Inhibition of leukotriene-B4 signalling-mediated host response to tuberculosis is a potential mode of adjunctive host-directed therapy.

Treatment of tuberculosis (TB) is faced with several challenges including the long treatment duration, drug toxicity and tissue pathology. Host-directed therapy provides promising avenues to find compounds for adjunctively assisting antimycobacterials in the TB treatment regimen, by promoting pathogen eradication or limiting tissue destruction. Eicosanoids are a class of lipid molecules that are potent mediators of inflammation and have been implicated in aspects of the host response against TB. Here, we have explored the blood transcriptome of pulmonary TB patients to understand the activity of leukotriene B4, a pro-inflammatory eicosanoid. Our study shows a significant upregulation in the leukotriene B4 signalling pathway in active TB patients, which is reversed with TB treatment. We have further utilized our in-house network analysis algorithm, ResponseNet, to identify potential downstream signal effectors of leukotriene B4 in TB patients including STAT1/2 and NADPH oxidase at a systemic as well as local level, followed by experimental validation of the same. Finally, we show the potential of inhibiting leukotriene B4 signalling as a mode of adjunctive host-directed therapy against TB. This study provides a new mode of TB treatment along with mechanistic insights which can be further explored in pre-clinical trials.

Mycobacterium tuberculosis elevates SLIT2 expression within the host and contributes to oxidative stress responses during infection.

During infection, Mycobacterium tuberculosis (Mtb) rewires distinct host signaling pathways that results in pathogen-favorable outcomes. Oxidative stress build-up is a key cellular manifestation that occurs due to the cumulative effect of elevated reactive oxygen species generation (ROS) and the inept ability of the cell to mitigate ROS levels. Here, we report the Mtb-induced expression of the neuronal ligand, SLIT2, to be instrumental in ROS accumulation during infection. Loss of function analysis revealed the heightened expression of SLIT2 to be dependent on the Mtb-mediated phosphorylation of the P38/JNK pathways. Activation of these kinases resulted in the loss of the repressive H3K27me3 signature on the Slit2 promoter. Furthermore, SLIT2 promoted the expression of Vanin1 (VNN1), that contributed to copious levels of ROS within the host. Thus, we dissect the pathway leading to the robust expression of SLIT2 during Mtb infection while outlining the potential consequences of SLIT2 upregulation in infected macrophages.

PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), triggers enhanced accumulation of lipids to generate foamy macrophages (FMs). This process has been often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in those involved in lipid efflux. Here, we define an Mtb-orchestrated modulation of the ubiquitination of lipid accumulation markers to enhance lipid accretion during infection. We find that Mtb infection represses the expression of the E3 ubiquitin ligase, ITCH, resulting in the sustenance of key lipid accrual molecules viz. ADRP and CD36, that are otherwise targeted by ITCH for proteasomal degradation. In line, overexpressing ITCH in Mtb-infected cells was found to suppress Mtb-induced lipid accumulation. Molecular analyses including loss-of-function and ChIP assays demonstrated a role for the concerted action of the transcription factor YY1 and the arginine methyl transferase PRMT5 in restricting the expression of Itch gene by conferring repressive symmetrical H4R3me2 marks on its promoter. Consequently, siRNA-mediated depletion of YY1 or PRMT5 rescued ITCH expression, thereby compromising the levels of Mtb-induced ADRP and CD36 and limiting FM formation during infection. Accumulation of lipids within the host has been implicated as a pro-mycobacterial process that aids in pathogen persistence and dormancy. In line, we found that perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in mouse peritoneal macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights into the role of PRMT5 and YY1 in augmenting mycobacterial pathogenesis. Thus, we posit that our observations could help design novel adjunct therapies and combinatorial drug regimen for effective anti-TB strategies.

Mycobacterium tuberculosis Calcium Pump CtpF Modulates the Autophagosome in an mTOR-Dependent Manner.

Calcium is a very important second messenger, whose concentration in various cellular compartments is under tight regulation. A disturbance in the levels of calcium in these compartments can play havoc in the cell, as it regulates various cellular processes by direct or indirect mechanisms. Here, we have investigated the functional importance of a calcium transporting P2A ATPase, CtpF of Mycobacterium tuberculosis (Mtb) in the pathogen's interaction with the host. Among its uncanny ways of dealing with the host with umpteen strategies for survival and persistence in humans, CtpF is identified as a new player. The levels of ctpF are upregulated in macrophage stresses like hypoxia, high nitric oxide levels and acidic pH. Using confocal microscopy and fluorimetry, we show that CtpF effluxes calcium in macrophages in early stages of Mtb infection. Downregulation of ctpF expression by conditional knockdown resulted in perturbation of host calcium levels and consequent decreased activation of mTOR. We present a mechanism how calcium efflux by the pathogen inhibits mTOR-dependent autophagy and enhances bacterial survival. Our work highlights how Mtb engages its metal efflux pumps to exploit host autophagic process for its proliferation.

Histone Methyltransferase SET8 Epigenetically Reprograms Host Immune Responses to Assist Mycobacterial Survival.

NQO1 and TRXR1 are important host reductases implicated in the regulation of inflammation and apoptosis. Although the transcriptional machinery governing these processes have been extensively investigated, the associated epigenetic regulatory events remain unclear. Here, we report that SET8, a histone H4 lysine 20 monomethylase (H4K20me1), is highly induced during Mycobacterium tuberculosis infection that orchestrates immune evasion strategies through the induction of NQO1 and TRXR1 in vivo. SET8, along with FoxO3a, mediates an active NQO1-PGC1-α complex, which promotes the anti-inflammatory M2 macrophage phenotype, and assists TRXR1-regulated arrest of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Strikingly, the loss-of-function analysis in an in vivo mouse tuberculosis model further corroborated the pivotal role of SET8-responsive NQO1 and TRXR1 in mycobacterial survival. Thus, augmenting host immune responses against Mycobacterium tuberculosis by harnessing the SET8-NQO1/TRXR1 axis with its specific and potent inhibitors could lead to promising host-directed therapeutic adjuvants for tuberculosis treatment.