Potassium ion channel Kir2.1 negatively regulates protective responses to Mycobacterium bovis BCG.

Tuberculosis caused by the pathogen Mycobacterium tuberculosis leads to increased mortality and morbidity worldwide. The prevalence of highly drug resistant strains has reinforced the need for greater understanding of host-pathogen interactions at the cellular and molecular levels. Our previous work demonstrated critical roles of calcium ion channels in regulating protective responses to mycobacteria. In this report we deciphered the roles of inwardly rectifying K+ ion channel Kir2.1 in epithelial cells. Data showed that infection of epithelial cells (and macrophages) increases the surface expression of Kir2.1. This increased expression of Kir2.1 results in higher intracellular mycobacterial survival, since either inhibiting or knocking down Kir2.1 results in mounting of a higher oxidative burst leading to a significant attenuation of mycobacterial survival. Further, inhibiting Kir2.1 also led to increased expression of T cell costimulatory molecules accompanied with increased activation of MAP Kinases and transcription factors NF-κB and pCREB. Furthermore, inhibiting Kir2.1 induced increased autophagy and apoptosis that could also contribute to decreased bacterial survival. Interestingly, an increased association of heat shock protein-70 with Kir2.1 was observed. The above results showed that mycobacteria modulate the expression and function of Kir2.1 in epithelial cells to its advantage.

Butyrate induces oxidative burst mediated apoptosis via Glucose-6-Phosphate Dehydrogenase (G6PDH) in macrophages during mycobacterial infection.

Microorganisms present in the gut modulate host defence responses against infections in order to maintain immune homeostasis. This host-microbe crosstalk is regulated by gut metabolites. Butyrate is one such small chain fatty acid produced by gut microbes upon fermentation that has the potential to influence immune responses. Here we investigated the role of butyrate in macrophages during mycobacterial infection. Results demonstrate that butyrate significantly suppresses the growth kinetics of mycobacteria in culture medium as well as inhibits mycobacterial survival inside macrophages. Interestingly, butyrate alters the pentose phosphate pathway by inducing higher expression of Glucose-6-Phosphate Dehydrogenase (G6PDH) resulting in a higher oxidative burst via decreased Sod-2 and increased Nox-2 (NADPH oxidase-2) expression. Butyrate-induced G6PDH also mediated a decrease in mitochondrial membrane potential. This in turn lead to an induction of apoptosis as measured by lower expression of the anti-apoptotic protein Bcl-2 and a higher release of Cytochrome C as a result of induction of apoptosis. These results indicate that butyrate alters the metabolic status of macrophages and induces protective immune responses against mycobacterial infection.

HSP-27 and HSP-70 negatively regulate protective defence responses from macrophages during mycobacterial infection.

Mycobacterium tuberculosis attenuates many defence responses from alveolar macrophages to create a niche at sites of infection in the human lung. Levels of Heat Shock Proteins have been reported to increase many folds in the serum of active TB patients than in latently infected individuals. Here we investigated the regulation of key defence responses by HSPs during mycobacterial infection. We show that infection of macrophages with M. bovis BCG induces higher expression of HSP-27 and HSP-70. Inhibiting HSP-27 and HSP-70 prior to mycobacterial infection leads to a significant decrease in mycobacterial growth inside macrophages. Further, inhibiting HSPs resulted in a significant increase in intracellular oxidative burst levels. This was accompanied by an increase in the levels of T cell activation molecules CD40 and IL-12 receptor and a concomitant decrease in the levels of T cell inhibitory molecules PD-L1 and IL-10 receptor. Furthermore, inhibiting HSPs significantly increased the expression of key proteins in the autophagy pathway along with increased activation of pro-inflammatory promoting transcription factors NF-κB and p-CREB. Interestingly, we also show that both HSP-27 and HSP-70 are associated with anti-apoptotic proteins Bcl-2 and Beclin-1. These results point towards a suppressive role for host HSP-27 and HSP-70 during mycobacterial infection.

Bcl2 negatively regulates Protective Immune Responses During Mycobacterial Infection.

We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apoptosis by upregulating the expression of Bcl2 and Inhibitor of Apoptosis (IAP). In addition, recent reports from our lab showed that stimulation of either macrophages or BMDCs results in the significant upregulation of Bcl2. In this report, we delineate the role of Bcl2 in mediating defense responses from dendritic cells (BMDCs) during mycobacterial infection. Inhibiting Bcl2 led to a significant decrease in intracellular bacterial burden in BMDCs. To further characterize the role of Bcl2 in modulating defense responses, we inhibited Bcl2 in BMDCs as well as human PBMCs to monitor their activation and functional status in response to mycobacterial infection and stimulation with M. tb antigen Rv3416. Inhibiting Bcl2 generated protective responses including increased expression of co-stimulatory molecules, oxidative burst, pro-inflammatory cytokine expression and autophagy. Finally, co-culturing human PBMCs and BMDCs with antigen-primed T cells increased their proliferation, activation and effector function. These results point towards a critical role for Bcl2 in regulating BMDCs defense responses to mycobacterial infection.