Activation of infected macrophages and inter-compartmental localization of mycobacteria within A.

<p>The plots depict reduced activation of H37RV/ARPC4 infected macrophages. <b>a)</b> Percentage of cells expressing PD1 among CD11B+ cells is shown in the dot plots with mean±STDEV. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B and PD1 antibodies and samples were acquired by flow cytometry. Data shown here are representative of three independent experiments. <b>b)</b> Cell death in H37Rv and H37Rv/ARPC4 Mtb infected macrophages. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains in 1∶10 ratio, were surface-stained with anti-CD11B, CD11C antibodies followed by PI staining for 20 minutes prior to acquisition by flow cytometry to assess cell death in infected macrophages. The percentage of cells expressing PI among CD11B±cells is shown with mean±STDEV. Data shown here are representative of three independent experiments. <b>c)</b> Expression of macrophage activation markers. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B, CD11C, MHCII, CD14, CD54, CD80, CD86 and CD69 antibodies and samples were acquired by flow cytometry. CD11B+ cells were gated for expression of MHCII, CD14, CD54, CD80 and CD86 and the percentage of cells expressing these markers are shown in the histogram overlay plots with mean±STDEV. Data shown here are representative of three independent experiments. <b>B.</b> Confocal Microscopy images showing that H37Rv/ARPC4 mycobacterial strain is less infectious and persistent in macrophage. FITC-labelled mycobacterial cells (green) were used for infecting macrophages (red) at an MOI of 5. At specific time-points, the cells were fixed, permeabilized and stained with anti-LAMP1 antibodies, followed by Alexa Fluor 594 goat anti-rat antibodies. (a)- (d) shows the infected macrophage cells at 0, 24, 48 and 72 hours' time-points post-infection, respectively. (e) and (f) are the graphical representation of the percentage of macrophage cells infected with FITC-labeled mycobacteria at different time-points and the percentage of bacteria localized in lysosomes in the infected cells, respectively. Experiments were repeated thrice and similar results were obtained.