Control Of Mycobacterium Tuberculosis Research Articles

CD4 T cell dysfunction is associated with bacterial recrudescence during chronic tuberculosis.

While most people contain Mycobacterium tuberculosis infection, some individuals develop active disease, usually within two years of infection. Why immunity fails after initially controlling infection is unknown. C57BL/6 mice control Mycobacterium tuberculosis for up to a year but ultimately succumb to disease. We hypothesize that the development of CD4 T cell dysfunction permits bacterial recrudescence. We developed a reductionist model to assess antigen-specific T cells during chronic infection and found evidence of CD4 T cell senescence and exhaustion. In C57BL/6 mice, CD4 T cells upregulate coinhibitory receptors and lose effector cytokine production. Single cell RNAseq shows that only a small number of CD4 T cells in the lungs of chronically infected mice are polyfunctional. While the origin and causal relationship between T- cell dysfunction and recrudescence remains uncertain, we propose T cell dysfunction leads to a feed-forward loop that causes increased bacillary numbers, greater T cell dysfunction, and progressive disease.

An exacerbated phosphate starvation response triggers Mycobacterium tuberculosis glycerol utilization at acidic pH.

The mechanisms controlling Mycobacterium tuberculosis (Mtb) replication and survival inside its human host remain ill-defined. Phagosome acidification and nutrient deprivation are common mechanisms used by macrophages to restrict the replication of intracellular bacteria. Mtb stops replicating at mildly acidic pH (<pH5.8), an adaptive process called "acid growth arrest," which is thought to play an important role in Mtb virulence. Using a genome-wide mutagenesis screen, we identified 95 genes whose disruption either decreases or increases Mtb fitness during acid growth arrest. We show that the virulence-associated inorganic phosphate (Pi) uptake system (Pst-1) regulates the ability of Mtb to replicate in acidic conditions. Deletion of pstA1, a gene coding for a subunit of the Pst-1 system, results in the overexpression of the Pi starvation regulator regX3, which is sufficient to restore Mtb growth in acid conditions. Our data further support the role of limited glycerol uptake and ROS-mediated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition in causing Mtb acid growth arrest. This study reveals an unexpected role of the Pi starvation response in regulating acid growth arrest and highlights the intricacy of the mechanisms regulating redox homeostasis, acid stress response, and nutrient utilization in Mtb.IMPORTANCEDespite the availability of antibiotic treatment, M. tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains a major infectious disease killer worldwide. A better understanding of the environments that Mtb faces during infection and the mechanisms Mtb employs to respond and adapt may help identify currently unexplored pathways and targets for the development of novel anti-TB drugs. Here, we demonstrate that Mtb growth in acid can be restored by the over-expression of the Pi starvation response regulator regX3. This work paves the way toward a better understanding of the mechanisms controlling Mtb growth at acidic pH and highlights the role of inorganic phosphate in this process.

Nucleotidyltransferase toxin MenT extends aminoacyl acceptor ends of serine tRNAs to control Mycobacterium tuberculosis growth.

Toxins of toxin-antitoxin systems use diverse mechanisms to inhibit bacterial growth. In this study, we characterize the translation inhibitor toxin MenT3 of Mycobacterium tuberculosis, the bacterium responsible for tuberculosis in humans. We show that MenT3 is a robust cytidine specific tRNA nucleotidyltransferase in vitro, capable of modifying the aminoacyl acceptor ends of most tRNA but with a marked preference for tRNASer, to which long stretches of cytidines are added. Furthermore, transcriptomic-wide analysis of MenT3 targets in M. tuberculosis identifies tRNASer as the sole target of MenT3 and reveals significant detoxification attempts by the essential CCA-adding enzyme PcnA in response to MenT3. Finally, under physiological conditions, only in the presence the native menAT3 operon, an active pool of endogenous MenT3 targeting tRNASer in M. tuberculosis is detected, likely reflecting the importance of MenT3 during infection.

Macrophage-derived extracellular vesicles from Ascaris lumbricoides antigen exposure enhance Mycobacterium tuberculosis growth control, reduce IL-1β, and contain miR-342-5p, miR-516b-5p, and miR-570-3p that regulate PI3K/AKT and MAPK signaling pathways.

Helminth coinfection with tuberculosis (TB) can alter the phenotype and function of macrophages, which are the major host cells responsible for controlling Mycobacterium tuberculosis (Mtb). However, it is not known whether helminth infection stimulates the release of host-derived extracellular vesicles (EVs) to induce or maintain their regulatory network that suppresses TB immunity. We previously showed that pre-exposure of human monocyte-derived macrophages (hMDMs) with Ascaris lumbricoides protein antigens (ASC) results in reduced Mtb infection-driven proinflammation and gained bacterial control. This effect was entirely dependent on the presence of soluble components in the conditioned medium from helminth antigen-pre-exposed macrophages. Our objective was to investigate the role of EVs released from helminth antigen-exposed hMDMs on Mtb-induced proinflammation and its effect on Mtb growth in hMDMs. Conditioned medium from 48-h pre-exposure with ASC or Schistosoma mansoni antigen (SM) was used to isolate EVs by ultracentrifugation. EVs were characterized by immunoblotting, flow cytometry, nanoparticle tracking assay, transmission electron microscopy, and a total of 377 microRNA (miRNA) from EVs screened by TaqMan array. Luciferase-expressing Mtb H37Rv was used to evaluate the impact of isolated EVs on Mtb growth control in hMDMs. EV characterization confirmed double-membraned EVs, with a mean size of 140 nm, expressing the classical exosome markers CD63, CD81, CD9, and flotillin-1. Specifically, EVs from the ASC conditioned medium increased the bacterial control in treatment-naïve hMDMs and attenuated Mtb-induced IL-1β at 5 days post-infection. Four miRNAs showed unique upregulation in response to ASC exposure in five donors. Pathway enrichment analysis showed that the MAPK and PI3K-AKT signaling pathways were regulated. Among the mRNA targets, relevant for regulating inflammatory responses and cellular stress pathways, CREB1 and MAPK13 were identified. In contrast, SM exposure showed significant regulation of the TGF-β signaling pathway with SMAD4 as a common target. Overall, our findings suggest that miRNAs in EVs released from helminth-exposed macrophages regulate important signaling pathways that influence macrophage control of Mtb and reduce inflammation. Understanding these interactions between helminth-induced EVs, miRNAs, and macrophage responses may inform novel therapeutic strategies for TB management.

Type I IFN signaling in the absence of IRGM1 promotes M. tuberculosis replication in immune cells by suppressing T cell responses

Polymorphisms in the IRGM gene are associated with susceptibility to tuberculosis in humans. A murine ortholog of Irgm, Irgm1, is also essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. Multiple processes have been associated with IRGM1 activity that could impact the host response to Mtb infection, including roles in autophagy-mediated pathogen clearance and expansion of activated T cells. However, what IRGM1-mediated pathway is necessary to control Mtb infection in vivo and the mechanistic basis for this control remains unknown. We dissected the contribution of IRGM1 to immune control of Mtb pathogenesis in vivo and found that Irgm1 deletion leads to higher levels of IRGM3-dependent type I interferon signaling. The increased type I interferon signaling precludes T cell expansion during Mtb infection. The absence of Mtb-specific T cell expansion in Irgm1−/− mice results in uncontrolled Mtb infection in neutrophils and alveolar macrophages, which directly contributes to susceptibility to infection. Together, our studies reveal that IRGM1 is required to promote T cell-mediated control of Mtb infection in neutrophils, which is essential for the survival of Mtb-infected mice. These studies also uncover new ways type I interferon signaling can impact TH1 immune responses.

Glycogen synthase kinase 3 inhibition controls Mycobacterium tuberculosis infection

Compounds targeting host control of infectious diseases provide an attractive alternative to antimicrobials. A phenotypic screen of a kinase library identified compounds targeting glycogen synthase kinase 3 as potent inhibitors of Mycobacterium tuberculosis (Mtb) intracellular growth in the human THP-1 cell line and primary human monocytes-derived macrophages (hMDM). CRISPR knockouts and siRNA silencing showed that GSK3 isoforms are needed for the growth of Mtb and that a selected compound, P-4423632 targets GSK3β. GSK3 inhibition was associated with macrophage apoptosis governed by the Mtb secreted protein tyrosine phosphatase A (PtpA). Phospho-proteome analysis of macrophages response to infection revealed a wide array of host signaling and apoptosis pathways controlled by GSK3 and targeted by P-4423632. P-4423632 was additionally found to be active against other intracellular pathogens. Our findings strengthen the notion that targeting host signaling to promote the infected cell's innate antimicrobial capacity is a feasible and attractive host-directed therapy approach.

Viperin inhibits interferon-γ production to promote Mycobacterium tuberculosis survival by disrupting TBK1-IKKε-IRF3-axis and JAK-STAT signaling

Objectives and designAs an interferon-inducible protein, Viperin has broad-spectrum antiviral effects and regulation of host immune responses. We aim to investigate how Viperin regulates interferon-γ (IFN-γ) production in macrophages to control Mycobacterium tuberculosis (Mtb) infection.MethodsWe use Viperin deficient bone-marrow-derived macrophage (BMDM) to investigate the effects and machines of Viperin on Mtb infection.ResultsViperin inhibited IFN-γ production in macrophages and in the lung of mice to promote Mtb survival. Further insight into the mechanisms of Viperin-mediated regulation of IFN-γ production revealed the role of TANK-binding kinase 1 (TBK1), the TAK1-dependent inhibition of NF-kappa B kinase-epsilon (IKKε), and interferon regulatory factor 3 (IRF3). Inhibition of the TBK1-IKKε-IRF3 axis restored IFN-γ production reduced by Viperin knockout in BMDM and suppressed intracellular Mtb survival. Moreover, Viperin deficiency activated the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, which promoted IFN-γ production and inhibited Mtb infection in BMDM. Additionally, a combination of the anti-TB drug INH treatment in the absence of Viperin resulted in further IFN-γ production and anti-TB effect.ConclusionsThis study highlights the involvement of TBK1-IKKε-IRF3 axis and JAK-STAT signaling pathways in Viperin-suppressed IFN-γ production in Mtb infected macrophages, and identifies a novel mechanism of Viperin on negatively regulating host immune response to Mtb infection.

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

Ex vivo and in vivo evidence that cigarette smoke-exposed T regulatory cells impair host immunity against Mycobacterium tuberculosis.

A strong epidemiologic link exists between cigarette smoke (CS) exposure and susceptibility to tuberculosis (TB). Macrophage and murine studies showed that CS and nicotine impair host-protective immune cells against Mycobacterium tuberculosis (MTB) infection. While CS and nicotine may activate T regulatory cells (Tregs), little is known about how CS may affect these immunosuppressive cells with MTB infection. We investigated whether CS-exposed Tregs could exacerbate MTB infection in co-culture with human macrophages and in recipient mice that underwent adoptive transfer of Tregs from donor CS-exposed mice. We found that exposure of primary human Tregs to CS extract impaired the ability of unexposed human macrophages to control an MTB infection by inhibiting phagosome-lysosome fusion and autophagosome formation. Neutralizing CTLA-4 on the CS extract-exposed Tregs abrogated the impaired control of MTB infection in the macrophage and Treg co-cultures. In Foxp3+GFP+DTR+ (Thy1.2) mice depleted of endogenous Tregs, adoptive transfer of Tregs from donor CS-exposed B6.PL(Thy1.1) mice with subsequent MTB infection of the Thy1.2 mice resulted in a greater burden of MTB in the lungs and spleens than those that received Tregs from air-exposed mice. Mice that received Tregs from donor CS-exposed mice and infected with MTB had modest but significantly reduced numbers of interleukin-12-positive dendritic cells and interferon-gamma-positive CD4+ T cells in the lungs, and an increased number of total programmed cell death protein-1 (PD-1) positive CD4+ T cells in both the lungs and spleens. Previous studies demonstrated that CS impairs macrophages and host-protective T effector cells in controlling MTB infection. We now show that CS-exposed Tregs can also impair control of MTB in co-culture with macrophages and in a murine model.

NCoR1 controls Mycobacterium tuberculosis growth in myeloid cells by regulating the AMPK-mTOR-TFEB axis.

Mycobacterium tuberculosis (Mtb) defends host-mediated killing by repressing the autophagolysosome machinery. For the first time, we report NCoR1 co-repressor as a crucial host factor, controlling Mtb growth in myeloid cells by regulating both autophagosome maturation and lysosome biogenesis. We found that the dynamic expression of NCoR1 is compromised in human peripheral blood mononuclear cells (PBMCs) during active Mtb infection, which is rescued upon prolonged anti-mycobacterial therapy. In addition, a loss of function in myeloid-specific NCoR1 considerably exacerbates the growth of M. tuberculosis in vitro in THP1 differentiated macrophages, ex vivo in bone marrow-derived macrophages (BMDMs), and in vivo in NCoR1MyeKO mice. We showed that NCoR1 depletion controls the AMPK-mTOR-TFEB signalling axis by fine-tuning cellular adenosine triphosphate (ATP) homeostasis, which in turn changes the expression of proteins involved in autophagy and lysosomal biogenesis. Moreover, we also showed that the treatment of NCoR1 depleted cells by Rapamycin, Antimycin-A, or Metformin rescued the TFEB activity and LC3 levels, resulting in enhanced Mtb clearance. Similarly, expressing NCoR1 exogenously rescued the AMPK-mTOR-TFEB signalling axis and Mtb killing. Overall, our data revealed a central role of NCoR1 in Mtb pathogenesis in myeloid cells.

Determining the mechanistic basis for Irgm1 mediated control of Mycobacterium tuberculosisinfection.

Abstract Irgm1 is a 47kDa IFN-γ inducible GTPase that has been shown to be essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. It was originally shown that Irgm1 is recruited to Mtb containing phagosomes in infected macrophages where it was proposed to be involved in autophagy-mediated clearance of Mtb. However, we have previously discovered that autophagy is not required in macrophages to control Mtb replication. In addition, it has since been reported that Irgm1 does not colocalize with phagosomes containing Mycobacterium bovis BCG in IFN-γ treated cells, suggesting that Irgm1 must have non-autophagic roles in TB infection that require detailed investigation. Irgm1 −/−mice were shown to have higher levels of type I interferon signaling, as well as CD4 +T cell survival defect, which could explain the susceptibility to Mtb infection, but this has yet to be determined. We infected Irgm1−/−mice with Mtb and found that they succumbed early to infection with higher bacterial burden, higher numbers of neutrophils, higher numbers of CD19 +B cells, and lower numbers of CD4 +T-lymphocytes in the lungs compared to wild-type (WT) mice. In addition, we found significantly increased expression of interferon-stimulated genes (ISGs) during Mtb infection of Irgm1−/−mice compared to WT. We also observed that T cell proliferation and lymph node responses were severely defective following Mtb infection in Irgm1−/−mice. Bone marrow chimera and transfer of Mtb-specific activated T lymphocytes into Irgm1−/−mice reverses the susceptibility to Mtb infection, indicating that a defect in T cell responses contributes to the susceptibility of Irgm1−/−mice to Mtb infection. This work was supported by Stephen I. Morse fellowship to Sumanta Kumar Naik and the Project supported by NIH AI142784 and AI132697 and BWF PATH Award to Dr. Christina Stallings.

Investigating the role of BHLHE40 in host responses to Mycobacterium tuberculosisinfection and myeloid cell development.

Abstract We currently lack a complete understanding of the immune responses required to control Mycobacterium tuberculosis (Mtb) infection. Our lab has shown that the transcription factor BHLHE40 is essential during Mtb infection. Deletion of Bhlhe40 in CD11c +lung macrophages and dendritic cells (DCs) is sufficient to increase mouse susceptibility to Mtb infection, though the role of BHLHE40 in these cells during infection is unknown. Murine bone marrow cultured in the presence of GM-CSF, an abundant cytokine in the lung, develop into populations of CD11c +macrophages, DCs, and granulocytes, all of which express Bhlhe40. Using this system, we examined cytokine and transcriptional responses before and after exposure to heat-killed Mtb to understand how BHLHE40 impacts cell development and inflammatory responses. We found that in WT mice, GM-CSF-cultured cells develop into 9 distinct populations and expression of Bhlhe40 was required for GM-CSF-mediated pro-inflammatory cytokine and transcriptional programs. Additionally, loss of Bhlhe40 led to decreased DCs accompanied by upregulation of the transcription factor C/EBPβ in DC progenitor populations, which negatively impacts DC development. ChIP-seq analysis revealed BHLHE40 binds directly to C/EBPβ’s gene locus, indicating that BHLHE40 may directly repress Cebpb. In contrast, loss of Bhlhe40 did not affect macrophage development, but led to a transcriptional shift towards an anti-inflammatory state. These findings demonstrate that in the presence of GM-CSF, BHLHE40 is required for DC development and inflammatory macrophage polarization. Defects in either of these aspects could contribute to the essential role for BHLHE40 in lung macrophages and DCs during Mtb infection in vivo.

Histone deacetylase (HDAC) inhibitors- based drugs are effective to control Mycobacterium tuberculosis infection and promote the sensibility for rifampicin in MDR strain.

Tuberculosis (TB) is a major public health problem, which has been aggravated by the alarming growth of drug-resistant tuberculosis. Therefore, the development of a safer and more effective treatment is needed. The aim of this work was repositioning and evaluate histone deacetylases (HDAC) inhibitors- based drugs with potential antimycobacterial activity. Using an in silico pharmacological repositioning strategy, three molecules that bind to the catalytic site of histone deacetylase were selected. Pneumocytes type II and macrophages were infected with Mycobacterium tuberculosis and treated with pre-selected HDAC inhibitors (HDACi). Subsequently, the ability of each of these molecules to directly promote the elimination of M. tuberculosis was evaluated by colony-forming unit (CFU)/mL. We assessed the expression of antimicrobial peptides and respiratory burst using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Aminoacetanilide (ACE), N-Boc-1,2-phenylenediamine (N-BOC), 1,3-Diphenylurea (DFU), reduce bacillary loads in macrophages and increase the production of β-defensin-2, LL-37, superoxide dismutase (SOD) 3 and inducible nitric oxide synthase (iNOS). While only the use of ACE in type II pneumocytes decreases the bacterial load through increasing LL-37 expression. Furthermore, the use of ACE and rifampicin inhibited the survival of intracellular multi-drug resistance M. tuberculosis. Our data support the usefulness of in silico approaches for drug repositioning to provide a potential adjunctive therapy for TB.

Impact of sensitisation programme on screening tests for latent tuberculosis infection among health-care workers: A questionnaire-based study

Background: Timely and accurate identification of people with latent tuberculosis infection (LTBI) is essential for controlling Mycobacterium tuberculosis. Hence, it is important that health-care workers have the latest and adequate knowledge about the existing diagnostic modalities. Methods: In this prospective, questionnaire-based study, various health-care workers were asked to respond to a 25-item questionnaire before the sensitisation programme (workshop) regarding knowledge, procedure and interpretation of screening tests for LTBI (tuberculin skin test [TST] and interferon-gamma release assay [IGRA]). The same questionnaire was repeated on the completion of the workshop to assess its impact. Results: The mean baseline knowledge among technicians, nurses, postgraduates and interns was 42.9%, 42.1%, 51.9% and 53.6%, which improved significantly to 84.2%, 83.4%, 89.4% and 89.5%, respectively after the workshop. Statistically, a significant difference was observed between pre- and post-workshop proprtion of all health-care workers with respect to the knowledge of TST and IGRA (P &lt; 0.05). Conclusions: This questionnaire-based study highlights the importance of training various health-care workers on diagnostic modalities for LTBI since the current study findings revealed that training increased the knowledge scores of the participants significantly.

Gene Silencing of Toll-like Receptor 2 Gene Expression as a Tactic to Control Mycobacterium Tuberculosis and Granuloma Formation.

Mycobacterium tuberculosis (MT) is the causative agent of tuberculosis (TB) in humans. Tuberculosis is one of the top 10 causes of mortality worldwide, resulting in 1.8 million deaths and 10.4 million new cases in 2016. Understanding the fundamental features of MT biology is critical to the eradication of MT in the future. Due to the increasing frequency of antimicrobial treatment resistance and problems in vaccine development, the pathogenesis of TB for its survival and growth is highly dependent on host lipids and stimulated-lipid droplets formation. Toll-like receptor 2 (TLR2) forms heterophilic dimers with TLR1 and TLR6, therefore, recognizing many MT components. Both of these receptors identify the invading antigen and activate downstream protein kinases. Some studies demonstrated that the cyclooxygenase-2 (COX-2) promoter-driven gene expression includes connecting sites for transcription factors, such as nuclear factor-kappa B, CREB, NFAT, and c/EBPβ. The current study aimed to investigate the role of the TLR2 receptor in positively regulating prostaglandin E2 production in M. bovis (BCG) infected macrophages in vivo using a human monocytic cell line THP-1. Our results revealed that MT infection triggers a time-dependent increase in COX-2 expression via pathways involving TLR2 receptor activation and enhances COX-2 expression, leading to an increase in lipid droplet formation and suppression of macrophage activation.

Autologous Cytokine-Induced Killer Cell Immunotherapy Enhances Chemotherapy Efficacy against Multidrug-Resistant Tuberculosis.

Objective Multidrug-resistant tuberculosis (MDR-TB) causes persistent infection and challenges tuberculosis control worldwide. T cell-mediated immunity plays a critical role in controlling Mycobacterium tuberculosis (Mtb) infection, and therefore, enhancing Mtb-specific T cell immune responses represents a promising therapeutic strategy against TB. Cytokine-induced killer (CIK) immunotherapy is based on autologous infusion of in vitro expanded bulk T cells, which include both pathogen-specific and nonspecific T cells from patient peripheral blood mononuclear cells (PBMC) into TB patients. Preclinical mouse studies have shown that the adoptive T cell therapy inhibited Mtb infection. However, the efficacy of CIK immunotherapy in the treatment of MDR-TB infection has not been evaluated in clinical trials. Methods We performed a retrospective study of MDR-TB patients who received CIK immunotherapy in combination with anti-TB chemotherapy and those who had standard chemotherapy. Results Our results showed that CIK immunotherapy in combination with anti-TB chemotherapy treatment increased the conversion rate of sputum smear and Mtb culture, alleviated symptoms, improved lesion absorption, and increased recovery. The kinetics of serology and immunology index monitoring data showed good safety profiles for the CIK treatment. Conclusion Our study has provided strong evidence that CIK immunotherapy in combination with anti-TB chemotherapy is beneficial for MDR-TB patients. A multicenter clinical trial is warranted to evaluate CIK as a new immune therapy for MDR-TB.

Early IL-10 promotes vasculature-associated CD4+ T cells unable to control Mycobacterium tuberculosis infection.

Cytokine-producing CD4+ T cells play a crucial role in the control of Mycobacterium tuberculosis infection; however, there is a delayed appearance of effector T cells in the lungs following aerosol infection. The immunomodulatory cytokine IL-10 antagonizes control of M. tuberculosis infection through mechanisms associated with reduced CD4+ T cell responses. Here, we show that IL-10 overexpression only before the onset of the T cell response impaired control of M. tuberculosis growth; during chronic infection, IL-10 overexpression reduced the CD4+ T cell response without affecting the outcome of infection. IL-10 overexpression early during infection did not, we found, significantly impair the kinetics of CD4+ T cell priming and effector differentiation. However, CD4+ T cells primed and differentiated in an IL-10–enriched environment displayed reduced expression of CXCR3 and, because they did not migrate into the lung parenchyma, their ability to control infection was limited. Importantly, these CD4+ T cells maintained their vasculature phenotype and were unable to control infection, even after adoptive transfer into low IL-10 settings. Together our data support a model wherein, during M. tuberculosis infection, IL-10 acts intrinsically on T cells, impairing their parenchymal migratory capacity and ability to engage with infected phagocytic cells, thereby impeding control of infection.

Comparative transcriptomic analysis of THP-1-derived macrophages infected with Mycobacterium tuberculosis H37Rv, H37Ra and BCG.

Tuberculosis (TB) remains a worldwide healthcare concern, and the exploration of the host‐pathogen interaction is essential to develop therapeutic modalities and strategies to control Mycobacterium tuberculosis (M.tb). In this study, RNA sequencing (transcriptome sequencing) was employed to investigate the global transcriptome changes in the macrophages during the different strains of M.tb infection. THP‐1 cells derived from macrophages were exposed to the virulent M.tb strain H37Rv (Rv) or the avirulent M.tb strain H37Ra (Ra), and the M.tb BCG vaccine strain was used as a control. The cDNA libraries were prepared from M.tb‐infected macrophages and then sequenced. To assess the transcriptional differences between the expressed genes, the bioinformatics analysis was performed using a standard pipeline of quality control, reference mapping, differential expression analysis, protein‐protein interaction (PPI) networks, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Q‐PCR and Western blot assays were also performed to validate the data. Our findings indicated that, when compared to BCG or M.tb H37Ra infection, the transcriptome analysis identified 66 differentially expressed genes in the M.tb H37Rv‐infected macrophages, out of which 36 genes were up‐regulated, and 30 genes were down‐regulated. The up‐regulated genes were associated with immune response regulation, chemokine secretion, and leucocyte chemotaxis. In contrast, the down‐regulated genes were associated with amino acid biosynthetic and energy metabolism, connective tissue development and extracellular matrix organization. The Q‐PCR and Western blot assays confirmed increased expression of pro‐inflammatory factors, altered energy metabolic processes, enhanced activation of pro‐inflammatory signalling pathways and increased pyroptosis in H37Rv‐infected macrophage. Overall, our RNA sequencing‐based transcriptome study successfully identified a comprehensive, in‐depth gene expression/regulation profile in M.tb‐infected macrophages. The results demonstrated that virulent M.tb strain H37Rv infection triggers a more severe inflammatory immune response associated with increased tissue damage, which helps in understanding the host‐pathogen interaction dynamics and pathogenesis features in different strains of M.tb infection.

Monkeying around with MAIT Cells: Studying the Role of MAIT Cells in SIV and Mtb Co-Infection.

There were an estimated 10 million new cases of tuberculosis (TB) disease in 2019. While over 90% of individuals successfully control Mycobacterium tuberculosis (Mtb) infection, which causes TB disease, HIV co-infection often leads to active TB disease. Despite the co-endemic nature of HIV and TB, knowledge of the immune mechanisms contributing to the loss of control of Mtb replication during HIV infection is lacking. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that target and destroy bacterially-infected cells and may contribute to the control of Mtb infection. Studies examining MAIT cells in human Mtb infection are commonly performed using peripheral blood samples. However, because Mtb infection occurs primarily in lung tissue and lung-associated lymph nodes, these studies may not be fully translatable to the tissues. Additionally, studies longitudinally examining MAIT cell dynamics during HIV/Mtb co-infection are rare, and lung and lymph node tissue samples from HIV+ patients are typically unavailable. Nonhuman primates (NHP) provide a model system to characterize MAIT cell activity during Mtb infection, both in Simian Immunodeficiency Virus (SIV)-infected and SIV-naïve animals. Using NHPs allows for a more comprehensive understanding of tissue-based MAIT cell dynamics during infection with both pathogens. NHP SIV and Mtb infection is similar to human HIV and Mtb infection, and MAIT cells are phenotypically similar in humans and NHPs. Here, we discuss current knowledge surrounding MAIT cells in SIV and Mtb infection, how SIV infection impairs MAIT cell function during Mtb co-infection, and knowledge gaps to address.

Repurposing Saquinavir for Host-Directed Therapy to Control Mycobacterium Tuberculosis Infection.

Despite the available antibiotics, tuberculosis (TB) has made its return since the 90’s of the last century as a global threat mostly due to co-infection with HIV, to the emergence of drug resistant strains and the lack of an effective vaccine. Host-directed strategies could be exploited to improve treatment efficacy, contain drug-resistant strains, improve immune responses and reduce disease severity. Macrophages in the lungs are often found infected with Mycobacterium tuberculosis (Mtb) and/or with HIV. The long-term survival of lung macrophages infected with Mtb or with HIV, together with their ability to produce viral particles, especially during TB, makes these niches major contributors to the pathogenicity of the infection. Among the available drugs to control HIV infection, protease inhibitors (PIs), acting at post-integrational stages of virus replication cycle, are the only drugs able to interfere with virus production and release from macrophages during chronic infection. For Mtb we recently found that the pathogen induces a general down-regulation of lysosomal proteases, helping bacteria to establish an intracellular niche in macrophages. Here we found that the PI saquinavir, contrary to ritonavir, is able to induce an increase of endolysosomal proteases activity especially of cathepsin S in Mtb infected macrophages and during co-infection with HIV. Our results indicate that saquinavir treatment of infected macrophages led not only to a significant intracellular killing of Mtb but also: (i) to an improved expression of the HLA class II antigen presentation machinery at the cell surface; (ii) to increased T-lymphocyte priming and proliferation; and (iii) to increased secretion of IFN-γ. All together the results indicate saquinavir as a potential host directed therapy for tuberculosis.