Effects Of The Alteration Of Th1/th2 And Th17/Treg Paradigm By Trichloroethylene Expose In BALB/c Mice

BackgroundTrichloroethylene(TCE), a widely used macromolecule organic solvents, has been used in industry as a degreaser and cleaning agent for metals and various parts of electronics. Because of its efficacy for grease remove and frequent usage, TCE has caused the pollution to the soil, air and water. TCE has become a major environmental pollutant and occupational toxicant. Workers exposed to TCE can lead to Occupational Dermatitis Medicamentosa-like of Trichloroethylene(ODMLT). It is widely considered ODMLT is a delayed type hypersensitivity mediated by antigen specific T lymphocytes. However, sensitization research cannot completely explain the mechanism of injury. The immune response induced by TCE is more strongly than other chemical irritants. It is suggested that TCE not only has sensitization, but also has immunotoxicity. Th1/Th2/Th17/Treg paradigm can effectively adjust the immune defense responses in human body, and associated with many autoimmune diseases. In this study, we aimed to explore possible alteration of Th1/Th2/Th17/Treg paradigm through examining variable cell types in TCE-exposed mice.ObjectiveThe study aimed to investigate that whether the alteration of Th1/Th2/Th17/Treg paradigm induced by TCE expourse is the pathogenesis of ODMLT by detecting the the proportion of Th cells(Th1, Th2, Th17 and Treg) and their specific cytokines(IFN-γ、IL-4、IL-17 and IL-10) and nuclear transcription factors(T-bet、GTAT-3、ROR-γt and Foxp3)in different groups and time-point.Method100 SPF female BALB/c mice aged 6-8w were acclimatized for a week. Then the mice were randomly divided into blank control group, vehicle control group(drinking water containing 1% DMSO), TCE low-dose group(2.5mg/ml) and TCE high-dose group(5.0mg/ml). After 2, 4, 8, 12, 16 weeks of TCE treatment, respectively, the mice were executed after blood was collected from ocular venous plexus and the spleen removed. Portions of spleen from control and TCE treated mice were prepared for flow cytometry to detect the proportion of Th cells(Th1, Th2, Th17 and Treg). The specific cytokines(IFN-γ、IL-4、IL-17 and IL-10) were detected by immunohistochemistry and nuclear transcription factors(T-bet、GTAT-3、ROR-γt and Foxp3)were assessed by real time quantitative–polymerase chain reaction(RT-PCR).Results1. Body Weight and Relative Organ WeightAll mice were in good condition during the whole study. Compared with blank control group, there was no obviously change of relative spleen weight in vehicle control group(P>0.05). There was also no significantly changed in TCE low-dose group which is compared with vehicle control group(P>0.05). Compared with vehicle control group, there was an obviously increase of relative spleen weight in TCE high-dose group, which was executed at 4 and 8 weeks(P<0.05).2. Expression of IFN-γ, IL-4, IL-17 A and IL-10 in spleenCompared with blank control group, the expression level of the specific cytokines in vehicle control group had no significant difference(P<0.05). In TCE low-dose group, the expression of IFN-γ and IL-4 were higher than vehicle control group at 4, 8,12 weeks, the difference was statistically significant(P<0.05); The expression of IL-17 was increased obviously while the expression of IL-10 was decreased markedly in every time-point, the differences were statistically significant(P<0.05). In TCE high-dose group, the expression of IFN-γ was increased obviously at 4, 8,12 weeks; the expression of IL-4 was increased obviously at 2, 4, 8,12 weeks; The expression of IL-17 was increased obviously while the expression of IL-10 was decreased markedly in every time-point, these differences were statistically significant(P<0.05), and these changes were more strongly than TCE low-dose group.3. The proportion of Th1, Th2, Th17 and Treg cells in spleenThe proportion of Th1, Th2, Th17 and Treg cells were detected by flow cytometry. The results showed: Compared with blank control group, vehicle control group shows no significant difference(P>0.05) in the proportion of Th cells. Compared with vehicle control group, the proportions of Th2 and Th17 cells increased, while the proportions of Treg decreased memorably in TCE low-group group, the differences were statistically significant(P<0.05); The proportions of Th1 cells in TCE low-dose group were much higher than vehicle control group at 2, 4, 8, 12 weeks, the differences were statistically significant(P<0.05). In TCE high-dose group, the chang tendency was similar to TCE low-dose group. Th1, Th2 and Th17 cells were increased while Treg cells were decreased at every time-point, these differences were statistically significant(P<0.05), and these changes were more strongly than TCE low-dose group.4. The alteration of Th1/Th2/Th17/Treg paradigmIn the present study, we found that the ratio of Th1/Th2 was decreased markedly and the ratio of Th17/Treg was increased obviously in both TCE low- and high-dose group, the differences were statistically significant(P<0.05). These changes were more strongly in TCE high-dose group.5. Expression of Transcription Factors T-bet, GATA3, RORγt and Foxp3 in spleenResult of RT-PCR showed: There was no significant difference between blank and vehicle control group in expression of nuclear transcription factors(P>0.05). In TCE low-dose group, the expression of T-bet and ROR-γt were higher than vehicle control group at 4, 8,12 weeks, the difference was statistically significant(P<0.05); The expression of GATA-3 was increased significantly at 2, 4, 8, 12 weeks(P<0.05); At 8, 12 weeks, the expression of Foxp3 was decreased in TCE low-dose group, the difference was statistically significant(P<0.05). In TCE high-dose group, the expression of T-bet was same to TCE low-dose group, but the level of alteration was much more strongly; The expression of GATA-3 was increased significantly at every point-time, the difference was statistically significant(P<0.05); At 4, 8,12 16 weeks, ROR-γt and Foxp3 were significant altered, which ROR-γt increased while Foxp3 decreased, the differences were statistically significant(P<0.05).6. Result of the ratio of T-bet/GTAT-3/ROR-γt/Foxp3The results showed that the ratio of T-bet/GATA-3 was decreased markedly in both TCE low- and high-dose group, the difference was statistically significant(P<0.05). The ratio of ROR-γt/Foxp3 was obvious increased at 4, 8, 12, 16 weeks in both TCE lowand high-dose group, the difference was statistically significant(P<0.05), and the alteration of the ratio of ROR-γt/Foxp3 was much more strongly in TCE high-dose group.Conclusions1. TCE has immunotoxicity. The proportion of Th cells(Th1, Th2, Th17 and Treg) in immune organs was altered after TCE exposure.2. Th1/Th2/Th17/Treg paradigm has been disordered during the TCE exposure. The disorder of immune function could be the molecular mechanisms underlying and pathogenesis of ODMLT.