Developmental perfluorooctane sulfonic acid exposure exacerbates house dust mite induced allergic responses in adult mice

Abstract

Perfluorooctane sulfonic acid (PFOS) is a long-chain per- and polyfluoroalkyl substance (PFAS), a persistent organic pollutant, which has been used in aqueous film-forming foams. Emerging epidemiological evidence indicates a significant body burden of PFOS is observed in the lungs. Furthermore, developmental PFOS exposure dysregulates lung development and exacerbates eosinophilic inflammation, which are critical risk factors for asthma. However, it is unknown whether PFOS exerts sex-dependent effects on house dust mite (HDM) induced asthmatic progression and allergic inflammation. In this study, timed pregnant Balb/cJ dams were dosed orally via PFOS (1.0 mg/kg/d) spiked or vehicle control mealworms from gestational day (GD) 0.5 to postnatal day (PND) 21. Subsequently, HDM (30 μg/day) was administered starting at PND 77-82 for 10 days, and the mice were sacrificed 48 h after their final treatment. The serum and lung PFOS concentrations were 3.391 ± 0.189 μg/mL and 3.567 ± 0.1676 μg/g in the offspring, respectively. Male mice exposed to PFOS + HDM showed higher total cell counts in bronchoalveolar lavage fluid (BALF), macrophage counts, and eosinophil counts compared to mice exposed to HDM alone. Female mice exposed to PFOS + HDM had increased BALF eosinophil percentage, mucous production, alternatively activated (M2) macrophage polarization, and M2-associated gene expression compared to female mice exposed to HDM alone. PFOS exposure had no significant effect on HDM-induced IL-4, IL-5, or IL-13, but RANTES was further elevated in female mice. Overall, our data suggest that developmental PFOS exposure increased the risk of exacerbated eosinophilic inflammation and M2 polarization, which were more severe in female mice, suggesting sex-dependent developmental effects of PFOS on allergic airway responses.

Keywords: Allergic inflammation; Developmental exposures; HDM; Macrophages; PFOS.

Figure 1:. PFOS Exposure Timeline and Internal Dosimetry.

(A) Timed pregnant Balb/c dams were exposed to PFOS from GD 0.5 to PND 21. The adult offspring were administered HDM (30 μg/day) for 10 days and sacrificed 48 h following the final HDM dose. PFOS concentrations were measured in (B) dam’s serum, (C) offspring’s serum and (D) lungs by LC-MS. Data are shown as mean ± SEM, n = 5–6 mice per sex/treatment group. Created in BioRender.

Figure 2:. PFOS Exacerbates Mucus Hypersecretion in Female Mice.

(A) Images of H&E, (B) PAS staining, and (C) percent (%) positive PAS area in the airway epithelium. Arrows indicate inflammatory cells (blue) or mucous (black). Total serum (D) IgE and (E) IgG concentrations are shown. Data are shown as mean ± SEM, n = 5–6 mice /sex/treatment group. *p<0.05, **p<0.001, ***p<0.0001 indicate statistical significance

Figure 3:. PFOS Alters BALF Differential cell counts in a Sex-Dependent Manner.

Total cell counts by AO/PI, and differential cell counts by Diff-Quik in (A-C) males and (D-F) females. Data are shown as mean percentage ± SEM, n = 7–10 mice /sex/treatment group. *p<0.05, **p<0.001, ***p<0.0001 indicate statistical significance compared to vehicle or indicated groups, #p<0.05 compared to HDM.

Figure 4:. PFOS Exacerbates HDM-induced M2 Macrophage Polarization in Female Mice.

Semi-automated counting of CD68⁺ and ARG1⁺ cells was performed on 20X imaged lung sections. Data are shown as mean counts per image ± SEM, n = 5–6 mice per sex/exposure group. *p<0.05, ***p<0.0001 indicate statistical significance

Figure 5:. PFOS Exposure Differentially Augments Allergic Inflammation Associated Gene Expression.

Total mRNA was isolated from the lungs of PFOS exposed and HDM exposed mice and analyzed with NanoString. (A) The heat maps generated in nSolver show selected target genes associated with Th2 immune response, macrophage polarization, and allergic inflammation. Normalized RNA counts for (B) Chil3, Arg1, Ccl11, Ccl24, Isg15, and Irf7 are shown. Data are shown as mean ± SEM, n = 5–6 mice per sex/exposure group. *p<0.05, **p<0.001, ***p<0.0001 indicate statistical significance.

Figure 6:. Analysis of PFOS and HDM-induced Gene Expression.

Unique and shared differentially expressed genes (DEGs) are shown in Venn diagrams within (A) HDM and (B) PFOS-containing groups. (C) Volcano plots following the same cutoff filters (10%-fold change and p < 0.05) are shown. Data was generated and analyzed in Rosalind.