Journal: PLoS ONE
Article Title: Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies
Figure Lengend Snippet: CD14 + monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC. ( A ) IFN-α production in CD14 + cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking endosomal acidification with 50nM Bafilomycin A 1 . ( B ) Ab-RSV-induced IFN-α production in CD14 + cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. ( C ) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3 neg. , CD14 neg. , CD19 neg. , CD16 neg. , CD56 neg. , Lin-1), MHC-II high , BDCA-4 + cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. ( D ) Purified pDC (obtained by negative selection removing CD3 + , CD19 + and CD16 + cells from fresh PBMC, followed by FACS purification of the BDCA-4 + cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. ( E ) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). ( F ) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P
Article Snippet: Inhibition assays To determine the involvement of specific innate immune pathways we employed inhibitors to block endosomal acidification (Bafilomycin A1 , 50nM, Calbiochem) and protein transport (Brefeldin A, 40nM, Sigma-Aldrich).
Techniques: Blocking Assay, Sequencing, Staining, Infection, Purification, Selection, FACS, Isolation