In this study, we aimed to investigate the modulating effects of pathogenic Mbv strains, differing in virulence-associated properties, on activation phenotypes in MΦ treated with the main cytokines regulating proinflammatory MΦ activation: IFN-γ and IL-10. Rapid growth of pathogenic mycobacteria in MΦ is one of the known factors contributing to bacterial virulence [18
]. Therefore, for this work, we selected two Mbv isolates differing significantly in the capacity to grow in MΦ. One of these isolates, strain B2, was capable of growing in BMDM at a rate similar to that of moderately virulent Mtb strain H37Rv, whereas the intracellular multiplication of other Mbv strain (MP287/03) was significantly faster. Additionally, we demonstrated that bacteria of MP287/03 strain continued to grow rapidly in cells activated by IFN-γ, whereas the growth of the strains B2 and H37Rv was significantly inhibited under this treatment. These data suggested that the MP287/03 strain was either more resistant to the bactericidal effects of macrophages classically activated by IFN-γ, or were able to inhibit MΦ activation induced by this cytokine.
The modulating effects of the Mbv strains were evaluated in comparison to those of the reference Mtb strain H37Rv, which was demonstrated in previous studies to induce in MΦ a proinflammatory activation and synergize with IFN-γ in induction of M1-type polarization of infected cells [7
]. In accordance with these observations, our data demonstrated that the Mtb strain H37Rv induced M1 type activation of the infected BMDM, although the level of activation was less pronounced in comparison with that induced by LPS. Activation of MΦ by the Mbv strains was even weaker than that induced by the H37Rv strain. The lowest level of proinflammatory cytokine expression was observed in MΦ infected with the fast growing Mbv strain MP287/03, although these cells produced high levels of MIP-2 chemokine. Additionally, these cells displayed increased levels of expression of M2 markers (Arg-1 and MR/CD206). Thus, the MP287/03 mycobacteria induced in MΦ an atypical, mixed M1/M2 activation phenotype that coincided with enhanced intracellular growth of the bacteria.
Most important was observation, that this strain induced weaker production of the key bactericidal factors, such as TNF-α and NO, even after pretreatment of MΦ with IFN-γ, priming these cells for M1-type activation. To study the mechanisms that could underlie the observed differences in RNI production, we looked at intracellular signaling pathways leading to NO production by the infected cells. The major regulators of NO production are iNOS and Arg -1, competitive enzymes which utilize a common substrate (L-arginine) to produce NO and citrulline, or urea and ornithine, respectively [21
]. In previous study [22
], induction of Arg-1 expression in MΦ by attenuated Mbv strain BCG was found to be essential for reduction of NO production, through the arginine substrate depletion mechanism, leading to promotion of the intracellular survival of these mycobacteria.
In this study, we demonstrated that pathogenic Mbv were also able to induce expression of Arg-1 in the infected MΦ. Importantly, the fast growing strain MP287/03 induced higher levels of the Arg-1, than any other studied strain, and strongly up-regulated expression of Arg-1 in IFN-γ-treated cells. Although all of the studied strains enhanced expression of iNOS, induced in cells by IFN-γ, in a similar manner, the increased level of Arg-1 observed in MΦ infected with the MP287/03 strain contributed to reduction of NO secretion by these cells. These data suggested that highly virulent Mbv, characterized by enhanced growth in MΦ could induce Arg-1 as a component of the strategy to subvert the antimicrobial activity of CAM, by hydrolyzing the substrate required for NO production.
Mechanisms leading to induction of Arg-1 expression by mycobacteria are only recently starting to be elucidated. Autocrine loop of secretion of IL-6, IL-10 and G-CSF, leading to phosphorylation of STAT3 was determined as an essential mechanism for induction of Arg-1 expression in BCG-infected MΦ [22
]. However, in our study, the increased Arg-1 expression induced by the strain MP287/03, coincided with low levels of IL-6 and IL-10 secretion by the infected MΦ. These data suggested that the signaling pathways, leading to the pronounced induction of the Arg-1 by highly virulent Mbv, could differ from those induced in the BCG-infected MΦ and should be investigated further in separate study.
Another mechanism, underlying the reduced microbicidity of MΦ infected with the strain MP287/03, could be associated with capacity of these bacteria to induce in the cells expression of MR. Infection with the strain H37Rv and incubation with IFN-γ, synergistically inhibited expression of MR gene in murine BMDM [7
], constitutively expressing high levels of MR [23
], resembling in this manner, alveolar macrophages [24
]. In line with these observations, infection of the cells pretreated with IFN-γ by the moderately virulent strains, H37Rv and B2, in our experiments resulted in down-regulation of MR expression. In contrast to these strains, infection of MΦ by the strain MP287/03 restored expression of MR reduced by the IFN-γ treatment. High and persistent levels of MR expression in the MΦ infected with strain MP287/03 in the presence or absence of IFN-γ suggested that these cells could be more susceptible to the deleterious effects of Mannosyl-capped lipoarabinomannan (ManLAM) expressed by the pathogenic mycobacteria. Interaction of Man-LAM with MR has been demonstrated to inhibit fusion of phagosomes with lysosomes in the infected MΦ, interfere with IFN-γ-mediated signaling in MΦ activation, as well as suppress TLR-dependent induction of expression of IL-12 and other proinflammatory cytokines [25
]. In line with this suggestion, the infected cells expressing higher levels of MR in our experiments were permissive to enhanced intracellular growth even in the presence of IFN-γ.
The ability of the strain MP287/03 to induce in MΦ some properties of the M2 cells, suggested that infection of the MΦ, pretreated with IL-10, by these bacteria may synergize in IL-10- dependent M2 polarization of these cells. The obtained results demonstrated that the treatment with IL-10 led to reduction of the proinflammatory MΦ activation by the studied mycobacterial strains. These cells displayed increased expression of the M2 markers, MR, IL-10 and Arg-1. The highest levels of Arg-1 were observed in the cells infected by MP287/03 mycobacteria, demonstrating that the treatment with IL-10 favored the M2-type activation of these cells.
Although the cells infected with MP287/03 strain displayed increased levels of the M2 markers in the presence or absence of regulating cytokines, these cells secreted high levels of the proinflammatory MIP-2 chemokine. In contrast to the MCP-1 chemokine, regulating monocyte recruitment which is essential for formation of functional granuloma, the continues production of MIP-2, and other chemokines attracting granulocytes, was demonstrated to cause excessive recruitment of neutrophils to the infected lungs, contributing to tissue damage in pulmonary tuberculosis, reviewed by [27
]. The high level of MIP-2 secretion and inappropriate proinflammatory MΦ activation, observed in the BMDM cultures infected with MP287/03 strain in this study, may have aggravating implications for in vivo
infection with these, fast-replicating intracellular bacteria. Verification of this important issue is currently under investigation in our laboratory.