Potential for TCDD to induce regulatory functions in B cells as part of the mechanism for T cell suppression in EAE.

Part of the mechanism by which 2,3,7.8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses immune function involves induction of regulatory T cells and suppression of effector T cells. The goal of this project was to examine whether TCDD's suppression of effector T cells was due in part to inducing B regulatory cells (Bregs). TCDD's potential to increase the percentage and/or function of CD24+CD38+ B cells was assessed in response to lipopolysaccharide (LPS) + interleukin (IL)-4 in vitro and in a mild model of experimental autoimmune encephalomyelitis (EAE) in vivo. In vitro, TCDD did not consistently increase the percentage of CD19+CD24+CD38+ cells using splenocytes, purified B cells or bone marrow (BM) cells. However, TCDD increased IL-10 in all three culture preparations, and TCDD increased the percentage of CD5+CD24+CD38+ cells producing IL-10. In EAE, TCDD did not affect the percentage of the CD24+CD38+ cell population in CD19, B220 or CD5 B cells in splenocytes (SPLC), lymph nodes (LN) nor BM cells at end-stage disease. On the other hand, TCDD increased the CD19+CD24+CD38+ percentage in the spinal cord (SC) in EAE. Moreover, TCDD-treated B cells isolated from spleens or TCDD-treated BM cells in EAE mice modestly reduced the ability of naïve effector T cells to express interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Together these data show that TCDD can induce regulatory functions in B cells, although it was not obvious simply by examining the expression of regulatory markers but by assessing function by cytokine production or mixed lymphocyte responses.

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD's effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD's EAE disease attenuation.

TCDD attenuates EAE through induction of FasL on B cells and inhibition of IgG production.

Previously we demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed experimental autoimmune encephalomyelitis (EAE), a model to study multiple sclerosis (MS), through induction of regulatory T cells (Tregs) and suppression of effector T cell function in the spleen. Since B cells and specifically regulatory B cells (Bregs) have been shown to be so critical in the pathology associated with EAE and MS, we wanted to determine whether TCDD could also induce Bregs. We specifically hypothesized that a Fas ligand (FasL)+ Breg population would be induced by TCDD in EAE thereby triggering apoptosis in Fas-expressing effector T cells as one mechanism to account for inhibition of T cell function by TCDD. TCDD (0.1-2.5 µg/kg/day administered orally for 12 days) modestly increased the percentage of FasL + B cells in the spleen and spinal cord in TCDD-treated EAE mice. However, we did not detect significant increases in percentages of FasL + B cells using TCDD in vitro in mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). Part of the modest effect by TCDD was likely related to the localized expression of FasL; for instance, in the spleen, FasL was more highly expressed by IgMhiIgDlo marginal zone (MZ) B cells, but IgMloIgDhi follicular (FO) B cells were more responsive to TCDD. Consistent with our observation of modest upregulation of FasL, we also observed modest changes in mitochondrial membrane potential in T cells co-cultured with isolated total B cells or IgM-depleted (i.e., FO-enriched) B cells from TCDD-treated EAE mice. These data suggest that while small microenvironments of apoptosis might be occurring in T cells in response to TCDD-treated B cells, it is not a major mechanism by which T cell function is compromised by TCDD in EAE. TCDD did robustly suppress IgG production systemically and in spleen and spinal cord B cells at end stage disease. Thus, these studies show that TCDD's primary effect on B cells in EAE is compromised IgG production but not FasL + Breg induction.