In Utero Ultrafine Particulate Exposure Yields Sex- and Dose-Specific Responses to Neonatal Respiratory Syncytial Virus Infection

Abstract

Exposure to particulate matter (PM) is associated with lower respiratory tract infections. The role of ultrafine particles (UFPs, ≤0.1 μm) in respiratory disease is not fully elucidated, especially in models of immunologically immature populations. To characterize the effects of maternal UFP exposure on neonatal infection, we exposed time-mated C57Bl/6n mice to filtered air or UFPs at a low dose (LD, ∼55 μg/m³) and high dose (HD, ∼275 μg/m³) throughout gestation. At 5 days of age, offspring were infected with a respiratory syncytial virus (RSV) strain known to mimic infant infection or sham control. Offspring body weights were significantly reduced in response to infection in the LD RSV group, particularly females. Pulmonary gene expression analysis demonstrated significantly increased levels of oxidative stress- and inflammation-related genes in HD-exposed male offspring in sham and RSV-infected groups. In males, the highest grade of inflammation was observed in the HD RSV group, whereas in females, the LD RSV group showed the most marked inflammation. Overall, findings highlight neonatal responses are dependent on offspring sex and maternal UFP dose. Importantly, infant RSV pathology may be enhanced following even low dose UFP exposure signifying the importance of preventing maternal exposure.

Keywords: lower respiratory tract infection; neonatal mouse model; prenatal exposure; respiratory syncytial virus; ultrafine particulate matter.

Figure 1

Ultrafine particle characterization showing the size and concentration distributions. (A) LD PM particle size (black) and concentration (gray) distribution, with a peak particle diameter of 0.034 μm (34 nm). (B) HD PM particle size (black) and concentration (gray) distribution, with a peak particle diameter of 0.052 μm (52 nm).

Figure 2

Offspring weights measured 9 days post-infection expressed as mean ± SEM. (A) Overall, offspring body weights were significantly lower in the LD RSV group, as compared with the HD RSV group (p = 0.0324). (B) Differences between body weights post-infection did not vary significantly in male offspring. (C) In females, offspring from the LD RSV group weighed significantly less than the FA Sham group (p = 0.0132). Offspring sample sizes from 2 to 6 litters, listed (n = male, female), include FA Sham (9, 2), LD Sham (16, 15), HD Sham (10, 9), FA RSV (16, 8), LD RSV (20, 13), and HD RSV (16, 16). Data analyzed using one-way ANOVA with Tukey’s multiple comparison test (*P < 0.05).

Figure 3

Genes related to oxidative stress and inflammatory response were evaluated in lung tissue collected 9 days post-infection. Males from the HD Sham and HD RSV goups showed significant increases in expression, up to 4 fold, from their controls for Nrf2 (A), Nqo1 (B), NF-κB (C), while no female groups displayed significantly different levels of expression. Offspring sample sizes from 3 to 6 litters, listed as (n = male, female) include FA Sham (6, 1), LD Sham (4, 3), HD Sham (7, 8), FA RSV (8, 7), LD RSV (5, 4), and HD RSV (7, 4). Error bars represent SEM. Data analyzed using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4

Scoring system was applied to evaluate severity of pulmonary inflammation. Representative photomicrographs of H&E-stained sections of lungs collected 9 days post-infection showing no inflammation (grade 0) in the FA Sham group, mild inflammation (grade 1) in the FA RSV group, moderate inflammation (grade 2) in the LD RSV group, and marked inflammation (grade 3) in the HD RSV group. Scale = 100 μm. Average scores by offspring sex shown in Table 1.

Figure 5

Flow cytometry analysis of offspring lungs 9 days post-infection, separated by sex. (A) CD8+ T cells. (B) CD4+ T cells differentiated by Th1 (IFN-γ+ cells) and Th2 (IL-4+ cells). (C) Th1/Th2 ratios. (D) T regulatory (Treg) cells (CD25+Foxp3+). Offspring sample sizes from 3 to 6 litters, listed as (n = male, female) for A–C and D, respectively, include FA RSV (3, 5) and (4, 6), LD RSV (2, 2) and (6, 6), and HD RSV (6, 1) and (3, 3). Error bars represent SEM. Data analyzed using one-way ANOVA with Tukey’s multiple comparison test.