Diesel exhaust particle-treated human bronchial epithelial cells upregulate Jagged-1 and OX40 ligand in myeloid dendritic cells via thymic stromal lymphopoietin

Abstract

Ambient particulate matter, including diesel exhaust particles (DEP), promotes the development of allergic disorders. DEP increase oxidative stress and influence human bronchial epithelial cell (HBEC)-dendritic cell interactions via cytokines, including thymic stromal lymphopoietin (TSLP). Upregulation of TSLP results in Th2 responses. Using primary culture HBEC and human myeloid dendritic cell (mDC) cocultures, we show in this study that DEP upregulation of Th2 responses occurred via HBEC-dependent mechanisms that resulted from oxidative stress. Moreover, DEP-treated HBEC and ambient particulate matter-treated HBEC upregulated OX40 ligand (OX40L) and the Notch ligand Jagged-1 mRNA and expression on mDC. Upregulation of OX40L as well as Jagged-1 on mDC required HBEC and did not occur in the presence of N-acetylcysteine. Furthermore, OX40L and Jagged-1 upregulation was inhibited when HBEC expression of TSLP was silenced. Thus, DEP treatment of HBEC targeted two distinct pathways in mDC that were downstream of TSLP expression. Upregulation of OX40L and Jagged-1 by mDC resulted in mDC-driven Th2 responses. These studies expand our understanding of the mechanism by which ambient pollutants alter mucosal immunity and promote disorders such as asthma.

Fig. 1. HBEC upregulate DC dependent Th2 polarization via ROS

Immature mDC were exposed (48h) to pHBEC treated with DEP (3 μg/cm²) or defined stimuli (A) or 16HBEC and defined stimuli (B). Myeloid DC were subsequently isolated and used in an MLR with allogeneic naïve CD4⁺ T cells. Supernatants were analyzed for IL-5 and IFNγ and data are expressed as the ratio of IL-5/IFNγ (mean ± SE, * = p < 0.05).

Fig. 2. Myeloid OX40L mRNA is upregulated by DEP-treated HBEC

Immature mDC were cultured (48h) with pHBEC (2A), 16HBEC (2B) or alone (2C) in the absence or presence of DEP (3 μg/cm²), carbon (3 μg/cm²), MF1 or MF2 and for mDC alone, recombinant TSLP. Myeloid DC were isolated and OX40L mRNA measured by RT-PCR. Data are expressed as relative mRNA expression of OX40L compared to GAPDH (2^ΔCt) (mean ± SE, n = 3, * = p < 0.05).

Fig. 3. Myeloid DC OX40L mRNA is upregulated by DEP-treated 16HBCE via TSLP and ROS

Myeloid DC were co-cultured (48h) with 16HBEC treated with DEP (3 μg/cm²) in the absence or presence of anti-TSLP, siTSLP or NAC and the appropriate controls. Myeloid DC were isolated and OX40L mRNA measured by RT-PCR. Data are expressed as relative mRNA expression of OX40L compared to GAPDH (2^ΔCt) (mean ± SE, n = 3, * = p < 0.05).

Fig. 4. Jagged-1 but not Dll4 is upregulated by DEP-treated HBEC

Immature mDC were cultured (48h) with DEP (3 μg/cm²), MF1, MF2, carbon (3 μg/cm²) or recombinant TSLP (15 ng/ml) in the presence of untreated or treated pHBEC (4A) or 16HBEC (4B) or alone (4C). Myeloid DC were isolated and Jagged-1 or Dll4 mRNA measured by RT-PCR. Data are expressed as relative mRNA expression of Jagged-1 or Dll4 compared to GAPDH (2^ΔCt) (mean ± SE, n = 3, * = p < 0.05).

Fig. 5. Myeloid DC Jagged-1mRNA is upregulated by DEP-treated 16HBEC via TSLP and ROS

Myeloid DC were co-cultured (48h) with 16HBEC treated with DEP (3 μg/cm²) in the absence or presence of anti-TSLP, siTSLP or NAC and the appropriate controls. Myeloid DC were isolated and Jagged-1 mRNA measured by RT-PCR. Data are expressed as relative mRNA expression of Jagged-1 compared to GAPDH (2^ΔCt) (mean ± SE, n = 3, * = p < 0.05).

Fig. 6. Ambient PM-treated 16HBEC upregulated the expression of mDC OX40L and Jagged-1, but not and Dll4 in mDC

Immature mDC were cultured (48h) with DEP, Hunter College, or South Bronx fine ambient PM (3 μg/cm²), in the presence of untreated or treated 16HBEC and mRNA for OX40L (6A), Jagged-1 (6B), or Dll4 (6C) measured by RT-PCR. Data are presented as a representative experiment performed in triplicate and expressed as relative mRNA expression of OX40L, Jagged-1 or Dll4 compared to GAPDH (2^ΔCt) (mean ± SE, in triplicate, * = p < 0.05).

Fig. 7. DEP-treated 16HBEC upregulated the expression of Jagged-1 and OX40L on the surface of mDC

Myeloid DC were cultured (48h) alone, or in the presence of untreated 16HBEC, or 16HBEC treated with DEP (3 μg/cm²), carbon (3 μg/cm²), or DEP and NAC. Cells were recovered and analyzed by flow cytometry. Jagged-1 and OX40L expression were detected as FITC and PE mean fluorescence intensity (MFI) on CD11c⁺ cells. (A) Relative expression (mean ± SE, n = 3, * = p < 0.05) was obtained by normalization against the MFI detected on mDC cultured alone. (B) % mDC expressing Jagged-1 alone, OX40L alone, or both Jagged-1 and OX40L, are shown in linear density plots for CD11c⁺ cells from one of three experiments.

Fig. 8. Th2 polarization by mDC exposed to DEP-treated HBEC is mediated via OX40L and Jagged-1

Myeloid DC were exposed to DEP (3 μg/cm²)-treated pHBEC (48 h) in the absence or presence of anti-OX40L or anti-Jagged-1 or appropriate control. DC were subsequently isolated and used in an MLR with allogeneic naïve CD4⁺ T cells. Supernatants were analyzed for IL-5 and IFNγ and data were expressed as the ratio of IL-5/IFNγ (mean ± SE, * = p < 0.05).