Research

Mycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis are extremely successful pathogens with the former afflicting more than one-third of the human population. Upon infection with M. tuberculosis, only about 10% of the infected populace goes on to develop active tuberculosis (TB) disease. The rest exhibit a clinically latent infection characterized by the persistence of, possibly, dormant bacilli. Approximately 10% of latently infected individuals progress to a highly contagious disease state, called reactivation TB. The reactivation risk is higher when confounded by immunosupression, for example, upon co-infection with HIV/AIDS.

The host-pathogen interplay is complex. Our research interest lies in studying the immunological parameters associated with host- Mycobacterium tuberculosis interactions during pulmonary-, latent- and reactivation TB. We are also investigating the signaling pathways and immunological responses orchestrated by various pathogen recognition receptors (PRRs) like Toll-like receptors (TLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs) viz Dectin-1. Mycobacteria-responsive epigenetic regulation of immune responses is another area of our interest. The following represent some of the research interests of our laboratory.

 

Epigenetic regulation of lipid homeostasis

Foamy macrophages (FMs) constituting lipid bodies nurse the mycobacterial survival in the granuloma. Balaji and colleagues show that mycobacteria induce a demethylase, JMJD3 in macrophages to activate the genes responsible for FM generation. Infection-triggered TLR2-NOTCH1-MUSASHI-JMJD3 pathway is essential for FM generation and concomitant M2 phenotypic responses.

 

 

Counter regulation of PRRs

Among a vast array of responder cells, macrophages are known to regulate cell-fate decisions during microbial challenges by careful tempering of various signaling events activated by innate receptors such as Dectin-1 or TLRs. We have demonstrated that, Dectin-1 activation cripples TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of Wnt signaling intermediate, beta-catenin via Syk-ROS signals contributing to the expression of WNT5A. Subsequently, WNT5A responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling (SOCS-1) mediated the downregulation of IRAK-1, IRAK-4 and MyD88 resulting in decreased expression of IL-12, IL-1beta and TNF-alpha. In vivo activation of Dectin-1 with pathogenic fungi or ligand resulted in increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus or Escherichia with concomitant decrease in TLRs-triggered proinflammatory cytokines. Altogether, our study established a new role for Dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit proinflammatory environment of the host.

Model depicting the molecular mechanism involved in counter-regulation of TLR induced inflammatory signature by Dectin-1

 

Mycobacteria-responsive epigenetic regulation of immune responses

Several pathogens have evolved strategies to evade host immune defences and clearly the study of evasion of autophagy is an emerging trend. We showed that pathogens such as Mycobacteria, Shigella, and Listeria but not Klebsiella, Staphylococcus, and Escherichia inhibit IFN-gamma-induced autophagy in macrophages by eliciting selective and robust activation of WNT and Sonic-hedgehog (SHH) pathways via mTOR-responsive miR-155 and miR-31. These post-translational modifiers were found to regulate cellular levels of PP2A, a phosphatase, to fine-tune autophagy. Diminished expression of PP2A led to inhibition of GSK-3beta, thus facilitating the prolonged activation of WNT and SHH signaling pathways. Sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases, which in tandem inhibited IFN-gamma-induced JAK-STAT signaling and contributed to evasion of autophagy. These results established a role for new host factors and inhibitory mechanisms employed by the pathogens to limit autophagy, which could be targeted for therapeutic interventions. We have also discovered that mycobacteria-induced miR-155 regulates the host macrophage apoptosis. M. bovis BCG was also found to regulate TLR2 responses through induction of SHH-responsive miRNAs. Further, M. bovis BCG-responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase or miR-150 and calibrate CIITA/MHC-II expression.

 

Model depicting Bacteria like Mycobacteria, Shigella, and Listeria
induce an MTOR-dependent upregulation in Mir155 and Mir31    
levels which target and regulate PP2A in the macrophages      

Dissection of signaling events activated in macrophages/dendritic cells upon infection with pathogenic mycobacteria

Pathogenic mycobacteria have evolved unique strategies to survive within the hostile environment of macrophages. In this intricate process, modulation of immune effectors like SOCS3, COX-2 and MMP-9 acts as an important factor influencing the overall host immune response. Our study demonstrates that M. bovis BCG triggered TLR2-dependent signaling leads to induced expression of SOCS3, COX-2 and MMP-9 in macrophages. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1, PI3K during M. bovis BCG triggered expression of multitude of immunological parameters including SOCS3, COX-2 and MMP-9. Intriguingly, Nitric Oxide assumes critical importance in M. bovis BCG mediated activation of Notch1 signaling as iNOS-/- macrophages exhibited compromised ability to execute M. bovis BCG triggered Notch1 signaling responses. These findings provide new insights into mechanisms by which Notch1, TLR2 and NO signals are integrated in a cross-talk that modulate defined set of effector functions in macrophages.

 

 

Model depicting M. bovis BCG triggered signaling pathways regulating COX-2 gene expression

 

Functional characterization of PE/PPE family antigens of M. tuberculosis

The PE/PPE gene family encodes proteins whose N-termini contain the characteristic motifs Pro-Glu (PE) or Pro-Pro-Glu (PPE) and C-termini have domains of varying length/sequence. Within the PE proteins, a subclass contains polymorphic GC-rich sequences (PGRS). Many members of PE/PPE multigene family are shown to be expressed upon infection of macrophages or in tubercle bacilli grown under low pH, stress condition etc. Specific expression of certain PE family genes suggests their possible role in pathogenesis or in virulence. In this regard, we have shown that PE_PGRS17 and PE_PGRS11 exhibit immunoreactivity in sera from adult pulmonary tuberculosis patients and children patients with pulmonary or extrapulmonary infection. Further, PE_PGRS17 and PE_PGRS11 elicited stronger IFN-g and IL-5 cytokine secretion from T cells in pulmonary TB patients compared to healthy subjects. Importantly, PE_PGRS17 and PE_PGRS11 recognized TLR2 and induced maturation and activation of human dendritic cells (DCs). The PE_PGRS proteins-mediated activation of DCs involved participation of ERK 1/2, p38 MAPK and NF-kB pathways and these observations suggested that by acting on APCs such as DCs, PE_PGRS17 and PE_PGRS11 proteins could potentially regulate immune responses to M. tuberculosis and the clinical course of TB.

 

 

 

 

Model depicting TLR2-, NOD1- and NOD2-driven integration of
NOTCH1-PI3K-PKC-MAPK-NF-kappa B signaling during      
maturation of human DCs                               

 

Other survival strategies employed by pathogenic mycobacteria

Alveolar macrophages form the first line of defense against inhaled droplets containing pathogenic mycobacteria, including M. tuberculosis, by controlling mycobacterial growth and regulating T cell responses. However, the ability of pathogenic mycobacteria to survive and persist amid a strong host immune response clearly implicates the importance of survival strategies employed by pathogenic microbes. We are interested in dissecting the survival strategies employed by pathogenic mycobacteria during infection of mouse/human macrophages and dendritic cells. Some of our results suggest various intracellular signaling pathways together with refractoriness to cytokine effects and effects of induced endogenous anti-inflammatory lipid mediators contribute to regulate either containment or pathological outcome of infection thereby boosting the immune evasion strategies of the pathogenic mycobacteria.

 

 
     
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