PTX, DEX and AZI demonstrate distinct inhibition of TLR-mediated cytokine mRNA and intracellular protein expression.

<p>Whole blood was pretreated for 2 hours with PTX (200 μM), DEX (10⁻⁷ M), AZI (20 μM) or vehicle control (V), either alone or in combination. Samples were stimulated with 10 ng/ml LPS or 1 μM R848, and cultured for 2 hours (mRNA expression) or 6 hours (flow cytometry) at 37°C in 5% CO₂, in the presence (TNF, IL-6 and IL-10) or absence (IL-1β) of Brefeldin A for flow cytometric assays. MFI of monocytes, gated with forward and side scatter as CD45⁺CD14⁺ cells, was measured as described in <i>Methods</i>. (a) LPS-induced relative mRNA expression (mean ± SEM) in cord and adult blood (n = 5 each, analyzed combined) in response to anti-inflammatory agents compared to LPS-stimulation alone, defined as 100%. Effects of anti-inflammatory treatment on (b) LPS- and (c) R848-induced intracellular cytokines in newborn monocytes (n = 8), plotted as MFI fold changes (± SEM) compared to TLRA stimulation alone (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196352#pone.0196352.s019" target="_blank">S6 Data file</a> for raw MFI data). Significant differences based on linear mixed models were indicated: *p<0.05, **p<0.01, ***p<0.001.