Evidence of sex differences in ozone-induced oxysterol and cytokine levels in differentiated human nasal epithelial cells

Acute exposure to ozone (O₃) causes upper and lower airway inflammation. We and others have previously demonstrated that O₃ oxidizes lipids, particularly cholesterol, into electrophilic oxysterols, such as secosterol B (SecoB), which can adduct proteins, thus altering cellular signaling pathways. To investigate how O₃-derived oxysterols influence cytokine and chemokine release, nasal epithelial cells (HNECs) from healthy donors (<i>n</i> = 18 donors) were exposed to 0.4 ppm O₃ for 4 h. Afterward, immune mediators in apical washes and basolateral supernatants were analyzed using ELISAs, whereas sterol and oxysterol levels were examined using liquid-chromatography mass spectrometry (LC-MS). O₃ exposure increased SecoB, 7-ketocholesterol (7Keto-Chol), 27-hydroxycholesterol (27OH-Chol), and epoxycholesterols in a sex-dependent manner. Female-derived HNECs had significant increases in SecoB, 27OH-Chol, and β-epoxycholesterol, whereas male-derived cells showed increases in 7Keto-Chol only. O₃ decreased the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-7 but increased interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), VEGF, and Eotaxin. Females exhibited O₃-induced IL-1β and VEGF increases, whereas males showed increased Eotaxin and reduced GM-CSF. Basolaterally, O₃ exposure decreased GM-CSF and thymus and activation-regulated chemokine (TARC) while raising IL-6, IL-13, IL-1β, IL-8, and TNFα. Females showed higher TNFα and IL-1β, but males did not. Oxysterols correlated differently with cytokines by sex. Females showed positive correlations between oxysterols and proinflammatory cytokines like IL-6 and IL-1β, whereas males displayed negative correlations with IL-6, IL-8, and TNFα. In conclusion, O₃-induced cytokine/chemokine responses and sterol/oxysterol levels in HNECs vary by sex, with donor-specific oxysterols associated with O₃-triggered inflammatory mediator release.<b>NEW & NOTEWORTHY</b> It is increasingly recognized that lung biology and responses to pollutant exposures differ in males and females. Using a model of differentiated nasal epithelial cells from male and female donors, our data demonstrate that pollutant-induced cytokine/chemokine responses and oxidized lipid levels vary by sex, with donor-specific oxidized lipids linked to inflammatory mediator release.