Developmental trichloroethylene exposure enhances predictive markers of autoimmunity in a sex-specific manner in disease-resistant female mice

Abstract

Trichloroethylene (TCE) is a widely used industrial chemical and common environmental pollutant. Exposure to TCE promotes CD4⁺ T cell-driven autoimmunity including autoimmune hepatitis (AIH) in both humans and female autoimmune-prone mice. Because the developing immune system is more sensitive during development, we predicted that non- autoimmune-prone, C57/Bl6 (B6) mice would exhibit some autoimmune-related changes using the Developmental Origins of Health and Disease (DOHaD) model of exposure. Both male and female mice were exposed to vehicle or an environmentally relevant dose of 5 μg/ml TCE (0.9 mg/kg/day) beginning at 2 weeks pre-conception and ending at weaning. CD4+ T cells were assessed for phenotypic markers by flow cytometry. An assessment of cytokines elicited ex vivo after 4d polarization from naïve to CD4⁺ T helper subsets (i.e., Th1, Th17, and T reg) was conducted. mRNA expression of liver genes associated with inflammation, regeneration/repair associated with AIH disease progression in autoimmune-prone mice were evaluated by qRT-PCR. The results demonstrated TCE’s ability to induce autoimmune- related biomarkers in B6 mice to an even greater degree in females compared to males when exposed during development.

1. INTRODUCTION

Trichloroethylene (TCE) is a chlorinated hydrocarbon best known for its use as an industrial solvent and degreasing agent. Among the noncancer outcomes associated with TCE exposure in humans includes its ability to adversely affect the immune system. Chronic TCE exposure in humans has been linked to autoimmune diseases (ADs) including, lupus, systemic sclerosis, and autoimmune hepatitis (AIH) [(Cooper et al., 2009; Parks and De Roos, 2014; Abbot et al., 2018)]. As most ADs are primarily CD4⁺ T cell-driven, occupational exposure studies in humans demonstrated TCE was associated with altered numbers of naïve and effector-like CD4s (Hosgood et al., 2011) commensurate with decreased serum levels of anti-inflammatory IL-10 and IL-4 and increased levels of proinflammatory IL-2 and IFN-γ reflective of a T helper 1 (Th1) phenotype (Iavicoli et al., 2005; Bassig et al., 2013). To model these observations in humans, we and others have exclusively studied the autoimmune-promoting effects of TCE in the MRL+/+ mouse strain. MRL+/+ mice are derived from intercrossings of several inbred mice to produce a genotype that permits a slow-progressing, spontaneous lupus-like disease and glomerulonephritis (Theofilopoulos and Dixon, 1985). However, TCE exposure accelerates AIH-like, T cell-mediated liver histopathology rather than lupus nephritis (Griffin et al., 2000; Cai et al., 2007).

Although T cell alterations can accompany autoimmune disease, they have been reported to exist prior to autoimmune disease pathology, including AIH in humans (Czaja, 2019). Previous studies in MRL+/+ mice have shown an increase in activated Th1-like CD4s were revealed early in life before the development of liver pathology when the exposure occurred during development (Gilbert et al., 2017a). The appearance of these T cell changes were accompanied by alterations in certain liver genes associated with inflammation and repair (Blossom et al., 2018) even in the absence of AIH-like liver pathology (Gilbert et al., 2009; Gilbert et al., 2014).

The idea that the developing immune system is adversely affected by environmental pollutants leading to autoimmune disease and other immune-mediated inflammatory disorders has gained attention in recent years (Weinstock et al., 2010; Howard, 2018). Therefore, one goal of the present study is to use the DOHaD exposure paradigm to determine whether exposure to TCE during this sensitive timeframe enhances autoimmune-related endpoints in C57/Bl6 (B6) mice. B6 mice represent a common laboratory mouse strain that neither develops autoimmunity nor possesses a genotypic variation common in major lupus-prone mice, including MRL+/+ mice (Pritchard et al., 2000) and are referred to as “disease-resistant.” We predicted that developmental exposure to TCE would promote early markers of disease in B6 mice. While females are more likely to develop ADs, idiopathic ADs appear to be rising in males across the U.S. (Dinse et al., 2020). Therefore, we also included male mice in the assessment of autoimmune-related disease endpoints related to TCE exposure.

2. MATERIALS AND METHODS

2.1. Animals, Treatment, and Experimental Design

This study was conducted at Arkansas Children’s Research Institute and approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences (Protocol # 3985; approval date 03/24/2020). The experimental design is depicted in Figure 1.

Figure 1. Experimental design.

Eight-ten-week-old C57/BL/6 mice were randomly assigned to one of two treatment groups (10 mice/treatment group). Each group consisted of either vehicle or trichloroethylene (TCE). All exposures began during mating (pre-conception) and continued during gestation and lactation. Female and male offspring were weaned at 3 weeks of age [postnatal day (PND) 21)] after which TCE was removed from the drinking water. Male and female offspring were euthanized at 10 weeks of age and tissues/cells assessed for endpoints as described in detail in the Materials and Methods section. This figure was created using software from BioRender.

The study uses the DOHaD approach where TCE is given only during development as described (Gilbert et al., 2017a; Byrum et al., 2019). Briefly, eight to ten-week-old male and female C57BL/6 (B6) mice purchased from Jackson Laboratories were allowed a 2-week acclimatization period in the animal facility and given ad libitum access to standard rodent chow and water prior to TCE exposure. Mice were group-housed in standard cages (4-5 mice/cage). Female B6 mice were randomly assigned to 1 of 2 groups. There were 10 females per group except for the TCE group (n=9), given an unexpected death of a female mouse during shipping. The groups consisted of control mice receiving drinking water mixed with vehicle (i.e., 1% Alkamuls EL-620), the reagent used to maintain TCE into solution, or mice receiving TCE + vehicle in the drinking water. All drinking water was ultrapure unchlorinated (MIlli-Q) to ensure that chlorination or its by-products did not confound the results. The female mice consumed this drinking water for 2 weeks prior to breeding in order to allow acclimatization to the taste of the TCE-containing water +/− vehicle containing mixture. After the 2-week period, two females were housed with one male breeder. The breeding triads then continued their respective drinking water treatment. Thus, male and female offspring used in this study were exposed developmentally to TCE + vehicle or vehicle only for approximately 8-9 weeks; 2 weeks prior to mating (pre-conception), 3 weeks during gestation, and an additional 3 weeks during lactation.

A dose of TCE shown in previous studies to be substantially lower than occupational exposure limits (0 or 5 μg/ml) was administered in the drinking water as previously described (Blossom et al., 2018). Starting at weaning (postnatal day [PND] 21) the resulting male and female pups were taken off their respective treatments and switched to untreated drinking water for an additional 7 weeks at which time the experiment was terminated. During the exposure period, the TCE-containing drinking water was changed 3 times/week to offset degradation of TCE. Non-TCE groups were given water containing only vehicle. The exposure to TCE was strictly pre-natal occurring during pre-conception, gestation and lactation. Thus, in order to estimate TCE exposure, dams were weighed throughout the study in conjunction with monitoring their water consumption. TCE exposure (μg/kg/day) was based on the average amount of TCE-containing water consumed per cage (2-4 mice/cage) divided by average mouse weight per cage and a previously calculated estimate of 20% degradation of TCE in the water bottles.

The day the pups were weaned, the dams were taken off drinking water containing vehicle +/− TCE and provided regular drinking water. After two weeks, the mice were weighed and euthanized for evaluation for endpoints for a separate study. For the offspring, at 10 weeks of age, one randomly selected male or female mouse per litter was selected for experimental endpoints (n=8 for controls and n=7 for TCE-treated group). Mice were euthanized using C02 followed by cervical dislocation, and examined for experimental endpoints as described below.

2.2. Flow Cytometry

Flow cytometric analysis of spleen cells isolated from individual mice was conducted. The phenotypic analysis of 10,000 events/sample was carried out using a Guava EasyCyte 12HT flow cytometer (Millipore Sigma). The data were presented as mean percentage ± SD and representative histograms. Nonviable cells, based on low forward scatter and side scatter, were excluded in each sample. Fluorescence Minus One and isotype Ig controls were included. Antibodies used were purchased from Thermofisher and included PE-anti-mouse B220 (clone RA3-6B2, rat IgG_2a); PerCP Cy5.5 anti-CD8 (clone 53-6.7, rat IgG_2a); FITC anti-CD4 (clone GK1.5, rat IgG_2b); and APC-anti-CD62L (clone F344, rat IgG_2a). All flow data were analyzed using FCS Express Software, version 7 (De Novo Software, Pasadena, CA).

2.3. CD4⁺ T cell isolation and polarization

Naïve CD4⁺ T cells were isolated from spleen cell suspensions using mouse naïve CD4⁺ T cell isolation kits from Miltinyi Biotec per manufacturer’s instruction. All CD4s subjected to subset polarization were resuspended in complete media into 6 well plates at a seeding density of 1x10⁶/ml and stimulated with immobilized anti-CD3 antibody (BioLegend; clone 145-2C11) plus soluble anti-CD28 antibody (BioLegend; clone 37.51) followed by the addition of reagents designed to skew the CD4 cells towards a Th1, Th17, or Treg phenotype according to manufacturer’s specifications from kits purchased from BioLegend. Briefly, for Th1 polarization, anti-mouse IL-4, clone 11B11 (10 μg/ml), recombinant mouse IL-2 (5 ng/ml) and recombinant mouse IL-12 (10 ng/ml) were added to the cultures. To promote differentiation into Th17 subsets, recombinant mouse IL-6 (50 ng/ml), recombinant human TGF-β (1 ng/ml), recombinant mouse IL-23 (5 ng/ml), anti-mouse IL-4 (10 μg/ml), and anti-mouse IFN-γ (10 μg/ml) were added to the cultures. For regulatory T cell (T reg) polarization, the cultures were treated with recombinant mouse IL-2 (5 ng/ml) and recombinant human TGF-β1 (5 ng/ml). On day 4, cells were harvested, and RNA were isolated for qRT-PCR assessment for subset-specific gene expression.

2.4. RNA isolation for qRT-PCR

RNA from the polarized CD4 cells were isolated as described using RNeasy Kits from Qiagen. Purity was examined on the NanoDrop 2000c for an A260/A280 range of 1.8-2.0. RNA isolated from both CD4s and liver tissue was reverse-transcribed into cDNA using the High Capacity cDNA Reverse transcriptase kit (Qiagen). mRNA was quantified using Applied Biosystems Custom Primers and SYBR Green Master Mix and run on ABI prism. Samples were run in duplicate and averaged to obtain mean fold change expression level of the subset-specific genes for Th1 (IFN-γ), Th17-(IL-17), and T reg (Foxp3) and compared with the housekeeping control gene (GAPDH) using 2^ΔΔCt method.

Flash frozen liver obtained at study terminus was homogenized with Trizol reagent (Life Technologies) and subjected to chloroform and 2-isopropanol treatment to extract RNA according to established protocols. After centrifugation, pellets were washed with 70% ethanol and dried for 1 min. After dissolving in nuclease-free water, RNA was stored in RNAse-free microcentrifuge tubes at −80°C until qRT-PCR.

2.5. qRT-PCR

mRNA levels were analyzed by qRT-PCR using RNA isolated from polarized CD4 subsets and liver using the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems). mRNA was quantified by qRT-PCR using predesigned TaqMan Gene Expression Assays (ThermoFisher). Samples were run in duplicate and averaged to obtain a mean fold change expression level expressed as mean (SD) fold-change values relative to unpolarized CD4 cells and normalized by GAPDH housekeeping gene. For liver, the fold change was calculated from one untreated control sample and normalized by GAPDH.

2.6. Statistical Analysis

Because the exposure occurred during development, the litter was considered to be the experimental “n.” Once mice were weaned at PND21 they were reassigned to cages according to their treatment group and identified based on their litter. Summary statistics such as mean and SD are presented for each treatment group. Data were initially evaluated with a two-way ANOVA with interactions to determine the overall effects of TCE and sex (main factors) and the interaction between them (Table 1). Comparisons of treatment means were carried out in the absence of statistically significant interaction effects as described (Wei et al., 2012). All analyses were followed by Tukey’s post-hoc tests to protect the overall significance level of 0.05. In order to compare responses within treatment (control vs. TCE) and within sex (male vs. female), four pre-specified pairwise comparisons included: 1) male control vs. female control; 2) male control vs. male TCE; 3) female control vs. female TCE, and 4) female control vs. female TCE. Statistical significant resulting from these comparisons are reported in graphs and tables. All analyses were completed in GraphPad Prism 9.0 (La Jolla, CA).

Table 1.

Two-factor ANOVA: Effects of sex and TCE treatment and their interaction.

	Interaction	Sex	TCE
Splenic immune cell subsets
% B cells (B220+)	0.72	0.001	0.97
% CD8 cells	0.56	0.14	0.13
% CD4 cells	0.28	0.005	0.54
% CD62Llo CD4 cells	0.03	0.006	0.43
Liver mRNA expression
Ccl5	0.05	0.04	0.0004
Ccl2	0.16	0.21	0.001
Tgfb	0.17	0.28	0.003
Tnfa	0.02	0.07	0.0001
IL1b	0.42	0.35	0.0003
Il6r	0.04	0.007	0.002
Il6	0.02	0.0005	0.01
GP130	0.0001	<0.0001	<0.0001
Egr1	0.93	0.76	0.01
Cyclind1	<0.0001	<0.0001	<0.0001
Ki67	<0.0001	<0.0001	<0.0001
Th1 mRNA expression
IFNg	<0.0001	<0.0001	<0.0001
Th17 mRNA expression
IL17	0.28	<0.0001	<0.0001
Treg mRNA expression
Foxp3	0.74	<0.0001	<0.0001

TCE, trichloroethylene.

Bold values indicate that the comparison is statistically significant, p < 0.05.

3. RESULTS

3.1. Pregnancy and Birth parameters

Neither terminal body weights nor a longitudinal evaluation of body weight during the treatment period revealed any statistically significant differences in the dams approximately 2 weeks after their exposure. As shown in Table 2, there were no statistically significant differences in terminal body weights between the TCE-exposed and control groups (both dams as well as offspring). In addition, TCE exposure did not appear to affect breeding effectiveness. The control group (n=10) had 80% pregnancy success as defined by 8/10 dams producing live litters compared to 77% pregnancy success rate in the TCE exposure group (7/9). The total number of offspring from all the litters equaled 42 pups from the control group and 37 pups from the TCE-exposure group. Interestingly, the average litter sizes were relatively small, (i.e., 5.25 pups/litter in controls vs. 5.28 pups/litter with TCE exposure. However, a recent study comparing litter sizes under different conditions among several inbred mouse strains showed that B6 mice generate anywhere between 5-11 pups per litter (Finlay et al., 2015). Importantly, TCE did not appear to play a role in litter size or any other birth parameters since litter sizes were similar between the groups. Terminal body weights were also similar between TCE and control groups.

Table 2.

Summary of Maternal and Offspring Data

	Control	TCE	P value (TCE vs. control)
Dams
Total no. dams	10	9
Total no. litters	8	7
Maternal terminal body weight (g)	25.0 (1.61)	25.7 (1.98)	0.23
Offspring
Litter size (no. of pups born)	5.25 (2.81)	5.28 (1.97)	0.97
Mean no. of females/litter	2.375 (1.84)	3.0 (2.16)	0.56
Mean no. of males/litter	2.875 (1.80)	2.29 (0.75)	0.42
Male offspring terminal body weight (g)	21.27 (4.09)	18.92 (1.88)	0.11
Female offspring terminal body weight (g)	23.77 (5.02)	19.41 (1.88)	0.09

Summary statistics of maternal and offspring data by treatment group. Unpaired t tests with Welch’s correction used to compare percentages represented as mean (SD) did not indicate statistical significance (p<0.05) when compared with controls. Where applicable, further assessment by two-factor ANOVA to test for main effects (sex x TCE treatment) did not reveal statistically significant differences in main effects or their interaction.

3.2. Higher percentage of CD62L^lo effector-like CD4s in TCE-exposed female mice compared to TCE-exposed males

Splenic T and B lymphocytes were immunophenotyped using flow cytometry. As shown in Figure 2A, the percentage of B cells from female mice that were exposed to TCE were significantly lower compared to B cells from male mice exposed to TCE (p=0.04). There were no statistically significant differences in the percentage of B cells when comparisons were made within each sex [female control vs. female TCE mice (p=0.99) and male control vs. male TCE exposure groups (p=0.99)], or between male and female control groups (p=0.08). A similar pattern was observed in CD4+ T cells. When comparing means, there were ~19% fewer CD4⁺ T cells from TCE-exposed females relative to TCE-exposed males (p=0.03). There were no statistically significant differences in the percentage of CD4 + T cells when comparisons were made within each sex [female control vs. female TCE mice (p=0.60) and male control vs. male TCE exposure groups (p=0.98)], or between male controls vs. female controls (p=0.31). In addition, there were no statistically significant differences in the mean percentage of CD8⁺ T cells among the groups.

Figure 2. TCE increased CD4 T cell activation markers in female B6 mice.

Spleen cells from individual male and female offspring (+/− TCE) were four-color stained with PE-anti-B220, PerCP Cy5.5 anti-CD8, FITC anti-CD4, and APC anti-CD62L and subjected to flow cytometry and analyzed as mentioned in materials and methods. (A) Percent of splenic B cells (B220, CD4 cells, CD8 cells. The percentage of CD62L^lo CD4⁺ T cells was determined by gating on the CD4+ cell population in each sample. The bars represent mean (SD) percent positive values. (B) Histograms represent the CD62L fluorescence intensity of one sample/group obtained from the graph showing %CD62L positive CD4 cells. Data were analyzed by two-way ANOVA to test for interaction and main effects of the two variables [trichloroethylene (TCE) and sex] and reported in Table 1. Shown in the graphs are p-values indicating statistical significance after pairwise Tukey’s post hoc analysis comparing the means in each group (**p<0.01, *p<0.05).

TCE-treated groups were different in terms of the percentage of activated or effector-like CD4+ T cells defined as CD62L^lo. TCE exposed females had 57% percent more effector-like CD4s (e.g., CD62L^lo) compared to TCE exposed males (p=0.007). There were no statistically significant differences when comparisons were made within each sex [female control vs. female TCE mice (p=0.16) and when comparing male control vs. male TCE exposure groups (p=0.69)], or between male controls vs. female controls (p=0.89). Figure 2B shows representative histograms taken from a data point in the graph of CD62L fluorescence intensity after gating on the CD4 T cells. In these samples, there were 31.93% CD62L^lo CD4s from control mice compared to 21.93% in TCE-treated mice from males. In contrast, CD4s from female mice had 52.77% CD62L^lo CD4s in the TCE group compared to only 26.06% in the control group.

3.3. TCE enhances Th1 polarization in female, but not male, mice

Naïve CD4⁺ T cells were purified from spleens and polarized using Th differentiation reagents designed to skew the cells towards a Th1, Th17, or Treg phenotype as described in the methods. The key genes that define each subset (i.e., IFN-γ for Th1, IL-17 for Th17, and the transcription factor, Foxp3 for T regs) were assessed by qRT-PCR on day 4 after polarization. As shown in Figure 3, the genes in each of the Th subsets were increased in all groups relative to undifferentiated CD4⁺ T cell controls as indicated by the dotted line on the graph. In terms of IFN-γ expression, there were significant within treatment and within sex differences. TCE enhanced the expression of IFN-γ in Th1 cells differentiated from female mice over that of Th1 cells from female controls by 4-fold (p<0.0001), and TCE-exposed males by 2.7-fold (p<0.001). However, unlike what was observed in females, there were no statistically significant differences in IFN-γ expression between TCE- and control-treated males (p=0.31).

Figure 3. TCE enhanced subset-specific cytokine production and generation of T regs in CD4 Th effectors when differentiated from naïve CD4+ T cells ex vivo.

CD4 T cells were purified from spleens using magnetic bead separation and incubated with reagents designed to polarize the cells towards Th1, Th17, and T reg cells as described in methods. After day 4, cells were harvested and processed for RNA and gene expression by qRT-PCR. Numbers in the bar graphs represent mean (SD) fold-change values relative to unstimulated CD4 cells and normalized by GAPDH housekeeping gene. Data were analyzed by two-way ANOVA to test for interaction and main effects of the two variables [trichloroethylene (TCE) and sex] and reported in Table 1. Shown in the graphs are p-values indicating statistical significance after pairwise Tukey’s post hoc analysis comparing the means in each group (****p<0.0001, ***p<0.005, **p<0.01, *p<0.05).

When the naïve CD4s were polarized to Th17 cells, the effect of TCE was retained in females, but also observed in males. Naïve CD4s from TCE-exposed male mice polarized to Th17 cells expressed 2-fold higher levels of IL-17 relative to control males (p=0.0014). Interestingly, polarized CD4s from female control mice had significantly lower levels of IL-17 compared male control (p=0.007). Yet, TCE exposure still significantly increased the expression in of IL-17 in females by 5-fold (p=0.04; compared to female controls). The effect of TCE appeared to be more robust in males. Male mice exposed to TCE had significantly higher IL-17 than female mice exposed to TCE (p=0.0002).

Interestingly, while expression of foxp3 was higher overall in the polarized T reg subsets compared to IFN-γ and IL-17 expression in Th1 and Th17 cells, respectively, the pattern was similar to that of IL-17 from polarized Th17 cells. Treatment differences within sex were observed. TCE significantly enhanced the expression of foxp3 in both males and females (by 1.7-fold in males and by 7.4-fold in females relative to male and female controls; p=0.01 and 0.004, respectively). Despite this robust increase by TCE exposure in females, the female control mice had significantly lower levels of Foxp3 compared to control male mice (p=0.002 vs. male controls). Likewise, TCE exposed males had higher levels of Foxp3 than TCE-exposed females (p=0.006).

3.4. Expression of liver biomarkers of inflammation and repair are higher in TCE-exposed females relative to males.

We assessed the expression of liver cytokines and chemokines associated with inflammation and autoimmunity including chemokines [C-C motif ligand 5 (ccl5)]; also known as RANTES, [C-C motif ligand 2 (ccl2)] known as monocyte chemotactic protein or MCP-1), and cytokines IL-1β, TNF-α, and TGF-β. There were striking treatment-related differences among all of the liver markers in females but not males. As shown in Table 3, TCE increased the expression of CCl2, CCl5, TNF-α, IL-1β, and TGF-β by 3.9-, 2.5-, 3.1-, 3.0-, 2.2-fold, respectively (TCE-treated vs. control females). However, there were no significant differences in TCE-treated vs. control-treated males. Within treatment differences were detected in only two liver markers where TCE exposure increased CCL-2 and TNF-α by 1.7-fold compared to expression in TCE-exposed males. There were no differences in the expression of these markers between male and female controls.

Table 3.

TCE increased liver cytokine/chemokine expression in female mice

					Comparing treatment effects within sex		Comparing sex effects within treatment group
	Control male (CM)	TCE male (TM)	Control female (CF)	TCE female (TF)	CM vs. TM	CF vs. TF	CM vs. CF	TM vs. TF
Ccl2	1.25 (0.78)	2.73 (0.38)	1.13 (0.41)	4.41 (2.82)	0.34	0.006	0.99	0.29
Ccl5	1.13 (0.55)	1.73 (0.43)	1.20 (0.76)	2.98 (0.53)	0.42	0.001	0.99	0.02
Tnfα	1.20 (0.64)	1.82 (0.35)	1.01 (0.61)	3.09 (0.82)	0.42	0.0003	0.95	0.03
Il1β	1.15 (0.53)	2.81 (0.56)	1.20 (0.49)	3.63 (2.02)	0.08	0.007	0.99	0.63
Tgfβ	1.22 (0.73)	1.71 (0.46)	1.16 (0.69)	2.60 (1.21)	0.77	0.04	0.06	0.36

TCE, trichloroethylene.

Numbers represent mean (SD) fold-change. p values are shown comparing treatment and sex effects as indicated. Bold p values indicate the comparison is statistically significant, p<0.05.

Components of the IL-6 signaling complex known to participate in regeneration and repair were also assessed at the mRNA level in liver. Significant within treatment and within sex in female mice were detected. As shown in Figure 4, TCE exposure increased the expression of IL-6 receptor (IL-6R), IL-6, and gp130 levels in female mice relative to female controls (p=0.001, p=0.008, and p<0.0001, respectively). Likewise, female mice exposed to TCE had significantly higher levels of these markers as compared to TCE-exposed male mice (p=0.004, p=0.001, and p<0.0001; IL-6R, IL-6, and gp130, respectively). In contrast, TCE exposure had no significant effect in males when compared with control males (p=0.70 for IL-6R; p= 0.99 for IL-6, and 0.81 for gp130). Likewise, no significant differences were observed between control males vs. control females (p=.0.89, p=0.59, 0.96; IL6R, IL-6, and gp130, respectively). Despite difference in other IL-6R signaling components, Egr1 expression was not significantly different among the groups.

Figure 4. Liver genes related to IL-6 signaling were increased in TCE-exposed females, but not males.

Expression of liver genes were measured by qRT-PCR as described in Materials and Methods. Results are represented in the graphs as mean (SD) fold-change. Data were analyzed by two-way ANOVA to test for significant interaction and main effects between the two variables (TCE and sex) as shown in Table 1. Shown in the graphs are p-values indicating statistical significance after pairwise Tukey’s post hoc analysis comparing the means in each group (****p<0.0001, ***p<0.005).

Because inflammation and liver regeneration are linked to increased proliferation and cell cycle progression, Ki-67 and Cyclin D1 mRNA expression was assessed in liver samples at the mRNA level (Núñez et al., 2017). Ki67 transcripts were again significantly increased in female mice exposed to TCE by 6.0-fold compared to female controls and by 6.5-fold compared to TCE-exposed males (Figure 5). Cyclind1 mRNA in liver was significantly increased by 6.3-fold in TCE-exposed female mice relative to control females and by 8.2- fold compared to TCE-exposed males. There were no significant within treatment effects in males (p=0.99 and p=0.98; ki67 and cyclind1, respectively), and responses between female and male controls did not differ (p=0.99 and 0.98 for Ki67 and Cyclind1, respectively).

Figure 5. Liver genes related to proliferation and cell cycle progression were increased in TCE-exposed females, but not males.

Expression of liver genes were measured by qRT-PCR as described in Materials and Methods. Results are represented in the graphs as mean (SD) fold-change. Data were analyzed by two-way ANOVA to test for significant interaction and main effects between the two variables (TCE and sex) as shown in Table 1. Shown in the graphs are p-values indicating statistical significance after pairwise Tukey’s post hoc analysis comparing the means in each group (****p<0.0001).

4. DISCUSSION

The goal of this study was determine whether autoimmune disease-related biomarkers commonly observed prior to the onset of liver pathology in TCE-exposed autoimmune-prone mice were similarly altered in autoimmune-resistant TCE-exposed B6 mice when exposure occurred during development. Because of the sensitive window of exposure, it was predicted that B6 mice would exhibit some early signs of autoimmunity, and perhaps the responses would be more robust in females. These predictions were confirmed. The reason for this sex difference in responses remains unclear but may be attributable to sex- hormones and responses to oxidative stress and inflammation in males vs. females. Further study is warranted to investigate sex-specific effects of TCE on immune-related endpoints and autoimmune disease.

Purified, naïve CD4 cells incubated with reagents to promote polarization ex vivo were examined in the current study. CD4⁺ T cells differentiate in vivo to many signals including pathogens. However, inappropriate activation and differentiation of CD4⁺ T cells differentiated CD4⁺ T cell subsets are related to autoimmunity. For instance, elevated frequencies of both Th1 and IL-17-secreting Th17 cells and a corresponding reduction of anti-inflammatory T reg numbers have been observed in both patients and mouse models of autoimmune disease. [Reviewed in (Pawlak et al., 2020)]. Therefore, the Th CD4⁺ subset phenotype can shape the nature of the immune response which, when dysregulated, can have detrimental consequences to human health, including Th1-and/or Th17-driven autoimmune responses. CD4⁺ T cell differentiation events are time-dependent, temporal, and difficult to elucidate in vivo, in part due to the short half-life of effector CD4⁺ T cells (van den Ham et al., 2013). For instance, previous transcriptomic analyses revealed IFN-γ (in 24h activated, unpolarized cells) was increased after 22 weeks of TCE exposure (Gilbert et al., 2016) yet was decreased at 40 weeks relative to controls (Gilbert et al., 2017b). This response is consistent with several studies documenting time-dependent inflammatory mediator fluctuations in autoimmune mouse models (Kuerten et al., 2010). This effect has also been shown in humans with autoimmune disease and correlates with early disease phase followed by a temporary recovery and then clinical relapse (Ryden et al., 2009). The biphasic response corresponding to the IFN-γ production by effector-like CD4⁺ T cells that we have previously observed in in TCE-treated mice may reflect a temporary compensatory response that is eventually overcome after continued exposure. This nonlinear response underscores potential difficulties with studying purified terminally differentiated cells isolated at one time point. Thus, in the current investigation, we predicted that a more accurate depiction of CD4⁺ T cell function would be gleaned by studying the gene expression patterns generated during differentiation from naïve to effector CD4⁺ T cells, rather than comparing expression levels in freshly isolated/already-differentiated CD4s. Our results showed that TCE had a significant effect on Th1, Th17 and Treg differentiation when polarized ex vivo. Studies in our lab that include a much more comprehensive assessment of other genes that may be altered by TCE exposure in differentiating Th cells using RNA sequencing methods in male and female mice and comparing autoimmune-prone and –resistant strains of mice are currently underway. Indeed, a more extensive kinetics study would need to be conducted in order to capture changes in gene expression over the time course of differentiation. Limitations included the evaluation of polarized subsets were only assessed at the gene expression level and, perhaps more importantly, not evaluated functionally. In addition, the CD4⁺ T cells were examined phenotypically using freshly isolated spleen cells with a limited number of markers. Future studies are planned to better characterize the immune cell populations in B6 mice exposed to TCE and to polarize the naïve CD4s prior to adoptive transfer in autoimmune mice to evaluate their impact on the course of disease.

We have previously used a panel of biomarkers to evaluate hepatic inflammatory and regenerative events in TCE-mediated liver injury over time [4-40 weeks of exposure (Gilbert et al., 2014)]. One of the more striking findings in this study was the effect of TCE on expression of cytokine and chemokine genes in the liver, as well as components of the IL-6 signaling complex involved in regeneration and repair. Chemokines are known to be increased in many different liver injury models including AIH and are important in recruiting immune cells to the liver (Chi et al., 2018). Responses to liver injury including hepatocellular carcinoma (Salama et al., 2019), Nonalcoholic fatty liver disease (Dey et al., 2019), chronic alcohol exposure (Chavez et al., 2011) and AIH (Huang et al., 2017) are also linked to proliferation and cell cycle progression. TCE-exposed females expressed higher levels of the proliferation and cell cycle markers, Ki67 and Cyclind1, relative to all other groups. Collectively, the results from the current study showed that B6 females, relative to B6 males, are more sensitive to TCE based on their expression of liver biomarkers. Based on these findings and the two-factor ANOVA results (Table 1), the significant interaction effect suggests that the actions of TCE and sex exert their effects additively rather than separately, and that female mice exposed to TCE showed the greatest alterations in the expression of liver genes important in inflammation, repair, and proliferation.

We and others have shown that adult mice chronically exposed to TCE demonstrated liver histopathology in the form of lymphoplasmacytic portal infiltrate and lobular inflammation with CD4⁺ T cells (Griffin et al., 2000; Gilbert et al., 2009; Chi et al., 2018). While CD4⁺ T cells participate in this response, other factors are likely responsible for this pathology. TCE is metabolized in the liver by cytochrome P450 (CYP) 2E1 or CYP2E1 and reports have described involvement of CYP2E1 in TCE-induced liver injury (Luo et al., 2018), including a recent study demonstrating enhanced serum anti-CYP2E1 in workers with occupational trichloroethylene hypersensitivity syndrome. (Nakajima et al., 2022). We did not assess liver histopathology since these effects are typically observed much later in older mice after chronic exposure to TCE (Griffin et al., 2000; Gilbert et al., 2017a). The mice in this study were only 10 weeks of age at study terminus, and would not be expected to exhibit signs of AIH liver pathology or serological marker of antibodies in the form of anti-liver antibodies against microsomal liver protein. Instead, we used increased mRNA expression of IL-6 signaling components as a biomarker of events in the liver that precede AIH pathology in our model (Gilbert et al., 2014).

One limitation of the current study, and many studies of rodent models, is extrapolating studies of TCE exposure in rodents to real human exposures and health outcomes. However, studies in rodents can identify potential underlying mechanisms of functional alterations observed in animals that will lead to potential functional endpoints and potential testable, therapeutic interventions humans. Furthermore, we administered TCE in a human relevant, low-dose drinking water exposure paradigm that recapitulates human exposure. Mice were given a low dose of TCE (5 μg/ml or approximately 5000 ppb) in the drinking water. This dose should yield a total mouse exposure approximating possible human environmental exposure. While pups were only exposed to TCE indirectly during pre-gestation, gestation, and lactation, the calculated TCE consumption in the dams averaged 0.93 mg/kg/day. To put the dose into perspective, the 8-h permissible exposure limit for occupational exposure to TCE has been established by the Occupational Safety and Health Administration at 76/mg/kg/day, and that also includes pregnant and lactating women. Although the dose in the current study is higher than the maximum contaminant level (MCL) for TCE in municipal water supplies (5 ppb or 0.005 μg/ml), it is not entirely outside the realm of actual human environmental exposure to TCE as exceedance of the MCL has been reported.

In summary, our findings have important implications for human health in terms of understanding sex-dependent responses to TCE in mice that do not develop spontaneous autoimmunity. These findings also underscore the need for additional studies that employ the DOHaD approach that will lead to additional mechanistic studies to understand how effects these effects persist even after toxicant exposures are discontinued. This finding could pave the way for future studies to evaluate the impact of gene/toxicant interactions using knockout mice that are widely available on the B6 background.

HIGHLIGHTS.

• Developmental exposure to Trichloroethylene promotes markers of autoimmunity in B6 mice

• Trichloroethylene enhanced Th1 polarization in female mice.

• Trichloroethylene increased liver markers that precede autoimmunity in female B6 mice.