IL-27 inhibits anti- Mycobacterium tuberculosis innate immune activity of primary human macrophages.

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen that primarily infects macrophages. Despite a robust anti-mycobacterial response, many times macrophages are unable to control M. tuberculosis. The purpose of this study was to investigate the mechanism by which the immunoregulatory cytokine IL-27 inhibits the anti-mycobacterial activity of primary human macrophages. We found concerted production of IL-27 and anti-mycobacterial cytokines by M. tuberculosis-infected macrophages in a toll-like receptor (TLR) dependent manner. Notably, IL-27 suppressed the production of anti-mycobacterial cytokines TNFα, IL-6, IL-1ß, and IL-15 by M. tuberculosis-infected macrophages. IL-27 limits the anti-mycobacterial activity of macrophages by reducing Cyp27B, cathelicidin (LL-37), LC3B lipidation, and increasing IL-10 production. Furthermore, neutralizing both IL-27 and IL-10 increased the expression of proteins involved in LC3-associated phagocytosis (LAP) pathway for bacterial clearance, namely vacuolar-ATPase, NOX2, and RUN-domain containing protein RUBCN. These results implicate IL-27 is a prominent cytokine that impedes M. tuberculosis clearance.

Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions.

For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelope integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of infections in mouse bone marrow-derived macrophages. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1ß and TNF-α. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants.

An intelligent peptide recognizes and traps Mycobacterium tuberculosis to inhibit macrophage phagocytosis.

Tuberculosis is a major public health concern worldwide, and it is a serious threat to human health for a long period. Macrophage phagocytosis of Mycobacterium tuberculosis (M. tuberculosis) is a crucial process for granuloma formation, which shelters the bacteria and gives them an opportunity for re-activation and spread. Herein, we report an intelligent anti-microbial peptide that can recognize and trap the M. tuberculosis, inhibiting the macrophage phagocytosis process. The peptide (Bis-Pyrene-KLVFF-WHSGTPH, in abbreviation as BFH) first self-assembles into nanoparticles, and then forms nanofibers upon recognizing and binding M. tuberculosis. Subsequently, BFH traps M. tuberculosis by the in situ formed nanofibrous networks and the trapped M. tuberculosis are unable to invade host cells (macrophages). The intelligent anti-microbial peptide can significantly inhibit the phagocytosis of M. tuberculosis by macrophages, thereby providing a favorable theoretical basis for inhibiting the formation of tuberculosis granulomas.

Mycobacterial acyl carrier protein suppresses TFEB activation and upregulates miR-155 to inhibit host defense.

Mycobacterial acyl carrier protein (AcpM; Rv2244), a key protein involved in Mycobacterium tuberculosis (Mtb) mycolic acid production, has been shown to suppress host cell death during mycobacterial infection. This study reports that mycobacterial AcpM works as an effector to subvert host defense and promote bacterial growth by increasing microRNA (miRNA)-155-5p expression. In murine bone marrow-derived macrophages (BMDMs), AcpM protein prevented transcription factor EB (TFEB) from translocating to the nucleus in BMDMs, which likely inhibited transcriptional activation of several autophagy and lysosomal genes. Although AcpM did not suppress autophagic flux in BMDMs, AcpM reduced Mtb and LAMP1 co-localization indicating that AcpM inhibits phagolysosomal fusion during Mtb infection. Mechanistically, AcpM boosted the Akt-mTOR pathway in BMDMs by upregulating miRNA-155-5p, a SHIP1-targeting miRNA. When miRNA-155-5p expression was inhibited in BMDMs, AcpM-induced increased intracellular survival of Mtb was suppressed. In addition, AcpM overexpression significantly reduced mycobacterial clearance in C3HeB/FeJ mice infected with recombinant M. smegmatis strains. Collectively, our findings point to AcpM as a novel mycobacterial effector to regulate antimicrobial host defense and a potential new therapeutic target for Mtb infection.

High Glucose and Carbonyl Stress Impair HIF-1-Regulated Responses and the Control of Mycobacterium tuberculosis in Macrophages.

Diabetes mellitus (DM) increases the risk of developing tuberculosis (TB), but the mechanisms behind diabetes-TB comorbidity are still undefined. Here, we studied the role of hypoxia-inducible factor-1 (HIF-1), a main regulator of metabolic and inflammatory responses, in the outcome of Mycobacterium tuberculosis infection of bone marrow-derived macrophages (BMM). We observed that M. tuberculosis infection of BMM increased the expression of HIF-1α and HIF-1-regulated genes. Treatment with the hypoxia mimetic deferoxamine (DFO) further increased levels of HIF-1-regulated immune and metabolic molecules and diminished the intracellular bacterial load in BMM and in the lungs of infected mice. The expression of HIF-1-regulated immunometabolic genes was reduced, and the intracellular M. tuberculosis levels were increased in BMM incubated with high-glucose levels or with methylglyoxal (MGO), a reactive carbonyl compound elevated in DM. In line with the in vitro findings, high M. tuberculosis levels and low HIF-1-regulated transcript levels were found in the lungs from hyperglycemic Leprdb/db compared with wild-type mice. The increased intracellular M. tuberculosis growth and the reduced expression of HIF-1-regulated metabolic and inflammatory genes in BMM incubated with MGO or high glucose were reverted by additional treatment with DFO. Hif1a-deficient BMM showed ablated responses of immunometabolic transcripts after mycobacterial infection at normal or high-glucose levels. We propose that HIF-1 may be targeted for the control of M. tuberculosis during DM. IMPORTANCE People living with diabetes who are also infected with M. tuberculosis are more likely to develop tuberculosis disease (TB). Why diabetic patients have an increased risk for developing TB is not well understood. Macrophages, the cell niche for M. tuberculosis, can express microbicidal mechanisms or be permissive to mycobacterial persistence and growth. Here, we showed that high glucose and carbonyl stress, which mediate diabetes pathogenesis, impair the control of intracellular M. tuberculosis in macrophages. Infection with M. tuberculosis stimulated the expression of genes regulated by the transcription factor HIF-1, a major controller of the responses to hypoxia, resulting in macrophage activation. High glucose and carbonyl compounds inhibited HIF-1 responses by macrophages. Mycobacterial control in the presence of glucose or carbonyl stress was restored by DFO, a compound that stabilizes HIF-1. We propose that HIF-1 can be targeted to reduce the risk of developing TB in people with diabetes.

Manipulation and exploitation of host immune system by pathogenic Mycobacterium tuberculosis for its advantage.

Mycobacterium tuberculosis (Mtb) can become a long-term infection by evading the host immune response. Coevolution of Mtb with humans has resulted in its ability to hijack the host's immune systems in a variety of ways. So far, every Mtb defense strategy is essentially dependent on a subtle balance that, if shifted, can promote Mtb proliferation in the host, resulting in disease progression. In this review, the authors summarize many important and previously unknown mechanisms by which Mtb evades the host immune response. Besides recently found strategies by which Mtb manipulates the host molecular regulatory machinery of innate and adaptive immunity, including the intranuclear regulatory machinery, costimulatory molecules, the ubiquitin system and cellular intrinsic immune components will be discussed. A holistic understanding of these immune-evasion mechanisms is of foremost importance for the prevention, diagnosis and treatment of tuberculosis and will lead to new insights into tuberculosis pathogenesis and the development of more effective vaccines and treatment regimens.

EST12 regulates Myc expression and enhances anti-mycobacterial inflammatory response via RACK1-JNK-AP1-Myc immune pathway.

c-Myc (Myc) is a well-known transcription factor that regulates many essential cellular processes. Myc has been implicated in regulating anti-mycobacterial responses. However, its precise mechanism in modulating mycobacterial immunity remains elusive. Here, we found that a secreted Rv1579c (early secreted target with molecular weight 12 kDa, named EST12) protein, encoded by virulent Mycobacterium tuberculosis (M.tb) H37Rv region of deletion (RD)3, induces early expression and late degradation of Myc protein. Interestingly, EST12-induced Myc was further processed by K48 ubiquitin proteasome degradation in E3 ubiquitin ligase FBW7 dependent manner. EST12 protein activates JNK-AP1-Myc signaling pathway, promotes Myc binding to the promoters of IL-6, TNF-α and iNOS, then induces the expression of pro-inflammatory cytokines (IL-6 and TNF-α)/inducible nitric oxide synthase (iNOS)/nitric oxide (NO) to increase mycobacterial clearance in a RACK1 dependent manner, and these effects are impaired by both Myc and JNK inhibitors. Macrophages infected with EST12-deficiency strain (H37RvΔEST12) displayed less production of iNOS, IL-6 and TNF-α. In conclusion, EST12 regulates Myc expression and enhances anti-mycobacterial inflammatory response via RACK1-JNK-AP1-Myc immune pathway. Our finding provides new insights into M.tb-induced immunity through Myc.

Commonalities of Mycobacterium tuberculosis Transcriptomes in Response to Defined Persisting Macrophage Stresses.

As the goal of a bacterium is to become bacteria, evolution has imposed continued selections for gene expression. The intracellular pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, has adopted a fine-tuned response to survive its host's methods to aggressively eradicate invaders. The development of microarrays and later RNA sequencing has led to a better understanding of biological processes controlling the relationship between host and pathogens. In this study, RNA-seq was performed to detail the transcriptomes of M. tuberculosis grown in various conditions related to stresses endured by M. tuberculosis during host infection and to delineate a general stress response incurring during persisting macrophage stresses. M. tuberculosis was subjected to long-term growth, nutrient starvation, hypoxic and acidic environments. The commonalities between these stresses point to M. tuberculosis maneuvering to exploit propionate metabolism for lipid synthesis or to withstand propionate toxicity whilst in the intracellular environment. While nearly all stresses led to a general shutdown of most biological processes, up-regulation of pathways involved in the synthesis of amino acids, cofactors, and lipids were observed only in hypoxic M. tuberculosis. This data reveals genes and gene cohorts that are specifically or exclusively induced during all of these persisting stresses. Such knowledge could be used to design novel drug targets or to define possible M. tuberculosis vulnerabilities for vaccine development. Furthermore, the disruption of specific functions from this gene set will enhance our understanding of the evolutionary forces that have caused the tubercle bacillus to be a highly successful pathogen.

Rapid GPR183-mediated recruitment of eosinophils to the lung after Mycobacterium tuberculosis infection.

Influx of eosinophils into the lungs is typically associated with type II responses during allergy and fungal and parasitic infections. However, we previously reported that eosinophils accumulate in lung lesions during type I inflammatory responses to Mycobacterium tuberculosis (Mtb) in humans, macaques, and mice, in which they support host resistance. Here we show eosinophils migrate into the lungs of macaques and mice as early as one week after Mtb exposure. In mice this influx is CCR3 independent and instead requires cell-intrinsic expression of the oxysterol receptor GPR183, which is highly expressed on human and macaque eosinophils. Murine eosinophils interact directly with bacilli-laden alveolar macrophages, which upregulate the oxysterol-synthesizing enzyme Ch25h, and eosinophil recruitment is impaired in Ch25h-deficient mice. Our findings show that eosinophils are among the earliest cells from circulation to sense and respond to Mtb infection of alveolar macrophages and reveal a role for GPR183 in the migration of eosinophils into lung tissue.

Mycobacterium tuberculosis-Induced Upregulation of the COX-2/mPGES-1 Pathway in Human Macrophages Is Abrogated by Sulfasalazine.

Macrophages are the primary human host cells of intracellular Mycobacterium tuberculosis (M.tb) infection, where the magnitude of inflammatory reactions is crucial for determining the outcome of infection. Previously, we showed that the anti-inflammatory drug sulfasalazine (SASP) significantly reduced the M.tb bactericidal burden and histopathological inflammation in mice. Here, we asked which genes in human inflammatory macrophages are affected upon infection with M.tb and how would potential changes impact the functional state of macrophages. We used a flow cytometry sorting system which can distinguish the dead and alive states of M.tb harbored in human monocyte-derived macrophages (MDM). We found that the expression of cyclooxygenase-2 and microsomal prostaglandin E2 synthase (mPGES)-1 increased significantly in tagRFP+ MDM which were infected with alive M.tb. After exposure of polarized M1-MDM to M.tb (H37Rv strain)-conditioned medium (MTB-CM) or to the M.tb-derived 19-kD antigen, the production of PGE2 and pro-inflammatory cytokines increased 3- to 4-fold. Upon treatment of M1-MDM with SASP, the MTB-CM-induced expression of COX-2 and the release of COX products and cytokines decreased. Elevation of PGE2 in M1-MDM upon MTB-CM stimulation and modulation by SASP correlated with the activation of the NF-κB pathway. Together, infection of human macrophages by M.tb strongly induces COX-2 and mPGES-1 expression along with massive PGE2 formation which is abrogated by the anti-inflammatory drug SASP.

A Blunted GPR183/Oxysterol Axis During Dysglycemia Results in Delayed Recruitment of Macrophages to the Lung During Mycobacterium tuberculosis Infection.

BACKGROUND: We previously reported that reduced GPR183 expression in blood from tuberculosis (TB) patients with diabetes is associated with more severe TB. METHODS: To further elucidate the role of GPR183 and its oxysterol ligands in the lung, we studied dysglycemic mice infected with Mycobacterium tuberculosis (Mtb). RESULTS: We found upregulation of the oxysterol-producing enzymes CH25H and CYP7B1 and increased concentrations of 25-hydroxycholesterol upon Mtb infection in the lungs of mice. This was associated with increased expression of GPR183 indicative of oxysterol-mediated recruitment of GPR183-expressing immune cells to the lung. CYP7B1 was predominantly expressed by macrophages in TB granulomas. CYP7B1 expression was significantly blunted in lungs from dysglycemic animals, which coincided with delayed macrophage infiltration. GPR183-deficient mice similarly had reduced macrophage recruitment during early infection. CONCLUSIONS: Taken together, we demonstrate a requirement of the GPR183/oxysterol axis for positioning of macrophages to the site of infection and add an explanation to more severe TB in diabetes patients.

CD84 is a Suppressor of T and B Cell Activation during Mycobacterium tuberculosis Pathogenesis.

Interest in host-directed therapies as alternatives/adjuncts to antibiotic treatment has resurged with the increasing prevalence of antibiotic-resistant tuberculosis (TB). Immunotherapies that reinvigorate immune responses by targeting immune checkpoints like PD-1/PD-L1 have proved successful in cancer therapy. Immune cell inhibitory receptors that trigger Mycobacterium tuberculosis-specific immunosuppression, however, are unknown. Here, we show that the levels of CD84, a SLAM family receptor, increase in T and B cells in lung tissues from M. tuberculosis-infected C57BL/6 mice and in peripheral blood mononuclear cells (PBMCs) from pulmonary TB patients. M. tuberculosis challenge experiments using CD84-deficient C57BL/6 mice suggest that CD84 expression likely leads to T and B cell immunosuppression during M. tuberculosis pathogenesis and also plays an inhibitory role in B cell activation. Importantly, CD84-deficient mice showed improved M. tuberculosis clearance and longer survival than M. tuberculosis-infected wild-type (WT) mice. That CD84 is a putative M. tuberculosis infection-specific inhibitory receptor suggests it may be a suitable target for the development of TB-specific checkpoint immunotherapies. IMPORTANCE Immune checkpoint therapies, such as targeting checkpoints like PD-1/PD-L1, have proved successful in cancer therapy and can reinvigorate immune responses. The potential of this approach for treating chronic infectious diseases like TB has been recognized, but a lack of suitable immunotherapeutic targets, i.e., immune cell inhibitory receptors that trigger immunosuppression specifically during Mycobacterium tuberculosis pathogenesis, has limited the application of this strategy in the development of new TB therapies. Our focus in this study was to address this gap and search for an M. tuberculosis-specific checkpoint target. Our results suggest that CD84 is a putative inhibitory receptor that may be a suitable target for the development of TB-specific checkpoint immunotherapies.

Sudapyridine (WX-081), a Novel Compound against Mycobacterium tuberculosis.

Bedaquiline (BDQ) was historically listed by the World Health Organization (WHO) in 2018 as the preferred option for rifampin-resistant tuberculosis (RR-TB) and multidrug-resistant tuberculosis (MDR-TB). However, when there is no other effective regimen, the side effects and weaknesses of BDQ limit its use of MDR-TB. There is a black box warning in the package insert of BDQ to warn patients and health care professionals that this drug may increase the risk of unexplained mortality and QT prolongation, which may lead to abnormal and potentially fatal cardiac rhythm. In addition, the phenomenon of elevated liver enzymes in clinical trials of BDQ is a potential sign of hepatotoxicity. Therefore, it is still a medical need to develop new compounds with better safety profiles, patient compliance, affordability, and the ability to retain the efficacy of BDQ. After extensive lead generation and optimization, a new analog, sudapyridine (WX-081), was selected as a potential new antituberculosis candidate to move into clinical trials. Here, we evaluated WX-081's overall preclinical profile, including efficacy, pharmacokinetics, and toxicology. The in vitro activity of WX-081 against drug-sensitive and drug-resistant tuberculosis was comparable to that of BDQ, and there was comparable efficacy between WX-081 and BDQ in both acute and chronic mouse tuberculosis models using low-dose aerosol infection. Moreover, WX-081 improved pharmacokinetic parameters and, more importantly, had no adverse effects on blood pressure, heart rate, or qualitative ECG parameters from nonclinical toxicology studies. WX-081 is under investigation in a phase 2 study in patients. IMPORTANCE This study is aimed at chemotherapy for multidrug-resistant tuberculosis (MDR-TB), mainly to develop new anti-TB drugs to kill Mycobacterium tuberculosis, a microorganism with strong drug resistance. In this study, the structure of a potent antituberculosis compound, bedaquiline (BDQ), was optimized to generate a new compound, sudapyridine (WX-081). This experiment showed that its efficacy was similar to that of BDQ, its cardiotoxicity was lower, and it had good kinetic characteristics. This compound will certainly achieve significant results in the control and treatment of tuberculosis in the future.

Mutation bias shapes the spectrum of adaptive substitutions.

Evolutionary adaptation often occurs by the fixation of beneficial mutations. This mode of adaptation can be characterized quantitatively by a spectrum of adaptive substitutions, i.e., a distribution for types of changes fixed in adaptation. Recent work establishes that the changes involved in adaptation reflect common types of mutations, raising the question of how strongly the mutation spectrum shapes the spectrum of adaptive substitutions. We address this question with a codon-based model for the spectrum of adaptive amino acid substitutions, applied to three large datasets covering thousands of amino acid changes identified in natural and experimental adaptation in Saccharomyces cerevisiae, Escherichia coli, and Mycobacterium tuberculosis Using species-specific mutation spectra based on prior knowledge, we find that the mutation spectrum has a proportional influence on the spectrum of adaptive substitutions in all three species. Indeed, we find that by inferring the mutation rates that best explain the spectrum of adaptive substitutions, we can accurately recover the species-specific mutation spectra. However, we also find that the predictive power of the model differs substantially between the three species. To better understand these differences, we use population simulations to explore the factors that influence how closely the spectrum of adaptive substitutions mirrors the mutation spectrum. The results show that the influence of the mutation spectrum decreases with increasing mutational supply ([Formula: see text]) and that predictive power is strongly affected by the number and diversity of beneficial mutations.

Macrophage global metabolomics identifies cholestenone as host/pathogen cometabolite present in human Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (M. tuberculosis) causes an enormous burden of disease worldwide. As a central aspect of its pathogenesis, M. tuberculosis grows in macrophages, and host and microbe influence each other's metabolism. To define the metabolic impact of M. tuberculosis infection, we performed global metabolic profiling of M. tuberculosis-infected macrophages. M. tuberculosis induced metabolic hallmarks of inflammatory macrophages and a prominent signature of cholesterol metabolism. We found that infected macrophages accumulate cholestenone, a mycobacterial-derived, oxidized derivative of cholesterol. We demonstrated that the accumulation of cholestenone in infected macrophages depended on the M. tuberculosis enzyme 3ß-hydroxysteroid dehydrogenase (3ß-Hsd) and correlated with pathogen burden. Because cholestenone is not a substantial human metabolite, we hypothesized it might be diagnostic of M. tuberculosis infection in clinical samples. Indeed, in 2 geographically distinct cohorts, sputum cholestenone levels distinguished subjects with tuberculosis (TB) from TB-negative controls who presented with TB-like symptoms. We also found country-specific detection of cholestenone in plasma samples from M. tuberculosis-infected subjects. While cholestenone was previously thought to be an intermediate required for cholesterol degradation by M. tuberculosis, we found that M. tuberculosis can utilize cholesterol for growth without making cholestenone. Thus, the accumulation of cholestenone in clinical samples suggests it has an alternative role in pathogenesis and could be a clinically useful biomarker of TB infection.

Nos2-/- mice infected with M. tuberculosis develop neurobehavioral changes and immunopathology mimicking human central nervous system tuberculosis.

BACKGROUND: Understanding the pathophysiology of central nervous system tuberculosis (CNS-TB) is hampered by the lack of a good pre-clinical model that mirrors the human CNS-TB infection. We developed a murine CNS-TB model that demonstrates neurobehavioral changes with similar immunopathology with human CNS-TB. METHODS: We injected two Mycobacterium tuberculosis (M.tb) strains, H37Rv and CDC1551, respectively, into two mouse strains, C3HeB/FeJ and Nos2-/- mice, either into the third ventricle or intravenous. We compared the neurological symptoms, histopathological changes and levels of adhesion molecules, chemokines, and inflammatory cytokines in the brain induced by the infections through different routes in different strains. RESULTS: Intra-cerebroventricular infection of Nos2-/- mice with M.tb led to development of neurological signs and more severe brain granulomas compared to C3HeB/FeJ mice. Compared with CDC1551 M.tb, H37Rv M.tb infection resulted in a higher neurobehavioral score and earlier mortality. Intra-cerebroventricular infection caused necrotic neutrophil-dominated pyogranulomas in the brain relative to intravenous infection which resulted in disseminated granulomas and mycobacteraemia. Histologically, intra-cerebroventricular infection of Nos2-/- mice with M.tb resembled human CNS-TB brain biopsy specimens. H37Rv intra-cerebroventricular infected mice demonstrated higher brain concentrations of inflammatory cytokines, chemokines and adhesion molecule ICAM-1 than H37Rv intravenous-infected mice. CONCLUSIONS: Intra-cerebroventricular infection of Nos2-/- mice with H37Rv creates a murine CNS-TB model that resembled human CNS-TB immunopathology, exhibiting the worst neurobehavioral score with a high and early mortality reflecting disease severity and its associated neurological morbidity. Our murine CNS-TB model serves as a pre-clinical platform to dissect host-pathogen interactions and evaluate therapeutic agents for CNS-TB.

Insights into the Complexity of a Dormant Mycobacterium tuberculosis Cluster Once Transmission Is Resumed.

Genotyping tools help identify the complexity in Mycobacterium tuberculosis transmission clusters. We carried out a thorough analysis of the epidemiological and bacteriological complexity of a cluster in Almería, Spain. The cluster, initially associated with Moroccan migrants and with no secondary cases identified in 4 years, then reappeared in Spanish-born individuals. In one case, two Mycobacterium tuberculosis clonal variants were identified. We reanalyzed the cluster, supported by the characterization of multiple cultured isolates and respiratory specimens, whole-genome sequencing, and epidemiological case interviews. Our findings showed that the cluster, which was initially thought to have restarted activity with just a single case harboring a small degree of within-host diversity, was in fact currently growing due to coincidental reactivation of past exposures, with clonal diversity transmitted throughout the cluster. In one case, within-host diversity was amplified, probably due to prolonged diagnostic delay. IMPORTANCE The precise study of the dynamics of tuberculosis transmission in socio-epidemiologically complex scenarios may require more thorough analysis than the standard molecular epidemiology strategies. Our study illustrates the epidemiological and bacteriological complexity present in a transmission cluster in a challenging epidemiological setting with a high proportion of migrant cases. The combination of whole-genome sequencing, refined and refocused epidemiological interviews, and in-depth analysis of the bacterial composition of sputa and cultured isolates was crucial in order to correctly reinterpret the true nature of this cluster. Our global approach allowed us to reinterpret correctly the unnoticed epidemiological and bacteriological complexity involved in the Mycobacterium tuberculosis transmission event under study, which had been overlooked by the usual molecular epidemiology approaches.

miR-495 Regulates Cellular Reactive Oxygen Species Levels by Targeting sod2 To Inhibit Intracellular Survival of Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis is a chronic infectious disease pathogen. To date, tuberculosis is a major infectious disease that endangers human health. To better prevent and treat tuberculosis, it is important to study the pathogenesis of M. tuberculosis. Based on early-stage laboratory research results, in this study, we verified the upregulation of sod2 in Bacillus Calmette-Guérin (BCG) and H37Rv infection. By detecting BCG/H37Rv intracellular survival in sod2-silenced and sod2-overexpressing macrophages, sod2 was found to promote the intracellular survival of BCG/H37Rv. miR-495 then was determined to be downregulated by BCG/H37Rv. BCG/H37Rv can upregulate sod2 expression by miR-495 to promote the intracellular survival of BCG/H37Rv through a decline in ROS levels. This study provides a theoretical basis for developing new drug targets and treating tuberculosis.

Myeloid-Derived Suppressor Cells Mediate T Cell Dysfunction in Nonhuman Primate TB Granulomas.

Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell population comprised of immature myeloid cells and myeloid progenitors with very potent immunosuppressive potential. MDSCs are reported to be abundant in the lungs of active tuberculosis (TB) patients. We sought to perform an in-depth study of MDSCs during latent TB infection (LTBI) and active TB (ATB) using the nonhuman primate (NHP) model of pulmonary TB. We found a higher proportion of granulocytic, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the lungs of ATB animals compared to those with LTBI or naive control animals. Active disease in the lung, but not LTBI, was furthermore associated with higher proliferation, expansion, and immunosuppressive capabilities of PMN-MDSCs, as shown by enhanced expression of Ki67, indoleamine 2,3-dioxygenase (IDO1), interleukin-10 (IL-10), matrix metallopeptidase 9 (MMP-9), inducible nitric oxide synthase (iNOS), and programmed death-ligand 1 (PD-L1). These immunosuppressive PMN-MDSCs specifically localized to the lymphocytic cuff at the periphery of the granulomas in animals with ATB. Conversely, these cells were scarcely distributed in interstitial lung tissue and the inner core of granulomas. This spatial regulation suggests an important immunomodulatory role of PMN-MDSCs by restricting T cell access to the TB granuloma core and can potentially explain dysfunctional anti-TB responses in active granuloma. Our results raise the possibility that the presence of MDSCs can serve as a biomarker for ATB, while their disappearance can indicate successful therapy. Furthermore, MDSCs may serve as a potential target cell for adjunctive TB therapy. IMPORTANCE Myeloid cells are immunocytes of innate origin that orchestrate the first response toward pathogens via immune surveillance (uptake and killing), antigen presentation, and initiation of adaptive immunity by T cell stimulation. However, MDSCs are a subset of innate immunocytes that deviate to an immunoregulatory phenotype. MDSCs possess strong immunosuppressive capabilities that are induced in autoimmune, malignant neoplastic, and chronic inflammatory diseases. Induction of MDSCs has been found in peripheral blood, bronchoalveolar lavage (BAL) fluid, and pleural effusions of active TB patients, but their precise localization in lung tissue and in TB granulomas remains unclear due to challenges associated with sampling lungs and granulomas from active TB patients. Nonhuman primates (NHPs) are an important animal model with TB granulomas that closely mimic those found in humans and can therefore be used for studies that are otherwise challenging with patient material. Herein, we study MDSC localization in the lungs of NHPs exhibiting latent and active TB. Our findings reveal that MDSCs localize and exert their immunosuppressive roles at the periphery rather than in the core of TB granulomas.