Modulation of the lung inflammatory response to ozone by the estrous cycle

Abstract

Emerging evidence suggests that sex differences exist in the control of lung innate immunity; however, the specific roles of sex hormones in the inflammatory response, and the mechanisms involved are unclear. Here, we investigated whether fluctuations in circulating hormone levels occurring in the mouse estrous cycle could affect the inflammatory response to air pollution exposure. For this, we exposed female mice (C57BL/6J, 8 weeks old) at different phases of the estrous cycle to 2 ppm of ozone or filtered air (FA) for 3 h. Following exposure, we collected lung tissue and bronchoalveolar lavage fluid (BAL), and performed lung function measurements to evaluate inflammatory responses and respiratory mechanics. We found a differential inflammatory response to ozone in females exposed in the luteal phase (metestrus, diestrus) versus the follicular phase (proestrus, estrus). Females exposed to ozone in the follicular phase had significantly higher expression of inflammatory genes, including Ccl2, Cxcl2, Ccl20, and Il6, compared to females exposed in the luteal phase (P < 0.05), and displayed differential activation of regulatory pathways. Exposure to ozone in the follicular phase also resulted in higher BAL neutrophilia, lipocalin levels, and airway resistance than exposure in the luteal phase (P < 0.05). Together, these results show that the effects of ozone exposure in the female lung are affected by the estrous cycle phase, and potentially hormonal status. Future studies investigating air pollution effects and inflammation in women should consider the menstrual cycle phase and/or circulating hormone levels.

Keywords: Air pollution; follicular phase; inflammation; luteal phase; sex hormones.

Figure 1

Study design. Estrous cycle stages were assessed daily for a minimum of 2 weeks prior to ozone exposure experiments. Female mice at different stages of the estrous cycle were exposed to 2 ppm of ozone or filtered air (FA, control) for 3 h between 11:00 am and 2:00 pm (n = 6–8 per phase/exposure group). Four hours after exposure (6:00 pm), blood and lung tissue samples were harvested. Lung function testing and BALF collection were performed at 24 h postexposure (11:00 am).

Figure 2

(A) Bronchoalveolar lavage (BAL) fluid cell counts measured at 24 h postexposure in female mice exposed for 3 h to 2 ppm ozone or filtered air in the luteal or follicular phase of the estrous cycle. (B) Total BAL (2.5 mL) cells and polymorphonuclear neutrophils (% of total) measured at 24 postexposure to ozone or filtered air. Results are expressed as means ± SEM of 5–8 mice per group (**P < 0.01, ***P < 0.001). FA: filtered air, O3: ozone.

Figure 3

Lipocalin‐2 (NGAL) levels measured by ELISA in BAL collected at 24 h postexposure from female mice exposed to ozone (2 ppm) or filtered air for 3 h in the luteal or follicular phase of the estrous cycle. Results are expressed as means ± SEM of 5–8 mice per group (**P < 0.01, ***P < 0.001). FA: filtered air, O3: ozone.

Figure 4

Heatmap (cluster analysis of log gene expression) of top differentially expressed genes measured by a PCR array in lung tissue collected at 4 h postexposure from female mice exposed to ozone (2 ppm) versus filtered air in the luteal and follicular estrous cycle phases (n = 6–8). FA, filtered air; O3, ozone; fol, follicular phase; lut, luteal phase.

Figure 5

Relative mRNA expression of selected genes (Ccl20, Cxcl2, Ccl2, Il6) analyzed by real‐time PCR in lung tissue collected at 4 h postexposure from female mice exposed to ozone (O3) or filtered air (FA) in the luteal or follicular phase of the estrous cycle (n = 12). Results are expressed as means ± SEM. Significant differences were analyzed by ANOVA (*P < 0.05, ***P < 0.001).

Figure 6

Comparison of networks affected by filtered air or ozone exposure in females exposed at different phases of the estrous cycle. (A) Diagram of biological networks of regulatory pathways whose expression were up‐ and downregulated in the lungs of animals exposed to filtered air (FA) versus ozone in the luteal phase. (B) Network analysis from animals exposed to FA versus ozone in the follicular phase. Both diagrams show reported direct (solid lines) and indirect (dashed lines) interactions. Molecules that are downregulated or upregulated are represented as a node in green or red, respectively.

Figure 7

Effects of ozone exposure on lung mechanical properties in female mice at different phases of the estrous cycle. (A) Respiratory system resistance (Rrs). (B) Newtonian resistance (Rn). (C) Tissue damping (G). Respiratory function parameters were measured with a flexiVent system in female mice at 24 h after exposure to ozone (solid line) or filtered air (dashed line) in the follicular (inverted triangle) or luteal (square) phase of the estrous cycle. Exposure to ozone significantly increased Rrs and Rn at higher doses of methacholine in mice the follicular phase but not the luteal phase. Results are expressed as mean ± SEM of data from 4 to 6 mice per group. FA, filtered air; O3, ozone; ^asignificantly different than luteal FA (P < 0.05), ^bsignificantly different than luteal O3 (P < 0.05).