Fig 1: PS ODNs inhibit TLR3 and TLR7. PBMCs from healthy donors (n=6–7) were treated with 1.4 µm IRS661, 2.8 µm IRS869, 2.8 µm cODN1, 2.8 µm cODN2 or untreated and then stimulated with 25 µg ml-1 Poly (I:C) (TLR3; a, b) or 0.3 µg ml-1 gardiquimod (TLR7; c, d) or left unstimulated for 24 h at 37 °C. Solid lines represent medians and dotted lines represent limit of detection. *P<0.05, **P<0.01, ***P<0.001 compared with TLR ligand-only treated condition. cODN, control ODN; IFN, interferon; IP-10, interferon gamma-induced protein 10; ODN, oligonucleotide.
Fig 2: Bafilomycin and chloroquine effectively inhibit TLR3-, TLR7- and TLR9-specific IP-10 and IFN-a. PBMCs from healthy donors (n=7–9) were treated with 50 µM bafilomycin, 6 µM chloroquine or untreated, and subsequently stimulated with 25 µg ml-1 Poly (I:C) (a, b), 0.3 µg mml-1 gardiquimod (c, d), 50 µM ODN2216 (e, f) for 24 h at 37 °C. IP-10 protein (pg ml-1; left panels) and IFN-a protein (pg ml-1; right panels) were measured by ELISA. Solid lines represent the median for each condition. Dotted lines represent the limit of detection (23.4 pg ml-1 for IP-10 and 4.9 pg ml-1 for IFN-a). *P<0.05, **P<0.01, ***P<0.001 compared with TLR ligand-only treated condition. IFN, interferon; IP-10, interferon gamma-induced protein 10; TLR, toll-like receptor; UT, untreated.
Fig 3: Bafilomycin significantly inhibits RV16-stimulated IP-10 and IFN-a/ß in asthma. PBMCs from asthmatic subjects (n=10) were treated with media alone, bafilomycin or chloroquine, and then stimulated with RV16 for 24 h at 37 °C. IP-10 protein (a), IFN-a (b) and IFN-ß (c) were measured by ELISA. Data represent mean values where the unstimulated control was subtracted, error bars represent s.e.m. ***P<0.001.
Fig 4: The ELISA for IFN-a,-ß and -? are shown in order in three graphs (a), (b) & (c). TLR-3 down regulated cells showed no increase in release of the soluble cytokines upon reovirus infection at a dose of 5 MOI. On the contrary, HCT116 control cells did show a rise in release of the three classes of interferons upon reovirus infection at 5 MOI. In case of IFN-a there was a significant increase (p= 0.05) of cytokine productions at all 4 time points namely 6, 12, 24 and 48 hours as represented by *. In case of IFN-ß the significance (p= 0.05) was observed in control cells treated and untreated at 6 and 12 hours where as the IFN-? showed a significant difference in the same group at 24 and 48 hours.
Fig 5: RV16 stimulation triggers significantly more IFN-a+CD303+ than IFN-a+CD14+ or IFN-a+CD1c+. PBMCs from healthy donors (n=9) were cultured in the presence of RV16 (multiplicity of infection=1) or media only (unstimulated control) for 24 h at 37 °C. Using intracellular cytokine staining, the percentage and/or proportion of IFN-a-producing cells in human PBMCs were evaluated. After gating on total live cells, monocytes (CD14+CD303-), mDCs (CD1c+CD14-) and pDCs (CD303+CD14-) were identified (a). Results for IFN-a-producing cells (b—from left to right, monocytes, mDCs or pDCs) are shown. The bar chart (c) shows the frequency of IFN-a-producing cells, and the plotted values represent the RV16-stimulated cells (b—bottom panels) minus the unstimulated cells (b—top panels). The proportion of IFN-a-producing cell was also evaluated (d). All data represent mean±s.e.m. *P<0.05, ***P<0.001. mono, monocytes; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell; IFN, interferon; RV16, rhinovirus-16.
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