S100A9 induces TLR mediated cytokine responses. (A) Primary bone marrow derived macrophages (BMDM) isolated from wild type, Trif-/- and Ptp1b-/- mice were infected with RSV and ELISA determined extracellular S100A9 protein levels, 24 hours later. (B) Expression of TRIF and PTP1B were silenced in human SAE cells and cells were infected with RSV. ELISA determined extracellular S100A9 levels compared to cells transfected with Scr. siRNA. (C) SAE cells were treated with 5 μg/ml S100A9 protein for 0, 15, 30 and 60 minutes and 24 hours. Trif, MyD88, IκBα, p38, ERK and β-Actin immunoblots were performed. Representative immunoblots demonstrate typical results from 3 separate assays. (D) S100A9 protein induced release of MCP-1, CXCL10, IL-8, RANTES and G-CSF from SAE human cells in a TLR4 dependent manner confirmed in SAE cells transfected with either Scr. siRNA or siRNA specific for TLR4. (E) S100A9 expression was silenced in human SAE cells and cells were treated with RSV to determine the impact of RSV-induced S100A9 on cytokine expression. Graphs are represented as mean ± S.E.M., where each measurement was performed 3 times on samples from 3 independent experiments. p values shown, comparing both treatments connected by a line. (F) Possible pathway for the regulation of S100A9-mediated lung damage during RSV-associated COPD exacerbations.