Chronic exposure to water pollutant trichloroethylene increased epigenetic drift in CD4+ T cells
Kathleen M. Gilbert*, Sarah J. Blossom,* Stephen W. Erickson*, Brad Reisfeld†, Todd J. Zurlinden†, Brannon Broadfoot*, Kirk West*, Shasha Bai*, and Craig A. Cooney‡
*University of Arkansas for Medical Sciences, Arkansas Children’s Hospital Research Institute, Little Rock, AR 72202; † Colorado State University, Fort Collins, CO 80523; ‡Central Arkansas Veterans Healthcare System, Little Rock, AR 72205
Aim: Autoimmune disease and CD4+ T cell alterations are induced in mice exposed to the water pollutant trichloroethylene (TCE). We examined here whether TCE altered gene-specific DNA methylation in CD4+ T cells as a possible mechanism of immunotoxicity. Materials & methods: Naive and effector/memory CD4+ T cells from mice exposed to TCE (0.5 mg/ml in drinking water) for 40 weeks were examined by bisulfite next generation DNA sequencing. Results: A probabilistic model calculated from multiple genes showed that TCE decreased methylation control in CD4+ T cells. Data from individual genes fitted to a quadratic regression model showed that TCE increased gene-specific methylation variance in both CD4 subsets. Conclusion: TCE increased epigenetic drift of specific CpG sites in CD4+ T cells.
Approximately 8% of the U.S. population has one or more autoimmune disease. The contribution of environmental stressors to autoimmune disease etiology is now known to far exceed genetic influences. One environmental contributor to autoimmunity is the common water pollutant TCE. Chronic TCE exposure in adults (occupational or environmental) has been linked to a variety of autoimmune diseases including lupus, scleroderma, hepatitis, and diabetes. We have been studying how TCE alters the immune system in a manner that promotes autoimmunity.
Mechanistic studies in animal models often focus on a single time point in toxicant-induced immune pathology, and thus fail to take into account the impact of chronicity. This study consisted of a longitudinal look at how adult exposure to the water pollutant TCE altered the immune system of female MRL+/+ mice. We examined phenotypic alterations as well as changes in DNA methylation associated with several functionally important genes in both naïve and effector/memory CD4+ T cells isolated every 6 weeks during a 40 week exposure to TCE.
The cytokine profiles and phenotype of differentiated CD4+ T cell subsets are normally fairly rigid due to carefully maintained levels of DNA methylation in the promoters of the pertinent genes (i.e. Ifng, Il17A, Il22, Ctla, Cdkn1a, Tnfsf14, and Foxp3)(1,2). The development of autoimmune disease may represent a disruption in the methylation patterns of differentiated CD4+ T cells, resulting in the demethylation and expression of genes that encode pro-inflammatory cytokines, chemokines or adhesion molecules (3-8). We found that TCE exposure loosened the normally tight epigenetic control mechanisms in CD4+ T cells, and increased what is known as “epigenetic drift”. Epigenetic drift is defined as increased variance in the level of methylation at specific CpG sites, and can result in altered gene expression and cellular phenotype. The TCE-induced increased epigenetic drift in CD4+ T cells was associated with increased variation in gene expression as demonstrated for the pro-inflammatory cytokine Ifng (Figure 1).
We have previously reported that in addition to its effects on CD4+ T cells, TCE, not surprisingly, impacts the liver. Specifically, we found that TCE inhibited pathways required for liver protection and regeneration.(9) TCE also promotes the production of anti-liver antibodies in association with its ability to generate autoimmune-hepatitis. We have combined the TCE effects on the liver and CD4+ T cells to generate the working model described in Figure 2. When TCE is metabolized in the liver it is converted into a reactive metabolite that can form adducts with liver proteins.(10) This altered liver protein is processed and presented by antigen presenting cells such as activated macrophages to CD4+ T cells specific for both altered and unaltered liver peptides. In addition, TCE, mostly likely via its metabolite TCAH (trichloroacetaldehyde hydrate), acts on CD4+ T cells to alter DNA methylation and gene expression. By virtue of their increased production of pro-inflammatory cytokines some of these altered effector/memory CD4+ T cells are pathogenic, and can act to promote inflammation in the liver via antibody production and direct cytotoxicity. Since TCE also inhibits mechanisms in the liver that would otherwise protect against this inflammation, tissue damage occurs in the form of autoimmune hepatitis.
Importance of this study: Defining the contribution of specific environmental factors to epigenetic drift is still in its infancy. Thus, this study is both novel and important since it was the first to demonstrate the impact of a common environmental pollutant, TCE, on epigenetic drift in effector/memory CD4+ T cells. Correlating TCE-induced epigenetic drift with changes in effector/memory CD4+ T cell function and with CD4+ T cell-mediated autoimmune pathology is an important step in defining how toxicants can have long-term effects on the immune response.
- Komori, H. K., T. Hart, S. A. LaMere, P. V. Chew, and D. R. Salomon. 2015. Defining CD4 T cell memory by the epigenetic landscape of CpG DNA methylation. J Immunol. 194: 1565-1579.
- Hashimoto, S., K. Ogoshi, A. Sasaki, J. Abe, W. Qu, Y. Nakatani, B. Ahsan, K. Oshima, F. H. Shand, A. Ametani, Y. Suzuki, S. Kaneko, T. Wada, M. Hattori, S. Sugano, S. Morishita, and K. Matsushima. 2013. Coordinated changes in DNA methylation in antigen-specific memory CD4 T cells. J Immunol. 190: 4076-4091.
- Lleo, A., W. Zhang, M. Zhao, Y. Tan, F. Bernuzzi, B. Zhu, Q. Liu, Q. Tan, F. Malinverno, L. Valenti, T. Jiang, L. Tan, W. Liao, R. Coppel, P. Invernizzi, Q. Lu, D. H. Adams, and M. E. Gershwin. 2015. DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis. Clin Epigenetics. 7: 61-0098.
- Altorok, N., P. Coit, T. Hughes, K. A. Koelsch, D. U. Stone, A. Rasmussen, L. Radfar, R. H. Scofield, K. L. Sivils, A. D. Farris, and A. H. Sawalha. 2014. Genome-wide DNA methylation patterns in naive CD4+ T cells from patients with primary Sjogren’s syndrome. Arthritis Rheumatol. 66: 731-739.
- Meyer, B., R. A. Chavez, J. E. Munro, R. C. Chiaroni-Clarke, J. D. Akikusa, R. C. Allen, J. M. Craig, A. L. Ponsonby, R. Saffery, and J. A. Ellis. 2015. DNA methylation at IL32 in juvenile idiopathic arthritis. Sci Rep. 5:11063. doi: 10.1038/srep11063.: 11063.
- Wang, Y., Y. Shu, Y. Xiao, Q. Wang, T. Kanekura, Y. Li, J. Wang, M. Zhao, Q. Lu, and R. Xiao. 2014. Hypomethylation and overexpression of ITGAL (CD11a) in CD4(+) T cells in systemic sclerosis. Clin Epigenetics. 6: 25-26.
- Wu, Z., X. Mei, D. Zhao, Y. Sun, J. Song, W. Pan, and W. Shi. 2015. DNA methylation modulates HERV-E expression in CD4+ T cells from systemic lupus erythematosus patients. J Dermatol Sci. 77: 110-116.
- Coit, P., P. Renauer, M. A. Jeffries, J. T. Merrill, W. J. McCune, K. Maksimowicz-McKinnon, and A. H. Sawalha. 2015. Renal involvement in lupus is characterized by unique DNA methylation changes in naive CD4+ T cells. J Autoimmun. 61:29-35. doi: 10.1016/j.jaut.2015.05.003. Epub;%2015 May 23.: 29-35.
- Gilbert, K. M., B. Reisfeld, T. J. Zurlinden, M. N. Kreps, S. W. Erickson, and S. J. Blossom. 2014. Modeling toxicodynamic effects of trichloroethylene on liver in mouse model of autoimmune hepatitis. Toxicol Appl Pharmacol. 279: 284-293.
- Halmes, N. C., E. J. Perkins, D. C. McMillan, and N. R. Pumford. 1997. Detection of trichloroethylene-protein adducts in rat liver and plasma. Toxicol Lett. 92: 187-194.
Acknowledgements: This work was supported by grants from the Arkansas Biosciences Institute, the National Institutes of Health (R01ES017286, R01ES021484), the Organic Compounds Property Contamination class action settlement (CV 1992-002603), and the UAMS Translational Research Institute (National Institutes of Health UL1RR029884).