Activated TLR4/MyD88 pathway promotes S100A9-mediated apoptosis and is required for optimal apoptosis of IAV infected cells.

<p>(<b>A</b>) Primary bone marrow derived macrophages (BMDM) isolated from wild-type (WT) and TLR4 knockout (KO) mice were incubated with purified recombinant mouse S100A9 protein (5 ug/mL) for 72 h. The apoptotic state of these cells was examined by FACS analysis of annexin V and PI stained cells. Apoptosis rate (% apoptosis) was calculated based on number of annexin V positive/PI negative cells (denoting early apoptosis)+number of annexin V positive/PI positive cells (denoting late apoptosis)/total number of cells. (<b>B</b>) WT and TLR4 KO BMDMs were infected with IAV (1 MOI). At 48 h post-infection, the apoptotic status was determined as described in (A). (<b>C</b>) IAV infected WT and TLR4 KO cells were subjected to TUNEL assay. TUNEL positive cells were analyzed by image J software. Percent TUNEL positive cells denotes ratio of number of TUNEL positive cells/total number of cells. (<b>D</b>) WT and MyD88 KO BMDMs were infected with IAV (1 MOI). At 48 h post-infection, the apoptotic status was determined. The values represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. Veh; cells incubated with HBSS buffer (vehicle control).