female mice

Sensitized female mice aged 8 weeks were anesthetized by intraperitoneal injection of a ketamine–medetomidine cocktail (55 mg/kg and 0.4 mg/kg, respectively) and underwent bilateral ovariectomy via a dorsolateral approach [57]. Ovariectomy was performed at least 4 weeks before the first OVA challenge to ensure the absence of endogenous ovarian hormones.

Preparation of murine and human seminal fluid

Murine EpF and SVF were collected from wild-type male C57BL/6Ncr mice aged 10 months. Briefly, we cut the right and left epididymis into three regions each (caput, corpus, and cauda) in 2 mL of PBS. The luminal content was squeezed from the epididymal tubules by using fine forceps and then centrifuged at 10,000g for 5 min at room temperature to obtain sperm-free EpF. Seminal vesicle secretions were collected surgically (approximately 100 μL per male mouse), dissolved in 5 mL of PBS (final 50-fold dilution), and then centrifuged at 10,000 xg for 5 min at room temperature to obtain SVF. Both murine EpF and SVF were passed through a nylon membrane syringe filter (pore size = 0.45 μm) to eliminate potential contamination of debris. To obtain human seminal fluid, fresh semen samples were collected from healthy volunteers (34-year-old to 54-year-old men with normal sperm counts and no infection with pathogenic viruses/microbes, n = 7) and diluted 1:2 in 90% Isolate (Irvine Scientific, CA, USA). They were then transferred on to the top of a 90% ISolate layer and centrifuged at 200 xg. The upper phase obtained was immediately frozen in liquid nitrogen and stored until use. The upper phase was then diluted 1:25 in PBS (final 50-fold dilution) for intraperitoneal injection. Written informed consent was obtained from all volunteers. All experimental protocols were approved by the ethical committees of the Research Institute of Pharmaceutical Sciences, Musashino University and Women’s Clinic Ooizumigakuen (human ethics number H27-02).

Exposure to murine and human seminal fluid

Thirty minutes before each OVA inhalation, 0.2-0.5 mL of the 50-fold-diluted murine or human seminal fluid samples was injected intraperitoneally into OVA-sensitized mice.

Measurement of airway responsiveness to methacholine

Airway responsiveness to methacholine challenge was determined 24 h after the last OVA inhalation, as described previously [59]. Briefly, mice were anesthetized and a tracheostomy was performed to insert a metal endotracheal tube (18-gauge needle). The endotracheal tube was connected to a computer-controlled volume ventilator (Flexi vent, SCIREQ, QC, Canada) to measure lung function. After the assessment of baseline measures of airway resistance in response to nebulized saline, changes in these parameters in response to challenge with increasing concentrations of nebulized methacholine (3.125, 6.25, 12.5, and 25 mg/mL) were analyzed.

Preparation of peripheral blood and BALF

Twenty-four hours after OVA challenge, mice were anesthetized as described above, and whole blood was obtained by heart puncture using a heparinized syringe to determine serum OVA-specific IgE levels. BALFs were obtained by cannulating the trachea through a small incision and flushing with 2 mL of saline; the fluids were then centrifuged at 540 xg for 10 min at 4 °C. The supernatant was collected for quantification of IL-13, and the pelleted bronchoalveolar lavage cells were resuspended in 1 mL of PBS to determine the total cell number by using a hemocytometer. To obtain counts of specific inflammatory cell types, no more than 50,000 cells from each BAL fluid were spun at 640 rpm for 2 min at room temperature onto glass microscope slides by using a Shandon Cytospin 4 (Thermo Electron, MA, USA). The cells were then stained with Diff-Quik (International Reagents Corporation, Osaka, Japan). At least 200 cells per mouse sample were counted; the total number of eosinophils was obtained by multiplying the percentage of eosinophils by the total number of cells.

Histology

Left lung lobes were collected after BALF preparation, fixed in 4% (w/v) buffered paraformaldehyde phosphate (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 24 h at 4 °C, and embedded in paraffin blocks. Coronal sections (8 μm thick) were prepared and stained with hematoxylin and eosin to examine inflammatory cell infiltration of the airway epithelium or PAS to examine mucus-producing cell hyperplasia.

Measurement of serum OVA-specific IgE and BALF IL-13

Serum OVA-specific IgE antibody levels were measured by using ELISA as described previously [58]. Briefly, OVA-coated 96-well plates (Maxisorp, Nunc, Roskilde, Denmark) were prepared, and then serially diluted serum samples (1:10–1:640) were added. The bound OVA-specific IgE antibody was labeled with a biotinylated rat anti-mouse IgE antibody (Yamasa Shoyu Co. Ltd., Chiba, Japan) and then treated with horseradish peroxidase-avidin (Dako Co., CA, USA). The chromogenic substrate 3,3’,5,5’-tetramethylbenzidine (BioFix, MD, USA) was added to the plate and the absorbance was measured with a Microwell plate reader (Tecan Group Ltd., Männedord, Switzerland) at a wavelength of 450 nm. The antibody titers of the samples were normalized against pooled standards generated in our laboratory and were expressed as units per milliliter (U/mL). The concentration of IL-13 in the BALF was determined by using a commercially available ELISA kit (R&D Systems, MN, USA) according to the manufacturer’s instructions.

Preparation of bone marrow-derived dendritic cells and antigen stimulation

Bone marrow cells were collected from femurs and tibias of 2-month-old male or female mice and cultured for 5–7 days in Petri dishes in RPMI1640 medium supplemented with 10% fetal calf serum, antibiotics, and 10 ng/mL GM-CSF to obtain BM-DCs. The BM-DCs were then stimulated with full-length OVA (500 μγ/mL) for 24 h to analyze the transcription level of the osteopontin gene. The effects of hSF on osteopontin expression in BM-DCs were examined by pre-incubating the cells with 10 μL of 10-fold-diluted hSF for 30 min before OVA stimulation. Total RNA was purified form BM-DCs by using a NucleoSpin kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions.

Real-time PCR

Right lung lobes were frozen in liquid nitrogen immediately after isolation and were then fractured with a Multi Bead Shocker (Yasui Kikai Co., Osaka, Japan). Total RNA was extracted from the lung powder as described previously [57]. For cDNA synthesis, 1.5 μg of total RNA was reverse-transcribed by using PrimeScript (Takara). Real-time PCR was performed for the genes encoding Foxp3, IL-17a, and osteopontin by using SYBR Premix Ex Taq II (Takara) in a total volume of 20 μL; β-actin served as the internal control. To calculate absolute copy numbers of target genes, were used serially diluted plasmids containing the target gene cDNAs to generate a standard curve for each target gene. Melting temperature was used to confirm the specificity of the reactions. The following forward and reverse primers were used:

β-actin (forward, 5’-CTC CTA GCA CCA TGA AGA TCA-; reverse, 5’-CCT GCT TGC TGA TCC ACA TC-3’)

IL-17A (forward, 5’- CGT CAC CCT GGA CTC TCC A-3’; reverse, 5’- CAG AAT TCA TGT GGT GGT CCA-3’)

Osteopontin (forward, 5’-AGA ATC TCC TTG CGC CAC AG-3’; reverse, 5’-ATC GTC ATC ATC GTC GTC CAT-3’)

Statistics

Data from experimental replicates were pooled and are presented as means ± SEM. All data were statistically analyzed by using Kruskal-Wallis tests with Dunn’s post-tests. A value of P < 0.05 was considered significant.