Exposure to a mixture of 23 chemicals associated with unconventional oil and gas operations alters immune response to challenge in adult mice

Abstract

The prevalence of unconventional oil and gas (UOG) operations raises concerns regarding the potential for adverse health outcomes following exposure to water tainted by mixtures of UOG associated chemicals. The potential effects that exposure to complex chemical mixtures has on the immune system have yet to be fully evaluated. In this study, effects on the immune system of adult mice exposed to a mixture of 23 chemicals that have been associated with water near active UOG operations were investigated. Female and male mice were exposed to the mixture via their drinking water for at least 8 weeks. At the end of the exposure, cellularity of primary and secondary immune organs, as well as an immune system function, were assessed using three different models of disease, i.e. house dust mite (HDM)-induced allergic airway disease, influenza A virus infection, and experimental autoimmune encephalomyelitis (EAE). The results indicated exposures resulted in different impacts on T-cell populations in each disease model. Furthermore, the consequences of exposure differed between female and male mice. Notably, exposure to the chemical mixture significantly increased EAE disease severity in females, but not in male, mice. These findings indicated that direct exposure to this mixture leads to multiple alterations in T-cell subsets and that these alterations differ between sexes. This suggested to us that direct exposure to UOG-associated chemicals may alter the adult immune system, leading to dysregulation in immune cellularity and function.

Keywords: Water pollutants; allergy; autoimmune; hydraulic fracturing; immunotoxicity; influenza.

Figure 1.. Effects of exposure to 23-chemical mixture on CD8⁺ T-cells and morbidity after viral infection.

Starting at 6 wk-of-age, C57Bl/6 mice were placed on drinking water containing an equimass mixture of 23 chemicals (Table 1) or vehicle. The final concentration of each chemical in the water was 0.1 μg/ml; control water contained 0.2% ethanol. Mice were maintained on these regimens for at least 8 wk prior to infection. After 8 weeks of exposure, 10 female and 10 male mice from each exposure group were infected intranasally (IN) with IAV (H3N2). Mice were maintained on their respective water treatment regimens throughout infection. (A,C) Mean change in body weight of (A) female and (C) male mice following infection. (B,D-H) CD8⁺ T-cells were examined 9 days post-infection in female mice, and 8 days after infection male mice, using flow cytometry. Mean number of CD8⁺ T-cells in mediastinal lymph nodes (MLN) of infected (B) female and (D) male mice. Mean number of cytotoxic T-lymphocytes (CTL; CD8⁺CD44^hiCD62L^lo cells) in IAV-infected (E) female and (G) male mice from each group. Mean number of IAV NP-specific CD8⁺ T-cells (D^bNP_366–375⁺CD8⁺ T-cells) in infected (F) female and (H) male mice. *Significantly different compared to same-sex control mice (p < 0.05; Student’s t-test). Data presented as means ± SEM. Numerical values that correspond to graphs, as well as p-values for each comparison, are listed in Tables S2 and S3.

Figure 2.. Consequences of mixture exposure on CD4⁺ T-cells during viral infection.

Starting at 6 wk-of-age, C57Bl/6 mice were placed on drinking water containing a mixture of 23 chemicals or containing vehicle control, and 10 female and 10 male mice from each exposure group were infected with IAV at least 8 wk later (see Figure 1). CD4⁺ T-cell responses were measured 9 days and 8 days after infection in, respectively, the female and male mice. Cell suspensions of MLN cells were prepared and stained for flow cytometry. CD4⁺ T-cells were defined as CD3⁺CD4⁺; CD4⁺ T-cell subsets were further defined using the following markers: TBet⁺ (T_H1 cells), RORγt⁺ (T_H17 cells), PD1⁺CXCR5⁺ (T_fh cells), and Foxp3⁺CD25⁺ (T_reg cells). Bar graphs shows numbers of (A,F) CD4⁺ T, (B,G) T_H1, (C,H) T_H17 (C,H), (D,I) T_reg, and (E,J) T_fh cells in, respectively, infected female and male mice. *Significantly different compared to same sex control mice (p < 0.05; Student’s t-test). Data presented as means ± SEM. Numerical values that correspond to graphs as well as p-values for each comparison are listed in Tables S2 and S3.

Figure 3.. EAE disease symptom onset and severity.

Following 8 wk of exposure to chemical mixture or vehicle control, 10 female and 10 male mice from each exposure group were immunized with CFA/MOG_35–55 emulsion. Disease progression was monitored and scored every other day for 42 days. Average disease score for each treatment group over time was determined in (A) female, and (D) male mice. Graphs also depict the day of disease onset (disease score ≥ 1) in (B) female, (E) male, and the average day of onset in (C) female and (F) male mice. 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. *Significantly different compared to control mice (p < 0.05; ANOVA). Data presented as means ± SEM.

Figure 4.. CD4+ T-cell subsets in EAE disease.

Following 8 wk of exposure to the mixture of 23 chemicals or water containing the vehicle control, 10 female and 10 male mice in each exposure group were immunized with CFA/MOG_35–55 emulsion to induce EAE (see Figure 3). All mice were euthanized 42 days after immunization, and cervical lymph nodes then obtained. (A,C,E,G) Mean number and (B,D,F,H) percentage of T_H1 (TBet⁺CD4⁺CD3⁺ cells) and T_H17 (RORγt⁺CD4⁺CD3⁺) cells based on flow cytometry. (I,L) Mean number of T_reg cells (Foxp3⁺CD25⁺ CD4⁺ CD3⁺) in (I) female and (L) male mice. (J,M) Mean T_reg:T_H1 ratios in (J) female and (M) male mice. (K,N) Mean T_reg:T_H17 ratios in (K) female and (N) male mice. *Significantly different compared to control mice (p < 0.05; Student’s t-test). Data presented as means ± SEM. Numerical values that corre-spond to graphs as well as p-values for each comparison are listed in Tables S4 and S5.

Figure 5.. Effects of exposure to chemical mixture on immune response in allergic airway disease model.

Following 10 wk of exposure to to the mixture or vehicle control, 10 female and 8 male mice in each exposure group were sensitized and challenged with HDM. To assess CD4⁺ T-cell subsets in their MLN, mice were euthanized 10 days after HDM administration. MLN of female mice: mean (A,G) number, (B,H) percentage, and (C,I) ratios of indicated CD4⁺ T-cell types based on flow cytometry. MLN of male mice: mean (D,J) number, (E,K) percentage, and (F,L) ratios of indicated CD4⁺ T-cell types. Airway leukocytes were examined via bronchoalveolar lavage (BAL); mean percentage neutrophil, lymphocyte, macrophage, and eosinophil in BAL from (M) female and (N) male mice. *Significantly different compared to control mice (p < 0.05; Student’s t-test). Data presented as means ± SEM. Numerical values that correspond to graphs as well as p-values for each comparison are listed in Tables S6 and S7.