Delineation of molecular events involved in Mycobacterium bovis BCG triggered expression of inducible Nitric Oxide Synthase and Nitric Oxide production : implication for Nitric Oxide mediated immune modulation

Abstract

Mycobacteria are successful intracellular pathogens capable of causing both acute and chronic diseases. The ability of mycobacteria to survive within the host depends upon their effectiveness in modulating or subverting robust host immune responses. The range of strategies employed by mycobacteria to evade the host immune response include, arrest of phagosome-lysosome fusion, induced secretion of immunosuppressive cytokines such as IL-10 and TGF-, inhibition of antigen processing and presentation to T cells, etc. Among various facets of host immunity, pro-inflammatory responses driven by type 1 T helper (Th-1) subset of CD4+ T cells assume significant importance. The initiation and vigorous activation of Th-1 responses against mycobacteria is orchestrated by antigen-presenting cells (APCs) such as macrophages and dendritic cells. APCs possess a wide range of innate receptors, among which, Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) are well-known sensors of mycobacteria or its components. The engagement of these innate receptors with mycobacteria or its components will trigger the induction of pro-inflammatory cytokines or chemokines that effectuate the activation and recruitment of CD4+ T cells to the site of infection. Importantly, the effector cytokine, interferon gamma (IFN-), is secreted by Th-1 CD4+ T cells, which are suggested to have a plethora of effects on infected host cells. In addition, APCs also induce production of reactive nitrogen intermediates (RNIs) and reactive oxygen intermediates (ROIs), which are known to have antimycobacterial functions. Thus, the outcome in terms of the containment or the development of disease symptoms is largely determined by a multitude of parameters that are associated with host-mycobacterial interactions.

In these complex processes, the role of nitric oxide (NO), a key antimicrobial as well as signaling molecule, remains intriguing. The NO production is catalyzed by the enzyme nitric oxide synthase (NOS), which primarily exhibits three isoforms: NOS I (endothelial nitric oxide synthase (eNOS)), NOS II (inducible nitric oxide synthase (iNOS)) and NOS III (neuronal nitric oxide synthase (nNOS)). Among the NOS, iNOS is present primarily in the macrophages and other immune cells. Its expression is induced during mycobacterial or other pathogenic infections resulting in increased NO production. NO, along with the other reactive intermediates, mounts a potent immune response against pathogenic mycobacteria, which results in effective containment of pathogenic mycobacterial infection. Interestingly, in addition to its antimicrobial properties, NO can modulate a wide range of signaling cascades in various cell types by nitration, nitrosation, and nitrosylation of key signaling molecules. These modulatory properties would have significant effects on factors that regulate cell-fate decisions of macrophages or dendritic cells during the course of infection with pathogenic mycobacteria. In view of these observations, mycobacteria-triggered iNOS expression/NO production assumes a critical function in the ensuing host immune responses.

Pattern recognition receptors (PRRs) like TLR2 and NOD2 act as important sensors for pathogenic mycobacteria during infection. TLR2 recognizes mycobacteria and mycobacterial cell wall antigens such as 19kDa lipoprotein and PIM2, while NOD2, an intracellular receptor, specifically recognizes muramyl dipeptide (MDP), an integral component of bacterial peptidoglycan. Notably, TLR2 and NOD2 activation is known to induce iNOS expression and NO production. Further, TLR2 or NOD2 receptor triggering results in the activation of downstream signaling molecules such as mitogen-activated protein kinases (MAPKs) and transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B). Albeit studies have reported the involvement of MAPKs or NF-B in regulation of iNOS expression, information in regard to receptor-proximal signaling events upon TLR2 or NOD2 receptor engagement remains scanty. In this context, we explored the role of TLR2 or NOD2-mediated signaling events triggered upon infection with M. bovis BCG during induced expression of iNOS in macrophages.

Our study demonstrated that M. bovis BCG triggered TLR2/NOTCH1 signaling cohorts playing a critical role in induced expression of iNOS and NO production. Infection of macrophages from TLR2-null mice showed significant reduction in NOTCH1 activation and abrogation of iNOS expression/NO generation. Further, pharmacological inhibitor and/or NOTCH1 specific siRNA-mediated interference significantly reduced M. bovis BCG’s ability to induce iNOS expression/NO production. Additionally, infection-induced expression of iNOS involved the participation of protein kinase C delta (PKC)-p38 MAPK signaling cascades, as pharmacological intervention or ectopic expression of dominant-negative mutant of PKC in macrophages severely inhibited M. bovis BCG’s capacity to trigger iNOS expression as well as NO production. Interestingly, NOTCH1-PKC-p38 MAPK signaling axis regulated iNOS expression by virtue of PKC-p38 MAPK-dependent activation of NF-B. In view of these observations, our current investigation comprehensively establishes a cross-talk between TLR2 and NOTCH1 to regulate PKC-p38 MAPK activation and NF-B recruitment to the iNOS promoter.

A latent infection of mycobacteria often marks a lifelong persistence of mycobacterial bacilli within the infected host, amid robust host immunity. The host immune responses involve the generation of mycobacterial antigen-specific CD4+ T cells that produce IFN-, which helps in the arrest of mycobacterial growth and halts the progression of infection. However, infected macrophages demonstrate strong refractoriness to T cell-mediated IFN- response. This observation clearly suggests a role for immune evasion mechanisms, wherein, mycobacteria and/or its cell components inhibit macrophage responses to IFN-, thereby facilitating its survival within macrophages. In this regard, our study demonstrated that M. bovis BCG infection compromised IFN-’s ability to induce class II transactivator (CIITA) and major histocompatibility complex (MHC) class II expression. Importantly, loss of iNOS activity either in iNOS-null macrophages or by pharmacological intervention in wild-type macrophages severely abrogated M. bovis BCG’s ability to suppress CIITA as well as MHC class II expression. On the contrary, treatment of macrophages with 3-morpholinosydnonimine (SIN-1), a NO donor, resulted in decreased expression of CIITA or MHC class II on the macrophage cell surface. Further, signaling perturbation experiments suggested the involvement of NOTCH1-triggered iNOS/NO in regulating macrophage responses to IFN-. Strikingly, bioinformatics analysis revealed conserved binding sites for Kruppel-like factor 4 (KLF4) transcription factor in the CIITA promoter. Furthermore, the ectopic expression of KLF4 severely abrogated CIITA expression and KLF4 dominant-negative constructs markedly reverted M. bovis BCG-mediated suppression of IFN--induced CIITA. Interestingly, M. bovis BCG-mediated KLF4 expression was severely compromised in iNOS-null macrophages and SIN1 treatment rescued KLF4 expression. Taken together, our study demonstrates that TLR2-NOTCH1 cross-talk modulates NO production, which is required for recruitment of KLF4 to the CIITA promoter, thus leading to downregulation of IFN--mediated expression of MHC class II. Our data, therefore, explains the molecular mechanisms utilized by mycobacteria to interfere with macrophage responses to IFN-.

In regard to the role for NOD2 receptor in immune responses to mycobacterial infections, our observations demonstrated that MDP-triggered NOD2 signaling induced iNOS expression and NO production. The induced iNOS expression involved activation of downstream signaling molecules like Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK), also termed as RIP2, and transforming growth factor -activated kinase (TAK1), that culminated in translocation of NF-B to the nucleus. Additionally, our study demonstrated important roles for NOD2 signaling in activation of Sonic hedgehog (SHH) signaling through induced expression of ligand SHH, patched-1 (PATCH1), smoothened (SMO), and transcription factors glioma-associated oncogene 1 (GLI1) and glioma-associated oncogene 2 (GLI2). Interestingly, iNOS/NO was involved in NOD2-mediated activation of SHH signaling. Treatment of iNOS-null macrophages with MDP showed a compromised ability to activate SHH signaling. On the contrary, treatment with the NO donor, SIN-1, triggered activation of SHH signaling in iNOS-null macrophages. The activity of SHH signaling is critically tuned by various negative regulators such as NUMB and glycogen synthase kinase 3 beta (GSK-3). In accordance with the above observations, NOD2 signaling activation by MDP led to decreased expression of NUMB and inactivation of GSK-3. Additionally, siRNA-mediated interference with NOD2 signaling significantly reverted MDP-mediated downregulation of NUMB expression or GSK-3 inactivation. The NUMB expression remained unaltered in macrophages from iNOS-null mice, and NO donor, SIN-1, treatment resulted in reduction in the expression of NUMB and GSK-3 inactivation. Interestingly, bioinformatics analysis of NUMB’s 3’ UTR predicted it to be a target for miRNA-146a (miR-146a), and our data demonstrated that NOD2-iNOS-NO mediates regulation of miR-146a expression. Further, NOD2/NO-induced expression of miR-146a led to a decrease in NUMB expression levels, which resulted in augmentation of SHH signaling. Collectively, NOD2/NO signaling axis activated SHH signaling in macrophages by upregulating SHH signaling effectors as well as by downregulating negative regulators of SHH signaling through induced expression of miR-146a. Further, we have demonstrated that SHH signaling modulates expression of proinflammatory cytokines and chemokines, which could gain importance in various pathophysiological conditions.

Collectively, our observations signify the potential role for TLR2 triggering by M. bovis BCG in activating receptor proximal NOTCH1 signaling during induced iNOS/NO production, which represents a crucial immune subversion mechanism employed by mycobacteria in order to suppress or attenuate host immune responses. Further, activation of NOD2-mediated SHH signaling in macrophages enhances our understanding of host-mycobacteria interactions and clearly paves the way towards the development of novel combinatorial therapeutics.