Trichloroethylene Alters Th1/Th2/Th17/Treg Paradigm in Mice: A Novel Mechanism for Chemically Induced Autoimmunity

Abstract

The role of environmental factors in autoimmune diseases has been increasingly recognized. While major advance has been made in understanding biological pathogen-induced autoimmune diseases, chemically triggered autoimmunity is poorly understood. Trichloroethylene (TCE), a common environmental pollutant, has recently been shown to induce autoimmunity. This study explored whether TCE could cause imbalance of T helper (Th) cell subsets which would contribute to the pathogenesis of TCE-induced medicamentosa-like dermatitis. BALB/c mice were treated with TCE via drinking water at doses of 2.5 or 5.0 mg/mL for 2, 4, 8, 12, and 16 weeks. Trichloroethylene exposure caused time- and dose-dependent increase in Th1, Th2, and Th17 and decrease in regulatory cell (Treg) in the spleen at 2, 4, 8, 12, and 16 weeks, with greatest changes mainly at 4 weeks. These effects were mirrored by similar changes in the expression of their corresponding cytokines interferon-γ, interleukin 4 (IL-4), IL-17A, and IL-10. Mechanistically, these phenotypic changes were accounted for by alterations to their respective master transcription factors T-box expressed in T cells, GATA-binding protein 3, Retinoic acid-related orphan receptor ct (RORct), and forkhead box P3. Of interest, TCE treatment shifted the ratios of Th1/Th2 and Th17/Treg; specifically, TCE increased Th17/Treg. These findings provide the first evidence that TCE exposure significantly changes the Th1/Th2/Th17/Treg paradigm and their specific cytokines driven by altered master transcription factors. This may promote autoimmune reactions in the pathogenesis of TCE-induced skin sensitization and associated damage to other tissues.

Keywords

trichloroethylene Th1/Th2/Th17/Treg paradigm IFN-γ IL-4 IL-17A IL-10

Introduction

The role of environmental factors in autoimmune diseases (AD) has been increasingly recognized.¹ While major advances have been made in understanding biological pathogen-induced AD, little is known about the autoimmunity caused by chemicals in the environment and workplace. One such chemical, which has public health significance and, recently, immunological importance, is trichloroethylene (TCE).

Trichloroethylene is a volatile organic solvent, widely used in industry as a degreaser and cleaning agent^2–3 and has become a major environmental pollutant and occupational hazard.^4,5 Trichloroethylene has been reported to cause multisystem damage including skin, liver, kidney and other organs, and nervous system through respiratory tract and skin exposures.⁶ Of special note, its skin lesion, occupational dermatitis medicamentosa-like of TCE (ODMLT) has become a major health problem in TCE-exposed workers,^7–8 and many cases of ODMLT have been reported in those regions with high TCE use.⁹ Occupational dermatitis medicamentosa-like of TCE is accompanied by severe liver and kidney injury, and even death.

Trichloroethylene-induced skin lesion ODMLT has been considered as a type IV allergic reaction mediated by antigen-specific T lymphocytes.^7,8 However, sensitization cannot fully explain the mechanism of injury.^10
–12 Recent studies show that TCE exposure, even at low level, is associated with the development of AD, suggesting TCE may cause immunological system damage.¹³ However, the immune mechanisms, especially the role of T-cell subtypes, remain unclear.^14
–16

Based on the surface phenotype and functional characteristics, the primary CD4⁺ T cells can be classified into T helper 1 (Th1), Th2, Th17, and Treg subsets.¹⁷ T helper 1 cells mainly secrete interferon-γ (IFN-γ), while Th2 cells produce interleukin-4 (IL-4) and IL-5, forming the major pro- and anti-inflammatory pair.¹⁸ T helper 17 cells release IL-17A (IL-17), IL-17F, IL-21, IL-22, and IL-23, which are involved in autoimmune response.¹⁹ Treg-related cytokines include IL-10 and transforming growth factor-β and play a major role in immunosuppression.²⁰ With the discovery of Th17 and Treg cells, the concept of Th1/Th2 counteracting balance has been expanded into the Th1/Th2/Th17/Treg paradigm in determining the net outcome of multifaceted immune responses. The Th1/Th2/Th17/Treg paradigm plays a crucial role in autoimmune processes, which has begun to be appreciated in immune diseases associated with exposure to environmental chemicals.

In our previous study, we found that the balance of Th1-specific cytokine IFN-γ and Th2-specific cytokine IL-4 was altered in TCE-sensitized BALB/c mice.²¹ We hypothesize that the imbalance of Th cell subsets in immune responses induced by TCE exposure contributes to the pathogenesis of ODMLT. The objective of the present study was, therefore, to explore changes inTh1/Th2/Th17/Treg paradigm in TCE action by examining these T-cell subsets in TCE-exposed mice.

Materials and Methods

Chemicals

Chemicals and reagents used and their sources were as follows: TCE (Sigma, San Francisco, USA); dimethyl sulfoxide (DMSO; Amresco, Washington, USA); anti-IFN-γ antibody, anti-IL-4 antibody, anti-IL-17A antibody, AND anti-IL-10 antibody (Santa Cruz Biotechnology, California, USA); Histostain Plus kit and Diaminobenzidine (DAB) substrate kit (ZSJQ,Shanghai, China); anti-mouse Fluorescein isothiocyanate(FITC)-conjugated CD3, anti-mouse P-phycoerythrin (PE)-Cy5-conjugated CD4, anti-mouse PE-conjugated IFN-γ, anti-mouse PE-conjugated IL-4, anti-mouse PE-conjugated IL-17A, anti-mouse PE-conjugated forkhead box P3 (Foxp3), and anti-mouse PE-conjugated IgG (eBioscience, California, USA); anti-mouse FITC-conjugated CD25 and Monensin Solution (Biolegend, San Diego, USA); IntraPrep Permeabilization Reagent (Beckman Coulter, Brea, USA); phorbol myristate acetate (PMA) and ionomycin (Sigma, San Francisco,USA); Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo, Waltham, USA); and LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland).

Animals and Treatment

Healthy and specific pathogen-free female BALB/c mice aged 7 to 9 weeks were obtained from Hunan Slac Jingda Laboratory Animal Co, Ltd (Hunan, China). The use of animals and the experimental protocol were approved by the Animal Care and Use Committee of Anhui Medicine University. All mice were offered standard lab chow and drinking water ad libitum, fed at 20°C and 50% to 60% relative humidity, and with a 12-hour light and 12-hour dark cycle. Trichloroethylene was dissolved in drinking water containing 1% DMSO. After a week of acclimation, 100 mice were randomly divided into blank control group, vehicle control group (drinking water containing 1% DMSO), 2.5 mg/mL TCE group, and 5.0 mg/mL TCE group. Each group contained 25 mice. The water was changed on alternate days to ensure dose maintenance. In the current study, TCE exposure was calculated from water consumption measured throughout the study. The mice were weighed once per week. After 2, 4, 8, 12, and 16 weeks of TCE treatment, respectively, blood was collected from ocular venous plexus; the mice were euthanized, and the spleen was quickly removed and weighed. One portion of spleen from control and TCE-treated mice was frozen for real-time quantitative polymerase chain reaction (RT-PCR). Another portion of spleen was fixed in 10% neutral-buffered formalin for 48 hours and embedded in paraffin. A further portion of spleen was used to isolate splenocytes. After the spleen was gently ground, the splenocytes were obtained after 200 mesh gauze filtration. And these cells were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented 10% fetal bovine serum.

Body Weight and Relative Organ Weight

The absolute weight of the spleen and the body weight (in grams) were directly measured. The relative organ weight (spleen to body weight ratio) was calculated as follows: relative spleen weight = (weight of spleen/body weight of the mouse on the day of euthanasia) ×100%.

Immunohistochemistry for IFN-γ, IL-4, IL-17A, and IL-10 in Spleen

For immunohistochemistry, the spleen sections were deparaffinized in xylene and rehydrated through a series of graded alcohols. In order to inhibit the endogenous peroxidase, the spleen sections were incubated with 3% H₂O₂ for 30 minutes. For antigen retrieval, the sections were put into 0.01 M sodium citrate buffer (pH = 6.0) in a microwave oven for 15 minutes. Then the sections were rinsed with phosphate buffered solution(PBS) after natural cooling. After blocking nonspecific binding with 5% normal rabbit serum in PBS for 15 minutes, anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-IL-10 antibodies (diluted 1:500) were added to the sections and incubated at 4°C overnight. On the second day, the sections were processed for peroxidase reactions according to the instructions of Histostain Plus kit and DAB substrate kit. Sections were counterstained with hematoxylin. Each tissue section was assessed on a score as follows: 0, the positive cells lower than 5%; 1, lower than 25%; 2, lower than 50%; 3, lower than 75%; and 4, greater than 75%. The staining intensity under the microscope was also graded as follows: 0 (no positive reaction), 1 (light brown), 2 (moderate brown), and 3 (dark brown). The final scores of immunoreactions were the combination of the score of positive reaction area and the score of reaction intensity. Five fields were selected in each section to assess the positive results. All sections were observed and scored by 3 pathologists.

Measurement of Th1, Th2, Th17, and Treg Cells in Spleen by Flow Cytometry

Suspensions of splenocytes were stimulated with 50 ng/mL PMA, 1 mg/mL ionomycin, and 5 mg/mL monensin at 37°C and 5% CO₂ for 8 hours. Splenocyte suspensions were washed in PBS and incubated with anti-mouse FITC-conjugated CD3, anti-mouse PE-Cy5-conjugated CD4, and anti-mouse FITC-conjugated CD25 for 20 minutes. Splenocytes were then fixed and permeabilized using IntraPrep Permeabilization Reagent. Intracellular cytokines (anti-mouse PE-conjugated IFN-γ, IL-4, IL-17A, and Foxp3) were stained for 20 minutes. Suspensions of splenocytes were analyzed by Beckman Coulter FC500 Flow Cytometer.

Determination of Transcription Factor Expression by RT-PCR

In order to determine the effect of TCE exposure on messenger RNA (mRNA) expression of master transcription factors for Th1/Th2/Th17/Treg differentiation, the mRNA levels of T-box expressed in T cells (T-bet), GATA3, Retinoic acid-related orphan receptor c(RORC), and Foxp3 were analyzed by RT-PCR. Total RNA was extracted from the spleen using Trizol reagent as per manufacturer’s instructions and the concentration and integrity of RNA were measured. RNase-free DNase was used to remove genomic DNA. The complementary DNAs were amplified by PCR with the following primers: mouse T-bet, forward primer 5′-ATTGGTTGGAGAGGAAGCGG-3′, and reverse primer 5′-GCACCAGGTTCGTGACTGTA-3′; mouse GATA3, forward primer 5′-AAGGCAGGGAGTGTGTGAAC-3′, and reverse primer 5′-TCGCTTGGGCTTGATAAGGG-3′; mouse RORC, forward primer 5′-ATCGTAGCCACCAGTACTCAG-3′, and reverse primer 5′-GTTGTGGAAGAACTCTGGGAA-3′; mouse Foxp3, forward primer 5′-TGGAACCACGGGCACTATCACA-3′, and reverse primer 5′-GAGGCTGCGTATGATCAGTTATGC-3′; GAPDH, forward primer 5′-ACCCCAGCAAGGACACTGAGCAAG-3′, and reverse primer 5′-GGCCCCTCCTGTTATTATGGGGGT-3′. Amplification program was as follows: initial denaturation was at 95°C for 10 minutes; then 45 cycles were started, including 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 20 seconds. The data analysis was performed using the Roche LightCycler 480 software(LightCycler480 software release 1.5.0).

Statistical Analysis

All data are presented as mean (standard deviation). Analysis of variance followed by the Student-Newman-Keuls test in SPSS version 16.0 was used for comparison among more than 2 groups. A value of P < 0.05 was considered statistically significant. The data of flow cytometry were analyzed by Beckman Coulter software (Beckman FC500).

Results

General Experimental Situation

All animals survived to the end of the study. The average water consumption was 0.12 mL/g body weight in each group. The dose of TCE exposure in 2.5 mg/mL TCE group and 5.0 mg/mL TCE group were 330 mg/kg/d and 620 mg/kg/d, respectively.

Body Weight and Relative Organ Weight of Mice

Compared with blank and vehicle control group, there was an increase of relative spleen weight in TCE high-dose group at 4 and 8 weeks (Figure 1). These findings indicate that the immune organ was enlarged during TCE exposure.

Figure 1.

Relative spleen weight in control and TCE treatment groups. Data are expressed as mean (standard deviation). Note: an increase of the spleen weight at 4 and 8 weeks; ^⋆ P < 0.05 compared with the vehicle control group. TCE indicates trichloroethylene.

Effect of TCE Exposure on Expression of IFN-γ, IL-4, IL-17A, and IL-10 in Spleen

The mean scores of the expression of IFN-γ, IL-4, IL-17A, and IL-10 in the spleen from different groups are shown in Table 1. Figures 2 to 5 are the representative micrographs of each cytokine. Compared with blank and vehicle control groups, the expression of IFN-γ, IL-4, and IL-17A was increased, while the expression of IL-10 was decreased clearly after TCE treatment. In addition, the changes of each cytokine in TCE high-dose group were greater than TCE low-dose group. As IFN-γ, IL-4, IL-17A, and IL-10 are the characteristic cytokines for Th1, Th2, Th17, and Treg, respectively, these data suggest that Th1/Th2/Th17/Treg paradigm could have been changed during TCE exposure.

Table 1.

Immunostaining Score for IFN-γ, IL-4, IL-17, and IL-10 in Spleen Sections.

Group	n	IFN-γ score of IFN-γ	IL-4 score	IL-17 score of IL-17	IL-10 score of IL-10
Blank control	25	1.33 (0.52)	1.31 (0.51)	1.67 (0.52)	4.17 (0.75)
Vehicle control	25	1.50 (0.55)	1.47 (0.55)	1.50 (0.84)	4.00 (0.63)
2W TCE low-dose group	5	1.67 (0.52)	1.83 (0.75)	2.83 (0.75)^a	3.17 (0.98)
4W TCE low-dose group	5	3.00 (0.63)^a	3.10 (0.61)^a	3.50 (0.84)^a	2.17 (0.75)^a
8W TCE low-dose group	5	2.67 (0.53)^a	3.67 (1.03)^a	3.17 (0.98)^a	2.50 (0.55)^a
12W TCE low-dose group	5	2.50 (0.54)^a	2.67 (0.82)^a	2.67 (0.52)^a	2.67 (0.52)^a
16W TCE low-dose group	5	1.83 (0.75)	2.00 (0.63)	2.50 (1.05)^a	3.50 (1.05)
2W TCE high-dose group	5	2.17 (0.75)	2.50 (0.84)^a	3.17 (0.75)^a	2.00 (0.63)^a
4W TCE high-dose group	5	3.83 (0.76)^a	3.50 (1.05)^a	3.83 (1.17)^a	1.83 (0.75)^a
8W TCE high-dose group	5	3.00 (0.64)^a	3.00 (0.89^a	3.17 (0.75)^a	2.33 (0.52)^a
12W TCE high-dose group	5	2.50 (0.55)^a	2.83 (1.17^a	2.83 (0.75)^a	2.50 (0.84)^a
16W TCE high-dose group	5	2.00 (0.63)	2.00 (0.89	2.67 (1.03)^a	2.33 (1.03)^a

Abbreviations: IFN-γ, interferon-gamma; IL, interleukin; TCE, trichloroethylene; W, week.

^a P < 0.05, compared with vehicle control group.

Figure 2.

Expression of IFN-γ following TCE treatment. Interferon-γ expression was detected by anti-IFN-γ antibody with Diaminobenzidine(DAB) kit (dark brown). A, blank control group; B, vehicle control group; C, 2 week (W) TCE low-dose group; D, 4W TCE low-dose group; E, 8W TCE low-dose group; F, 12W TCE low-dose group; G, 16W TCE low-dose group; H, 2W TCE high-dose group; I, 4W TCE high-dose group; J, 8W TCE high-dose group; K, 12W TCE high-dose group; and L, 16W TCE high-dose group (magnification 400). Note: IFN-γ expression was increased in all TCE treatment groups in a time- and dose-dependent manner. IFN-γ indicates interferon-γ; TCE, trichloroethylene.

Figure 3.

Expression of IL-4 following TCE treatment. Interleukin-4 expression was detected by anti-IL-4 antibodies with Diaminobenzidine(DAB). A, Blank control group; B, vehicle control group; C, 2 week (W) TCE low-dose group; D, 4W TCE low-dose group; E, 8W TCE low-dose group; F, 12W TCE low-dose group; G, 16W TCE low-dose group; H, 2W TCE high-dose group; I, 4W TCE high-dose group; J, 8W TCE high-dose group; K, 12W TCE high-dose group; L, 16W TCE high-dose group (magnification 400). There was a time- and dose-dependent increase of IL-4 expression following TCE treatment. IL indicates interleukin; TCE, trichloroethylene.

Figure 4.

Expression of IL-17 in response to TCE treatment. Expression of IL-17 was detected by IL-17 antibody with Diaminobenzidine (DAB) reaction. A, Blank control group; B, vehicle control group; C, 2 week (W) TCE low-dose group; D, 4W TCE low-dose group; E, 8W TCE low-dose group; F, 12W TCE low-dose group; G, 16W TCE low-dose group; H, 2W TCE high-dose group; I, 4W TCE high-dose group; J, 8W TCE high-dose group; K, 12W TCE high-dose group; L, 16W TCE high-dose group (magnification 400). Trichloroethylene treatment caused an increase of IL-17 expression with time- and dose-dependence. IL indicates interleukin; TCE, trichloroethylene.

Figure 5.

Changes in the expression of IL-10 by TCE treatment. Interleukin-10 expression was determined by immunostaining with IL-10 antibody with Diaminobenzidine (DAB) detection. A, Blank control group; B, vehicle control group; C, 2 week (W) TCE low-dose group; D, 4W TCE low-dose group; E, 8W TCE low-dose group; F, 12W TCE low-dose group; G, 16W TCE low-dose group; H, 2W TCE high-dose group; I, 4W TCE high-dose group; J, 8W TCE high-dose group; K, 12W TCE high-dose group; L, 16W TCE high-dose group (magnification 400). Trichloroethylene exposure reduced IL-10 expressions with time- and dose-dependence. IL indicates interleukin; TCE, trichloroethylene.

Effect of TCE Exposure on Th1, Th2, Th17, and Treg Cells in Spleen of Mice

Flow cytometry was performed to directly quantify Th1, Th2, Th17, and Treg subsets and determine the Th1/Th2/Th17/Treg paradigm following TCE exposure. The data from Table 2 and Figure 6 show that, compared to blank and vehicle control groups, the proportions of Th1 (CD3⁺CD4⁺IFN-γ⁺), Th2 (CD3⁺CD4⁺IL-4⁺), and Th17 (CD4⁺IL-17A⁺) cells were significantly increased, while the proportions of Treg (CD4⁺CD25⁺Foxp3⁺) were significantly decreased in TCE treatment group, resulting in greater ratios of Th17/Treg cells. Interestingly, the ratio of Th1/Th2 was clearly decreased. This implies that Th1/Th2/Th17/Treg paradigm was skewed to Th1, Th2, and Th17 cell in TCE exposure group, especially in Th2 and Th17 cells. These data further demonstrate that the Th1/Th2/Th17/Treg paradigm is involved in TCE-mediated autoimmunity.

Table 2.

The Percentage of Th1, Th2, Th17, and Treg Cells in Each Group by Flow Cytometry.

Group	n	Th1 (%)	Th2 (%)	Th17 (%)	Treg (%)
Blank control group	25	2.21 (0.10)	1.24 (0.09)	2.24 (0.17)	3.81 (0.05)
Vehicle control group	25	2.24 (0.12)	1.32 (0.14)	2.30 (0.13)	3.82 (0.04)
2W TCE low-dose group	5	2.97 (0.09)^a	2.23 (0.05)^a	3.15 (0.30)^a	2.50 (0.12)^a
4W TCE low-dose group	5	3.42 (0.13)^a	2.68 (0.05)^a	3.40 (0.17)^a	2.37 (0.12)^a
8W TCE low-dose group	5	3.20 (0.12)^a	2.90 (0.11)^a	3.19 (0.23)^a	2.61 (0.09)^a
12W TCE low-dose group	5	2.96 (0.13)^a	2.54 (0.10)^a	2.97 (0.09)^a	2.69 (0.13)^a
16W TCE low-dose group	5	2.40 (0.04)	1.99 (0.10)^a	2.90 (0.10)^a	2.53 (0.17)^a
2W TCE high-dose group	5	3.14 (0.06)^a	2.28 (0.07)^a	3.18 (0.11)^a	2.02 (0.13)^a
4W TCE high-dose group	5	3.68 (0.12)^a	2.95 (0.14)^a	3.85 (0.14)^a	1.76 (0.05)^a
8W TCE high-dose group	5	3.48 (0.08)^a	3.06 (0.15)^a	3.27 (0.06)^a	2.09 (0.04)^a
12W TCE high-dose group	5	3.25 (0.06)^a	2.57 (0.05)^a	3.17 (0.08)^a	2.10 (0.08)^a
16W TCE high-dose group	5	3.08 (0.11)^a	2.24 (0.10)^a	3.08 (0.08)^a	2.12 (0.17)^a

Abbreviations: TCE, trichloroethylene; Th, T helper; W, week.

^a P < 0.05, compared with vehicle control group.

Figure 6.

The ratio of Th1/Th2 and Th17/Treg. A, Ratio of Th1/Th2; B, ratio of Th17/Treg. Data are expressed as mean (standard deviation). P < 0.05 compared with the vehicle control group.

Mechanisms of Action—Effect of TCE Exposure on Expression of T-bet, GATA3, RORct, and Foxp3 in Spleen of Mice

To determine the molecular mechanisms underlying the change in Th1/Th2/Th17/Treg paradigm, the expression of master transcription factors for Th1/Th2/Th17/Treg cell differentiation (T-bet, GATA-binding protein 3 [GATA3], RORct, and Foxp3) was assessed by RT-PCR. As shown in Figures 7 and 8, compared with blank and vehicle control groups, the expression of T-bet, GATA3, and RORct mRNA was increased. Conversely, the expression of Foxp3 mRNA was decreased after TCE exposure. Consistent with the flow cytometry data (Table 2, Figure 6) on Th cell subsets, the ratio of T-bet/GATA3 was decreased significantly, while the ratio of RORct/Foxp3 was markedly increased. The data indicate that TCE exposure promoted the expression of master transcription factors directed to the differentiation of Th1, Th2, and Th17 cells, specifically upregulating the master transcription factors for differentiation of Th2 and Th17 cells, but downregulating the master transcription factor directed to differentiation of Treg cells. Moreover, the alteration in the RORct/Foxp3 ratio was more pronounced in TCE high-dose group than in TCE low-dose group (P < 0.05), further supporting the key role of autoimmune mechanism.

Figure 7.

The expression levels of transcription factors T-bet, GATA3, RORct, and Foxp3 in spleen. Measurement of mRNA expression by quantitative RT-PCR (A) T-bet, (B) GATA3, (C) RORC, and (D) Foxp3. Data are expressed as mean (standard deviation). P < 0.05 compared with the vehicle control group. Foxp3 indicates forkhead box P3; GATA3, GATA binding protein 3; mRNA, messenger RNA; RT-PCR, real-time quantitative polymerase chain reaction; T-bet, T-box expressed in T cells.

Figure 8.

The ratio of T-bet/GATA3 and RORC/Foxp3. A, Ratio of T-bet/GATA3; B, ratio of RORC/Foxp3. Data are expressed as mean (standard deviation). P < 0.05 compared with the vehicle control group. Foxp3 indicates forkhead box P3; GATA3, GATA binding protein 3; T-bet, T-box expressed in T cells.

Discussion

Beyond the classic metabolic and type IV allergic reactions, recent studies suggest that TCE exposure is related to the development of AD. Compared with other types of chemical sensitization, the immune response caused by TCE is more severe. It has been suggested that TCE not only causes sensitization but also produces immunotoxicity. Recent advances show that the imbalance of T-cell subsets plays important roles in a variety of AD.²² Effector CD4⁺ T cells can be divided into Th1, Th2, Th17, and Treg subsets according to their differentiation and function. These subsets secrete different cytokines to form networks. T helper 1 cells produce large quantities of IFN-γ and activate macrophages and promote the cell-mediated immunity. Th2 cells produce mainly IL-4 cytokines, suppress Th1 cell activation, and contribute to humoral immunity. T helper 1- and Th2-mediated immunities are reciprocally regulated, and a balance is maintained in immune-mediated disease. Thanks to the discovery of Th17 and Treg cells, the concept of Th1/Th2 paradigm has been expanded into the Th1/Th2/Th17/Treg paradigm²³ to provide better insight into the network of immunological regulations. T helper 17 cells, which produce the hallmark cytokine IL-17A, have been found to play vital roles in the pathogenesis of inflammatory and AD. In contrast to Th17 cells, Treg cells serve as the most important counter regulator in the immune system.²⁴ Treg cells have been shown to inhibit the differentiation of Th17 via Treg-specific transcription factor Foxp3 or downregulation of IL-23 and IL-17 expression. Thus, Th17 and Treg subsets are reciprocally regulated and maintain a balance in vivo.²⁵ The Th1/Th2/Th17/Treg paradigm can effectively adjust the immune defense responses in human body and is associated with many AD, such as systemic lupus erythematosus.²³ It has been reported that occupational exposure to TCE led to a significant rise in CD4⁺ T cells in peripheral blood.²⁶ Our previous study also showed that the proportions of Th1- and Th2-specific cytokines IFN-γ and IL-4 were significantly changed in TCE-sensitized BALB/c mice.²¹ This suggests that the imbalance of Th1/Th2/Th17/Treg cells may be involved in the pathogenesis of ODMLT.

In order to ascertain the direct evidence for a change in the expression level of cytokines in spleen tissue in TCE-treated BALB/c mice, the current investigation examined IFN-γ, IL-4, IL-17A, and IL-10 characteristic of Th1, Th2, Th17, and Treg cells, respectively, in this tissue. Our data show that these cytokines were significantly altered by TCE exposure. This suggests an imbalance of these cytokines, favoring autoimmune response, evidenced by a high IL17A/IL-10 ratio. These data suggested that TCE exposure may lead to the imbalance of Th1/Th2/Th17/Treg paradigm. Indeed, direct quantification of Th cell subsets further showed that these changes to the above Th subset-specific cytokines were mirrored by alterations to their corresponding Th cell subsets Th1, Th2, Th17, and Treg. These corroborative 2 lines of evidence provide strong support for the involvement of imbalanced Th1/Th2/Th17/Treg paradigm in TCE-induced immune response. This argument is given further weight by a dose- and time-response relationship in changes to these specific cytokines and Th cell subsets.

Type IV allergic reaction is known to be typical cellular immunity mediated by T lymphocytes. Cellular immunity is mainly mediated by Th1 cells while humoral immunity is mediated by Th2 cells.^27,28 The shift of balance of Th1/Th2 paradigm toward Th2 observed from the present study suggests that TCE-mediated autoimmunity involves both humoral immunity and cellular immunity, and humoral immunity plays a more important role in this process. This mode of action provides a new immunological mechanism for ODMLT which cannot be fully explained by type IV allergic reaction.

The differentiation of initial CD4⁺ T cells into Th1, Th2, Th17, or Treg cells is critically regulated by master transcription factors.²⁹ T-box expressed in T cells and GATA3 are the 2 master transcription factors that regulate the differentiation of Th1 and Th2 cells and the expression of their cytokine genes, respectively.³⁰ T helper 17 and Treg cells are 2 novel T-cell subsets which have recently been shown to originate from the same developmental lineage.³¹ The master gene for the differentiation from CD4⁺ T cells to Treg cells is a transcription factor Foxp3, while the development of Th17 cells is controlled by the transcription factor retinoic acid-RORct.³² To gain further insight into molecular mechanisms to the altered Th1/Th2/Th17/Treg paradigm in TCE-mediated autoimmunity, we examined the expression of master transcription factors for Th1/Th2/Th17/Treg cell differentiation—T-bet, GATA3, RORct, and Foxp3 by RT-PCR. Changes to these master transcription factors indeed mirrored those of the above Th cell subsets. These data indicate that changes in differentiation of Th1/Th2/Th17/Treg subsets is driven by changes of the master transcription factors.

Autoimmunity and its link to environmental factors is a challenging issue in immunology and modern medicine. While molecular mimicry has been suggested as a mechanism for biological pathogen induced and exacerbated AD,³³ the mechanisms for chemical-triggered autoimmunity are poorly understood. Some chemicals may bind to tissue proteins and form neoantigens.³⁴ Trichloroethylene can bind covalently to tissue proteins³⁵; this could be a potential mechanism for its dermal sensitization. However, this is the first observation for a chemical to activate the master transcription factors for these Th cell subsets in vivo. We propose an interaction of environmental chemicals with Th cell master transcription factors as a novel mechanism for chemical-induced autoimmunity.

In summary, the present study demonstrates for the first time that changes in the Th1/Th2/Th17/Treg paradigm and their corresponding cytokines contribute to TCE-mediated autoimmunity. These changes can be attributed to upregulation of the expression of master transcription factors promoting the differentiation of Th1, Th2, and Th17 cells, but suppressing the differentiation of Treg cells, resulting in the Th1-, Th2-, and T17-biased immunity. These pathways may serve as novel target for prevention and treatment of ODMLT in workers and general population exposed to TCE.

Footnotes

Authors’ Contributions

Shu-long Li and Yun Yu contributed equally to this article. S. Li, Y. Yu, and Q. Zhu contributed to conception and design; contributed to acquisition, analysis, and interpretation; drafted the manuscript; and critically revised the manuscript. P. Yang and J. Zhang contributed to conception, contributed to acquisition and analysis, and critically revised manuscript. H. Wang contributed to conception, acquisition and analysis, and drafted manuscript. C. Zhang contributed to conception, acquisition, and critically revised the manuscript. T. Shen and C. Wu contributed to conception and design, interpretation, and critically revised the manuscript. M. Liu contributed to conception, acquisition, and drafted the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Natural Science Foundation of China (81673141), and School Funded Project of Anhui Medical University (2017xkj026). C.W. gratefully acknowledges support from Biotechnology and Biological Sciences Research Council (BBSRC) (BB/I025379/1; BB/P004695/1), Age UK and Henry Smith Charity, and National Institute of Aging (NIA, 1R01AG049321-01A1).

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