17β-Estradiol affects lung function and inflammation following ozone exposure in a sex-specific manner

Abstract

Inflammatory lung diseases affect men and women disproportionately, suggesting that fluctuations of circulating hormone levels mediate inflammatory responses. Studies have shown that ozone exposure contributes to lung injury and impairment of innate immunity with differential effects in men and women. Here, we hypothesized that 17β-estradiol enhances inflammation and airway hyperresponsiveness (AHR), triggered by ozone exposure, in the female lung. We performed gonadectomy and hormone treatment (17β-estradiol, 2 wk) in C57BL/6J female and male mice and exposed animals to 1 ppm of ozone or filtered air for 3 h. Twenty-four hours later, we tested lung function, inflammatory gene expression, and changes in bronchoalveolar lavage fluid (BALF). We found increased AHR and expression of inflammatory genes after ozone exposure. These changes were higher in females and were affected by gonadectomy and 17β-estradiol treatment in a sex-specific manner. Gonadectomized male mice displayed higher AHR and inflammatory gene expression than controls exposed to ozone; 17β-estradiol treatment did not affect this response. In females, ovariectomy reduced ozone-induced AHR, which was restored by 17β-estradiol treatment. Ozone exposure also increased BALF lipocalin-2, which was reduced in both male and female gonadectomized mice. Treatment with 17β-estradiol increased lipocalin-2 levels in females but lowered them in males. Gonadectomy also reduced ozone-induced expression of lung IL-6 and macrophage inflammatory protein-3 in females, which was restored by treatment with 17β-estradiol. Together, these results indicate that 17β-estradiol increases ozone-induced inflammation and AHR in females but not in males. Future studies examining diseases associated with air pollution exposure should consider the patient's sex and hormonal status.

Keywords: air pollution; estrogen; lung inflammation; sex differences; sex hormones.

Fig. 1.

Experimental design. Sham and gonadectomized [ovariectomized (OVX), orchiectomized (ORX)] female and male C57BL/6J mice received daily 17β-estradiol (10 μg·kg body wt⁻¹·day⁻¹). The hormone or vehicle control was mixed with nut cream and administered orally for 2 wk. Animals were then exposed to ozone (1 ppm; O₃) or filtered air (FA) for 3 h.

Fig. 2.

Serum levels of 17β-estradiol and testosterone measured by ELISA. A and B: 17β-Estradiol levels in female (A) and male (B) mice are shown. C: testosterone levels in male mice. D: progesterone levels in female mice. Animals were exposed for 3 h to 1 ppm ozone (O₃) or filtered air (FA). OVX, ovariectomized mice; ORX, orchiectomized mice; OVX+E2, 17β-estradiol-treated ovariectomized mice; ORX+E2, 17β-estradiol-treated orchiectomized mice. Values are means ± SE of n = 5–8 mice per group (*P < 0.05, **P < 0.005, ***P < 0.0005).

Fig. 3.

Bronchoalveolar lavage fluid (BALF) fluid cell counts. Total cells in female (A) and male (B) mice exposed for 3 h to 1 ppm ozone (O₃) or filtered air (FA). Polymorphonuclear neutrophils (% of total) in females (C) and males (D) were measured at 24 h postexposure to O₃ or FA. OVX, ovariectomized mice; ORX, orchiectomized mice; OVX+E2, 17β-estradiol-treated ovariectomized mice; ORX+E2, 17β-estradiol-treated orchiectomized mice. Values are means ± SE of n = 5–8 mice per group (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001).

Fig. 4.

Lipocalin-2 [neutrophil gelatinase-associated lipocalin (NGAL)] levels in bronchoalveolar lavage fluid (BALF) of treated mice. Lipocalin was measured by ELISA in BALF from female (A) and male (B) mice exposed to ozone (1 ppm; O₃) or filtered air (FA) for 3 h. OVX, ovariectomized mice; ORX, orchiectomized mice; OVX+E2, 17β-estradiol-treated ovariectomized mice; ORX+E2, 17β-estradiol-treated orchiectomized mice. Values are means ± SE of n = 5–8 mice per group (*P < 0.05, **P < 0.005, ****P < 0.0001).

Fig. 5.

Relative mRNA expression of Nos2 and Cxcl2 (macrophage inflammatory protein-2α, MIP-2α) in lung tissue. Gene expression was analyzed by real-time PCR in whole lung tissue extracts from female (A and C) and male (B and D) mice exposed to ozone (O₃) or filtered air (FA). OVX, ovariectomized mice; ORX, orchiectomized mice; OVX+E2, 17β-estradiol-treated ovariectomized mice; ORX+E2, 17β-estradiol-treated orchiectomized mice. Values are means ± SE of n = 5–8 mice per group (*P < 0.03, **P < 0.006, ***P < 0.001).

Fig. 6.

Relative mRNA expression of IL-6 and Ccl20 (macrophage inflammatory protein-2α, MIP-2α). Gene expression was analyzed by real-time PCR in whole lung tissue extracts from female (A and C) and male (B and D) mice exposed to ozone (O₃) or filtered air (FA). OVX, ovariectomized mice; ORX, orchiectomized mice; OVX+E2, 17β-estradiol-treated ovariectomized mice; ORX+E2, 17β-estradiol-treated orchiectomized mice. Values are means ± SE of n = 5–8 mice per group (*P < 0.05, **P < 0.002, ***P < 0.001).

Fig. 7.

Effect of ozone exposure and 17β-estradiol treatment on airway hyperresponsiveness in females. Airway resistance was measured by flexiVent in female mice exposed to ozone (O₃; dashed line) or filtered air (FA; solid line). A–C: respiratory system (whole lung) resistance (Rrs) in sham (control) (A), ovariectomized (OVX) mice (B), and 17β-estradiol-treated ovariectomized (OVX+E2) mice (C). D: comparison of Rrs at 50 mg/mL of methacholine among groups. Values are means ± SE of data from n = 4–6 mice per group (*P < 0.05, **P < 0.005, ***P < 0.001).

Fig. 8.

Effect of ozone (O₃) exposure and 17β-estradiol treatment on airway hyperresponsiveness in males. Airway resistance was measured by flexiVent in male mice exposed to O₃ (dashed line) or filtered air (FA; solid line). A–C: respiratory system (whole lung) resistance (Rrs) in sham (control; A), orchiectomized (ORX) mice (B), and 17β-estradiol- treated orchiectomized (ORX+E2) mice (C). D: comparison of Rrs at 50 mg/mL of methacholine among groups. Values are means ± SE of data from n = 4–6 mice per group (***P < 0.001).

Fig. 9.

Tissue damping (G) in male and female mice exposed to ozone (O₃) or filtered air (FA). G (constant phase model) was measured by flexiVent in mice exposed to O₃ (dashed line) or FA (solid line). A–C: females: sham (control) (A), ovariectomized (OVX) (B), and 17β-estradiol-treated ovariectomized (OVX+E2) (C) mice. D–F: males: sham (control) (D), orchiectomized (ORX) (E), and 17β-estradiol- treated orchiectomized (ORX+E2) (F) mice. Values are means ± SE of data from n = 4–6 mice per group (*P < 0.05).

Fig. 10.

Respiratory system elastance in female mice. Respiratory system (whole lung) elastance (Ers) was measured by flexiVent in female mice exposed to ozone (O₃; dashed line) or filtered air (FA; solid line). A–C: Ers in sham (control) (A), ovariectomized (OVX) mice (B), and 17β-estradiol-treated ovariectomized (OVX+E2) (C) mice. D: comparison of maximum Ers at 50 mg/mL of methacholine among groups. Values are means ± SE of data from n = 4–6 mice per group (**P < 0.005).

Fig. 11.

Respiratory system elastance in male mice. Respiratory system (whole lung) elastance (Ers) was measured by flexiVent in male mice exposed to ozone (O₃; dashed line) or filtered air (FA; solid line). A–C: Ers in sham (control) (A), ovariectomized (OVX) mice (B), and 17β-estradiol-treated ovariectomized (OVX+E2) (C) mice. D: comparison of maximum Ers at 50 mg/mL of methacholine. Values are means ± SE of data from n = 4–6 mice per group (*P < 0.05, **P < 0.003, ***P < 0.001).

Fig. 12.

Tissue elastance (H) in male and female mice exposed to ozone (O₃) or filtered air (FA). H (constant phase model) was measured by flexiVent in mice exposed to O₃ (dashed line) or FA (solid line). A–C: females: sham (control) (A), ovariectomized (OVX) (B), and 17β-estradiol-treated ovariectomized (OVX+E2) (C) mice. D–F: males: sham (control) (D), orchiectomized (ORX) (E), and 17β-estradiol- treated orchiectomized (ORX+E2) (F) mice. Values are means ± SE of data from 4–6 mice per group.