Induction of WNT5A expression is TLR and NF-κB dependent. Cells were stimulated for 4 hours. Total RNA was isolated, reverse transcribed, and analyzed by quantitative RT-PCR. (A, top) Human macrophages from 3 individual donors were stimulated with M avium (M av), M tuberculosis (M tb; MOI 3), or LPS (10 ng/mL) in the presence of an anti-TLR2 antibody or an isotype control antibody (10 μg/mL or as indicated). Statistical analysis was performed on data obtained from 3 independent experiments by normalizing WNT5A expression in the absence of antibody to 100% (ctrl) (*P < .05). (A, bottom) For the dose-response curve, means ± SD of duplicate determinations of 1 representative experiment of 3 is shown. (B, top) Human macrophages from 3 individual donors were stimulated with M avium (M av), M tuberculosis (Mtb; MOI 3), or LPS (10 ng/mL) in the presence of BAY11-7082 (inhibitor of IκB-α-phosphorylation), dimethyl sulfoxide (DMSO) as solvent control, or left untreated (ctrl), and WNT5A mRNA expression was analyzed. For statistical analyses, results from 3 individual donors in the presence of an optimal inhibitor concentration of 3 μM were compared to DMSO-treated cultures normalized to 100% (*P < .05; **P < .005). (C) Detection of LPS-induced (10 ng/mL) WNT5A expression in human macrophages in the presence of Compound 406 (1-100 ng/mL). Mean ± SD of duplicate determinations of 1 representative experiment of 3 is shown. (D) Human macrophages were stimulated for 4 hours with recombinant human TNF-α, LPS (each 10 ng/mL), or M avium (MOI 10) in the presence of anti–TNF-α antibodies (10 μg/mL). Total RNA was isolated, reverse transcribed, and analyzed for WNT5A expression. Mean ± SD of duplicate determination of 1 representative experiment of 3 are shown.