Figure 4 The activation profiles of macrophages treated with IFN-γ or IL-10 and infected with pathogenic mycobacteria. BMDM were pretreated, or not, with murine r-IFN-γ or r-IL-10 for 2 h, infected with the studied mycobacterial strains at a MOI of 5:1, washed, treated again with the cytokines and incubated for an additional 48 h. The cells stimulated with LPS and r-IFN-γ

for 48 h, or left untreated, were used as a positive and negative controls of classical proinflammatory activation, respectively. To evaluate markers of M1-type activation, the culture supernatants were tested for proinflammatory mediator levels (A-C) and the adhered cells were tested for expression of iNOS (D). Measurement of TNF-α, IL-6, MCP-1, MIP-2 and IL-12 concentrations was performed by Bioplex test, and HDAC activation NO production was evaluated by Griess reaction Assays were completed with duplicate samples, and results are expressed as a mean of three independent experiments. To evaluate markers of M2-type activation, expression of Arginase 1 and MR/CD206 in the adhered cells was tested by Western blotting (E) and secretion of IL-10 was quantified by Bioplex assay (F). Lower panels in D and

E, quantification of the protein levels by densitometric analysis of immunoreactive bands. Asterisks in A, B and F indicate the infected cultures treated with recombinant IFN-γ or IL-10, for which the induced cytokine production differed significantly from that in the corresponding cultures incubated without the presence of exogenic cytokines (*p < 0.05; **p < 0.01; ***p < 0.001). Lines over bars in A and B indicate the Mbv isolates for C188-9 supplier which the induced cytokine or NO production differed significantly Urocanase from that induced by H37Rv (#p < 0.01; ##p < 0.001). To verify whether signaling pathways leading to NO production were differentially modulated by the mycobacterial strains, we evaluated induction of iNOS, the essential enzyme for the conversion of arginine to citrulline and NO. The results obtained showed that treatment with IFN-γ induced iNOS expression in the cultured macrophages, and subsequent infection of these cells with bacteria enhanced the level

of enzyme expression in a similar manner (Figure 4D), demonstrating no strain-specific difference in the regulation of IFN-γ-dependent signaling which leads to transactivation of the iNOS gene. Evaluation of expression of M2 markers in the cells pretreated with IFN-γ demonstrated Q-VD-Oph research buy suppression of Arg-1 expression induced by the strains B2 and H37Rv, but not those infected with strain MP287/03 (Figure 4E). Expression of MR by MΦ was slightly inhibited in the cell cultures treated with IFN-γ, and further reduced after infection of these cells with the strains B2 or H37Rv. In contrast, infection with the strain MP287/03 restored a high level of expression of this receptor (Figure 4E), suggesting induction of MR gene transcription due to mycobacteria in these cells.