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The cytokine IFNγ is a principal effector of macrophage activation and immune resistance to mycobacterial infection; however, pathogenic mycobacteria are capable of surviving in IFNγ-activated macrophages by largely unknown mechanisms. In this study, we find that pathogenic mycobacteria, including M. bovis BCG and M. tuberculosis can sense IFNγ to promote their proliferative activity and virulence phenotype. Moreover, interaction with the host intracellular environment increases the susceptibility of mycobacteria to IFNγ through upregulating expression of mmpL10, a mycobacterial IFNγ receptor, thereby facilitating IFNγ-dependent survival and growth of mycobacteria in macrophages. Transmission electron microscopy analysis reveals that IFNγ triggers the secretion of extracellular vesicles, an essential virulence strategy of intracellular mycobacteria, while proteomics identifies numerous pivotal IFNγ-induced effectors required for mycobacterial infection in macrophages. Our study suggests that sensing host IFNγ is a crucial virulence mechanism used by pathogenic mycobacteria to survive and proliferate inside macrophages., (© 2024. The Author(s).)

Programmed cell death protein 4 (PDCD4) is instrumental in regulating a range of cellular processes such as translation, apoptosis, signal transduction, and inflammatory responses. There is a notable inverse correlation between PDCD4 and the mammalian target of rapamycin (mTOR) pathway, which is integral to cellular growth control. Activation of mTOR is associated with the degradation of PDCD4. Although the role of PDCD4 is well established in oncogenesis and immune response regulation, its function in mycobacterial infections and its interplay with the mTOR pathway necessitate further elucidation. This study investigates the modulation of PDCD4 expression in the context of mycobacterial infections, revealing a consistent pattern of downregulation across diverse mycobacterial species. This observation underscores the potential utility of PDCD4 as a biomarker for assessing mTOR pathway activation during such infections. Building on this finding, we employed a novel approach using PDCD4-based mTOR (Tor)-signal-indicator (TOSI) reporter cells for the high-throughput screening of FDA-approved drugs, focusing on mTOR inhibitors. This methodology facilitated the identification of several agents, inclusive of known mTOR inhibitors, which upregulated PDCD4 expression and concurrently exhibited efficacy in impeding mycobacterial proliferation within macrophages. These results not only reinforce the significance of PDCD4 as a pivotal marker in the understanding of infectious diseases, particularly mycobacterial infections, but also illuminate its potential in the identification of mTOR inhibitors, thereby contributing to the advancement of therapeutic strategies., Importance: This study emphasizes the critical role of the mammalian target of rapamycin (mTOR) pathway in macrophage responses to mycobacterial infections, elucidating how mycobacteria activate mTOR, resulting in PDCD4 degradation. The utilization of the (Tor)-signal-indicator (TOSI) vector for real-time monitoring of mTOR activity represents a significant advancement in understanding mTOR regulation during mycobacterial infection. These findings deepen our comprehension of mycobacteria's innate immune mechanisms and introduce PDCD4 as a novel marker for mTOR activity in infectious diseases. Importantly, this research laid the groundwork for high-throughput screening of mTOR inhibitors using FDA-approved drugs, offering the potential for repurposing treatments against mycobacterial infections. The identification of drugs that inhibit mTOR activation opens new avenues for host-directed therapies, marking a significant step forward in combating tuberculosis and other mycobacterial diseases., Competing Interests: The authors declare no conflict of interest.

Mycobacterium tuberculosis ( Mtb ) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis ( M. bovis ) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage 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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest with the content of this article., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Human inherited disorders of interferon-gamma (IFN-γ) immunity underlie severe mycobacterial diseases. We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to interleukin-23 (IL-23) and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN-γ by innate-like adaptive mucosal-associated invariant T cells (MAIT) and γδ T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-γ., Competing Interests: Declaration of interests J.-L.C. serves on the scientific advisory boards of ADMA Biologics Inc., Kymera Therapeutics, and Elixiron Immunotherapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest with the content of this article., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium ( M. avium ) infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we showed that macrophages infected with M. avium switch to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle volume or changes in the energy charge. Instead, we found that pyruvate import contributes to hyperpolarization of mitochondria in infected cells and increases production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduces the macrophage burden of M. avium . While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Røst, Louet, Bruheim, Flo and Gidon.)

Pathogenic mycobacteria including Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, manipulate host macrophages to persist and cause disease. In mycobacterial infection, highly plastic macrophages, shift between inflammatory M1 and permissive M2 phenotypes which alter the disease outcome and allow bacteria to survive intracellularly. Here we examine the impact of MAP infection on polarised macrophages and how increased lipid availability alters macrophage phenotype and bacterial persistence. Further, we assess if host microRNA (miRNA) are sensitive to macrophage polarisation state and how MAP can drive their expression to overcome innate responses. Using in vitro MAP infection, we find that increasing lipid availability through supplementing culture media with exogenous lipid increases cellular nitric oxide production. Lipid-associated miRs -19a, -129, -24, and -24-3p are differentially expressed following macrophage polarisation and lipid supplementation and are further regulated during MAP infection. Collectively, our results highlight the importance of host lipid metabolism in MAP infection and demonstrate control of miRNA expression by MAP to favour intracellular persistence., (© 2022. The Author(s).)

Mycobacterium mucogenicum ( Mmuc ), a rapidly growing nontuberculous mycobacterium (NTM), can infect humans (posttraumatic wound infections and catheter-related sepsis). Similar to other NTM species, Mmuc exhibits colony morphologies of rough ( Mmuc -R) and smooth ( Mmuc -S) types. Although there are several case reports on Mmuc infection, no experimental evidence supports that the R-type is more virulent. In addition, the immune response and metabolic reprogramming of Mmuc have not been studied on the basis of morphological characteristics. Thus, a standard ATCC Mmuc strain and two clinical strains were analyzed, and macrophages were generated from mouse bone marrow. Cytokines and cell death were measured by ELISA and FACS, respectively. Mitochondrial respiration and glycolytic changes were measured by XF seahorse. Higher numbers of intracellular bacteria were found in Mmuc -R-infected macrophages than in Mmuc -S-infected macrophages. Additionally, Mmuc -R induced higher levels of the cytokines TNF-α, IL-6, IL-12p40, and IL-10 and induced more BMDM necrotic death. Furthermore, our metabolic data showed marked glycolytic and respiratory differences between the control and each type of Mmuc infection, and changes in these parameters significantly promoted glucose metabolism, extracellular acidification, and oxygen consumption in BMDMs. In conclusion, at least in the strains we tested, Mmuc -R is more virulent, induces a stronger immune response, and shifts bioenergetic metabolism more extensively than the S-type. This study is the first to report differential immune responses and metabolic reprogramming after Mmuc infection and might provide a fundamental basis for additional studies on Mmuc pathogenesis.

The inflammasome complex is important for host defense against intracellular bacterial infections. Mycobacterium tuberculosis (Mtb) is a facultative intracellular bacterium which is able to survive in infected macrophages. Here we discuss how the host cell inflammasomes sense Mtb and other related mycobacterial species. Furthermore, we describe the molecular mechanisms of NLRP3 inflammasome sensing of Mtb which involve the type VII secretion system ESX-1, cell surface lipids (TDM/TDB), secreted effector proteins (LpqH, PPE13, EST12, EsxA) and double-stranded RNA acting on the priming and/or activation steps of inflammasome activation. In contrast, Mtb also mediates inhibition of the NLRP3 inflammasome by limiting exposure of cell surface ligands via its hydrolase, Hip1, by inhibiting the host cell cathepsin G protease via the secreted Mtb effector Rv3364c and finally, by limiting intracellular triggers (K + and Cl - efflux and cytosolic reactive oxygen species production) via its serine/threonine kinase PknF. In addition, Mtb inhibits the AIM2 inflammasome activation via an unknown mechanism. Overall, there is good evidence for a tug-of-war between Mtb trying to limit inflammasome activation and the host cell trying to sense Mtb and activate the inflammasome. The detailed molecular mechanisms and the importance of inflammasome activation for virulence of Mtb or host susceptibility have not been fully investigated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rastogi and Briken.)

Background: Extracellular vesicles (EVs) play a key role in cell communication and the pathogenesis of some diseases. EVs may accelerate cell death during the course of mycobacterial infection and are also considered as a new vaccine design, drug delivery, and biomarker candidates. The current study evaluates the effects of EVs from serum samples of mycobacteria-infected patients on THP-1 monocytes and PBMC cells., Method: EVs were purified from the serum, then cultured separately with THP-1 monocytes and PBMCs. The cell death was determined through annexin V-FITC and PI staining. GW4869, an EVs inhibitor, was used to determine if EVs released from serum could increase THP-1 monocytes cell death., Results: The cell death was significantly increased in the presence of 10 µg/ml and 5 µg/ml concentrations of the purified EVs (p < 0.05). Minimal cell death was determined in 2.5 µg/ml and 1.2 µg/ml (p < 0.05). Up to 85% of the cells were viable in the presence of the GW4869 inhibitor (p < 0.05)., Conclusion: Direct infection of the cells with EVs released from mycobacteria-infected patients samples, the multiplicity of infection with the EVs, and virulent or avirulent mycobacteria may change the status of the cell death. The isolated EVs  from serum samples of patients with mycobacterial  infection accelerated cell death, which means that they might   not be considered as an optimal tool for developing drug delivery and vaccine against tuberculosis., (© 2022. The Author(s).)

The mycobacterium genus contains a broad range of species, including the human pathogens M. tuberculosis and M. leprae . These bacteria are best known for their residence inside host cells. Neutrophils are frequently observed at sites of mycobacterial infection, but their role in clearance is not well understood. In this review, we discuss how neutrophils attempt to control mycobacterial infections, either through the ingestion of bacteria into intracellular phagosomes, or the release of neutrophil extracellular traps (NETs). Despite their powerful antimicrobial activity, including the production of reactive oxidants such as hypochlorous acid, neutrophils appear ineffective in killing pathogenic mycobacteria. We explore mycobacterial resistance mechanisms, and how thwarting neutrophil action exacerbates disease pathology. A better understanding of how mycobacteria protect themselves from neutrophils will aid the development of novel strategies that facilitate bacterial clearance and limit host tissue damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Parker, Forrester, Kaldor, Dickerhof and Hampton.)

Mycobacterial diseases are a major public health challenge. Their causative agents include, in order of impact, members of the Mycobacterium tuberculosis complex (causing tuberculosis), Mycobacterium leprae (causing leprosy), and non-tuberculous mycobacterial pathogens including Mycobacterium ulcerans. Macrophages are mycobacterial targets and they play an essential role in the host immune response to mycobacteria. This review aims to provide a comprehensive understanding of the immune-metabolic adaptations of the macrophage to mycobacterial infections. This metabolic rewiring involves changes in glycolysis and oxidative metabolism, as well as in the use of fatty acids and that of metals such as iron, zinc and copper. The macrophage metabolic adaptations result in changes in intracellular metabolites, which can post-translationally modify proteins including histones, with potential for shaping the epigenetic landscape. This review will also cover how critical tuberculosis co-morbidities such as smoking, diabetes and HIV infection shape host metabolic responses and impact disease outcome. Finally, we will explore how the immune-metabolic knowledge gained in the last decades can be harnessed towards the design of novel diagnostic and therapeutic tools, as well as vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Llibre, Dedicoat, Burel, Demangel, O’Shea and Mauro.)

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., (© 2021 Aayushi Singh et al., published by De Gruyter.)

The mycobacterial cell wall is composed of large amounts of lipids with varying moieties. Some mycobacteria species hijack host cells and promote lipid droplet accumulation to build the cellular environment essential for their intracellular survival. Thus, lipids are thought to be important for mycobacteria survival as well as for the invasion, parasitization, and proliferation within host cells. However, their physiological roles have not been fully elucidated. Recent studies have revealed that mycobacteria modulate the peroxisome proliferator-activated receptor (PPAR) signaling and utilize host-derived triacylglycerol (TAG) and cholesterol as both nutrient sources and evasion from the host immune system. In this review, we discuss recent findings that describe the activation of PPARs by mycobacterial infections and their role in determining the fate of bacilli by inducing lipid metabolism, anti-inflammatory function, and autophagy.

Mycobacterium tuberculosis ( M. tb ) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-α, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2α, IRE1α, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. Recombinant Mycobacterium smegmatis ( M. smegmatis ) containing pe6 displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host-pathogen interaction during M. tb  infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sharma, Shariq, Quadir, Singh, Sheikh, Hasnain and Ehtesham.)

Tumor necrosis factor (TNF) is one of the main cytokines regulating a pro-inflammatory environment. It has been related to several cell functions, for instance, phagocytosis, apoptosis, proliferation, mitochondrial dynamic. Moreover, during mycobacterial infections, TNF plays an essential role to maintain granuloma formation. Several effector mechanisms have been implicated according to the interactions of the two active forms, soluble TNF (solTNF) and transmembrane TNF (tmTNF), with their receptors TNFR1 and TNFR2. We review the impact of these interactions in the context of mycobacterial infections. TNF is tightly regulated by binding to receptors, however, during mycobacterial infections, upstream activation signalling pathways may be influenced by key regulatory factors either at the membrane or cytosol level. Detailing the structure and activation pathways used by TNF and its receptors, such as its interaction with solTNF/TNFRs versus tmTNF/TNFRs, may bring a better understanding of the molecular mechanisms involved in activation pathways which can be helpful for the development of new therapies aimed at being more efficient against mycobacterial infections.

Background: Host-directed therapy is considered a novel anti-tuberculosis strategy in tackling the tuberculosis burden through autophagy induction by various inducers to curtail the growth of intracellular Mycobacterium tuberculosis., Methods: In this study, we investigated the anti-tubercular role of soybean lectin, a lectin isolated from Glycine max (Soybean). Effect of SBL on intracellular mycobacterial viability through autophagy and the mechanism involved in differentiated THP-1 cells was studied using different experimental approaches., Results: We initially performed a time kinetic experiment with the non-cytotoxic dose of SBL (20 μg/ml) and observed autophagy induction after 24 h of treatment. Abrogation of autophagy in the presence of 3-MA and an increase in LC3 puncta formation upon Baf-A1 addition elucidated the specific effect on autophagy and autophagic flux. SBL treatment also led to autophagy induction in mycobacteria infected macrophages that restricted the intracellular mycobacterial growth, thus emphasizing the host defensive role of SBL induced autophagy. Mechanistic studies revealed an increase in P2RX7 expression, NF-κB activation and reactive oxygen species generation upon SBL treatment. Inhibition of P2RX7 expression suppressed NF-κB dependent ROS level in SBL treated cells. Moreover, SBL induced autophagy was abrogated in the presence of either different inhibitors or P2RX7 siRNA, leading to the reduced killing of intracellular mycobacteria., Conclusion: Taken together, these results conclude that SBL induced autophagy exerts an anti-mycobacterial effect in P2RX7-NF-κB dependent manner through the generation of ROS., General Significance: This study has provided a novel anti-mycobacterial role of SBL, which may play an important role in devising new therapeutic interventions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mycobacterial pathogens pose a sustained threat to human health. There is a critical need for new diagnostics, therapeutics, and vaccines targeting both tuberculous and nontuberculous mycobacterial species. Understanding the basic mechanisms used by diverse mycobacterial species to cause disease will facilitate efforts to design new approaches toward detection, treatment, and prevention of mycobacterial disease. Molecular, genetic, and biochemical approaches have been widely employed to define fundamental aspects of mycobacterial physiology and virulence. The recent expansion of genetic tools in mycobacteria has further increased the accessibility of forward genetic approaches. Proteomics has also emerged as a powerful approach to further our understanding of diverse mycobacterial species. Detection of large numbers of proteins and their modifications from complex mixtures of mycobacterial proteins is now routine, with efforts of quantification of these datasets becoming more robust. In this review, we discuss the "genetic proteome," how the power of genetics, molecular biology, and biochemistry informs and amplifies the quality of subsequent analytical approaches and maximizes the potential of hypothesis-driven mycobacterial research. Published proteomics datasets can be used for hypothesis generation and effective post hoc supplementation to experimental data. Overall, we highlight how the integration of proteomics, genetic, molecular, and biochemical approaches can be employed successfully to define fundamental aspects of mycobacterial pathobiology., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium is known for subverting the host defense machinery, and one such mechanism is the inhibition of autophagy. Here, we have demonstrated that Mycobacterium tuberculosis (MTB) secretes a virulence factor; an early secretory antigenic target protein (ESAT-6) into the phagosome, which induces the expression and activity of mitochondrial superoxide dismutase (SOD-2) of macrophages. Using a series of experiments, and Mycobacterium bovis BCG as a model strain (where ESAT-6 protein is not expressed), we have delineated that the protein regulates SOD-2 of macrophages. The expression and augmentation of SOD-2 activity were confirmed by either incubating the macrophages with ESAT-6 protein, transfection of macrophage by esat6 gene using a eukaryotic promoter vector, or by infection with different mycobacterial strains. The induction of acidification of phagosomal compartment containing bacteria was observed in cells that express low levels of SOD-2. This was further confirmed by observing a significant decrease in the M. bovis BCG intracellular load in the sod-2 knocked-down macrophages., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lipid droplets (LDs) are organelles that have multiple roles in inflammatory and infectious diseases. LD act as essential platforms for immunometabolic regulation, including as sites for lipid storage and metabolism, inflammatory lipid mediator production, and signaling pathway compartmentalization. Accumulating evidence indicates that intracellular pathogens may exploit host LDs as source of nutrients and as part of their strategy to promote immune evasion. Notably, numerous studies have demonstrated the interaction between LDs and pathogen-containing phagosomes. However, the mechanism involved in this phenomenon remains elusive. Here, we observed LDs and PLIN2 surrounding M. bovis BCG-containing phagosomes, which included observations of a bacillus cell surrounded by lipid content inside a phagosome and LAM from mycobacteria co-localizing with LDs; these results were suggestive of exchange of contents between these compartments. By using beads coated with M.tb lipids, we demonstrated that LD-phagosome associations are regulated through the mycobacterial cell wall components LAM and PIM. In addition, we demonstrated that Rab7 and RILP, but not Rab5, localizes to LDs of infected macrophages and observed the presence of Rab7 at the site of interaction with an infected phagosome. Moreover, treatment of macrophages with the Rab7 inhibitor CID1067700 significantly inhibited the association between LDs and LAM-coated beads. Altogether, our data demonstrate that LD-phagosome interactions are controlled by mycobacterial cell wall components and Rab7, which enables the exchange of contents between LDs and phagosomes and may represent a fundamental aspect of bacterial pathogenesis and immune evasion., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020. Published by Elsevier B.V.)

ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-β-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PL pro , have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Advances in microarray, RNA-seq and omics techniques, thousands of long non-coding RNAs (lncRNAs) with unknown functions have been discovered. LncRNAs have presented a diverse perspective on gene regulation in diverse biological processes, especially in human immune response. Macrophages participate in the whole phase of immune inflammatory response. They are able to shape their phenotype and arouse extensive functional activation after receiving physiological and pathological stimuli. Emerging studies indicated that lncRNAs participated in the gene regulatory network during complex biological processes of macrophage, including macrophage-induced inflammatory responses. Here, we reviewed the existing knowledges of lncRNAs in the processes of macrophage development and polarization, and their roles in several different inflammatory diseases. Specifically, we focused on how lncRNAs function in macrophage, which might help to discover some potential therapeutic targets and diagnostic biomarkers., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Extracellular vesicles are membrane-bound structures released by living cells and present in body fluids. Their composition includes proteins, lipids, carbohydrates, and nucleic acids and are involved in transfers between cells. Extracellular vesicles can deliver molecules to cells and tissues even if distant. As a consequence, they have a role in information transmission and in the modulation of the biological function of recipient cells. Among other things, they are involved in antigen presentation and the induction of secretion events by immune cells. Thus, extracellular vesicles participate in the regulation of immune responses during infections. We will discuss their potential as effectors and disease biomarkers concerning only mycobacterial infections.

Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH 4 ) modulates IL-1β production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polarization of naive T cells into interferon (IFN)-γ-producing T helper 1 (Th1) cells is an essential event in the inflammatory response to pathogens. Herein, we identify the RNA binding protein Sam68 as a specific modulator of Th1 differentiation. Sam68-knockout (ko) naive T cells are strongly defective in IL-12-mediated Th1 polarization and express low levels of T-bet and Eomes. Consequently, Sam68-ko Th1 cells are significantly impaired in IFN-γ production. Moreover, we found that Sam68 is required for the induction of an inflammatory Th1 response during Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, thus limiting bacterial dissemination in the lungs. Mechanistically, Sam68 directly binds to the microRNA miR-29, a negative regulator of Th1 response, and inhibits its expression during BCG infection. These findings uncover a novel post-transcriptional mechanism required for the Th1-mediated defense against intracellular pathogens and identify a new function for Sam68 in the regulation of the immune response.

Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline-generating (i.e., iNOS) and l-citrulline-using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either Ass1 or Asl resulted in increased Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis H 37 R v burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium bovis , the causative agent of tuberculosis in cattle and humans, infects host macrophages and induces endoplasmic reticulum stress (ERS), mitochondrial damage, and interleukin (IL)-1β production. The relationship between these phenotypes is yet to be elucidated. In this study, we investigated the role of ERS in mitochondrial damage and IL-1β production in macrophages during infection with a virulent M. bovis strain. We found that ERS activates the inflammasome via NOD-like receptor family, pyrin domain-containing 3 (NLRP3)-caspase-8 and that IFN-inducible protein absent in melanoma 2 (AIM2) triggered mitochondrial damage. ERS increased reactive oxygen species (ROS), which promoted translocation of the inflammasome to the mitochondria. NLRP3, but not AIM2, was involved in the ERS-induced cleavage of caspase-8 and Bid, leading to mitochondrial damage, which was required for the production of mature IL-1β. Our data suggest that ERS induces macrophages to produce mature IL-1β during infection with virulent M. bovis through a positive feedback loop between mitochondrial damage and inflammasome activation. To the best of our knowledge, this is the first evidence of the involvement of ERS and mitochondrial damage in inflammasome activation during M. bovis infection.

The induction and action of type I interferon (IFN) is of fundamental importance in human immune defenses toward microbial pathogens, particularly viruses. Basic discoveries within the molecular and cellular signaling pathways regulating type I IFN induction and downstream actions have shown the essential role of the IFN regulatory factor (IRF) and the signal transducer and activator of transcription (STAT) families, respectively. However, the exact biological and immunological functions of these factors have been most clearly revealed through the study of inborn errors of immunity and the resultant infectious phenotypes in humans. The spectrum of human inborn errors of immunity caused by mutations in IRFs and STATs has proven very diverse. These diseases encompass herpes simplex encephalitis (HSE) and severe influenza in IRF3- and IRF7/IRF9 deficiency, respectively. They also include Mendelian susceptibility to mycobacterial infection (MSMD) in STAT1 deficiency, through disseminated measles infection associated with STAT2 deficiency, and finally staphylococcal abscesses and chronic mucocutaneous candidiasis (CMC) classically described with Hyper-IgE syndrome (HIES) in the case of STAT3 deficiency. More recently, increasing focus has been on aspects of autoimmunity and autoinflammation playing an important part in many primary immunodeficiency diseases (PID)s, as exemplified by STAT1 gain-of-function causing CMC and autoimmune thyroiditis, as well as a recently described autoinflammatory syndrome with hypogammaglobulinemia and lymphoproliferation as a result of STAT3 gain-of-function. Here I review the infectious, inflammatory, and autoimmune disorders arising from mutations in IRF and STAT transcription factors in humans, highlightning the underlying molecular mechanisms and immunopathogenesis as well as the clinical/therapeutic perspectives of these new insights.

Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host-mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms&#95;AcpM) and a strain transformed with an empty vector (Ms&#95;Vec). Ms&#95;AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms&#95;AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms&#95;Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection., (Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Purpose: Inborn errors of IFN-γ-mediated immunity underlie Mendelian Susceptibility to Mycobacterial Disease (MSMD), which is characterized by an increased susceptibility to severe and recurrent infections caused by weakly virulent mycobacteria, such as Bacillus Calmette-Guérin (BCG) vaccines and environmental, nontuberculous mycobacteria (NTM)., Methods: In this study, we investigated four patients from four unrelated consanguineous families from Isfahan, Iran, with disseminated BCG disease. We evaluated the patients' whole blood cell response to IL-12 and IFN-γ, IL-12Rβ1 expression on T cell blasts, and sequenced candidate genes., Results: We report four patients from Isfahan, Iran, ranging from 3 months to 26 years old, with impaired IL-12 signaling. All patients suffered from BCG disease. One of them presented mycobacterial osteomyelitis. By Sanger sequencing, we identified three different types of homozygous mutations in IL12RB1. Expression of IL-12Rβ1 was completely abolished in the four patients with IL12RB1 mutations., Conclusions: IL-12Rβ1 deficiency was found in the four MSMD Iranian families tested. It is the first report of an Iranian case with S321* mutant IL-12Rβ1 protein. Mycobacterial osteomyelitis is another type of location of BCG infection in an IL-12Rβ1-deficient patient, notified for the first time in this study.

Purpose: Inborn errors of IFN-γ immunity underlie Mendelian susceptibility to mycobacterial disease (MSMD). Autosomal recessive complete IL-12Rβ1 deficiency is the most frequent genetic etiology of MSMD. Only two of the 84 known mutations are copy number variations (CNVs), identified in two of the 213 IL-12Rβ1-deficient patients and two of the 164 kindreds reported. These two CNVs are large deletions found in the heterozygous or homozygous state. We searched for novel families with IL-12Rβ1 deficiency due to CNVs., Methods: We studied six MSMD patients from five unrelated kindreds displaying adverse reactions to BCG vaccination. Three of the patients also presented systemic salmonellosis, two had mucocutaneous candidiasis, and one had disseminated histoplasmosis. We searched for CNVs and other variations by IL12RB1-targeted next-generation sequencing (NGS)., Results: We identified six new IL-12Rβ1-deficient patients with a complete loss of IL-12Rβ1 expression on phytohemagglutinin-activated T cells and/or EBV-transformed B cells. The cells of these patients did not respond to IL-12 and IL-23. Five different CNVs encompassing IL12RB1 (four deletions and one duplication) were identified in these patients by NGS coverage analysis, either in the homozygous state (n = 1) or in trans (n = 4) with a single-nucleotide variation (n = 3) or a small indel (n = 1). Seven of the nine mutations are novel. Interestingly, four of the five CNVs were predicted to be driven by nearby Alu elements, as well as the two previously reported large deletions. The IL12RB1 locus is actually enriched in Alu elements (44.7%), when compared with the rest of the genome (10.5%)., Conclusion: The IL12RB1 locus is Alu-enriched and therefore prone to rearrangements at various positions. CNVs should be considered in the genetic diagnosis of IL-12Rβ1 deficiency.

Murine leprosy, caused by Mycobacterium lepraemurium (MLM), is a chronic disease that closely resembles human leprosy. Even though this disease does not directly involve the nervous system, we investigated a possible effect on working memory during this chronic infection in Balb/c mice. We evaluated alterations in the dorsal region of the hippocampus and measured peripheral levels of cytokines at 40, 80, and 120 days post-infection. To evaluate working memory, we used the T-maze while a morphometric analysis was conducted in the hippocampus regions CA1, CA2, CA3, and dentate gyrus (DG) to measure morphological changes. In addition, a neurochemical analysis was performed by HPLC. Our results show that, at 40 days post-infection, there was an increase in the bacillary load in the liver and spleen associated to increased levels of IL-4, working memory deterioration, and changes in hippocampal morphology, including degeneration in the four subregions analyzed. Also, we found a decrease in neurotransmitter levels at the same time of infection. Although MLM does not directly infect the nervous system, these findings suggest a possible functional link between the immune system and the central nervous system.

During mycobacteria infection, anti-inflammatory responses allow the host to avoid tissue damage caused by overactivation of the immune system; however, little is known about the negative modulators that specifically control mycobacteria-induced immune responses. Here we demonstrate that integrin CD11b is a critical negative regulator of mycobacteria cord factor-induced macrophage-inducible C-type lectin (Mincle) signaling. CD11b deficiency resulted in hyperinflammation following mycobacterial infection. Activation of Mincle by mycobacterial components turns on not only the Syk signaling pathway but also CD11b signaling and induces formation of a Mincle-CD11b signaling complex. The activated CD11b recruits Lyn, SIRPα and SHP1, which dephosphorylate Syk to inhibit Mincle-mediated inflammation. Furthermore, the Lyn activator MLR1023 effectively suppressed Mincle signaling, indicating the possibility of Lyn-mediated control of inflammatory responses. These results describe a new role for CD11b in fine-tuning the immune response against mycobacterium infection.

Mycobacterium tuberculosis (M. tuberculosis) invading and activating microglia causes the most serious subtypes of tuberculosis called tubercular meningitis. However, the developmental process of tubercular meningitis, especially the early phase, is poorly understood due to lacking well-established and well-accepted visible models in vitro and in vivo. Here, consistent with one recent report, we found Mycobacterium marinum (M. marinum) invade the zebrafish brain and subsequently cause granuloma-like structures. We further showed that M. marinum, which shares similar characteristics with M. tuberculosis, can invade microglia and replicate in microglia, which subsequently promote the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. M. marinum infection in microglia can also promote autophagy, which conversely limits the replication of M. marinum. Thus, pharmacological activation of autophagy by rapamycin could prevent M. marinum replication. Our study provides in vivo and in vitro models to study underlying pathogenic mechanisms of tubercular meningitis by using M. marinum. Our results also showed that activation of autophagy could be a meaningful way to prevent tubercular meningitis.

Granulomatous inflammation causes severe tissue damage in mycobacterial infection while redox status was reported to be crucial in the granulomatous inflammation. Here, we used a NADPH oxidase 2 (NOX2)-deficient mice (Ncf1-/-) to investigate the role of leukocyte-produced reactive oxygen species (ROS) in mycobacterium-induced granulomatous inflammation. We found poorly controlled mycobacterial proliferation, significant body weight loss, and a high mortality rate after M. marinum infection in Ncf1-/- mice. Moreover, we noticed loose and neutrophilic granulomas and higher levels of interleukin (IL)-1β and neutrophil chemokines in Ncf1-/- mice when compared with those in wild type mice. The lack of ROS led to reduced production of IL-1β in macrophages, whereas neutrophil elastase (NE), an abundant product of neutrophils, may potentially exert increased inflammasome-independent protease activity and lead to higher IL-1β production. Moreover, we showed that the abundant NE and IL-1β were present in the caseous granulomatous inflammation of human TB infection. Importantly, blocking of IL-1β with either a specific antibody or a recombinant IL-1 receptor ameliorated the pulmonary inflammation. These findings revealed a novel role of ROS in the early pathogenesis of neutrophilic granulomatous inflammation and suggested a potential role of IL-1 blocking in the treatment of mycobacterial infection in the lung.

Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen that can live within macrophages owning to its ability to arrest phagolysosome biogenesis. The guanine nucleotide exchange factor H1 (GEF-H1) may contribute to the phagocytosis of bacteria by macrophages through mediating the crosstalk between microtubules and the actin cytoskeleton. Its role in Shigella infection has been determined but little is known about the role of GEF-H1 in mycobacterial infection. In the present study, we demonstrated that GEF-H1 functioned as a key regulator of the macrophage-mediated anti-mycobacterial response. We found that both mRNA and protein expression levels of GEF-H1 were significantly upregulated in macrophage during mycobacterial infection. Moreover, silencing of GEF-H1 with specific siRNAs reduced the phosphorylation of p38 mitogen-activated protein kinase and TANK binding kinase 1 as well as the expression of interleukin-1β (IL-1β), IL-6, and interferon-β (IFN-β), without affecting nitric oxide production or autophagy. Importantly, GEF-H1 depletion attenuated macrophages-mediated mycobacterial phagocytosis and elimination. Taken together, our data supported that GEF-H1 was a novel regulator of inflammatory cytokine production and mycobacterial elimination, and may serve as a novel potential target for clinical treatment of tuberculosis.

Animal mycobacterioses are among the most important zoonoses worldwide. These are generally caused by either Mycobacterium tuberculosis (MTB), M. bovis (MBO) or M. avium (MAV). To test the hypothesis that different species of pathogenic mycobacteria isolated from varied anatomic locations or animal species differ in virulence and pathogenicity, we performed experiments with three mycobacteria strains (NTSE-3(MTB), NTSE-4(MBO) and NTSE-5 (MAV)) obtained from animal species. Spoligotyping analysis was used to confirm both MTB and MBO strains while the MAV strain was confirmed by 16s rDNA sequencing. BALB/c mice were intranasally infected with the three strains at low and high CFU doses to evaluate variations in pathogenicity. Clinical and pathological parameters were assessed. Infected mice were euthanized at 80 days post-inoculation (dpi). Measures of lung and body weights indicated that the MBO infected group had higher mortality, more weight loss, higher bacterial burden and more severe lesions in lungs than the other two groups. Cytokine profiles showed higher levels of TNF-α for MBO versus MTB, while MAV had the highest amounts of IFN-β in vitro and in vivo. In vitro levels of other cytokines such as IL-1β, IL-10, IL-12, IL-17, and IFN-β showed that Th1 cells had the strongest response in MBO infected mice and that Th2 cells were inhibited. We found that the level of virulence among the three isolates decreased in the following order MBO>MTB>MAV.

Toll-like receptors are transmembrane glycoproteins predominantly expressed in tissues with immune function. They are considered one of the most important pattern recognition receptor families discovered at the end of 20th century and a key aspect of the innate immune system response to infectious disease. Here we present a review of the current knowledge of individual Toll-like receptors, 1 through 13, with a focus on their role in the immune system response to mycobacterial infection. We present literature to date about the Toll-like receptors structure, localization and expression, signaling pathways, and function. The Toll-like receptor family may have proven an important role in the immune system response to mycobacterial infections, including M. tuberculosis and non-tuberculous (NTM) organisms., (Published by Elsevier B.V.)

According to most models of mycobacterial infection, inhibition of the pro-inflammatory macrophage immune responses contributes to the persistence of bacteria. Mycobacterium avium subsp. paratuberculosis (MAP) is a highly successful pathogen in cattle and sheep and is also implicated as the causative agent of Crohn's disease in humans. Pathogenic mycobacteria such as MAP have developed multiple strategies to evade host defence mechanisms including interfering with the macrophages' capacity to respond to IFN-γ, a feature which might be lacking in non-pathogenic mycobacteria such as M. smegmatis. We hypothesized that pre-sensitisation of macrophages with the pro-inflammatory cytokine IFN-γ would help in overcoming the inhibitory effect of MAP or its antigens on macrophage inflammatory responses. Herein we have compared a series of macrophage activation parameters in response to MAP and M. smegmatis as well as mycobacterial antigens. While IFN-γ did overcome the inhibition in immune suppressive mechanisms in response to MAP antigen as well as M. smegmatis, we could not find a clear role for IFN-γ in overcoming the inhibition of macrophage inflammatory responses to the pathogenic mycobacterium, MAP. We demonstrate that suppression of macrophage defence mechanisms by pathogenic mycobacteria is unlikely to be overcome by prior sensitization with IFN-γ alone. This indicates that IFN-γ signaling pathway-independent mechanisms may exist for overcoming inhibition of macrophage effector functions in response to pathogenic mycobacteria. These findings have important implications in understanding the survival mechanisms of pathogenic mycobacteria directed towards finding better therapeutics and vaccination strategies.

Mycobacterium avium (M. avium) causes significant pulmonary infection, especially in immunocompromised hosts. Alveolar macrophages (AMs) represent the first line of host defense against infection in the lung. Interferon gamma (IFN-γ) activation of AMs enhances in vitro killing of pathogens such as M. avium We hypothesized that airway delivery of AMs into the lungs of immunodeficient mice infected with M. avium will inhibit M. avium growth in the lung and that this macrophage function is in part IFN-γ dependent. In this study, normal BALB/c and BALB/c SCID mice received M. avium intratracheally while on mechanical ventilation. After 30 days, M. avium numbers increased in a concentration-dependent manner in SCID mice compared with normal BALB/c mice. Airway delivery of IFN-γ-activated BALB/c AMs or J774A.1 macrophages overexpressing IFN-γ into the lungs of SCID mice resulted in a significant decrease in M. avium growth (P < 0.01, both comparisons) and limited dissemination to other organs. In addition, airway delivery of IFN-γ activated AMs and macrophages overexpressing IFN-γ increased the levels of IFN-γ and TNF-α in SCID mice. A similar protective effect against M. avium infection using J774A.1 macrophages overexpressing IFN-γ was observed in IFN-γ knockout mice. These data suggest that administration of IFN-γ activated AMs or macrophages overexpressing IFN-γ may partially restore local alveolar host defense against infections like M. avium, even in the presence of ongoing systemic immunosuppression., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that forms a complex with glucose-regulated protein 78 (GRP78) to induce apoptosis. Previously, we reported that ER stress-induced apoptosis is a critical host defense mechanism against Mycobacterium tuberculosis (Mtb). We sought to understand the role of Par-4 during ER stress-induced apoptosis in response to mycobacterial infection. Par-4 and GRP78 protein levels increased in response Mtb (strain: H37Ra) infection. Furthermore, Par-4 and GRP78 translocate to the surface of Mtb H37Ra-infected macrophages and induce apoptosis via caspase activation. NF-κB activation, Mtb-mediated ER stress, and Par-4 production were significantly diminished in macrophages with inhibited ROS production. To test Par-4 function during mycobacterial infection, we analyzed intracellular survival of Mtb H37Ra in macrophages with Par-4 overexpression or knockdown. Mtb H37Ra growth was significantly reduced in Par-4 overexpressing macrophages and increased in knockdown macrophages. We also observed increased Par-4, GRP78, and caspases activation in Bacillus Calmette-Guérin (BCG)-infected prostate cancer cells. Our data demonstrate that Par-4 is associated with ER stress-induced apoptosis resulting in reduced intracellular survival of mycobacteria. BCG treatment increases Par-4-dependent caspase activation in prostate cancer cells. These results suggest ER stress-induced Par-4 acts as an important defense mechanism against mycobacterial infection and regulates cancer.

Foamy macrophages (FM)s harbor lipid bodies that not only assist mycobacterial persistence within the granulomas but also are sites for intracellular signaling and inflammatory mediators which are essential for mycobacterial pathogenesis. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs during mycobacterial infection are not clear. Here, we report for the first time that jumonji domain containing protein (JMJD)3, a demethylase of the repressive H3K27me3 mark, orchestrates the expression of M. tuberculosis H37Rv-, MDR-JAL2287-, H37Ra- and M. bovis BCG-induced genes essential for FM generation in a TLR2-dependent manner. Further, NOTCH1-responsive RNA-binding protein MUSASHI (MSI), targets a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein, to positively regulate infection-induced JMJD3 expression, FM generation and M2 phenotype. Investigations in in vivo murine models further substantiated these observations. Together, our study has attributed novel roles for JMJD3 and its regulators during mycobacterial infection that assist FM generation and fine-tune associated host immunity.

Mycobacterium tuberculosis (Mtb) is the most common co-infection in HIV patients and a serious co-epidemic. Apart from increasing the risk of reactivation of latent tuberculosis (TB), HIV infection also permits opportunistic infection of environmental non-pathogenic mycobacteria. To gain insights into mycobacterial survival inside host macrophages and identify mycobacterial proteins or processes that influence HIV propagation during co-infection, we employed proteomics approach to identify differentially expressed intracellular mycobacterial proteins during mono- and HIV co-infection of human THP-1 derived macrophage cell lines. Of the 92 proteins identified, 30 proteins were upregulated during mycobacterial mono-infection and 40 proteins during HIV-mycobacteria co-infection. We observed down-regulation of toxin-antitoxin (TA) modules, up-regulation of cation transporters, Type VII (Esx) secretion systems, proteins involved in cell wall lipid or protein metabolism, glyoxalate pathway and branched chain amino-acid synthesis during co-infection. The bearings of these mycobacterial factors or processes on HIV propagation during co-infection, as inferred from the proteomics data, were validated using deletion mutants of mycobacteria. The analyses revealed mycobacterial factors that possibly via modulating the host environment, increased viral titers during co-infection. The study provides new leads for investigations towards hitherto unknown molecular mechanisms explaining HIV-mycobacteria synergism, helping address diagnostics and treatment challenges for effective co-epidemic management.

Infection of humans with Mycobacterium tuberculosis (Mtb) results in diverse outcomes that range from acute disease to establishment of persistence and to even clearance of the pathogen. These different outcomes represent the combined result of host heterogeneity on the one hand, and virulence properties of the infecting strain of pathogen on the other. From the standpoint of the host, the balance between PGE2, LXA4 and LTB4 represents at least one of the factors that dictates the eventual pathophysiology. We therefore built an ODE model to describe the host-pathogen interaction and studied the local stability properties of the system, to obtain the parametric conditions that lead to different disease outcomes. We then modulated levels of the pro- and anti-inflammatory lipid mediators to better understand the convergence between host phenotype and factors that relate to virulence properties of the pathogen. Global sensitivity analysis, using the variance-based method of extended Fourier Amplitude Sensitivity Test (eFAST), revealed that disease severity was indeed defined by combined effects of phenotypic variability at the level of both host and pathogen. Interestingly here, [PGE2] was found to act as a switch between bacterial clearance and acute disease. Our mathematical model suggests that development of more effective treatments for tuberculosis will be contingent upon a better understanding of how the intrinsic variability at the level of both host and pathogen contribute to influence the nature of interactions between these two entities., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Human tuberculosis remains a huge global public health problem with an estimated 1/3rd of the population being infected. Defensins are antibacterial cationic peptides produced by a number of cell types, most notably neutrophil granulocytes and epithelial cells. All three defensin types ( α -, β -, and θ -defensins) have antibacterial activities, mainly through bacterial membrane permeabilization. Defensins are effective against Gram-negative and Gram-positive bacteria including mycobacteria and are active both intra- and extracellularly. Mycobacterial resistance has never been demonstrated although the mprF gene encoding resistance in Staphylococcus aureus is present in the Mycobacterium tuberculosis genome. In addition to their antibacterial effect, defensins are chemoattractants for macrophages and neutrophils. There are many cases for their use for therapy or prophylaxis in tuberculosis as well. In conclusion, we propose that there is considerable scope and potential for exploring their use as therapeutic/prophylactic agents and more comprehensive survey of defensins from different species and their bioactivity is timely., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Unlike conventional polymeric drug delivery systems, where drugs are entrapped in polymers, this study focuses on the incorporation of the drug into the polymer backbone to achieve higher loading and sustained release. Crosslinked, biodegradable, xylitol based polyesters have been synthesized in this study. The bioactive drug moiety, p-aminosalicylic acid (PAS), was incorporated in xylitol based polyesters to impart its anti-mycobacterial activity. To understand the influence of the monomer chemistry on the incorporation of PAS and its subsequent release from the polymer, different diacids have been used. Controlled release profiles of the drug from these polyesters were studied under normal physiological conditions. The degradation of the polyesters varied from 48% to 76% and the release of PAS ranged from 54% to 65% of its initial loading in 7days. A new model was developed to explain the release kinetics of PAS from the polymer that accounted for the polymer degradation and drug concentration. The thermal, mechanical, drug release and cytocompatibility properties of the polymers indicate their suitability in biomedical applications. The released products from these polymers were observed to be pharmacologically active against Mycobacteria. The high drug loading and sustained release also ensured enhanced efficacy. These polymers form biocompatible, biodegradable polyesters where the sustained release of PAS may be tailored for potential treatment of mycobacterial infections., Statement of Significance: In the present work, we report on novel polyesters with p-aminosalicylic acid (PAS) incorporated in the polymer backbone. The current work aims to achieve controlled release of PAS and ensures the delivered PAS is stable and pharmacologically active. The novelty of this work primarily involves the synthetic chemistry of polymerization and detailed analysis and efficacy of active PAS delivery. A new kinetic model has been developed to explain the PAS release profiles. These polymers are biodegradable, cytocompatible and anti-mycobacterial in nature., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Mycobacteria have a complex cell wall with a high lipid content that confers unique advantages for bacterial survival in the hostile host environment, leading to long-term infection. There is a wealth of evidence suggesting the role cell wall-associated lipid antigens play at the host-pathogen interface by contributing to bacterial virulence. One pathway that pathogenic mycobacteria use to subvert host immune pathways to their advantage is host cholesterol/lipid homeostasis. This review focuses on the possible role of pathogen- and host-associated lipids in the survival and persistence of pathogenic mycobacteria with emphasis on Mycobacterium avium subsp. paratuberculosis. We draw upon literature in diverse areas of infectious and metabolic diseases and explain a mechanism by which mycobacterial-induced changes in the host cellular energy state could account for phenomena that are a hallmark of chronic mycobacterial diseases.