Fig 3.

1,25D₃ increases TLR signaling but not TLR expression in HIV⁺ U1 macrophages. (A and B) Differentiated human U937 and HIV⁺ U1 macrophages were incubated with the TLR ligands lipid A (LA) (for TLR4), PamCys (for TLR2/1), and 19-kDa lipoprotein from M. tuberculosis (19 kDa MTb; 1 μg/ml) (for TLR2/1) for 24 h in the presence or absence of 1,25D₃ pretreatment. Cell-free culture supernatants were assayed for TNF by ELISA (n = 3). (C) Differentiated U937 and HIV⁺ U1 macrophages were incubated for 24 h in the presence or absence of 1,25D₃ pretreatment and then stained with PE-labeled anti-TLR antibodies or isotype control antibody. Surface expression was measured by flow cytometry. Left panels show isotype control (gray lines)- and TLR (black lines)-labeled cells; right panels show TLR-labeled cells (gray lines) and 1,25D₃-treated TLR-labeled cells (black lines). Representative histograms for independent experiments with similar results (n = 3) are shown. (D) Specific TLR2 and TLR4 mRNAs were detected by real-time PCR (n = 3). Quantitative data represent means ± SEM. *, P < 0.05.