Mechanistic And Functional Insights Into Mycobacterium Bovis BCG Induced Expression Of Cyclooxygenase-2 : Implications For Immune Evasion Strategies
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Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Protective immunity against pathogenic mycobacteria depends principally on cell-mediated immunity executed by efficient anti-infectious functions of type 1 T helper (Th1) subset of CD4+ T cells. The polarization of Th1 responses is orchestrated by IL-12 secreted by antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). A hallmark of Th1 type CD4+ T cells is the production of IFN-γ that activates plethora of innate cell-mediated immunity. It is well known that cytokines such as IFN-γ, IL-12 and TNF-α are required for control of mycobacterial infection in humans as well as in mice. However, it remains unclear that why the immune response controls mycobacteria, but does not eradicate infection suggesting critical roles for series of survival strategies employed by pathogenic mycobacteria. In general, these evasion strategies 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, induced secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. that ultimately suppress the secretion of IL-12 and IFN-γ from APCs and T cells respectively, culminating in a skewed Th1/Th2 balance towards unprotective Th2 responses. Th2 cells secrete IL-4, IL-5, IL-9, IL-10 and IL-13 but are deficient in clearing intracellular infections including pathogenic mycobacteria. This eventually leads to inhibition of host’s immuno-protective responses with concomitant increase in the vulnerability to chronic mycobacterial infection. In this intricate process, modulation of cyclooxygenase-2 (COX-2) levels, a key enzyme catalyzing the rate-limiting step in the inducible production of prostaglandin E2 (PGE2), by mycobacteria like Mycobacterium bovis BCG assumes critical importance in influencing the overall host immune response. PGE2, an immunosuppressive member of prostaglandin family, is known to restrain production of IL-12, as well as reactive oxygen intermediates. PGE2-mediated inhibition of IL-12R, diminishes IL-12 responsiveness of macrophages and dendritic cells. PGE2 also inhibits the secretion of IFN-γ, which is important in activating T cells and macrophages. In contrast, PGE2 promotes IL-10 production by macrophages, dendritic cells and Th1-to-Th2 shift of acquired immune responses by inhibiting IL-2 and enhancing IL-4 production. Albeit, mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways are generally believed to be involved, little is known about the signaling molecules playing significant roles upstream of MAPK and NF-κB pathways during mycobacteria triggered COX-2 expression. Further, information on early receptor proximal signaling mechanisms essential during mycobacteria mediated induction of COX-2 remains scanty. In this regard, signaling cascade triggered upon recognition of mycobacterial components by pattern recognition receptors (PRR) signify as critical event in overall regulation of cell fate decisions. PRR like Toll like receptor (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) are two nonredundant recognition mechanisms of pathogenic mycobacteria. Several components of mycobacteria have been identified as being responsible for TLR2-dependent activation including 19-kDa lipoprotein, lipomannan etc.; while NOD2 recognizes mycobacterial peptidoglycans through its interaction with muramyl dipeptide (MDP). Interestingly, although mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), trehalose 6,6′-dimycolate (TDM; cord factor), PE/PPE family proteins etc., are released and traffic out of the mycobacterial phagosome platform into endocytic compartments. Importantly, these antigens could gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represents a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. Further, PIM2 is a known TLR2 agonist and reported to activate NF-κB, AP-1, and MAPK suggesting that mycobacterial envelope antigen PIM2 could modulate the inflammatory responses similar to mycobacteria bacilli. In this context, we explored the signaling events modulated by M. bovis BCG, and role for TLR2 and NOD2 in this intricate process, to trigger the expression of COX-2 in macrophages. Our studies demonstrated that M. bovis BCG triggered TLR2-dependent signaling leads to COX-2 expression and PGE2 secretion in vitro in macrophages and in vivo in mice. Further, the presence of PGE2 could be demonstrated in sera or CSF of tuberculosis patients. Similarly, mycobacterial TLR2 agonist PIM2 and NOD2 ligand MDP triggered COX-2 expression in macrophages. The induced COX-2 expression in macrophages either by M. bovis BCG or PIM2 or MDP was dependent on NF-κB activation, which was in turn mediated by iNOS/NO and Wnt-β-Catenin dependent participation of the members of Notch1-PI3K signaling cascade. Importantly, loss of iNOS activity either in iNOS null macrophages or by pharmacological intervention in wild type macrophages severely abrogated M. bovis BCG ability to trigger the generation of Notch1 intracellular domain (NICD) as well as activation of PI3K signaling cascade. On contrary, treatment of macrophages with SIN-1, an NO donor, resulted in a rapid increase in generation of NICD, activation of PI3K pathway as well as the expression of COX-2. Interestingly, pharmacological inhibition as well as siRNA mediated knockdown of Wnt-β-Catenin signaling compromised ability of M. bovis BCG to induce activation of Notch1-PI3K signaling and drive COX-2 expression. Concomitantly, activation of Wnt-β-Catenin signaling by LiCl triggered activation of Notch1 and PI3K pathway as well as COX-2 expression. Stable expression of NICD in RAW 264.7 macrophages resulted in augmented expression of COX-2. Further, signaling perturbation experiments suggested involvement of the cross-talk of Notch1 with PI3K signaling cascade. In this perspective, we propose TLR2 and NOD2 as two major receptors involved in mycobacteria mediated activation of Notch1PI3K signaling, and the activation of iNOS/NO and Wnt-β-Catenin signaling axis as obligatory early receptor proximal signaling events during mycobacteria induced COX-2 expression in macrophages. Functional characterization of mycobacterial antigens that are potent modulators of host immune responses to pathogens by virtue of induced expression of COX-2 assumes critical importance for deciphering pathogenesis of mycobacterial diseases as well as to identify novel therapeutic targets to combat the disease. In this context, a group of novel antigens carried by M. tuberculosis that are expressed upon infection of macrophages belong to PE and PPE family of proteins. Ten percent of the coding capacity of M. tuberculosis genome is devoted to the PE and PPE gene family members, exemplified by the presence of Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many members of the PE family exhibit multiple copies of polymorphic guanine-cytosine– rich sequences (PGRS) at the C-terminal end, which are designated as the PE_PGRS family of proteins. A number of PE/PPE proteins associate with the cell wall and are known to induce strong T & B cell responses in humans. However information related to effects of PE/PPE antigens on the maturation and functions of human dendritic cells and eventual modulation of T cell responses as well as underlying signaling events remains obscure. Our results demonstrated that two cell wall associated/secretory PE_PGRS proteins PE_PGRS 17, PE_PGRS 11 and PPE family protein PPE 34 recognize TLR2, induce maturation and activation of human dendritic cells and enhance the ability of dendritic cells to stimulate CD4+ T cells. In addition, tuberculosis patients were found to have a high frequency of T cells specific to PE_PGRS and PPE antigens. We further found that PE/PPE proteins-mediated activation of dendritic cells involves participation of ERK1/2, p38 MAPK and NF-κB signaling pathways. While, PE_PGRS antigens-matured dendritic cells secreted high amounts of inflammatory cytokine IL-12, PPE 34 triggered maturation of dendritic cells was associated with secretion of high amounts of anti-inflammatory cytokine IL-10 but not the Th1-polarizing cytokine IL-12. Consistent with these results, PPE 34-matured dendritic cells favored secretion of IL-4, IL-5 and IL-10 from CD4+ T cells and contributed to Th2 skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, PPE 34-skewed Th2 immune response involved induced expression of COX-2 in dendritic cells. Our results suggest that by inducing differential maturation and activation of human dendritic cells, PE/PPE proteins could potentially modulate the initiation of host immune responses against mycobacteria. Taken together, our observations clearly signify the potential role for TLR2 and NOD2 triggering by M. bovis BCG in activating receptor proximal Notch1-PI3K signaling during induced COX-2/PGE2 expression which represents a crucial immune subversion mechanism employed by mycobacteria in order to suppress or attenuate host immune responses. Further, differential maturation of human dendritic cells by PE_PGRS and PPE antigens as well as their ability to stimulate CD4+ T cells towards Th1 and Th2 phenotype respectively, improves our understanding about host-mycobacteria interactions and clearly paves a way towards the development of novel combinatorial therapeutics.