Background/Purpose: Plasmacytoid dendritic cells (pDCs) constitute a rare blood cell subset exquisitely attuned for production of large quantities of interferon-alpha (IFN-α), and these cells have been implicated in several autoimmune diseases.  There is therefore much interest in understanding how IFN-α is regulated in pDCs.  Prostaglandin E2 (PGE2) is known to have an inhibitory effect on pDCs via receptors EP2 and EP4, but the mechanism(s) underlying this inhibition is not known.  The goal of this study was to elucidate the molecular pathways involved in the inhibition of IFN-α by PGE2.

Methods: Human peripheral blood mononuclear cells (PBMCs), isolated “untouched” primary pDCs, or a pDC line were cultured in the presence of lupus immune complexes (ICs) or agonists of TLR7 (Loxoribine) or TLR9 (CpG-A, ODN 2216) with or without PGE2 (1, 10, and 100 ng/mL), dibutyryl-cAMP (100 μM), or Rapamycin (10 ng/mL).  At 1h, cells were harvested and fixed to slides, then stained with DAPI and fluorescently-labeled antibody against IRF7, and analyzed by confocal or fluorescence microscopy.  At 20h, IFN-α, IL-6, and TNF in the supernatants were quantified by ELISA, and cells were analyzed by flow cytometry using fluorescently-labeled antibodies to identify pDCs (CD123+BDCA2+), and quantify cell death (Annexin V, propidium iodide).

Results: Lupus IC and TLR7 or 9 agonists induced IFN-α by PBMC which could be inhibited by PGE2 in a dose-dependent manner, whereas production of IL-6 and TNF were unaffected.  This was also true for isolated pDC stimulated by CpG-A, and this was not due to pDC death.  By confocal microscopy of primary pDCs, IRF7 translocation was increased by TLR9 agonist stimulation and significantly reduced by addition of PGE2 but not carrier control (DMSO).  By fluorescence microscopy of primary pDC or the pDC line, the TLR9 agonist increased the IRF7 signal and the intensity of staining was reduced by PGE2.  Because PGE2 action on pDC is believed to be via the receptors EP2 and EP4, which drive an increase in cAMP and activation of PKA in other cell types, we addressed whether dibutyryl-cAMP, a cell-permeable cAMP analog, could mimic the effects of PGE2.  Indeed, dibutyryl-cAMP reduced the production of IFN-α by 56.7 ± 9.8 % (p < 0.04).  Lastly, to determine if mTOR played a role in IFN-α regulation, we treated PBMCs with a suppressor of mTOR, Rapamyin, and found that it, too, could robustly inhibit IFN-α production (76.8 ± 5.2 %, p < 0.004) but not IL-6 or TNF, without inducing pDC death.

Conclusion: PGE2 is able to inhibit pDC production of IFN-α in response to both lupus IC and TLR agonist stimulation without affecting other pro-inflammatory cytokines or inducing pDC death.  We show for the first time that PGE2 can inhibit pDC IRF7 protein expression and nuclear translocation.  Consistent with a role for PGE2 receptors EP2 and EP4 in this inhibition, a cAMP analog could also inhibit IFN-α.  Lastly, mTOR plays a significant role in IFN-α production, as treatment with its suppressor, Rapamycin, significantly inhibits IFN-α but not other pro-inflammatory cytokines IL-6 and TNF.  These results are consistent with a role of PGE2 in inhibition of the mTOR pathway in pDCs, and may represent a novel target for immune modulation in type 1 IFN-associated diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-plasmacytoid-dendritic-cells-by-prostaglandin-e2/