Suppression of TLR2-Induced IL-12, Reactive Oxygen Species, and Inducible Nitric Oxide Synthase Expression byMycobacterium tuberculosisAntigens Expressed inside Macrophages during the Course of Infection

Abstract

We report the enrichment of and immune responses mediated by genes expressed by Mycobacterium tuberculosis inside macrophages as a function of time. Results indicate that M. tuberculosis expresses different genes at different times postinfection. Genes expressed early (day 1) following infection enhance M. tuberculosis-mediated activation of dendritic cells (DCs), whereas genes expressed later (day 5) in the infection prevent DC activation. However, all genes downmodulated MHC class I and II expression on infected macrophages, thus compromising their ability to interact with Ag-specific T cells. Day-1 and -5 genes downmodulated proinflammatory cytokine production from DCs, thus impairing signal 3 during DC–T cell cognate interactions. Consequently, T cells activated by Ag-experienced DCs secreted low levels of IFN-γ and IL-17 but maintained high IL-10 secretion, thus inducing suppressor responses. Further characterization revealed that day-1 and -5 genes increased TLR2-induced expression of suppressors of cytokine signaling 1 from DCs and downmodulated IL-12 expression. In addition, day-1 and -5 genes prevented the generation of reactive oxygen species in DCs. In contrast, although day-5 genes increased TLR2-mediated suppressors of cytokine signaling 1 expression in macrophages, day-1 genes downmodulated the expression of inducible NO synthase 2. Similar downregulation of immune responses was observed upon exogenous stimulation with day-1 or -5 Ags. Finally, day-1 and -5 genes promoted enhanced survival of M. tuberculosis inside DCs and macrophages. These results indicate that M. tuberculosis genes, expressed inside infected macrophages as a function of time, collectively suppress protective immune responses by using multiple and complementary mechanisms.

Suppression of Dendritic Cell-mediated Responses by Genes in Calcium and Cysteine Protease Pathways during Mycobacterium tuberculosis Infection

“…Dichloroflourescin diacetate (DCFH-DA) and FITC-tagged Alexa Fluor 488 were obtained from Molecular Probes (Eugene, OR). Recombinant M. tuberculosis antigens Rv2463 and Rv3416 were expressed and purified as described recently (23). TLR2 ligand Pam 3 Csk 4 was purchased from Invivogen (San Diego, CA).…”

“…Generation of DCs-DCs were differentiated with GM-CSF as described previously (23,24). Briefly, bone marrow from the tibias and femurs of BALB/c mice were flushed out, and lymphocytes and I-A ϩ cells were depleted following magnet-assisted cell sorting.…”

“…Flow Cytometry-Cells were stained for the surface levels of CD80, CD86, CD54, and CD40 using FITC-tagged monoclonal antibodies and analyzed by flow cytometry on FACS Calibur (BD Biosciences) as described previously (23). The data were plotted and analyzed using CellQuest Pro software.…”

“…Measurement of Cytokines-Cytokines in the culture supernatants were measured by employing a sandwich ELISA as described previously (23,24). The samples were diluted to obtain absorbance in the linear range of the standards.…”

Exploring the Use of Lipid Based Nano-Formulations for the Management of Tuberculosis

“…In the granulomas, CD 4 cells T-Lymphocytes secretes cytokines which activates the macrophages to destroy the bacteria with which they are infected previously. Within macrophages, MTb is able to survive through hallmark mechanism of survival which includes suppression of intracellular Ca 2+ concentrations by the mycobacterium, inhibition of reactive oxygen and nitrogen production, suppression of production of pro-inflammatory cytokines and chemokines, and prevention of phagosome maturation [22].…”