Aryl hydrocarbon receptor (AhR) is well known for mediating the toxic effects of dioxin-containing pollutants, but has also been shown to be involved in the natural regulation of the immune response. In this study, we investigated the effect of AhR activation by its endogenous ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the differentiation, maturation and function of monocyte-derived DCs in Behçet's disease (BD) patients. In this study, we showed that AhR activation by FICZ and ITE down-regulated the expression of co-stimulatory molecules including human leucocyte antigen D-related (HLA-DR), CD80 and CD86, while it had no effect on the expression of CD83 and CD40 on DCs derived from BD patients and normal controls. Lipopolysaccharide (LPS)-treated dendritic cells (DCs) from active BD patients showed a higher level of interleukin (IL)-1β, IL-6, IL-23 and tumour necrosis factor (TNF)-α production. FICZ or ITE significantly inhibited the production of IL-1β, IL-6, IL-23 and TNF-α, but induced IL-10 production by DCs derived from active BD patients and normal controls. FICZ or ITE-treated DCs significantly inhibited the T helper type 17 (Th17) and Th1 cell response. Activation of AhR either by FICZ or ITE inhibits DC differentiation, maturation and function. Further studies are needed to investigate whether manipulation of the AhR pathway may be used to treat BD or other autoimmune diseases.
The aryl hydrocarbon receptor (AhR) belongs to the basic-helix-loop-helix-PER-ARNT-SIM family and is a ligand-dependent transcription factor which is involved in the detection of intracellular or environmental changes, sensing light, oxygen and redox potentials . Upon activation by ligands, AhR can dimerize with the constitutively expressed AhR nuclear transporter (Arnt) and then translocates into the nucleus, inducing target gene expression . The AhR, well known for its role in mediating the toxicity of xenobiotics such as 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) and benzo(a)pyrene (B(a)P), has recently been found to play a role in the regulation of the immune response [3-5]. It has been reported that AhR-deficient macrophages showed a higher level of proinflammatory cytokines upon lipopolysaccharide (LPS) stimulation compared with wild-type (WT) cells, and that AhR-deficient mice were more susceptible to LPS-induced lethal shock than WT mice , indicating that AhR plays a regulatory role in the inflammatory response induced by LPS. Activation of the AhR by environmental toxins differs from that seen following stimulation with its natural ligands. AhR activation of T cells by TCDD, for instance, was shown to inhibit autoimmunity by inducing the generation of regulatory T cells (Treg), whereas it worsened autoimmunity following activation by 6-formylindolo[3,2-b]carbazole (FICZ), an endogenous ligand derived from photoproducts of tryptophan . 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methylester (ITE), an endogenous non-toxic AhR ligand isolated originally from lung , has been reported to be able to inhibit experimental autoimmune encephalomyelitis (EAE) severity by inducing forkhead box protein 3 (FoxP3+) Treg cells and tolerogenic dendritic cells . Whether the endogenous ligands FICZ or ITE can also affect the human immune response, and more specifically the human DC immune response, has not yet been clarified.
Behçet's disease (BD) is a chronic systemic autoinflammatory disease, characterized by recurrent uveitis, oral and genital mucous ulcers and skin lesions . A number of studies have demonstrated that an enhanced T helper type 1 (Th1) and Th17 cell immune response and associated molecules were involved in the pathogenesis of BD [9-11]. Because current treatment in uveitis including non-specific immunosuppressive agents (corticosteroids, cyclosporin, cyclophosphamide and others) is associated with serious side effects, many studies have focused on other means of T cell immunomodulation, or immune reprogramming, thereby focusing on dendritic cells (DCs), as they play a critical role in modulating autoreactive T cells.
DCs are widely recognized as the most potent antigen-presenting cells, having the unique function in regulating immunity and tolerance [12, 13]. Upon recognition of pathogens with its pattern recognition receptors, DCs up-regulate surface major histocompatibility complex (MHC)-II and co-stimulatory molecules (CD80, CD86 and CD40), and produce proinflammatory cytokines that drive naive T cells into different effector T cells, including Th1 and Th17 effector cells, which can lead to autoimmune pathology [14, 15]. In addition to the initiation of the immune response as mentioned above, DCs have also been reported to be able to suppress immune responses and induce immune tolerance dependent upon the stage of the differentiation, maturation and function [16-18]. As AhR may affect the function of DCs and thereby modulate the inflammation associated with an autoinflammatory disease such as BD, we decided to focus on the role of AhR activation in this disease and to provide data that might explain the possible mechanisms involved.
The aim of our work was to study the effects of AhR activation by endogenous FICZ or ITE on human monocyte-derived DC differentiation, maturation and functional activities in BD patients. We found that the AhR ligands FICZ or ITE inhibited the expression of co-stimulatory molecules human leucocyte antigen D-related (HLA-DR), CD80 and CD86. Furthermore, AhR activation suppressed the production of proinflammatory cytokines, and induced anti-inflammatory cytokine production by DCs from BD patients and normal controls. Finally, we showed that FICZ or ITE-treated DCs significantly inhibited Th17 and Th1 cell polarization.
Materials and methods
Eighteen BD patients with active uveitis (11 men and seven women, with an average age of 34·5 years) and 26 healthy controls (15 men and 11 women, with an average age of 36·7) were included into this study. The healthy control subjects had no clinical history of uveitis or other systemic diseases. Patients were seen in our uveitis centre of the First Affiliated Hospital of Chongqing Medical University between February 2012 and October 2012. The diagnosis of BD was made according to the diagnostic criteria of the International Study Group for BD . At the time of sampling, the patients had active intraocular inflammation, but they were not being treated with systemic therapies, such as corticosteroids, cyclosporin or cyclophosphamide. Active intraocular inflammation was evidenced by the presence of anterior chamber flare and cells (100%), non-granulomatous keratic precipitates (100%), vitreous cells (80%) and retinal vasculitis (100%). Typical systemic findings included recurrent oral aphthous ulcers (100%), skin lesions (80%), arthritis (32%) and recurrent genital ulcers (40%). The study followed the tenets of the Declaration of Helsinki and was approved by the Clinical Ethical Research Committee of Chongqing Medical University, and informed consent was obtained from all the BD patients and normal controls.
Peripheral blood samples were obtained from BD patients and healthy volunteers. CD14+ monocytes were separated from peripheral blood mononuclear cells (PBMCs) using human microbeads (purity >90%; Miltenyi Biotec, Bergisch Gladbach, Germany). The generation of immature monocyte-derived moDCs was described earlier . Immature moDCs were stimulated with 0·05% dimethylsulphoxide (DMSO) or with AhR ligands (FICZ (100 nmol/l) (Enzo Life Sciences, Ann Arbor, MI, USA) or ITE (100 nmol/l; Tocris Bioscience, Minneapolis, MN, USA) in the presence of LPS (Sigma-Aldrich, St Louis, MO, USA) for 24 h. Cells were subsequently harvested for surface marker fluorescence activated cell sorter (FACS) analysis or functional assays. The supernatants were collected for cytokine [interleukin (IL)-1β, IL-6, IL-23, IL-10, IL-12 and tumour necrosis factor (TNF)-α] measurement by enzyme-linked immunosorbent assay (ELISA). All our experiments included the DMSO vehicle as a control and the final concentration of DMSO was 0·05%, which has been widely used in a large number of previous papers to study the effect of chemicals that must be dissolved in DMSO [21, 22]. Matured DCs were washed three times before co-culturing with CD4+ T cells. Co-cultures were performed with a DC : T cell ratio of 1:5 in 96-well plates for 4 days; the cell culture supernatants were then collected for IL-17 and interferon (IFN)-γ measurement. The cells were harvested for intracellular detection of IL-17 and IFN-γ using flow cytometry.
To study the effect of AhR activation on the surface markers of DCs, the following monoclonal antibodies (mAbs) were used: anti-human HLA-DR-peridinin chlorophyll-cyanin 5·5 (PerCP-Cy5·5), anti-human CD86-phycoerythrin (PE), anti-human CD80-fluorescein isothiocyanate (FITC), anti-human CD40-allophycocyanin (APC) and anti-human CD83-FITC. All these fluorescein-conjugated and isotype-matched antibodies were purchased from BD Biosciences (Biosciences, Sunnyvale, CA, USA). The cells were incubated for 30 min 4°C with the above fluorescein-conjugated antibodies or isotype control antibodies in staining buffer, avoiding exposure to light. Cells were then washed, resuspended and subjected to FACS analysis. FACS aanalysis was performed on a FACS Aria cytometer (BD Biosciences) and analysis was performed using FlowJo software (Tree Star Software, San Carlos, CA, USA). The results were expressed as the percentage difference compared with isotype control (IC) using the formula [mean fluorescence intensity (MFI) of each marker – MFI of IC]/MFI of IC × 100%, which refers to the methods described previously [16, 23].
For analysis of the frequency of Th1 and Th17, the cells were stimulated by adding phorbol myristate acetate (PMA) (50 ng/ml; Sigma-Aldrich) and ionomycin (1 ug/ml; Sigma) for 1 h at 37°C. Brefeldin A (10 ug/ml; Sigma) was then added for another 4 h, the cells were fixed and permeabilized using the eBioscience Cytofix/Cytoperm kit, according to the manufacturer's instructions, and then incubated with anti-CD3-PerCP-Cy5·5, anti-human CD8-APC, anti-human IL-17A-PE, anti-human IFN-γ -FITC (BD Biosciences) and isotype control antibodies.
Supernatants from control DCs, FICZ-treated DCs or ITE-treated DCs, the co-cultures of DCs and CD4+T cells were harvested and stored at −80°C until cytokine determination. IL-17, IFN-γ, IL-1β, IL-6, IL-23, IL-10, IL-12 and TNF-α levels in the cell culture supernatants were measured with Duoset ELISA development kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's protocols.
The statistical significance of differences was determined by independent-sample t-test, Mann–Whitney U-test, Wilcoxon's matched-pairs test or paired-samples t-test. Differences with a P-value less than 0·05 were considered statistically significant. All analyses were performed using commercially available statistical software (spss 12·0; SPSS Inc., Chicago, IL, USA).
FICZ and ITE inhibit LPS-induced HLA-DR, CD80 and CD86 expression on DCs derived from BD patients and normal controls
To investigate the effect of AhR activation on DC differentiation from monocytes, we examined the effect of FICZ and ITE on the DC surface phenotype. Immature DCs were stimulated with LPS with or without FICZ or ITE for 24 h, after which the phenotype was analysed by flow cytometry. We did not detect a difference in the expression of the tested surface molecules in LPS-treated DCs between BD patients and normal controls. FICZ and ITE significantly down-regulated HLA-DR, CD80 and CD86 expression in LPS-treated DCs derived from BD patients and normal controls. No detectable difference was observed concerning the inhibitory effect between the two tested ligands. The ligands had no significant effect on the expression of the co-stimulatory molecules CD40 and CD83 (Fig. 1). These data suggest that AhR activation by FICZ or ITE may act as a negative regulator of LPS-triggered functional differentiation of DCs by suppressing the surface expression of HLA-DR, CD80 and CD86.
FICZ and ITE modulate the production of cytokines by DCs derived from BD patients and normal controls
The types of cytokines produced by DCs define the outcome of the different classes of Th cells generated. For example, DC-secreted IL-1β, IL-6, IL-23 and TNF-α can induce the differentiation of Th17 cells, while IL-12 can induce Th1 differentiation. DC-secreted IL-10 initiates the differentiation of type 1 regulatory T cells (Tr1 cells), but inhibits Th1 and Th17 differentiation. Our following set of experiments was therefore aimed at investigating the effect of FICZ and ITE on the cytokine profile produced by DCs. The results showed that LPS-treated DCs from active BD patients showed a higher level of IL-1β, IL-6, IL-23 and TNF-α production compared to that from normal controls. However, there was no difference with regard to the IL-10 expression by LPS-treated DCs between active BD patients and normal controls (Fig. 2). FICZ or ITE significantly inhibited the production of IL-1β, IL-6, IL-23 and TNF-α, but induced a significant increase in the production of anti-inflammatory IL-10 by DCs derived from active BD patients and normal controls (Fig. 3). The levels of IL-12P70 were under the detection limit in the supernatants of FICZ- or ITE-treated DCs or control DCs derived from active BD patients and normal controls. Together, these data indicate that AhR activation by FICZ or ITE may act as a negative regulator of the LPS-stimulated innate response of DCs by suppressing the production of proinflammatory cytokines such as IL-1β, IL-6, IL-23 and TNF-α, and inducing the production of the anti-inflammatory cytokine IL-10.
FICZ and ITE inhibit DC-initiated Th1 and Th17 polarization from CD4+ T cells
DCs are potent inducers of T cell immune responses. We next investigated whether culturing DCs with AhR ligands had an effect on the development of a Th1 or Th17 response. We co-cultured CD4+ T cells with control DCs, FICZ- or ITE-treated DCs for 4 days. IFN-γ and IL-17 in the cell culture supernatants were measured by ELISA and the frequency of Th1 and Th17 cells was determined by FACS. The results showed that the production of IFN-γ and IL-17 was markedly decreased in the culture supernatants of CD4+T cells co-cultured with FICZ- or ITE-treated DCs compared with that co-cultured with untreated control DCs (Fig. 4a–d). Consistent with the ELISA results, the FACS results showed that the frequency of IFN-γ- and IL-17-expressing CD4+ T cells was also decreased in the CD4+ T cells co-cultured with FICZ- or ITE-treated DCs compared with those cultured with untreated control DCs (Fig. 4e–g).
In this study, we found that AhR activation by the endogenous ligands FICZ or ITE inhibited the expression of co-stimulatory molecules on DCs obtained from active BD patients and normal controls. Moreover, we found that DCs from active BD patients showed a higher level of IL-1β, IL-6, IL-23 and TNF-α production. FICZ or ITE inhibited the production of these cytokines, while they induced the secretion of the anti-inflammatory cytokine IL-10. FICZ or ITE-treated DCs were shown to inhibit both the Th1 as well as the Th17 cell immune response. Our data are in accord with and provide a theoretical foundation for earlier observations describing that ITE could inhibit the development of experimental autoimmune uveoretinitis (EAU) and the related Th1 and Th17 cell immune response . Our results extend these observations and suggest that the potent immunosuppressive capacity of ITE on EAU may be caused by an effect on the activation and function of DCs. Our findings are also in agreement with a Japanese study showing that another AhR ligand, TCDD, can suppress the severity of EAU, but its high toxicity makes it unsuitable for human uveitis treatment .
Previous studies have reported that AhR was expressed on DCs , a finding that was confirmed in the present study for human DCs. Our experiments extend further studies from the literature showing that the effect of the ligands FICZ and ITE that we used were not seen in AhR-deficient mice [7, 27]. These findings indicate that both the effect of ITE and FICZ on DCs was dependent on binding and activation of AhR. We used one concentration of FICZ to perform our experiments, which was based on a literature search, whereby the concentration of FICZ being used in human studies ranges between 20 nmol/l and 200 nmol/l . We therefore decided to use a concentration of 100 nmol/l to study the role of FICZ in the activation and function of human monocyte-derived dendritic cells. As there have been no published reports on the effect of ITE on human immune cells, we decided to use a concentration (100 nml/l) of ITE that was used earlier in mouse studies . Whether there is a dose-dependent effect of FICZ and ITE on DC activation and function needs further study.
DCs serve as professional APCs and provide the first line of defence against pathological infections. Interaction of DCs with TLR ligands results in the secretion of a number of proinflammatory cytokines and induction of the differentiation of naive T cells into effector T cells . In addition, it has been reported that DCs can present organ-specific antigens from necrotic cells under inflammatory conditions to activate autoreactive T cells, which can lead to autoimmune disease . It has been proposed that DC deregulation is implicated in the pathogenesis of BD . A number of research groups have adopted this idea and have attempted to develop tolerogenic DCs for the treatment of autoimmune disease [16, 32, 33]. Recently, a number of studies have emerged showing a role of AhR activation in the generation of DCs. The exogenous AhR ligand VAF347 was shown to inhibit the generation of Langerhans cells (LC) from precursors, but did not affect LC maturation and monocyte-derived DC differentiation . Others showed that AhR activation of DCs by ITE induced a tolerogenic type of DCs in mice . The dioxin agonist TCDD was shown to be able to induce inflammatory cytokines by human monocytes and bone marrow-derived DCs (BMDCs) via its association with AhR [34, 35]. The studies mentioned above concerning the role of AhR activation were all performed in mouse models, and as yet studies with human DCs are scarce. Because murine cells may respond in a different manner to AhR ligands, we decided to repeat the experiments concerning the role of endogenous ITE in DC activation and function using human DCs.
Successful T cell activation requires not only an interaction between the T cell receptor and antigen-associated MHC-II, but also co-stimulation via interactions between accessory molecules, such as CD28–CD86 and CD40L–CD40. We therefore first addressed the role of AhR activation by FICZ or ITE on the important immunoregulatory surface molecules of DCs. In the process of DC generation in our study, we used granulocyte–macrophage colony-stimulating factor (GM-CSF) (100 ng/ml) and IL-4 (50 ng/ml) to stimulate the differentiation of DCs from monocytes, The IL-4 level we used may be somewhat high, but it has been demonstrated to be effective in DC differentiation and well used in our previous studies as well as other's studies [36-39]. The effect of LPS on DCs has been shown previously in the literature [40-42]. In this study, we investigated the effect of AhR ligands on the already existing effect of LPS on DCs. We found that the AhR ligands FICZ and ITE inhibited the expression of HLA-DR, CD80 and CD86 in LPS-treated DCs derived from both BD patients and normal controls. The response of DCs between uveitis patients and controls to AhR stimulation was similar, and we failed to find an effect on the expression of the co-stimulatory molecules CD40 and CD83. These results suggest that AhR activation by FICZ and ITE could inhibit the differentiation and maturation of DCs by selectively suppressing surface expression of HLA-DR, CD80 and CD86, further contributing to defective T cell activation and subsequent immune suppression. Our findings are in disagreement with results showing that FICZ or ITE inhibited CD11c expression and increased the levels of MHC-II and CD86 on GM-CSF-stimulated BMDCs in mouse studies . Our results are also inconsistent with other animal studies , which showed that ITE did not affect the number or the phenotype of BMDCs. As mentioned above, these discrepancies may be due to species differences (mouse versus human) or to differences in the DCs (BMDCs stimulated GM-CSF versus monocyte-derived DCs stimulated with GM-CSF, IL-4 and LPS) used. It is worth pointing out that experiments using non-LPS-stimulating DCs may provide further information on the role of AhR in the activation and function of human monocyte-derived DCs.
Mature DCs produce cytokines that regulate the differentiation of naive T cells into various Th cell subsets. CD4+ Th cell subsets, such as Treg and Th17/Th1 cells, contribute to either tolerance or autoimmunity. We next examined the effect of FICZ and ITE on the cytokines expressed by DCs. We found that LPS-induced DCs from active BD patients showed a significantly higher level of IL-1β, IL-6, IL-23 and TNF-α compared to normal controls, which further confirmed the hyperactivity of DCs in BD. As IL-1β, IL-6, IL-23 and TNF-α are critical for Th17 differentiation and Th17 cells were reported to be involved in the pathogenesis of BD, the increased expression of these proinflammatory cytokines produced by DCs may explain why Th17 cells are increased in the peripheral blood of BD patients . Our finding, that AhR activation by endogenous ligands led to a significant inhibition of proinflammatory cytokine production, is different from that seen when AhR is activated by the dioxin TCDD. In these studies, TCDD led to up-regulated LPS-induced IL-6 and TNF-α production by bone marrow-derived DCs .
The difference confirms earlier observations showing that AhR activation by environmental pollutants leads to a different response on the immune system when compared to the natural endogenous ligands. Vogel et al. reported recently that AhR activation by TCDD could promote the differentiation and maturation of bone-marrow-derived DCs (BMDC) and AhR-dependent nuclear accumulation of RelB. Furthermore, they showed that the increased accumulation of RelB in the nucleus was associated with increased DNA-binding activity of RelB and AhR on a RelB AhRE element and an interaction of AhR with RelB [43, 44]. It has been reported that RelB is a critical factor in the development and maturation of DCs, and that it is indispensable for the up-regulation of MHC-II and CD80/86 and co-stimulatory capacity [45, 46]. These results suggest that the TCDD-mediated effect on DC differentiation and function is mediated by the induction of RelB expression following the interaction of AhR with RelB. In the present study, we found that FICZ and ITE could inhibit the differentiation and maturation of DCs, but whether RelB was also involved in this process was not investigated and needs further study.
Further in-vitro experiments showed that Th1 and Th17 cell responses were inhibited when CD4+ T cells were co-cultured with FICZ- or ITE-treated DCs compared to control DCs. These findings, concerning the role of AhR activation by ITE on the function of DCs, are in agreement with those reported earlier in mouse studies . In our study, we found that AhR activation with FICZ could inhibit Th1 and Th17 cell responses by affecting DC maturation and function. Our findings with FICZ are in agreement with the results of Ramirez et al., who found that in humans FICZ could inhibit IL-17 and IFN-γ production by CD4+T cells [28, 47]. However, they are different from results showing that an enhanced IL-17A production by CD4+T cells was observed in the presence of FICZ in murine studies [48-50]. These contradictory phenomena further stress a difference between human and mouse, and demonstrate that the response to AhR activation by FICZ depends on the cell type and species used in the studies. A previous study performed by Duarte et al. reported that systemic application of TCDD and FICZ could confer protection from EAE through inhibiting Th17 cell differentiation, but they were able to promote IL-17 and IL-22 protein expression in vitro. This discrepancy between the in-vivo and in-vitro effects of AhR also indicates the complexity of the AhR pathway . Quintana et al. reported that EAE was aggravated by FICZ, whereas it could be inhibited by TCDD. The reasons as to the discrepancy of the effect of AhR activation by FICZ and TCDD are not clear. Quintana et al. suggested that the promotion by AHR of both Treg and TH17 cell differentiation might stem from its capacity to interact with different transcriptional partners in different cellular contexts . Veldhoen and Duarte thought that the different outcome seemed to be partly dependent upon the stability of the ligand used. TCDD is a high-affinity, non-degradable exogenous ligand , which might explain its different mode of action compared to the endogenous ligands such as FICZ and ITE. Whether these theories are correct awaits further study.
In conclusion, our results suggest that FICZ and ITE inhibit the activation and function of DCs from active BD patients and normal controls and support the important role of AhR activation as a negative modulator of the Th1 and Th17 cell response by interfering with the maturation and function of DCs. AhR activation may offer a possible novel therapeutic approach for BD and other autoimmune diseases which are mediated by an aberrant Th1 and Th17 cell immune response.
C. W. designed the study, performed experiments, analysed the data and wrote the manuscript. Z. Y. performed experiments, analysed the data and wrote the manuscript. A. K. revised the manuscript. Y. Z. collected the samples. P. Y. designed the study and revised the manuscript. All authors have read and approved the final manuscript.
This work was supported by Natural Science Foundation Major International (Regional) Joint Research Project (81320108009), Key Project of Natural Science Foundation (81130019), National Natural Science Foundation Project (31370893), National Key Clinical Specialties Construction Program of China, Basic Research program of Chongqing (cstc2013jcyjC10001), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), Key Project of Health Bureau of Chongqing (2012-1-003) and Fund for PAR-EU Scholars Program. The authors thank all donors enrolled in the present study.
None of the authors has a proprietary or financial interest in any product mentioned.