Developmental Exposure to a Mixture of 23 Chemicals Associated With Unconventional Oil and Gas Operations Alters the Immune System of Mice

Abstract

Chemicals used in unconventional oil and gas (UOG) operations have the potential to cause adverse biological effects, but this has not been thoroughly evaluated. A notable knowledge gap is their impact on development and function of the immune system. Herein, we report an investigation of whether developmental exposure to a mixture of chemicals associated with UOG operations affects the development and function of the immune system. We used a previously characterized mixture of 23 chemicals associated with UOG, and which was demonstrated to affect reproductive and developmental endpoints in mice. C57Bl/6 mice were maintained throughout pregnancy and during lactation on water containing two concentrations of this 23-chemical mixture, and the immune system of male and female adult offspring was assessed. We comprehensively examined the cellularity of primary and secondary immune organs, and used three different disease models to probe potential immune effects: house dust mite-induced allergic airway disease, influenza A virus infection, and experimental autoimmune encephalomyelitis (EAE). In all three disease models, developmental exposure altered frequencies of certain T cell sub-populations in female, but not male, offspring. Additionally, in the EAE model disease onset occurred earlier and was more severe in females. Our findings indicate that developmental exposure to this mixture had persistent immunological effects that differed by sex, and exacerbated responses in an experimental model of autoimmune encephalitis. These observations suggest that developmental exposure to complex mixtures of water contaminants, such as those derived from UOG operations, could contribute to immune dysregulation and disease later in life.

Figure 1.

Experimental design and average values for maternal and litter parameters. A, The mixture of 23 chemicals added, in equimass proportions, to the drinking water. B, Pregnant C57Bl/6 dams were placed on control water (0.2% ethanol) or water containing 0.1 or 1 μg/ml of the mixture on day of pregnancy (GD0), through weaning (PND21). There were 10 dams in each group. At maturity (6–8 weeks), offspring of separate dams from each treatment group were randomly assigned to one of four assessment groups for the indicated assessments: immune organ cellularity, HDM-induced airway disease, IAV infection, or EAE. Within each immunological assessment, there were at least 8 nonsibling males and 8 nonsibling females from each developmental exposure group. C, Average daily water consumption by dams. D, Average number of male and female offspring per litter according to treatment group. E, F, Average body weight of adult (8-week) female and male offspring by exposure group. Error bars represent SEM.

Figure 2.

Immunological effects of developmental exposure in a model of allergic airway disease. At maturity (6–8 weeks of age), 9–10 female and 9–10 male offspring from each developmental exposure group were sensitized and challenged with HDM. Within each group, offspring of the same sex were from different dams. A–F, Representative dot plots from flow cytometric analyses of CD4⁺ T cell subsets from MLN 48 h after HDM challenge, and mean percentages ± SEM are depicted according to sex and treatment group for Th2 cells (A–B, GATA3⁺CD4⁺ T cells), Th17 cells (C–D, RORγt⁺CD4⁺ T cells), and Tregs (E–F, Foxp3⁺CD25⁺CD4⁺ T cells). All dot plots are gated on CD4⁺ T cells. G–H, Mean ratio (±SEM) of Treg:Th2 cells and Treg:Th17 cells in MLN from female offspring. I, Mean number (±SEM) of eosinophils, macrophages, lymphocytes, and neutrophils in BAL from female offspring. J–K, Mean ratio (±SEM) of Treg:Th2 cells and Treg:Th17 cells in MLN from male offspring. L, Mean number (±SEM) of eosinophils, macrophages, lymphocytes, and neutrophils in BAL from male offspring. An * represents a p-value ≤ .05 compared with same sex control.

Figure 3.

Effects of developmental exposure to chemicals associated with UOG on T cells and body weight change after viral infection. At maturity (6–8 weeks of age), 9–10 female and 9–10 male offspring from each exposure group infected with IAV. Within each group, offspring of the same sex were from different dams. A–F, Representative dot plots depict flow cytometric analyses of T cell subsets from MLN 9 days after infection: IAV NP-specific CD8⁺ T cells (A–B, D^bNP_366-375⁺CD8⁺ T cells, gated on CD3⁺CD8⁺ cells), Th1 cells (C–D, TBet⁺CD4⁺ cells, gated on CD3⁺CD4⁺ cells), and Tregs (E–F, Foxp3⁺CD25⁺CD4⁺ cells; gated on CD3⁺CD4⁺ cells). The mean percentages (±SEM) of the indicated T cell sub-types in each exposure group and for both sexes are denoted on the plots. G–L, The mean ratios (±SEM) for Treg:Th1 cells, Treg:Th17 cells, and Treg:NP-specific CD8⁺ T cells for each exposure group and separated by sex. M–N, Mean (±SEM) body weight change following infection for female (M) and male (N) offspring. An * represents a p-value ≤ .05 compared with sex matched offspring of control dams.

Figure 4.

CD4+ T cell subset proportions during EAE disease progression. Twenty-six adult (6–8 weeks of age) female and male offspring from each exposure group were immunized with a CFA/MOG_35–55 emulsion to induce EAE. To enumerate CD4⁺ T cells in cervical lymph nodes, 8–10 female and 8–10 male mice from each group were sacrificed 7, 21, or 42 days after immunization. Mice were randomly assigned to each time point. A–D, The bar graphs depict the mean Treg:Th1 ratios (A–B) and mean Treg:Th17 ratios (±SEM) (C–D). Same sex offspring at each timepoint are from different dams. An * represents a p-value ≤ .05 compared with same sex control on the same day post immunization.

Figure 5.

EAE symptom onset and severity following immunization with MOG peptide. Ten adult (6–8 weeks of age) female and male offspring from each exposure group were immunized with a CFA/MOG_35–55 emulsion to induce EAE. Disease progression was monitored and scored every other day for 42 days. A, D, Kaplan Meier plots show the day of disease onset (disease score ≥1) in female (A) and male (D) offspring. B, E, The bar graphs depict the average day of onset for female (B) and male (E) developmentally exposed offspring. C, F, The average EAE disease score (±SEM) according to treatment group and day post immunization in female (C) and male (F) offspring. Disease scores were 0 = normal mouse, 1 = limp tail, 2 = limp tail and hind limb weakness, 3 = partial hind limb paralysis, 4 = complete hind limp paralysis, 5 = moribund. An * represents a p-value ≤ .05 compared with same sex control offspring.