Ambient particulate matter activates the aryl hydrocarbon receptor in dendritic cells and enhances Th17 polarization

Abstract

The objective of this study was to explore the role of the aryl hydrocarbon receptor (AhR) in ambient particulate matter (PM)-mediated activation of dendritic cells (DCs) and Th17-immune responses in vitro. To assess the potential role of the AhR in PM-mediated activation of DCs, co-stimulation, and cytokine expression, bone marrow (BM)-derived macrophages and DCs from C57BL/6 wildtype or AhR knockout (AhR^-/-) mice were treated with PM. Th17 differentiation was assessed via co-cultures of wildtype or AhR^-/- BMDCs with autologous naive T cells. PM_2.5 significantly induced AhR DNA binding activity to dioxin responsive elements (DRE) and expression of the AhR repressor (AhRR), cytochrome P450 (CYP) 1A1, and CYP1B1, indicating activation of the AhR. In activated (OVA sensitized) BMDCs, PM_2.5 induced interleukin (IL)-1β, CD80, CD86, and MHC class II, suggesting enhanced DC activation, co-stimulation, and antigen presentation; responses that were abolished in AhR deficient DCs. DC-T cell co-cultures treated with PM and lipopolysaccharide (LPS) led to elevated IL-17A and IL-22 expression at the mRNA level, which is mediated by the AhR. PM-treated DCs were essential in endowing T cells with a Th17-phenotype, which was associated with enhanced expression of MHC class II and cyclooxygenase (COX)-2. In conclusion, PM enhances DC activation that primes naive T cell differentiation towards a Th17-like phenotype in an AhR-dependent manner.

Keywords: Aryl hydrocarbon receptor (AhR); Dendritic cells (DCs); Particulate matter (PM); Polycyclic aromatic hydrocarbons (PAHs); Th17 cells.

Figure 1

Sacramento PM_2.5 induces DRE luciferase reporter activity in HepG2 cells. HepG2 cells were incubated with PBS (control), PM (50 μg/mL), the standard reference material (SRM) 1649a urban dust particles (UDP; 50 μg/mL), or TCDD (10 nM), an AhR agonist, for 4 hrs (A). Data are from three independent experiments and presented as mean ± SEM. Bars indicate a significant difference of p < 0.05 between groups. Sacramento PM_2.5 activated AhR and DNA binding activity in BM-derived macrophages from wildtype (AhR^+/+), but not from AhR^−/− C57BL/6 mice (B). Macrophages were incubated with PBS (control), PM (50 μg/mL) or TCDD (10 nM, positive control) for 1 h. Nuclear extracts were used in electrophoretic-mobility-shift assay (EMSA) with a consensus DRE-binding element.

Figure 2

Macrophages fail to become activated by PM or OVA in the absence of the AhR. BM-derived macrophages from wildtype (wt, white bars) or AhR^−/− (black bars) C57BL/6 (B6) mice were treated for 24 hrs with PBS, PM (50 μg/mL), OVA (10 μg/mL), or OVA+PM. Gene expression of various activation markers (A–F) was assessed via qPCR and normalized to Gapdh or Efe1a1. Data represent three independent experiments and are presented as mean ± SEM. A significant difference of p < 0.05 is indicated by solid bars between wildtype vs. AhR^−/− genotype groups, asterisks between wildtype PBS control vs. wildtype treatment groups, or by dashed lines between wildtype OVA vs. wildtype OVA+PM treatment groups.

Figure 3

PM enhances DC maturation and antigen presentation capabilities. BMDCs from wildtype (wt, white bars) or AhR^−/− (black bars) C57BL/6 (B6) mice were treated for 24 hrs with PBS, PM (50 μg/mL), OVA (10 μg/mL), or OVA+PM. Gene expression of various activation markers (A–I) was assessed via q-PCR and normalized to Gapdh or Efe1a1. Data represent three independent experiments and are presented as mean ± SEM. A significant difference of p < 0.05 is indicated by solid bars between wildtype vs. AhR^−/− genotype groups, asterisks between wildtype PBS control vs. wildtype treatment groups, or by dashed lines between wildtype OVA vs. wildtype OVA+PM treatment groups.

Figure 4

PM acts through DCs to enhance IL-17A gene expression in T cells, an effect mediated completely by the AhR in DCs. Wildtype (wt, white bars) or AhR^−/− (black bars) BMDCs derived from C57BL/6 (B6) mice pre-treated with PBS (PBS/−) or LPS (10 ng/mL; LPS/−) for 6 hrs were co-cultured with autologous naive CD4⁺CD62L⁺ T cells at a 1:10 ratio (2.5×10⁴ DCs: 2.5×10⁵ T cells/well) and treated with PBS (PBS/PBS or LPS/PBS), PM (LPS/PM, 50 μg/mL), UDP (LPS/UDP, 50 μg/mL), or TCDD (LPS/TCDD, 2nM) for 4 days with re-stimulation on day 3. On day 4, cellular RNA was isolated to analyze gene expression patterns (A–D). Gene expression was assessed via q-PCR and normalized to Efe1a1. Data represent three independent experiments and are presented as mean ± SEM. A significant difference of p < 0.05 is indicated by solid bars between wildtype vs. AhR^−/− genotype groups or asterisks between wildtype PBS control vs. wildtype treatment groups.

Figure 5

PM acts through DCs to enhance IL-17A protein levels in T cells, an effect mediated completely by the AhR in DCs. Wildtype (wt, white bars) or AhR^−/− (black bars) BMDCs derived from C57BL/6 (B6) mice pre-treated with PBS (PBS/−) or LPS (10 ng/mL; LPS/−) for 6 hrs were co-cultured with autologous naive CD4⁺CD62L⁺ T cells at a 1:10 ratio (2.5×10⁴ DCs: 2.5×10⁵T cells/well) and treated with PBS (PBS/PBS or LPS/PBS), PM (LPS/PM, 50 μg/mL), UDP (LPS/UPD, 50 μg/mL), or TCDD (LPS/TCDD, 2nM) for 4 days with re-stimulation on day 3. On day 4, cell culture supernatants were analyzed via ELISA to determine (A) IL-17A and (B) IL-22 protein levels. Data represent three independent experiments and are presented as mean ± SEM. A significant difference of p < 0.05 is indicated by solid bars between wildtype vs. AhR^−/− genotype groups or asterisks between wildtype PBS control vs. wildtype treatment groups.

Figure 6

In the absence of DCs, CD4⁺CD62L⁺ T cells treated with PM fail to differentiate towards a Th17-like phenotype despite AhR activation. CD4⁺CD62L⁺ T cells derived from C57BL/6 (B6) mice were activated using MACSiBeads conjugated to anti-CD3ε and anti-CD28 to mimic antigen presenting cell activation signals in the presence of PBS, PM (50 μg/mL), UDP (50 μg/mL), or TCDD (2nM) for 4 days with re-stimulation on day 3. On day 4, cellular RNA was isolated to analyze gene expression patterns (A). Gene expression was assessed via q-PCR and normalized to Efe1a1. Cell culture supernatants were analyzed via ELISA to determine IL-17A protein levels (B). Data represent three independent experiments and are presented as mean ± SEM. Bars indicate a significant difference of p < 0.05 between groups.

Figure 7

PM treatment of DCs does not lead to secretion of Th17-inducing cytokines, rather PM enhances MHC class II and COX-2 expression in DCs. Wildtype (wt, white bars) or AhR^−/− (black bars) BMDCs derived from C57BL/6 (B6) mice were pre-treated with PBS (PBS/−) or LPS (10 ng/mL; LPS/−) for 6 hrs and then treated with PBS (PBS/PBS or LPS/PBS), PM (LPS/PM, 50 μg/mL), UDP (LPS/UPD, 50 μg/mL), or TCDD (LPS/TCDD, 2nM) for 24 hrs. Cellular RNA was isolated to analyze gene expression patterns (A–L). Gene expression was assessed via q-PCR and normalized to Efe1a1. Data represent three independent experiments and are presented as mean ± SEM. A significant difference of p < 0.05 is indicated by solid bars between wildtype vs. AhR^−/− genotype groups or asterisks between wildtype PBS control vs. wildtype treatment groups.