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Keywords:

  • dendritic cell;
  • endocrine-disrupting chemical;
  • epigenetics;
  • interferon;
  • phthalate

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References

Background

Exposure to environmental endocrine-disrupting chemicals (EDCs) is associated with allergy, chronic inflammation, and immunodeficiency. Phthalates, the common EDCs used in plastic industry, may act as adjuvants to disrupt immune system and enhance allergy. Plasmacytoid DCs (pDCs) are predominant cells secreting type I interferon (IFN) against infection and are professional antigen-presenting cells in regulating adaptive immunity. However, the effects of phthalates on the function of pDCs are unknown.

Methods

Circulating pDCs were isolated from healthy subjects, were pretreated with diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP), and were stimulated with Toll-like receptor (TLR)-9 agonist CpG. IFN-α/IFN-β levels, surface markers, and T-cell stimulatory function were investigated using ELISA, flow cytometry, and pDC/T-cell coculture assay. Mechanisms were investigated using receptor antagonists, pathway inhibitors, Western blotting, and chromatin immunoprecipitation.

Results

Diethylhexyl phthalate and butyl benzyl phthalate suppressed CpG-induced IFN-α/IFN-β expression in pDCs, and the effect was reversed by aryl hydrocarbon receptor (AHR) antagonist. Diethylhexyl phthalate suppressed CpG-activated mitogen-activated protein kinase (MAPK)-MEK1/2-ERK-ELK1 and NFκB signaling pathways. Diethylhexyl phthalate suppressed CpG-induced interferon regulatory factor (IRF)-7 expression by suppressing histone H3K4 trimethylation at IRF7 gene promoter region through inhibiting translocation of H3K4-specific trimethyltransferase WDR5 from cytoplasm into nucleus. Butyl benzyl phthalate or diethylhexyl phthalate-treated pDCs suppressed IFN-γ but enhanced IL-13 production by CD4+ T cells.

Conclusion

Phthalates may interfere with immunity against infection and promote the deviation of Th2 response to increase allergy by acting on human pDCs via suppressing IFN-α/IFN-β expression and modulating the ability to stimulate T-cell responses.

Endocrine-disrupting chemicals (EDCs) are ubiquitous in the environment and have been reported to be associated with allergy, chronic inflammation, and immunodeficiency [1]. Phthalate, one of EDCs, is a commonly used plasticizer in plastic industry and may act as an adjuvant to enhance allergic responses [2]. Recently, the association between the level of phthalates from polyvinyl chloride (PVC) surface materials in the home and the risk of asthma and allergies is reported [3].

Allergy and asthma are regarded as chronic inflammatory diseases driven mainly by allergen-specific T helper type 2 (Th2) immune response [4]. Dendritic cells (DCs) are professional antigen-presenting cells with a critical role in initiation and regulation of adaptive immune responses and participate in the pathogenesis of allergy and asthma [5]. Plasmacytoid DCs (pDCs) are predominant cells secreting type I interferon (IFN), such as IFN-α and IFN-β, and are critical in host Th1 responses in immunity against viral infection [6]. In addition to direct antiviral activity, type I IFN also increases the survival of T cells, the differentiation of Th1 and cytotoxic T lymphocytes, as well as the activity of natural killer cells, leading to overall boosting of antiviral T-cell activity [7]. In the presence of type I IFN,the Th2 immune response is decreased [8]. In asthmatic airways, allergen-sensitized pDCs inhibit both Th1 and Th2 immunity and subsequently induce tolerance to allergens [9]. While phthalate may disrupt the immune system and promote allergic response, the effects of phthalate on the function of human pDCs are unknown.

In this study, we investigated the effects of two phthalates, BBP, and DEHP on the function of human pDCs by examining the expression of type I IFN, the maturation markers, and the ability of T-cell polarization. We provided the novel evidence for the impact of phthalates on the function of human pDCs and the detailed mechanisms including epigenetic regulation.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References

Isolation and analysis of pDCs

The study of human subjects was approved by the Institutional Review Board of Kaohsiung Medical University, Kaohsiung, Taiwan. Ten healthy adults were enrolled, and peripheral blood mononuclear cells (PBMCs) were isolated after obtaining informed consent. Circulating pDCs were magnetically sorted by BDCA-4 cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer's instructions. The purity of isolated pDCs was more than 96%. Purified pDCs (1 × 105 cells/condition) were pretreated with BBP or DEHP (Sigma-Aldrich, St. Louis, MO, USA) for 2 h and then stimulated with Toll-like receptor (TLR)-9 agonist of CpG oligodeoxynucleotide (ODN)-2216 (10 μg/ml) in the presence of IL-3 (10 ng/ml) as previously reported [10, 11] to investigate the expression of IFN-α and IFN-β. To examine the involvement of the estrogen receptor (ER), aryl hydrocarbon receptor (AHR), pregnane X receptor (PXR), or constitutive androstane receptor (CAR), pDCs were pretreated with the selective ER antagonist ICI 182780, the AHR antagonist CH223191, the natural AHR antagonist (apigenin and kaempferol; two flavonoids), the PXR antagonist (sulforaphane), and the CAR antagonist (androstanol) for 1 h and then treated with BBP or DEHP. All nuclear receptor antagonists were purchased from Calbiochem (San Diego, CA, USA). Apigenin and kaempferol were purchased from Sigma-Aldrich (Sigma-Aldrich). To investigate the cell signaling, pDCs were pretreated with IκB kinase (IKK) inhibitor BAY 117085 (Calbiochem, Cambridge, MA, USA), p38-mitogen-activated protein kinase (MAPK) inhibitor (SB203580), ERK-MAPK inhibitor (PD98059), or JNK-MAPK inhibitor (SP600125) (Cayman Chemical, Ann Arbor, MI, USA) 1 h before the stimulation of CpG in the presence of IL-3. To investigate epigenetic regulation, pDCs were pretreated with anacardic acid (AA; a histone acetyltransferase inhibitor) or methylthioadenosine (MTA; a histone methyltransferase inhibitor) 1 h before treatment of the cells with BBP or DEHP. AA and MTA were purchased from Calbiochem (San Diego, CA, USA). Supernatants were collected at indicated time point.

Cell viability assay

Purified pDCs were incubated with or without DEHP or BBP at 10−7 M or vehicle solution for 48 h. Cell viability was examined using CytoScan WST-1 Cell Proliferation Assay (G-Biosciences, Maryland Heights, MO, USA) according to the instruction of the manufacturer. The cell viability in each group was expressed as a percentage of the control value.

Flow cytometry analysis

After the treatment of BBP or DEHP for 48 h, pDCs were stained with fluorescence-conjugated monoclonal antibodies including anti-HLA-DR, anti-CD40, anti-CD80, and anti-CD86 (eBioscience, San Diego, CA, USA). The surface markers of pDCs were analyzed using a FACScan flow cytometer and CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA).

Western blotting analysis

Purified pDCs were pretreated with BBP or DEHP for 2 h and were stimulated with CpG (10 μg/ml) in the presence of IL-3 (10 ng/ml) for indicated time points. The cells were lysed with equal volumes of ice-cold 150-μl lysis buffer. After centrifugation at 13 000 × g for 15 min, equal amounts of cell lysates were analyzed by Western blotting using anti-p65/anti-phospho-p65, anti-MAPK (p38, ERK and JNK)/anti-phospho-MAPK (pp38, pERK, and pJNK), anti-MKK1/2 and anti-phospho-MKK1/2, and anti-ELK1/anti-phospho-ELK1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-IRF-7 antibody and anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, USA).

In some conditions, purified pDCs were treated with DEHP for 2 h and then stimulated with CpG in the presence of IL-3 for indicated time point. Cytosolic and nuclear protein extractions were performed as described in our previous work [12-14]. The cytosolic and nuclear protein lysates were used for Western blotting analysis with anti-WDR5 and anti-H3 antibodies (Upstate Biotechnology, Waltham, MA, USA). Immunoreactive bands were visualized using horseradish peroxidase–conjugated secondary antibody and the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Sunnyvale, CA, USA).

RNA extraction and real-time PCR

Isolated pDCs were pretreated with DEHP (10−8M) for 2 h and were stimulated with CpG in the presence of IL-3. Total RNAs were extracted from cells 4 h after CpG stimulation using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. RNA of each sample was reverse-transcribed to first-strand cDNA using a SuperScript™ First-Strand Synthesis System with a RT-PCR kit (Invitrogen). Measurement was taken by a ABI PRISM 9700 HT sequence detection system (Applied Biosystems, Foster City, CA, USA) using a predeveloped TaqMan probe/primer combination for IRF-7 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the same cDNA samples. TaqMan PCR was performed using AmpliTaq Gold polymerase and universal master mix (Applied Biosystems). Threshold cycle numbers were transformed using the comparative threshold cycle and relative value methods according to the manufacturer's recommendation and were expressed relative to GAPDH.

T-cell stimulation assay

CD4+ T cells were purified from PBMCs with human CD4 magnetic beads (Miltenyi Biotec) according to the manufacturer's instructions. After treatment with BBP or DEHP for 48 h in the presence of CpG and IL-3, pDCs (1 × 105) were washed with PBS 3 times and were cocultured with CD4+ T cells (1 × 106) for 5 day in the presence of anti-CD3 and anti-CD28 antibodies (eBioscience). Cell supernatants were collected for IFN-γ, IL-13, and IL-17A measurement.

Enzyme-linked immunosorbent assay (ELISA)

The level of cytokines including IFN-α, IFN-β, IFN-γ, IL-13, and IL-17A of the cell supernatants was determined by ELISA (R & D systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation assay (ChIP) was performed as described previously [12-14]. Briefly, purified pDCs were treated with DEHP (10−8 M) for 2 h and then stimulated with CpG in the presence of IL-3. After 30 min, cells were collected and treated with 1% formaldehyde for 10 min at room temperature and were sonicated and immunoprecipitated overnight at 4°C with anti-H3K4 trimethylation antibody (Upstate Biotechnology) or anti-IRF-7 antibody (Santa Cruz Biotechnology). Immune complexes were collected, and the DNA amounts were analyzed using PCR with designed primers and probes for specific regions. DNA from immune complex by anti-H3K4 trimethylation antibody was quantified using designed primer for IRF7 promoter region encompassing the following subregions relative to the transcription start sites: IRF7-1 (−335/+11), IRF7-2 (−690/−489), and IRF7-3 (+102/+252) according to the previous report [15]. DNA from immune complex by anti-IRF-7 antibody was quantified using designed primer for IFNA10 and IFNB promoter regions encompassing the following subregions relative to the transcription start sites: IFNA10 (−248/+4, including IRF-2/-1 and CRE-BP site) and IFNB (−268/−59, including PRDII/PRDI site) according to the previous report [16]. PCRs were run on the ABI 7700 TaqMan thermocycler (Applied Biosystems).

Statistical analysis

All data are presented as mean ± SD. Differences between control and experimental groups were analyzed by using the Wilcoxon signed rank test. A P value less than 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References

DEHP and BBP suppressed CpG plus IL-3-induced IFN-α and IFN-β expression in human pDCs via the AHR

Figure 1 revealed that DEHP (Fig. 1A) and BBP (Fig. 1B) suppressed CpG plus IL-3-induced IFN-α expression in pDCs at 48 h after CpG stimulation. DEHP or BBP alone had no effect on IFN-α or IFN-β expression (data not shown). Next, we verified whether the suppressive effect of DEHP and BBP on CpG plus IL-3-induced IFN-α expression was due to the cytotoxic effect of DEHP and BBP on pDCs. As shown in Fig. 1C, DEHP and BBP had no effect on cell viability of pDCs. The addition of natural relative AHR antagonists (apigenin and kaempferol) and selective AHR antagonist (CH223191) partly reversed the suppressive effect of DEHP on CpG plus IL-3-induced IFN-α in pDCs (Fig. 1D and E). During the culture time period, DEHP and BBP had no effect on CpG plus IL-3-induced expression of costimulatory molecules HLA-DR, CD40, CD80, or CD86 in pDCs as judged by flow cytometry (data not shown). Figure 2 revealed that DEHP and BBP also suppressed CpG plus IL-3-induced IFN-β expression (Fig. 2A, B), and the suppressive effect was reversed by apigenin, kaempferol (Fig. 2C), and CH223191 (Fig. 2D). The addition of ER, PXA, and CAR antagonists did not reverse the suppressive effect of DEHP on the CpG plus IL-3-induced IFN-α and IFN-β expression in pDCs (data not shown). These findings suggested that DEHP and BBP suppressed CpG plus IL-3-induced IFN-α and IFN-β expression via a mechanism that involves the AHR in human pDCs.

image

Figure 1. Diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) suppressed IFN-α expression via the AHR in human pDCs. CpG plus IL-3-induced IFN-α expression in pDCs was suppressed by varying doses of two phthalates (A) DEHP and (B) BBP at 48 h after CpG stimulation. (C) DEHP and BBP (10−7 M) had no cytotoxic effects on pDCs. (D) Apigenin and kaempferol, the natural aryl hydrocarbon receptor (AHR) antagonists, partially reversed the suppressive effect of DEHP on CpG plus IL-3-induced IFN-α in pDCs. (E) CH223191, the selective AHR receptor antagonist, reversed the suppressive effect of DEHP on CpG plus IL-3-induced IFN-α in pDCs. Results presented in (A), (B), (D), and (E) are the mean ± SD of 10 independent experiments using 10 subjects' pDCs. Results presented in (C) are from 4 independent experiments using four subjects' pDCs. *P < 0.05 compared with vehicle and CpG plus IL-3-treated cells. #P < 0.05 compared with DEHP and CpG plus IL-3-treated cells.

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image

Figure 2. Diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) suppressed IFN-β expression via the AHR in human pDCs. (A) DEHP or (B) BBP suppressed CpG plus IL-3-induced IFN-β expression in pDCs at 48 h after CpG stimulation. (C) Apigenin and kaempferol, and (D) CH223191 partially reversed the suppressive effect of DEHP on CpG plus IL-3-induced IFN-β in pDCs. Results presented are the mean ± SD of 10 independent experiments using 10 subjects' pDCs. *P < 0.05 compared with vehicle and CpG plus IL-3-treated cells. #P < 0.05 compared with DEHP and CpG plus IL-3-treated cells.

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DEHP suppressed CpG plus IL-3-induced IFN-α expression in pDCs via the MKK 1/2-ERK-ELK1 pathway and the NFκB pathway

Figure 3 revealed that SB203580 (the MAPK-p38 inhibitor), SP600125 (the MAPK-JNK inhibitor), PD98059 (the MAPK-ERK inhibitor), and BAY11-7082 (the IκBα inhibitor) suppressed CpG plus IL-3-induced IFN-α (Fig. 3A) and IFN-β (Fig. 3B) expressions, suggesting that all three MAPK and NFκB pathways are involved in CpG plus IL-3-induced IFN-α and IFN-β expression in pDCs. Western blotting analysis revealed that DEHP suppressed CpG plus IL-3-induced phosphorylation of MAPK-ERK, its up-stream signaling MKK1/2, and its down-stream signaling ELK1 in pDCs (Fig. 3C, D). However, DEHP had no effect on CpG plus IL-3-induced phosphorylation of MAPK-p38 and pJNK expression in pDCs (Fig. 3E). DEHP also suppressed CpG plus IL-3-induced phosphorylation of p65 (Fig. 3F). These findings suggested the involvement of the MKK1/2-ERK-ELK1 pathway and the NFκB-p65 pathways in the suppressive effect of DEHP on CpG plus IL-3-induced IFN-α and IFN-β expression in pDCs.

image

Figure 3. Diethylhexyl phthalate (DEHP) suppressed IFN-α and IFN-β expression in human pDCs via the MKK1/2-ERK-ELK1 and the NFκB-p65 pathway. The levels of CpG plus IL-3-induced (A) IFN-α and (B) IFN-β expression were suppressed in the presence of 10 μM pathway inhibitors including SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), PD98059 (ERK inhibitor), and BAY11-7082 (NFκB inhibitor). Western blotting revealed that DEHP suppressed CpG plus IL-3-induced phosphorylation of MKK1/2, ERK, and ELK-1 (C and D), but had no effect on CpG plus IL-3-induced phosphorylation of p38 and JNK (E). Diethylhexyl phthalate suppressed CpG plus IL-3-induced phosphorylation of p65 (F). Results presented are the mean ± SD of five independent experiments using five subjects' pDCs. For Western blotting analyses, one experiment representative of five is shown. *P < 0.05 compared with vehicle and CpG plus IL-3-treated cells.

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DEHP suppressed CpG plus IL-3-induced IFN-α and IFN-β expression in pDCs via the IRF-7 pathway

IRF-7 is the master transcriptional factor for IFN-α and IFN-β in response to TLR activation [17]. Next, we investigated whether DEHP suppressed CpG plus IL-3-induced type I IFN via modulating the expression of IRF-7. Figure 4 revealed that that DEHP suppressed CpG plus IL-3-induced IRF-7 mRNA (Fig. 4A) and protein (Fig. 4B) in pDCs. AHR antagonist (CH223191) reversed the suppressive effect of DEHP on CpG plus IL-3-induced IRF-7 expression (Fig. 4C). The MAPK-ERK inhibitor (PD98059) suppressed CpG plus IL-3-induced IRF-7 expression (Fig. 4D), suggesting the involvement of the MAPK-ERK pathway in CpG plus IL-3-induced IRF-7 expression. Because DEHP suppress the MAPK-ERK pathway, these findings may suggest that DEHP may firstly suppress CpG plus IL-3-induced MKK1/2-ERK-ELK1 pathway and subsequently inhibit the IRF-7 expression. ChIP assay with anti-IRF-7 antibody revealed that DEHP suppressed the binding of IRF-7 to IFNA10 and IFNB gene promoter regions. Taken together, the evidence suggested the involvement of the AHR, the MAPK-ERK, and the IRF-7 pathways in the suppressive effect of DEHP on CpG plus IL-3-induced IFN-α and IFN-β expression in pDCs.

image

Figure 4. Diethylhexyl phthalate (DEHP) suppressed CpG plus IL-3-induced IRF-7 expressions in human pDCs. Diethylhexyl phthalate suppressed CpG plus IL-3-induced IRF-7 (A) mRNA and (B) protein expressions in human pDCs. (C) CH223191 partially reversed the suppressive effect of DEHP on CpG plus IL-3-induced IRF-7 expression in pDCs. (D) CpG plus IL-3-induced IRF-7 expression was suppressed by PD98059 (ERK inhibitor). (E) DEHP suppressed the binding of IRF-7 to IFNA10 and IFNB gene promoter region. Results presented are the mean ± SD of five independent experiments using five subjects' pDCs for RT-PCR and ChIP analyses. For Western blotting analyses, one experiment representative of five is shown. *P < 0.05 compared with vehicle and CpG plus IL-3-treated cells.

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DEHP suppressed CpG plus IL-3-induced IRF-7 expression by suppressing histone H3K4 trimethylation at IRF7 promoter region through inhibiting the translocation of H3K4-specific methyltransferase WDR5

Epigenetic regulation such as histone modifications is recently regarded as one of important mechanisms for gene expression [18]. Histone acetylation by histone acetyltransferases or histone trimethylation at H3K4, H3K36, and H3K79 by specific methyltransferases is associated with gene activation [19]. In the present study, we found that MTA (the histone methyltransferase inhibitor; Fig. 5A), but not AA (the histone acetyltransferase inhibitor; data not shown), suppressed CpG plus IL-3-induced IRF-7 expression. ChIP assay revealed that DEHP suppressed CpG plus IL-3-induced histone H3K4 trimethylation at the proximal promoter subregion of IRF7-2 (Fig. 5B). Western blotting revealed that the expression of the H3K4-specific methyltransferase WDR5 was decreased in cytoplasm but was increased in nucleus in CpG plus IL-3-treated pDCs, and DEHP reversed the effect of CpG plus IL-3 on WDR5 expression (Fig. 5C). These findings suggested that DEHP may suppress IRF-7 expression by suppressing histone H3K4 trimethylation at IRF7 gene promoter region through inhibiting the translocation of H3K4-specific methyltransferase WDR5.

image

Figure 5. Diethylhexyl phthalate (DEHP) suppressed CpG plus IL-3-induced histone H3K4 trimethylation at the IRF7 gene promoter region via inhibiting the translocation of H3K4-specific methyltransferase WDR5 in human pDCs. (A) CpG plus IL-3-induced IRF-7 expression was suppressed by methylthioadenosine (MTA; the histone methyltransferase inhibitor). (B) ChIP analyses revealed that DEHP suppressed CpG plus IL-3-induced H3K4 trimethylation at the IRF7 gene promoter subregion IRF7-2. (C) DEHP suppressed CpG plus IL-3-induced translocation of H3K4-specific methyltransferase WDR5 from cytoplasm into nucleus. Results presented are the mean ± SD of five independent experiments using five subjects' pDCs for ChIP analyses. For Western blotting analyses, one experiment representative of five is shown. *P < 0.05 compared with vehicle and CpG plus IL-3-treated cells.

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DEHP- and BBP-treated pDCs suppressed IFN-γ but enhanced IL-13 production by CD4+ T cells

We next examined whether DEHP and BBP modulate the T-cell stimulation function of pDCs using pDC/CD4+ T-cell coculture assay. After treatment with BBP or DEHP for 48 h in the presence of CpG and IL-3, pDCs were cocultured with CD4+ T cells for 5 day in the presence of anti-CD3 and anti-CD28 antibodies. DEHP- and BBP-treated pDCs suppressed IFN-γ (Fig. 6A) and enhanced IL-13 (Fig. 6B) but had no effect on IL-17A (data not shown) production by CD4+ T cells.

image

Figure 6. Butyl benzyl phthalate (BBP)- and Diethylhexyl phthalate (DEHP)-treated pDCs suppressed IFN-γ but enhanced IL-13 production by CD4+ T cells. After treatment with BBP or DEHP for 48 h in the presence of CpG and IL-3, pDCs (105) were cocultured with CD4+ T cells (106) for 5 day in the presence of anti-CD3 and anti-CD28 antibodies. The level of (A) IFN-γ was decreased, but the level of (B) IL-13 was increased in CD4+ T cells. Results presented are the mean ± SD of 10 independent experiments using 10 subjects' pDCs and autologous CD4+ T cells. *P < 0.05 compared with vehicle-treated cells in the presence of CpG and IL-3.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References

Phthalates are potent modulators of immune systems and may contribute to the allergic response and the development of asthma [3]. Plasmacytoid DCs (pDCs) are the predominant cells to secret type I interferon (IFN), which can limit viral infection [6] and suppress Th2 immune responses [8]. In addition, pDCs participates in the pathogenesis of asthma by limiting both Th1/Th2 responses [9]. In the present study, we demonstrated, for the first time, the modulatory effects of phthalates on the function of human pDCs and the detailed mechanisms including epigenetic regulations. We found that phthalates suppressed type I IFN expression via the AHR, the MKK1/2-ERK-ELK1 pathway, the NFκB-p65 pathway, and the IRF-7 pathway through epigenetic regulation in pDCs. We also found that phthalates modulated the T-cell stimulation function of pDCs to promote Th2 polarization by suppressing IFN-γ and enhancing IL-13 expression by T cells. These important novel findings provided further insights to understand how phthalates disrupt the immune system by their impact on pDCs.

Respiratory viral infection, such as rhinovirus or respiratory syncytial virus, contributes to the development and exacerbation of asthma [20, 21] and is positively associated with hospitalization in patients with asthma [22]. In response to viral infection, pDCs are activated with robust expression of type I IFN, which subsequently limits the viral replication and accelerates the elimination of virus [23]. In addition, plasmacytoid DCs also play an immune regulatory role in reducing pulmonary inflammation and inhibiting the development of airway hyper-responsiveness [5, 24]. In the present study, type I IFN (IFN-α/β) expression by pDCs was suppressed by phthalate, and type III (IFN-γ) production by T cells was also suppressed by phthalate-treated pDCs. Interestingly, pDCs from asthmatic patients are impaired to producing IFN-α in response to TLR9 stimulation [25]. The inhibitory effect of phthalates on IFN-α and IFN-β expression in pDCs may further suppress immunity against viral infections and subsequently prolong the viral elimination, resulting in more severe clinical symptoms and poorer outcomes during exacerbation of asthma.

Nuclear receptors, such as ER and AHR, are the major receptors reported to mediate the effects of EDCs [1]. In our previous study, we demonstrated that two common EDCs (nonylphenol and 4-octylphenol) increase inflammation by up-regulating pro-inflammatory cytokine TNF-α and down-regulating anti-inflammatory cytokine IL-10 via the ER in human myeloid DCs [12]. In the present study, we found that the AHR, but not ER, mediated the suppressive effect of phthalates on type I IFN expression in pDCs. Other immunosuppressive effect of AHR activation, including suppression of IL-12 and induction of indoleamine-2,3-dioxygenase and TGF-β3 in DCs, is also reported [26]. These findings suggest that different nuclear receptors may have different roles in regulating the function of immune cells, and EDCs may influence the immune system via different nuclear receptor-activated pathways. Interestingly, we found that two flavonoids namely apigenin and kaempferol, the natural AHR antagonists with antioxidant and anti-inflammatory effects [27, 28], were capable of reversing the suppressive effect of phthalates on type I IFN expression in pDCs. These findings suggest that flavonoids may be the natural compounds, which can protect against the toxicity of phthalates. The preventive or therapeutic effects of flavonoids on the toxicity of phthalates and the mechanism deserve further investigation.

One of the novelties in the present study is that we provided the detailed intracellular mechanisms including epigenetic regulation for the suppressive effect of phthalate on type I IFN expression in pDCs. Proteins in the IRF family control many aspects of activity of DCs, particular in type I IFN induction [29]. IRF-7, activated upon TLR signaling, is required for IFN-α and IFN-β induction in pDCs [30]. In the present study, we firstly demonstrated that DEHP suppressed CpG plus IL-3-induced IRF-7 mRNA (Fig. 4A) and protein (Fig. 4B) expression, which can be reversed by AHR antagonist (Fig. 4C). We secondly demonstrated CpG plus IL-3-induced IRF-7 was abrogated by the MAPK-ERK inhibitor PD 98059 (Fig. 4D) and the methyltransferase inhibitor MTA (Fig. 5A). Thirdly, we demonstrated that DEHP suppressed CpG plus IL-3-induced histone H3K4 trimethylation at IRF7 promoter region. We also found that DEHP inhibited the translocation of H3K4-specific methyltransferase WDR5, which may contribute to, at least partly, the reduction in H3K4 trimethylation at IRF7 promoter region. Intriguingly, it has been reported that some genes can be marked by a unique bivalent domain, which contain both the H3K4 methylation for gene activation and H3K27 methylation for gene regression together [31]. Because the level of reduced translocation of WDR5 seemed not as much as that of reduced H3K4 trimethylation at IRF7 promoter region, potent factors other than WDR5 may result in the reduction in H3K4 trimethylation at IRF7 promoter region, and therefore, further investigations to elucidate DEHP perturbation of epigenetic regulation of IRF-7 are warranted. Furthermore, we also demonstrated DEHP not only suppressed the production of IRF-7 but also suppressed the binding of IRF-7 to IFNA and IFNB promoter regions (Fig. 4E). To our knowledge, this is the first report in the literature providing evidence that EDCs can epigenetically modulate type I IFN in human pDCs.

In conclusion, the present study demonstrated the immunomodulatory effect of phthalates on human pDCs. Phthalates suppressed type I IFN expression in pDCs via the AHR, the MKK1/2-ERK-ELK1 pathway, the NFκB-p65 pathways, and the IRF-7 pathway by the modification of histone H3K4 trimethylation at IRF7 promoter region. Phthalates attenuated the capability of pDCs to stimulate IFN-γ, but enhanced the capability of pDCs to stimulate IL-13 production by T cells. Phthalates may disrupt immunity against infection by suppressing type I IFN and Th1 immune response and may enhance allergic inflammation by increasing Th2 polarization.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References

The study is supported by the grant from the National Health Research Institutes (NHRI-102A1-PDCO-03010201, EOPP10-014 and EOSP07-014), the grant from the Center of Excellence for Environmental Medicine, Kaohsiung Medical University Research Foundation KMU-EM-99-4-1, the grant from National Science Council (NSC 99-2314-B-037-014-MY3), the grants from Kaohsiung Medical University Hospital Research Foundation KMUH99-9I08 and KMUH100-0M23 and a grant from Department of Health, Taiwan (EODOH01).

Conflict of interest

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References

The authors declare no conflict of interest in relation to the work and the manuscript.

Author contributions

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References

Kuo CH contributed to design of the experiments, acquisition of data, analysis and interpretation of data, and writing the manuscript. Hsieh CC, Kuo HF, Huang MY, Yang SN, and Chen LC contributed to acquisition, analysis, and interpretation of data. Huang SK contributed to conception of the study, and analysis and interpretation of data. Hung CH contributed to conception and design of the study, analysis and interpretation of data, writing the manuscript, and revising the manuscript critically for important intellectual content. All authors contributed to final approval of the version to be published.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. Author contributions
  9. References