Dendritic cells are professional antigen presenting cells that play an important role in both the innate and adaptive arms of the immune system. In this study, we have characterized the consequences of AhR activation on the innate and adaptive functions of inflammatory BMDCs and examined the role of DRE-mediated events in the generation of these effects. We demonstrate that AhR activation alters BMDC differentiation and induces a regulatory phenotype, but surprisingly without altering the ability of the DCs to initiate antigen-specific activation of naïve, CD4+ T cells.
Dendritic cells exist in multiple, functionally distinct stages of differentiation. Several investigators have linked TCDD-induced modulation of DC differentiation to the generation of defective T cell responses (Vorderstrasse and Kerkvliet, 2001
; Lee et al., 2007
). In our study, TCDD decreased expression of CD11c but increased MHC II and CD86 levels on BMDCs. Engagement of B7 (CD86) molecules on a DC with CD28 or CTLA4 on the surface of the T cell can activate or impair T cell responses, respectively (Krummel and Allison, 1995
; Lenschow et al., 1996
). Successful T cell activation involves not only interactions between the TcR on the T cells and antigen-associated MHC class II on antigen presenting cells, but also costimulation via interactions between accessory molecules such as CD28 and CD86. In this context, studies have linked TCDD-induced increases in CD86 expression on DCs with increased T cell activation in an MLR response (Lee et al., 2007
). However, AhR activation has also been shown to induce CTLA-4 expression on T cells which can bind to CD86 on a DC with high affinity and impair T cell responses (Funatake et al., 2005
). Thus, in the context of AhR activation, ligation of CD86 on DCs with CTLA-4 on T cells could alternatively contribute towards the generation of immune suppression. Furthermore, consistent with previous studies documenting TCDD-induced modulation of the adhesion molecules CD11a and CD54, TCDD decreased the expression of both of these molecules on inflammatory BMDCs (Shepherd et al., 2001
; Vorderstrasse and Kerkvliet, 2001
). The decreased expression of adhesion molecules on DCs could contribute to defective T cell activation and subsequent immune suppression. TCDD-induced immune suppression has also been linked to increased CD25 expression on T cells (Funatake et al., 2005
). More recently, CD25 was also identified as a phenotypic marker for regulatory DCs (DCregs) (von Bergwelt-Baildon et al., 2006
; Driesen et al., 2008
). TCDD increased CD25 expression on the BMDCs, suggesting the induction of a regulatory phenotype in these inflammatory BMDCs. Taken together, TCDD-induced alteration of molecules involved in antigen presentation, costimulation and adhesion could result in defective T cell activation contributing to immune suppression. Recently, ligand-specific activation of the AhR has been shown to generate Tregs or Th17 cells (Quintana et al., 2008
). However, the effects of ligand-specific activation in inflammatory DCs had not been previously described. Interestingly, the AhR ligands, FICZ and ITE generated phenotypical alterations in BMDCs similar to TCDD, demonstrating a lack of differential responsiveness of DCs to an array of AhR ligands.
While the AhR is essential for TCDD-induced immunotoxicity, no information exists regarding the role of DREs in TCDD-induced alterations in DCs. We investigated the role of the AhR and DREs in TCDD-induced alteration of DC differentiation using BMDCs from AhR null, AhRnls/nls
mice. While the AhR null mice, lacking functional AhR receptor, enabled us to determine the role of the AhR in the generation of TCDD-induced alterations in DCs, involvement of DRE-mediated events was assessed using the AhRnls/nls
mice expressing a mutant AhR that binds ligand but fails to translocate into the nucleus or bind DREs, respectively. Our results show that the effects of AhR activation in BMDCs are strictly AhR-dependent but not exclusively DRE-mediated. TCDD-treated BMDCs from AhRdbd/dbd
mice displayed increased MHC class II and CD86 expression when compared to vehicle-treated AhRdbd/dbd
BMDCs. However, TCDD-induced increases in MHC class II and CD86 expression on AhR dbd/dbd
BMDCs did not reach similar levels to those observed on TCDD-treated BMDCs from AhRdbd/-
control mice. This suggested that a non-DRE-mediated mechanism was contributing to the TCDD-induced differentiation of the DCs. Of the several non-DRE-mediated mechanisms implicated in TCDD immunotoxicity, interactions of the activated AhR with NF-kB signaling components can induce defects in DCs following TCDD exposure (Ruby et al., 2002
; Vogel and Matsumura, 2008
). Thus, one possible explanation for the partial effects of TCDD on BMDC differentiation as observed in DCs from AhRdbd/dbd
mice could be due to altered NF-kB signaling.
A primary function of DCs is the recognition of pathogen associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) such as TLRs. Based on the observed TCDD-induced modulation of DC differentiation, we assessed whether TCDD-exposed BMDCs were responsive to PAMPs via TLR activation. TCDD increased BMDC responsiveness to TLR stimulation via TLR4 and TLR9 without affecting the basal mRNA levels of these receptors. Following activation, DCs secrete cytokines that orchestrate developing immune responses. In our study, TCDD increased LPS- and CpG-induced IL-6 and TNF-α production by BMDCs but decreased their production of NO. Multiple possibilities could account for these observed effects. The NF-kB independent transcription factor, LPS-Induced TNF-α Factor (LITAF), is a transcription factor that has been implicated in the LPS-induced expression of TNF-α and other inflammatory cytokines (Sun et al., 2004
; Tang et al., 2005
). LITAF contains 7 DRE sequences and upregulation of LITAF in DCs could underlie our observed TCDD-induced increases in LPS-induced pro-inflammatory cytokine secretion (Sun et al., 2004
). However, recent studies have documented the ability of activated AhR to negatively regulate inflammatory responses in human monocyte-derived DCs and murine macrophages (Lawrence et al., 2008
; Kimura et al., 2009
). Discrepancies in these results could be due to differences in the AhR agonists used in the respective studies (VAGF347 versus TCDD) or in the cells that were examined (human monocyte-derived DCs versus murine DCs or macrophages). In addition, activation of inflammatory DCs by LPS or CpG usually induces upregulation of inducible nitric oxide synthase (iNOS) and subsequent increases in NO production. AhR activation by TCDD has been shown to increase pulmonary iNOS levels contributing to an enhanced IFN-γ production in the lungs (Neff-LaFord et al., 2007
). Contrary to these reports, in our study TCDD decreased NO production by both unstimulated and TLR-activated BMDCs. Additional investigations are thus warranted to further understand the effects of TCDD on NO production by BMDCs.
A previous study examining the effects of TCDD on BMDCs reported decreased mRNA levels of IL-10 but not IL-12 following LPS-stimulation (Lee et al., 2007
). Contrary to this study, our data indicate that TCDD does not alter the production of IL-10 and decreases CpG-induced IL-12 production by BMDCs. Different DC preparations (unpurified versus purified BMDCs) used in these two studies could account for the observed differences. Under in vivo
conditions, inflammatory DCs function to limit infection by their secretion of inflammatory cytokines. Induction of inflammation is a hallmark feature of TCDD-induced immunotoxicity and our results suggest a role for DCs in TCDD-induced inflammatory responses. However, how TCDD-induced alterations of inflammatory DCs impacts the development of acute inflammation remains to be determined.
NF-kB is a major signaling pathway involved in the differentiation of DCs and their responsiveness to TLR stimulation. Furthermore, several studies have linked physical interactions of activated AhR with NF-kB p65 and RelB in the generation of TCDD-induced cellular dysfunction and immune suppression (Ruby et al., 2002
; Camacho et al., 2005
; Vogel et al., 2007a
; Vogel et al., 2007b
; Vogel and Matsumura, 2008
). Because TCDD alters the differentiation of DCs and their responsiveness to TLRs, we investigated the effects of AhR activation on NF-kB signaling in inflammatory BMDCs. TCDD suppressed basal p65 activity in unstimulated BMDCs as well as BMDCs activated by LPS and CpG. Furthermore, a trend towards increased RelB activity was also observed in TCDD-treated BMDCs. Binding of RelB/AhR to specific response elements (RelBAhRE) in certain target genes has been linked to the regulation of cytokines and chemokines such as IL-8 and BLC following TCDD exposure (Vogel et al., 2005
; Vogel et al., 2007b
; Vogel and Matsumura, 2008
). It would be of interest to determine if the enhanced production of IL-6 and TNF-α by TCDD-treated BMDCs is also regulated by increased interactions between activated AhR and RelB. Overall, our data suggest that TCDD-induced modulation of DC differentiation and their responsiveness to TLR stimulation may be mediated via altered NF-kB signaling.
Dendritic cells capture antigens via distinct mechanisms. While soluble antigen uptake primarily involves macropinocytic or receptor-mediated endocytic events, particulate antigens are internalized primarily via phagocytosis (Banchereau et al., 2000
). TCDD decreased the uptake of the soluble antigens ovalbumin and LDL but increased phagocytosis of latex beads by inflammatory DCs. These results suggest that TCDD differentially impacts distinct mechanisms of antigen uptake. Studies have shown macrophage mannose receptor and scavenger receptor A to mediate the endocytosis of ova and acetylated LDL, respectively (Pearson et al., 1993
; Becker et al., 2006
; Burgdorf et al., 2007
). Therefore, based on our results, we hypothesize that decreased expression of these receptors in BMDCs following exposure to TCDD leads to decreased uptake of soluble antigens. Conversely, TCDD increased the uptake of latex beads by BMDCs. So far, only one study has described effects of TCDD on the uptake of latex beads. In this in vivo
study, uptake of latex beads by splenic DCs was unaffected following TCDD exposure (Vorderstrasse et al., 2003
). The observed discrepancy in our results could be attributed to distinct antigen uptake mechanisms in the two functionally distinct DC populations (steady-state versus inflammatory), which could be differentially affected by TCDD.
Because our data demonstrated that TCDD altered antigen uptake, we assessed the effects of TCDD on the T cell stimulatory capacity of inflammatory BMDCs. Surprisingly, TCDD-treated BMDCs generated normal antigen-specific T cell responses in vivo
. These results were unexpected, as TCDD not only induced a regulatory phenotype in BMDCs, but also increased their expression of several regulatory mediators including IDO1, IDO2 and TGF-β3. Several possibilities could account for the lack of T cell suppression by TCDD-treated BMDCs. Though our results demonstrated that TCDD alters antigen uptake of ovalbumin by BMDCs, it is possible that the observed decreases in antigen uptake do not reach a physiological threshold below which T cell activation is affected. Additionally, TCDD may increase the expression, but not the function or activity of the suppressive mediators IDO1, IDO2 and TGF-β3. In this case, we would not expect TCDD-treated BMDCs to contribute to the suppression of T cell activation. Recent studies have linked AhR activation in DCs with suppression of T cell responses (Hauben et al., 2008
). In these studies, AhR-activated BMDCs suppressed T cell responses only following LPS stimulation; immature BMDCs did not alter T cell responses. Thus, we also evaluated the expression of the suppressive mediators in BMDCs following LPS activation. TCDD-treated BMDCs stimulated with LPS displayed increased expression of IDO1, IDO2, TGF-β1, TGF-β2 and Thbs1. However, future experiments are needed to determine whether TCDD-treated BMDCs stimulated with LPS can affect antigen-specific T cell responses.
Collectively, we show that AhR activation alters inflammatory DC differentiation, generating DCs that are hyperresponsive to TLR stimulation and exhibit a regulatory phenotype in vitro. Surprisingly, these BMDCs fail to alter antigen-specific T cell activation. However, further studies are needed to examine the potential for AhR-activated DCs to contribute to the generation of regulatory T cells and the suppression of T cell-mediated immune responses. One of the novelties of our data is the identification of biomarkers of exposure to TCDD and TCDD-like chemicals on a highly sensitive and extremely important immune cell population, the inflammatory DCs. Moreover, these effects were detected at very low, environmentally relevant levels of exposure. Because our results indicate that specific modulation occurs in these cells, it would be of great value to evaluate human inflammatory DCs. Similarly, our data further define mechanisms of action of TCDD on the immune system. This information may be useful for the generation of focused therapeutic approaches to treat humans exposed to environmental chemicals that can activate the AhR. Furthermore, it highlights the potential to develop selective AhR modulators that may be beneficial in the treatment of allergies and autoimmune diseases.