Genetic and hormonal mechanisms underlying sex-specific immune responses in tuberculosis.

Tuberculosis (TB), the world's deadliest bacterial infection, afflicts more human males than females, with a male/female (M/F) ratio of 1.7. Sex disparities in TB prevalence, pathophysiology, and clinical manifestations are widely reported, but the underlying biological mechanisms remain largely undefined. This review assesses epidemiological data on sex disparity in TB, as well as possible underlying hormonal and genetic mechanisms that might differentially modulate innate and adaptive immune responses in males and females, leading to sex differences in disease susceptibility. We consider whether this sex disparity can be extended to the efficacy of vaccines and discuss novel animal models which may offer mechanistic insights. A better understanding of the biological factors underpinning sex-related immune responses in TB may enable sex-specific personalized therapies for TB.

Sex and Gender Differences in Tuberculosis Pathogenesis and Treatment Outcomes.

Tuberculosis remains a daunting public health concern in many countries of the world. A consistent observation in the global epidemiology of tuberculosis is an excess of cases of active pulmonary tuberculosis among males compared with females. Data from both humans and animals also suggest that males are more susceptible than females to develop active pulmonary disease. Similarly, male sex has been associated with poor treatment outcomes. Despite this growing body of evidence, little is known about the mechanisms driving sex bias in tuberculosis disease. Two dominant hypotheses have been proposed to explain the predominance of active pulmonary tuberculosis among males. The first is based on the contribution of biological factors, such as sex hormones and genetic factors, on host immunity during tuberculosis. The second is focused on non-biological factors such as smoking, professional exposure, and health-seeking behaviors, known to be influenced by gender. In this chapter, we review the literature regarding these two prevailing hypotheses by presenting human but also experimental animal studies. In addition, we presented studies aiming at examining the impact of sex and gender on other clinical forms of tuberculosis such as latent tuberculosis infection and extrapulmonary tuberculosis, which both appear to have their own specificities in relation to sex. We also highlighted potential intersections between sex and gender in the context of tuberculosis and shared future directions that could guide in elucidating mechanisms of sex-based differences in tuberculosis pathogenesis and treatment outcomes.

CRISPR Correction of the GBA Mutation in Human-Induced Pluripotent Stem Cells Restores Normal Function to Gaucher Macrophages and Increases Their Susceptibility to Mycobacterium tuberculosis.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the ß-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.

Therapeutic potential of coumestan Pks13 inhibitors for tuberculosis.

Polyketide synthase 13 (Pks13) is an important enzyme found in Mycobacterium tuberculosis (M. tuberculosis) that condenses two fatty acyl chains to produce α-alkyl ß-ketoesters, which in turn serve as the precursors for the synthesis of mycolic acids that are essential building blocks for maintaining the cell wall integrity of M. tuberculosis Coumestan derivatives have recently been identified in our group as a new chemotype that exert their antitubercular effects via targeting of Pks13. These compounds were active on both drug-susceptible and drug-resistant strains of M. tuberculosis as well as showing low cytotoxicity to healthy cells and a promising selectivity profile. No cross-resistance was found between the coumestan derivatives and first-line TB drugs. Here we report that treatment of M. tuberculosis bacilli with 15 times the MIC of compound 1, an optimized lead coumestan compound, resulted in a colony forming unit (CFU) reduction from 6.0 log10 units to below the limit of detection (1.0 log10 units) per mL culture, demonstrating a bactericidal mechanism of action. Single dose (10 mg/kg) pharmacokinetic studies revealed favorable parameters with a relative bioavailability of 19.4%. In a mouse infection and chemotherapy model, treatment with 1 showed dose-dependent mono-therapeutic activity, whereas treatment with 1 in combination with rifampin showed clear synergistic effects. Together these data suggest that coumestan derivatives are promising agents for further TB drug development.

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma.

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.

Design, Synthesis and Biological Evaluation of N-phenylindole Derivatives as Pks13 Inhibitors againstMycobacterium tuberculosis.

Polyketide synthase 13 (Pks13), an essential enzyme for the survival of Mycobacterium tuberculosis (Mtb), is an attractive target for new anti-TB agents. In our previous work, we have identified 2-phenylindole derivatives against Mtb. The crystallography studies demonstrated that the two-position phenol was solvent-exposed in the Pks13-TE crystal structure and a crucial hydrogen bond was lost while introducing bulkier hydrophobic groups at indole N moieties. Thirty-six N-phenylindole derivatives were synthesized and evaluated for antitubercular activity using a structure-guided approach. The structure-activity relationship (SAR) studies resulted in the discovery of the potent Compounds 45 and 58 against Mtb H37Rv, with an MIC value of 0.0625 µg/mL and 0.125 µg/mL, respectively. The thermal stability analysis showed that they bind with high affinity to the Pks13-TE domain. Preliminary ADME evaluation showed that Compound 58 displayed modest human microsomal stability. This report further validates that targeting Pks13 is a valid strategy for the inhibition of Mtb and provides a novel scaffold for developing leading anti-TB compounds.

Effective Host-Directed Therapy for Tuberculosis by Depletion of Myeloid-Derived Suppressor Cells and Related Cells Using a Diphtheria Toxin Fusion Protein.

Myeloid-derived suppressor cells (MDSCs) are present in elevated numbers in tuberculosis patients and have been found to be permissive for Mycobacterium tuberculosis proliferation. To determine whether depletion of MDSCs may improve host control of tuberculosis, we used a novel diphtheria toxin-based fusion protein DABIL-4 that targets and depletes interleukin 4 (IL-4) receptor-positive cells. We show that DABIL-4 depletes both polymorphonuclear MDSCs and monocytic MDSCs, increases interferon-γ + T cells, and reduces the lung bacillary burden in a mouse tuberculosis model. These results indicate that MDSC-depleting therapies targeting the IL-4 receptor are beneficial in tuberculosis and offer an avenue towards host-directed tuberculosis therapy.

Design, synthesis and antimycobacterial evaluation of novel adamantane and adamantanol analogues effective against drug-resistant tuberculosis.

The treacherous nature of tuberculosis (TB) combined with the ubiquitous presence of the drug-resistant (DR) forms pose this disease as a growing public health menace. Therefore, it is imperative to develop new chemotherapeutic agents with a novel mechanism of action to circumvent the cross-resistance problems. The unique architecture of the Mycobacterium tuberculosis (M. tb) outer envelope plays a predominant role in its pathogenesis, contributing to its intrinsic resistance against available therapeutic agents. The mycobacterial membrane protein large 3 (MmpL3), which is a key player in forging the M. tb rigid cell wall, represents an emerging target for TB drug development. Several indole-2-carboxamides were previously identified in our group as potent anti-TB agents that act as inhibitor of MmpL3 transporter protein. Despite their highly potent in vitro activities, the lingering Achilles heel of these indoleamides can be ascribed to their high lipophilicity as well as low water solubility. In this study, we report our attempt to improve the aqueous solubility of these indole-2-carboxamides while maintaining an adequate lipophilicity to allow effective M. tb cell wall penetration. A more polar adamantanol moiety was incorporated into the framework of several indole-2-carboxamides, whereupon the corresponding analogues were tested for their anti-TB activity against drug-sensitive (DS) M. tb H37Rv strain. Three adamantanol derivatives 8i, 8j and 8l showed nearly 2- and 4-fold higher activity (MIC = 1.32 - 2.89 µM) than ethambutol (MIC = 4.89 µM). Remarkably, the most potent adamantanol analogue 8j demonstrated high selectivity towards DS and DR M. tb strains over mammalian cells [IC50 (Vero cells) ≥ 169 µM], evincing its lack of cytotoxicity. The top eight active compounds 8b, 8d, 8f, 8i, 8j, 8k, 8l and 10a retained their in vitro potency against DR M. tb strains and were docked into the MmpL3 active site. The most potent adamantanol/adamantane-based indoleamides 8j/8k displayed a two-fold surge in potency against extensively DR (XDR) M. tb strains with MIC values of 0.66 and 0.012 µM, respectively. The adamantanol-containing indole-2-carboxamides exhibited improved water solubility both in silico and experimentally, relative to the adamantane counterparts. Overall, the observed antimycobacterial and physicochemical profiles support the notion that adamantanol moiety is a suitable replacement to the adamantane scaffold within the series of indole-2-carboxamide-based MmpL3 inhibitors.

Relative and Quantitative Phosphoproteome Analysis of Macrophages in Response to Infection by Virulent and Avirulent Mycobacteria Reveals a Distinct Role of the Cytosolic RNA Sensor RIG-I in Mycobacterium tuberculosis Pathogenesis.

Comparative phosphoproteomics of Mycobacterium tuberculosis (Mtb)- and Mycobacterium bovis BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylation status, herein, we compared the phosphorylation profile of macrophages upon exposure to Mtb and BCG. We observed a significant dephosphorylation of proteins following Mtb infection relative to those with uninfected or BCG-infected cells. A comprehensive tandem mass tag mass spectrometry (MS) approach detected ∼10% phosphosites on a variety of host proteins that are modulated in response to infection. Interestingly, the innate immune-enhancing interferon (IFN)-stimulated genes were identified as a class of proteins differentially phosphorylated during infection, including the cytosolic RNA sensor RIG-I, which has been implicated in the immune response to bacterial infection. We show that Mtb infection results in the activation of RIG-I in primary human macrophages. Studies using RIG-I knockout macrophages reveal that the Mtb-mediated activation of RIG-I promotes IFN-ß, IL-1α, and IL-1ß levels, dampens autophagy, and facilitates intracellular Mtb survival. To our knowledge, this is the first study providing exhaustive information on relative and quantitative changes in the global phosphoproteome profile of host macrophages that can be further explored in designing novel anti-TB drug targets. The peptide identification and MS/MS spectra have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013171.

Matrix Metalloproteinase Inhibition in a Murine Model of Cavitary Tuberculosis Paradoxically Worsens Pathology.

Matrix metalloproteinases (MMPs) degrade extracellular matrix and are implicated in tuberculosis pathogenesis and cavitation. In particular, MMP-7 is induced by hypoxia and highly expressed around pulmonary cavities of Mycobacterium tuberculosis-infected C3HeB/FeJ mice. In this study, we evaluated whether administration of cipemastat, an orally available potent inhibitor of MMP-7, could reduce pulmonary cavitation in M. tuberculosis-infected C3HeB/FeJ mice. We demonstrate that, compared with untreated controls, cipemastat treatment paradoxically increases the frequency of cavitation (32% vs 7%; P = .029), immunopathology, and mortality. Further studies are needed to understand the role of MMP inhibitors as adjunctive treatments for pulmonary tuberculosis.

Revisiting the ß-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach.

The suboptimal effectiveness of ß-lactam antibiotics against Mycobacterium tuberculosis has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a ß-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R. Sergysels, et al., Int J Tuberc Lung Dis 16:558-560, 2012, https://doi.org/10.5588/ijtld.11.0414). We hypothesized that the innate resistance of M. tuberculosis to ß-lactams is mediated in part by noncanonical accessory proteins that are not considered the classic targets of ß-lactams and that small-molecule inhibitors of those accessory targets might sensitize M. tuberculosis to ß-lactams. In this study, we screened an NIH small-molecule library for the ability to sensitize M. tuberculosis to meropenem. We identified six hit compounds, belonging to either the N-arylindole or benzothiophene chemotype. Verification studies confirmed the synthetic lethality phenotype for three of the N-arylindoles and one benzothiophene derivative. The latter was demonstrated to be partially bioavailable via oral administration in mice. Structure-activity relationship studies of both structural classes identified analogs with potent antitubercular activity, alone or in combination with meropenem. Transcriptional profiling revealed that oxidoreductases, MmpL family proteins, and a 27-kDa benzoquinone methyltransferase could be the targets of the N-arylindole potentiator. In conclusion, our compound-compound synthetic lethality screening revealed novel small molecules that were capable of potentiating the action of meropenem, presumably via inhibition of the innate resistance conferred by ß-lactam accessory proteins. ß-Lactam compound-compound synthetic lethality may be an alternative approach for drug-resistant tuberculosis.

Advancing the Therapeutic Potential of Indoleamides for Tuberculosis.

Indole-2-carboxamide derivatives are inhibitors of MmpL3, the cell wall-associated mycolic acid transporter of Mycobacterium tuberculosis In the present study, we characterized indoleamide effects on bacterial cell morphology and reevaluated pharmacokinetics and in vivo efficacy using an optimized oral formulation. Morphologically, indoleamide-treated M. tuberculosis cells demonstrated significantly higher numbers of dimples near the poles or septum, which may serve as the mechanism of cell death for this bactericidal scaffold. Using the optimized formulation, an expanded-spectrum indoleamide, compound 2, showed significantly improved pharmacokinetic (PK) parameters and in vivo efficacy in mouse infection models. In a comparative study, compound 2 showed superior efficacy over compound 3 (NITD-304) in a high-dose aerosol mouse infection model. Since indoleamides are equally active on drug-resistant M. tuberculosis, these findings demonstrate the therapeutic potential of this novel scaffold for the treatment of both drug-susceptible and drug-resistant tuberculosis.

Diverse Cavity Types and Evidence that Mechanical Action on the Necrotic Granuloma Drives Tuberculous Cavitation.

Effacement of normal lung parenchyma by cavities is an important sequela of pulmonary tuberculosis. Despite its clinical significance, the pathogenesis of tuberculous cavitation is poorly understood, with controversy as to whether the fundamental mechanism involves matrix depletion, lipid pneumonia, or mechanical factors. In this study, a repetitive aerosol infection model using Mycobacterium tuberculosis was used to generate cavities in 20 New Zealand white rabbits. Serial computed tomography was performed to monitor cavity progression over 14 weeks. Three-dimensional reconstructions were compiled for each time point, allowing comprehensive four-dimensional cavity mapping. Terminally, cavities were processed for histopathology. Cavities progressed rapidly from areas of consolidation, and often showed a pattern of explosive growth followed by gradual contraction. Cavities formed preferentially in the caudodorsal lung fields, and frequently were subpleural. Cavitation was associated invariably with necrosis. Histomorphology showed four distinct cavity types that provide mechanistic clues and insight on early cavity development. Our study shows that cavitation is a highly dynamic process with preferential formation at sites of high mechanical stress. These findings suggest a model for the pathogenesis of tuberculous cavitation in which mechanical stress acts on the necrotic granuloma to produce acute tears in structurally weakened tissue, with subsequent air trapping and cavity expansion.

Inhibition of innate immune cytosolic surveillance by an M. tuberculosis phosphodiesterase.

Mycobacterium tuberculosis infection leads to cytosolic release of the bacterial cyclic dinucleotide (CDN) c-di-AMP and a host-generated CDN, cGAMP, both of which trigger type I interferon (IFN) expression in a STING-dependent manner. Here we report that M. tuberculosis has developed a mechanism to inhibit STING activation and the type I IFN response via the bacterial phosphodiesterase (PDE) CdnP, which mediates hydrolysis of both bacterial-derived c-di-AMP and host-derived cGAMP. Mutation of cdnP attenuates M. tuberculosis virulence, as does loss of a host CDN PDE known as ENPP1. CdnP is inhibited by both US Food and Drug Administration (FDA)-approved PDE inhibitors and nonhydrolyzable dinucleotide mimetics specifically designed to target the enzyme. These findings reveal a crucial role of CDN homeostasis in governing the outcome of M. tuberculosis infection as well as a unique mechanism of subversion of the host's cytosolic surveillance pathway (CSP) by a bacterial PDE that may serve as an attractive antimicrobial target.

Repetitive Aerosol Exposure Promotes Cavitary Tuberculosis and Enables Screening for Targeted Inhibitors of Extensive Lung Destruction.

Background: Cavitation is a serious consequence of tuberculosis. We tested the hypothesis that repetitive exposure to the same total bacterial burden of Mycobacterium tuberculosis drives greater lung destruction than a single exposure. We also tested whether inhibition of endogenous matrix metalloproteinase-1 (MMP-1) may inhibit cavitation during tuberculosis. Methods: Over a 3-week interval, we infected rabbits with either 5 aerosols of 500 colony-forming units (CFU) of M. tuberculosis or a single aerosol of 2500 CFU plus 4 sham aerosols. We administered the MMP-1 inhibitor cipemastat (100 mg/kg daily) during weeks 5-10 to a subset of the animals. Results: Repetitive aerosol infection produced greater lung inflammation and more cavities than a single aerosol infection of the same bacterial burden (75% of animals vs 25%). Necropsies confirmed greater lung pathology in repetitively exposed animals. For cipemastat-treated animals, there was no significant difference in cavity counts, cavity volume, or disease severity compared to controls. Conclusions: Our data show that repetitive aerosol exposure with M. tuberculosis drives greater lung damage and cavitation than a single exposure. This suggests that human lung destruction due to tuberculosis may be exacerbated in settings where individuals are repeatedly exposed. MMP-1 inhibition with cipemastat did not prevent the development of cavitation in our model.

Bacterial subversion of cAMP signalling inhibits cathelicidin expression, which is required for innate resistance to Mycobacterium tuberculosis.

Antimicrobial peptides such as cathelicidins are important components of innate immune defence against inhaled microorganisms, and have shown antimicrobial activity against Mycobacterium tuberculosis in in vitro models. Despite this, little is known about the regulation and expression of cathelicidin during tuberculosis in vivo. We sought to determine whether the cathelicidin-related antimicrobial peptide gene (Cramp), the murine functional homologue of the human cathelicidin gene (CAMP or LL-37), is required for regulation of protective immunity during M. tuberculosis infection in vivo. We used Cramp-/- mice in a validated model of pulmonary tuberculosis, and conducted cell-based assays with macrophages from these mice. We evaluated the in vivo susceptibility of Cramp-/- mice to infection, and also dissected various pro-inflammatory immune responses against M. tuberculosis. We observed increased susceptibility of Cramp-/- mice to M. tuberculosis as compared with wild-type mice. Macrophages from Cramp-/- mice were unable to control M. tuberculosis growth in an in vitro infection model, were deficient in intracellular calcium influx, and were defective in stimulating T cells. Additionally, CD4+ and CD8+ T cells from Cramp-/- mice produced less interferon-ß upon stimulation. Furthermore, bacterial-derived cAMP modulated cathelicidin expression in macrophages. Our results demonstrate that cathelicidin is required for innate resistance to M. tuberculosis in a relevant animal model and is a key mediator in regulation of the levels of pro-inflammatory cytokines by calcium and cyclic nucleotides. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

"Depupylation" of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates.

Ubiquitin (Ub) provides the recognition and specificity required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic ubiquitin-like protein (Pup) is functionally analogous to Ub in Mycobacterium tuberculosis (Mtb), as it dooms proteins to the Mtb proteasome. Studies suggest that Pup and Ub do not share similar mechanisms of activation and conjugation to target proteins. Dop (deamidase of Pup; Mtb Rv2112c/MT2172) deamidates the C-terminal glutamine of Pup to glutamate, preparing it for ligation to target proteins by proteasome accessory factor A (PafA). While studies have shed light on the conjugation of Pup to proteins, it was not known if Pup could be removed from substrates in a manner analogous to the deconjugation of Ub from eukaryotic proteins. Here, we show that Mycobacteria have a "depupylase" activity provided by Dop. The discovery of a depupylase strengthens the parallels between the Pup- and Ub-tagging systems of prokaryotes and eukaryotes, respectively.

Crosstalk between Mycobacterium tuberculosis and the host cell.

The successful establishment and maintenance of a bacterial infection depend on the pathogen's ability to subvert the host cell's defense response and successfully survive, proliferate, or persist within the infected cell. To circumvent host defense systems, bacterial pathogens produce a variety of virulence factors that potentiate bacterial adherence and invasion and usurp host cell signaling cascades that regulate intracellular microbial survival and trafficking. Mycobacterium tuberculosis, probably one of the most successful pathogens on earth, has coexisted with humanity for centuries, and this intimate and persistent connection between these two organisms suggests that the pathogen has evolved extensive mechanisms to evade the human immune system at multiple levels. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are hijacked to prevent the generation of an effective immune response thus benefiting the survival of M. tuberculosis within the host cell. Here, we describe several of these mechanisms, with an emphasis on the cyclic nucleotide signaling and subversion of host responses that occur at the intracellular level when tubercle bacilli encounter macrophages, a cell that becomes a safe-house for M. tuberculosis although it is specialized to kill most microbes.

Suppressor Cell-Depleting Immunotherapy With Denileukin Diftitox is an Effective Host-Directed Therapy for Tuberculosis.

Host-directed therapies that augment host immune effector mechanisms may serve as important adjunctive therapies for tuberculosis treatment. We evaluated the activity of denileukin diftitox in an acute mouse model of tuberculosis (TB) infection and analyzed the cellular composition and bacterial burden in lungs and spleens. These in vivo studies show that denileukin diftitox potentiates standard TB treatment in the mouse model, an effect which may be due to depletion of T-regulatory and myeloid-derived suppressor cells during TB infection. Our results indicate that denileukin diftitox and other suppressor cell-depleting therapies may be useful adjunctive, host-directed therapies for TB.