Suppression of Protective Responses upon Activation of L-Type Voltage Gated Calcium Channel in Macrophages during Mycobacterium bovis BCG Infection

Abstract

The prevalence of Mycobacterium tuberculosis (M. tb) strains eliciting drug resistance has necessitated the need for understanding the complexities of host pathogen interactions. The regulation of calcium homeostasis by Voltage Gated Calcium Channel (VGCCs) upon M. tb infection has recently assumed importance in this area. We previously showed a suppressor role of VGCC during M. tb infections and recently reported the mechanisms of its regulation by M. tb. Here in this report, we further characterize the role of VGCC in mediating defence responses of macrophages during mycobacterial infection. We report that activation of VGCC during infection synergistically downmodulates the generation of oxidative burst (ROS) by macrophages. This attenuation of ROS is regulated in a manner which is dependent on Toll like Receptor (TLR) and also on the route of calcium influx, Protein Kinase C (PKC) and by Mitogen Activation Protein Kinase (MAPK) pathways. VGCC activation during infection increases cell survival and downmodulates autophagy. Concomitantly, pro-inflammatory responses such as IL-12 and IFN-γ secretion and the levels of their receptors on cell surface are inhibited. Finally, the ability of phagosomes to fuse with lysosomes in M. bovis BCG and M. tb H37Rv infected macrophages is also compromised when VGCC activation occurs during infection. The results point towards a well-orchestrated strategy adopted by mycobacteria to supress protective responses mounted by the host. This begins with the increase in the surface levels of VGCCs by mycobacteria and their antigens by well-controlled and regulated mechanisms. Subsequent activation of the upregulated VGCC following tweaking of calcium levels by molecular sensors in turn mediates suppressor responses and prepare the macrophages for long term persistent infection.

Suppression of Toll-like receptor 2–mediated proinflammatory responses by Mycobacterium tuberculosis protein Rv3529c

“…It has been reported that an increase in calcium ion concentration inside Mfs increases phagosome-lysosome fusion and that mycobacteria depletes Ca 2+ concentration in Mfs to prevent this fusion [17]. We have also recently demonstrated the role of voltage-gated calcium channel regulated calcium influx in regulating phagolysosome fusion [11]. As shown in Fig.…”

“…To that end, cells were incubated with biopharmacologic inhibitors that prevent calcium release from intracellular stores or calcium influx from an external medium and inhibitors to PKC and ERK-MAPK, followed by stimulation with Pam 3 Csk 4 , in the presence or absence of Rv3529c. Recent reports from our laboratory have identified significant roles for these signaling intermediates in modulating defense responses of Mfs in M. tuberculosis [9][10][11][12]. As shown in Fig.…”

“…PMA-treated THP-1 cells (0.5 3 10 5 /ml) were stimulated with 15 mg/ml Rv3529c for 24 h. Mycobacterium bovis BCG or M. tuberculosis H37Rv was labeled with FM4-64 dye for 2 min on ice, followed by thorough washes, as recently described [11]. Rv3529c-stimulated THP-1-derived Mfs were then infected with either 2 MOI of FM464-labeled M. bovis BCG or M. tuberculosis H37Rv for 4 h, with or without pretreatment with 2 mM of ionomycin for 45 min before infection.…”

Modulation of macrophage defense responses by Mycobacterial persistence protein MprA (Rv0981) in human THP-1 cells: effect of single amino acid variation on host-pathogen interactions

“…The fact that no significant change was observed for IFN-γ, and IL-6, indicates that MprA and MprA* may not regulate the responses mediated by these cytokines. Gupta et al,(Gupta et al, 2010) can prolong the survival of the pathogen in the macrophage (Malik et al, 2000;Sharma et al, 2016). It is known that MprA has a role in persistence of infection (Zahrt & Deretic, 2001).…”

“…THP-1 cells were maintained in RPMI-1640 (P04-18047Pan Biotech,Germany) supplemented with 10% Fetal Bovine Serum (Gibco) in 5% CO 2 in humidified environment at 37°C. For all the experiments, THP-1 monocytes were differentiated into macrophages with 50ng/ml PMA (Phorbol 12-myristate 13-acetate) (Sigma) for 16 hours followed by 24 hours of incubation in complete medium Sharma et al, 2016). The optimum concentration of protein for maximum activity was standardized using titration assay for MprA and MprA* ( Supplementary Fig 2).The differentiated THP-1 cells were treated with 15 μ g/ml of MprA and MprA* in all the subsequent experiments unless otherwise mentioned.…”