Reduction of TLR4 on the macrophage surface inhibits A2t-driven cytokine production. (A) Seven-day macrophages from 3 donors were transfected with small interfering RNAs: 2 separate sequences specific for TLR4 were used, either alone or in combination (“TLR4 a” and “TLR4 b”), as well as negative control siRNA, and an siRNA for an unrelated gene (glyceraldehyde-3-phosphate dehydrogenase). Three days after transfection, cells were stained with fluorescein isothiocyanate–labeled mouse anti–human TLR4 antibody, and surface expression of TLR4 was analyzed by FACS. Results represent the mean ± SEM(after subtraction of fluorescence from the isotype control) of 3 experiments with separate donors. (B) Macrophages (transfected as in panel A) were treated 2 days posttransfectionwith A2t (5nM), LPS (10 ng/mL), or Pam₃CSK₄ (100 ng/mL) for 24 hours, and concentration of IL-6 was measured in the supernatants by luminex and expressed as the percentage of the value of IL-6 induced by LPS for that donor. Results are the mean ± SEM; *P < .05.