Figure 2 The capacity of pathogenic mycobacteria to grow intracellularly in macrophages treated with IFN-γ or IL-10. Cultures of BMDM were pretreated with exogenic murine r-IFN -γ or r-IL-10 for 2 h, infected with the mycobacterial strains at a MOI of 1, as indicated in the legend to Figure 1, and incubated in the presence of these cytokines for an additional 6 days. The intracellular CFU numbers determined at day 0 and day 6 are presented. The data of

three independent experiments Dorsomorphin concentration are shown as mean ± SD of samples in triplicate. Asterisks represent statistical significance (p < 0.05) compared to infected cells cultured without addition of the cytokines. Innate macrophage activation by the pathogenic mycobacterial strains differing in growth kinetics in macrophages To study the effects of pathogenic Mbv isolates on MΦ activation, we evaluated characteristic markers of M1- and M2- type macrophage polarization induced in infected BMDM, in the presence or absence of IFN-γ and IL-10. First, we investigated the innate MΦ activation induced by infection. Evaluation of expression of the M1 proinflammatory markers, including factors mediating recruitment of the phagocytic cells (MCP-1/CCL2 and MIP-2/CXCL2), and contributing to the MΦ microbicidity (TNF-α, IL-12, IL-6 and NO), demonstrated

that the studied pathogenic mycobacterial strains induced different patterns of cytokine secretion check details by the BMDM (Figure 3A). Both clinical isolates of Mbv induced less IL-6 and MCP-1, and, additionally, the Mbv strain MP287/03 induced less TNF-α, Protirelin than the reference strain H37Rv. In contrast, the level of secretion of MIP-2, an important chemokine regulating migration of granulocytes, was significantly increased in cultures infected with the Mbv strains. These cells secreted 10-fold more MIP-2 than the cells infected by H37Rv strain, and 3-fold more than those infected by the strain B2. Neither mycobacterial strain tested in this study was

able to induce in MΦ the production of NO or IL-12, although production of these mediators was induced by the LPS (Figure 3A). Figure 3 The activation profiles of macrophages infected with pathogenic mycobacteria. BMDM were infected with the studied mycobacterial strains at a MOI of 5:1, washed and incubated for an additional 48 h. The cells left untreated and cells stimulated with LPS for 48 h were used as a negative and positive controls of proinflammatory activation, respectively. To evaluate markers of M1-type activation (A), the culture supernatants of infected cultures were harvested and tested for TNF-α, IL-6, MCP-1, MIP-2 and IL-12 by Bioplex test, and for NO production by Griess reaction. Assays were completed with duplicate samples, and results are expressed as a mean of three independent experiments.