Chronic TCDD exposure results in the dysregulation of gene expression in splenic B-lymphocytes and in the impairments in T-cell and B-cell differentiation in mouse model

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure in humans is associated with marked immune suppressions and increased incidence of lymphoblastic diseases. To elucidate mechanisms of impairments in humoral immune responses, we used a murine model. Following a 20-week administration of low doses of TCDD, we observed severely reduced antibody titers, dramatically decreased number of splenic Th1 and Th2 cells and an increase in CD19⁺ B cells. Transcriptional profiling of CD19⁺ B cells showed that markers of pre-B cells were significantly elevated, indicating delayed B cell maturation. These changes in B cells were accompanied by decreases of T helper cell numbers and reduced IgM and IgG titers. A transcriptome analysis of splenic B cells followed by Ingenuity Pathway Analysis (IPA) revealed a set of differentially expressed genes known to play roles in tumorigenesis, cell-proliferation and cell-migration. The most up-regulated transcript gene was Eph receptor A2 (EphA2), a known oncogene, and the most down-regulated transcript was ZBTB16 that codes for a negative transcriptional regulator important in epigenetic chromatin remodeling. IPA identified cAMP-responsive element modulator (CREM) and cAMP-responsive element binding protein 1 (CREB1) as top upstream regulators. Consistently, a MAPPER promoter database analysis showed that all top dysregulated genes had CREM and/or CREB1 binding sites in their promoter regions. In summary, our data showed that chronic TCDD exposure in mice caused suppressed humoral immunity accompanied with profound dysregulation of gene expression in splenic B-lymphocytes, likely through cAMP-dependent pathways. This dysregulation resulted in impairments in T-cell and B-cell differentiation and activation of the tumorigenic transcription program.

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that targets various organs including the immune system in both acute and chronic exposure. Even at doses with no obvious signs of toxicity, TCDD alters the immune functions in virtually every species studied so far (Holsapple et al., 1991, Kerkvliet, 2002). TCDD acts via the activation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that belongs to the basic helix-loop-helix superfamily whose members play key roles in gene expression networks underlying many essential physiological and developmental processes. Human and animal exposures to TCDD result in immune suppression that is not observed in AhR null mice (Vorderstrasse et al., 2001a). AhR is involved in both innate and acquired immune responses and has been shown to be essential for the differentiation and maintenance of the intestinal intraepithelial lymphocytes (IELs) and the innate lymphoid cells (ILCs) (Li et al., 2011, Spits and Di Santo, 2011). The role of AhR in the Th17 and T regulatory (Treg) cell development is firmly established (Ho and Steinman, 2008). B cells are also highly sensitive targets of TCDD (Sulentic and Kaminski, 2011b). Epidemiologic evidence shows that TCDD exposure depresses humoral immunity in human subjects. As such, both Seveso accident subjects with the highest TCDD exposure and Vietnam Veterans exhibited decreased plasma IgG levels and increased incidence of lymphatic tumors, especially non-Hodgkin's lymphoma (NHL) (Sulentic and Kaminski, 2011b).

TCDD has been demonstrated to suppress the primary IgM response in purified splenic B cells (Dooley and Holsapple, 1988). This suppression, both in vivo and in vitro, involved the activation of AhR (Vorderstrasse et al., 2001b). Another TCDD effect on immune system is a shift in the Th1/Th2 balance, an important measure of the immune system fitness (Kidd, 2003). A large body of data demonstrates the effects of AhR activation on the differentiation of the T-cell subsets (Nguyen et al., 2013), including a number of genome-wide expression studies on T-cells (Li et al., 2013, Stubbington et al., 2015). While the roles of AhR in B cell differentiation are demonstrated (De Abrew et al., 2011, Zhang et al., 2013), there is a dearth of data that allows a better mechanistic understanding of the long-term consequence of the chronic TCDD exposure on humoral immunity (e.g., B cells) that underlie referenced above adverse outcomes of TCDD exposure (Kerkvliet, 2002). With the application of transcriptome analysis, we could gain a full view of the effects of TCDD on B lymphocytes at the transcriptional level. Transcriptomic studies have been performed on B cell lines (De Abrew et al., 2010); however, to our knowledge, there were no such in vivo studies reported regarding B lymphocytes isolated from mice chronically treated with TCDD. In the current study, we used a murine model that recapitulates a chronic human exposure to TCDD to evaluate the effects of TCDD on the immune phenotype and the splenic B cells transcriptome. Specifically, splenic B lymphocytes were isolated after a 20-week TCDD exposure at an environmentally relevant dose for transcriptomic analysis. Our analysis provides an important insight into the possible mechanisms of the impairments in the humoral immunity and into the increased incidence of lymphatic tumors resulting from the chronic TCDD exposure in humans.