Figure 4.

IL-27 inhibits anti-mycobacterial activity of MDSC. To determine that IL-27 inhibits anti-mycobacterial activity of MDSC, HIV (-) MDSC and HIV MDSC were isolated as in Figure 1 using flow cytometry and (A) infected with Mtb-GFP at MOI of 1:5, in the presence of neutralizing IL-27 or isotype matched control antibody for 2-hrs. During last 30-mins of infection 1 µM of CellROX deep read reagent was added and subsequently stained with aqua fluorescent LIVE/DEAD stain. Expression of ROS (Cell ROS) in Mtb-GFP+ Live cells was determined by flow cytometry and Mean Fluorescence Intensity (MFI) calculated. Net ROS expression was calculated as in Materials and Methods. (B) MDSC were infected for 3-hrs, washed with PBS to remove extracellular bacteria and cultured with neutralizing IL-27 or isotype matched control antibody. M tuberculosis growth (colony forming units (CFU)/ml) was determined in the cellular lysates at day-0 and -3 post-infection (p.i.). (C) MDSC were transfected with empty (Control) or MyD88 expressing plasmid using Lipofectamine 3000. After 16-18 hrs post-transfection, were infected with M tuberculosis at MOI of 1:5 for 3-hrs washed with PBS to remove extracellular bacteria and cultured with neutralizing IL-27 or isotype matched control antibody. M tuberculosis growth (colony forming units (CFU)/ml) was determined in the cellular lysates at day-0 and -3 post-infection (p.i.). (D) Regulation of immunity in HIV-Mtb co-infection: HIV-infected individuals on cART with suppressed HIV replication have increased IL-27 and MDSC in these individuals exhibit (1) higher expression of IL-27R, (2) lower expression of MyD88 which abrogates TLR-signaling and (3) reduced expression of Reactive Oxide Species (ROS) in response to M tuberculosis. The histograms show mean values +/- SEM; N=4 donors. *p < 0.05.