Delineation Of Signaling Events Regulating Mycobacterium Bovis BCG Induced Expression Of MMR-9 And SPI6 : Possible Implications For Immune Subversion Mechanisms

Abstract

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.