<i>Mycobacterium tuberculosis H37Rv</i> infection disrupts the proinflammatory function of CCR5 in macrophages to produce high level of IL-10.

<p>Bone marrow derived macrophages (2×10⁶ cells/ml) were either treated with CCR5 siRNA or Control siRNA followed by <i>Mycobacterium tuberculosis H37Rv</i> (MOI = 1∶10) infection for 24 h and assayed for the levels of IL-10 (A), TGF-β (B), TNF-α (C), IL-12 (D) and IFN-γ (E) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means ± standard deviations of values from triplicate experiments that yielded similar observations. ***<i>P</i><.001 and **<i>P</i><.05 compared to that of the infected macrophages. In a separate set of experiment, siRNA pre treated macrophages were infected with <i>H37Rv</i> for 3 h. The extracellular bacteria were removed from the culture plate and the macrophages were incubated for another 3 hrs. RNA was isolated and semi quantitative RT-PCR analyses for TNF-α, IL-12, IFN-γ, IL-10, TGF-β and GAPDH were done. Data represented here are from one of three independent experiments, all of which yielded similar results (F). In separate experimental sets, Murine macrophages were pretreated with either control siRNA, CCR5-specific siRNA for 24 h or with Lyn inhibitor PP2, ERK inhibitor PD for 2 h, washed and then infected with <i>Mycobacterium</i> tuberculosis and changes in messenger RNA (mRNA) expression of IL-10 and GAPDH were determined by semi quantitative RT-PCR (G). Murine macrophages were transfected with siRNA and infected as mentioned above for 24 h and assayed for the levels of IL-10 (H) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. ***<i>P</i><.001 and **<i>P</i><.05 compared to that of the control siRNA treated infected macrophages. Murine macrophages (1×10⁶cells/ml) were treated as described previously and then subsequently followed by <i>Mycobacterium</i> tuberculosis infection for 45 min. After 45 min of incubation, ChIP assays were conducted as described in Materials and Methods. Immunoprecipitations were performed using Abs specific to phosphorylated H3 (IP phospho-H3) (I) or acetylated H3 (IP acetyl-H3) (J), and conventional RT-PCR was performed using primers specific to the IL-10 promoter. Data represented here are from one of three independent experiments, all of which yielded similar results.