Mtb Burden Research Articles

Identification of an Isoxazole Derivative as an Antitubercular Compound for Targeting the FadD Enzymes of Mycobacterium tuberculosis.

FadD32, a fatty acyl-AMP ligase, plays an indispensable role in mycobacterial mycolic acid synthesis and is a validated target for tuberculosis (TB) drug development. The crystal structure of Mycobacterium tuberculosis (Mtb)FadD32 has laid the foundation of structure-based drug discovery against this crucial enzyme. Here, we screened the "isoxazole" scaffold containing molecules against MtbFadD32 and identified a compound 2,4-dibromo-6-[3-(trifluoromethyl)-1,2-oxazol-5-yl]phenol (M1) with specific inhibitory activity against Mtb. Kinetics experiments showed that M1 inhibits MtbFadD32 and MtbFadD28 activity. The transcriptomics response of Mtb disclosed M1-mediated regulation of mycobacterial decisive genes involved in cell wall synthesis, consequently creating unfavorable conditions for Mtb survival. Further, M1 curtails the Mtb survival in infected macrophages and reduces Mtb burden and tubercular granulomas in a chronic infection model of BALB/c mice. Our findings provide an effective chemical scaffold to inhibit MtbFadD32 with the potential to inhibit multiple MtbFadD family of enzymes for further development as a promising candidate for treating TB.

Pharmacological inhibition of macrophage triglyceride biosynthesis pathways does not improve Mycobacterium tuberculosis control in infected mice.

Tuberculosis (TB) necrotic granulomas contain triglyceride-rich macrophages (foam cells) with reduced antimicrobial functions. We assessed the ability of two compounds to reduce triglyceride content and Mycobacterium tuberculosis (Mtb) burden in infected human monocyte-derived macrophages and in the lungs of Mtb-infected C3HeB/FeJ mice: A-922500 (DGATi), an inhibitor of diacylglycerol acyltransferase 1; and LY2584702 (p70S6Ki), an inhibitor of p70 S6 kinase. DGATi and p70S6Ki significantly reduced the lipid content and bacillary burden in Mtb-infected macrophages. Each inhibitor reduced the cellular triglyceride content in bronchoalveolar lavage samples of Mtb-infected mice. After 6 weeks of treatment, p70S6Ki alone reduced the lung bacterial burden in Mtb-infected mice. However, DGATi alone, and DGATi or p70S6Ki in combination with isoniazid did not reduce lung bacterial burden or alter lung inflammation. These findings provide further insight into the role of foam cells in tuberculosis pathogenesis and the utility of interventions targeting these cell populations as adjunctive host-directed therapies.

5-Fluoroindole Reduces the Bacterial Burden in a Murine Model of Mycobacterium tuberculosis Infection.

Tuberculosis is a disease caused by a single pathogen that leads to a death toll estimated to be more than a million per year. Mycobacterium tuberculosis (Mtb), which affects mainly the lungs, spreads by airborne transmission when infectious respiratory particles from an infected human enter the respiratory tract of another person. Despite diagnosis and treatment being well established, the rise of cases of patients infected with Mtb strains with multidrug resistance to the antibiotics used in the regimen against the disease is alarming. Indole used as a core molecule has been described as a promising structure to treat several diseases. 5-Fluoroindole (5-FI) compound, evaluated in the free base and in the hydrochloride (5-FI.HCl) forms, inhibited the growth of pan-sensitive Mtb H37Rv strain in the same range (4.7-29.1 μM) of clinical isolates that have resistance to at least two first-line drugs. Although 5-FI showed no cytotoxicity in Vero and HepG2 cells, high permeability (2.4.10-6 cm/s) in the PAMPA assay, and high metabolic stability (Clint 9.0 mL/min/kg) in rat liver microsomes, limited solubility at plasmatic and intestinal pH values prompted formation and employment of its salt form (5-FI.HCl). Although the 5-FI.HCl compound showed increased solubility at pH values of 7.4 and 9.1 and increased stability in aqueous solutions, data for intrinsic clearance (Clint = 48 mL/min/kg) and a half-life (t 1/2 = 12 min) showed decreased metabolic stability. As 5-FI.HCl showed both good absorption and ability to reach the systemic circulation of animals without the need to use vehicles containing cosolvents or surfactants, it was chosen to evaluate its effectiveness in the model of tuberculosis in mice. The in vivo results showed the concentration of the compound in plasma increasing within 30 min in the systemic circulation and the capacity of reducing the Mtb burden in the lungs at the concentration of 200 μmol/kg after 21 days of infection, with no toxicity in mice.

Antigen specificity shapes antibody functions in tuberculosis.

Tuberculosis (TB) is the number one infectious disease cause of death worldwide due to an incomplete understanding of immunity. Emerging data highlight antibody functions mediated by the Fc domain as immune correlates. However, the mechanisms by which antibody functions impact the causative agent Mycobacterium tuberculosis (Mtb) are unclear. Here, we examine how antigen specificity determined by the Fab domain shapes Fc effector functions against Mtb. Using the critical structural and secreted virulence proteins Mtb cell wall and ESAT-6 & CFP-10, we observe that antigen specificity alters subclass, antibody post-translational glycosylation, and Fc effector functions in TB patients. Moreover, Mtb cell wall IgG3 enhances disease through opsonophagocytosis of extracellular Mtb . In contrast, polyclonal and a human monoclonal IgG1 we generated targeting ESAT-6 & CFP-10 inhibit intracellular Mtb . These data show that antibodies have multiple roles in TB and antigen specificity is a critical determinant of the protective and pathogenic capacity.

Single Cell Analysis of Peripheral TB-Associated Granulomatous Lymphadenitis.

We successfully employed a single cell RNA sequencing (scRNA-seq) approach to describe the cells and the communication networks characterizing granulomatous lymph nodes of TB patients. When mapping cells from individual patient samples, clustered based on their transcriptome similarities, we uniformly identify several cell types that known to characterize human and non-human primate granulomas. Whether high or low Mtb burden, we find the T cell cluster to be one of the most abundant. Many cells expressing T cell markers are clearly quantifiable within this CD3 expressing cluster. Other cell clusters that are uniformly detected, but that vary dramatically in abundance amongst the individual patient samples, are the B cell, plasma cell and macrophage/dendrocyte and NK cell clusters. When we combine all our scRNA-seq data from our current 23 patients (in order to add power to cell cluster identification in patient samples with fewer cells), we distinguish T, macrophage, dendrocyte and plasma cell subclusters, each with distinct signaling activities. The sizes of these subclusters also varies dramatically amongst the individual patients. In comparing FNA composition we noted trends in which T cell populations and macrophage/dendrocyte populations were negatively correlated with NK cell populations. In addition, we also discovered that the scRNA-seq pipeline, designed for quantification of human cell mRNA, also detects Mtb RNA transcripts and associates them with their host cell's transcriptome, thus identifying individual infected cells. We hypothesize that the number of detected bacterial transcript reads provides a measure of Mtb burden, as does the number of Mtb-infected cells. The number of infected cells also varies dramatically in abundance amongst the patient samples. CellChat analysis identified predominating signaling pathways amongst the cells comprising the various granulomas, including many interactions between stromal or endothelial cells and the other component cells, such as Collagen, FN1 and Laminin,. In addition, other more selective communications pathways, including MIF, MHC-1, MHC-2, APP, CD 22, CD45, and others, are identified as originating or being received by individual immune cell components.

Myriocin enhances the clearance of M. tuberculosis by macrophages through the activation of PLIN2.

Myriocin is an inhibitor of de novo synthesis of sphingolipids and ceramides. In this research, we showed myriocin could significantly reduce Mtb burden and histopathological inflammation in mice. However, the underlying mechanism remains unclear. RNA-seq analysis revealed a significant increase in gene expression of PLIN2/CD36/CERT1 after myriocin treatment. The reduced bactericidal burden was only reversed after silencing the lipid droplets (LDs) surface protein PLIN2. This suggests that myriocin enhances the ability of macrophages to clear Mtb depending on the PLIN2 gene, which is part of the PPARγ pathway. Indeed, we observed a significant increase in the number of LDs following myriocin treatment.IMPORTANCEMycobacterium tuberculosis has the ability to reprogram host cell lipid metabolism and alter the antimicrobial functions of infected macrophages. The sphingolipids, such as ceramides, are the primary host lipids utilized by the bacteria, making the sphingomyelinase/ceramide system critical in Mtb infections. Surprisingly, the antimicrobial effect of myriocin was found to be independent of its role in reducing ceramides, but instead, it depends on the lipid droplets surface protein PLIN2. Our findings provide a novel mechanism for how myriocin enhances Mtb clearance in macrophages.

The Influence of Body Fat Dynamics on Pulmonary Immune Responses in Murine Tuberculosis: Unraveling Sex-Specific Insights.

The World Health Organization (WHO) highlights a greater susceptibility of males to tuberculosis (TB), a vulnerability attributed to sex-specific variations in body fat and dietary factors. Our study delves into the unexplored terrain of how alterations in body fat influence Mycobacterium tuberculosis (Mtb) burden, lung pathology, immune responses, and gene expression, with a focus on sex-specific dynamics. Utilizing a low-dose Mtb-HN878 clinical strain infection model, we employ transgenic FAT-ATTAC mice with modulable body fat to explore the impact of fat loss (via fat ablation) and fat gain (via a medium-fat diet, MFD). Firstly, our investigation unveils that Mtb infection triggers severe pulmonary pathology in males, marked by shifts in metabolic signaling involving heightened lipid hydrolysis and proinflammatory signaling driven by IL-6 and localized pro-inflammatory CD8+ cells. This stands in stark contrast to females on a control regular diet (RD). Secondly, our findings indicate that both fat loss and fat gain in males lead to significantly elevated (1.6-fold (p ≤ 0.01) and 1.7-fold (p ≤ 0.001), respectively) Mtb burden in the lungs compared to females during Mtb infection (where fat loss and gain did not alter Mtb load in the lungs). This upsurge is associated with impaired lung lipid metabolism and intensified mitochondrial oxidative phosphorylation-regulated activity in lung CD8+ cells during Mtb infection. Additionally, our research brings to light that females exhibit a more robust systemic IFNγ (p ≤ 0.001) response than males during Mtb infection. This heightened response may either prevent active disease or contribute to latency in females during Mtb infection. In summary, our comprehensive analysis of the interplay between body fat changes and sex bias in Mtb infection reveals that alterations in body fat critically impact pulmonary pathology in males. Specifically, these changes significantly reduce the levels of pulmonary CD8+ T-cells and increase the Mtb burden in the lungs compared to females. The reduction in CD8+ cells in males is linked to an increase in mitochondrial oxidative phosphorylation and a decrease in TNFα, which are essential for CD8+ cell activation.

FIP200 promotes Mycobacterium tuberculosis pathogenesis independent of its role in autophagy

Abstract Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a critical health issue. Xenophagy, the targeting of Mtb to autophagosomes within infected cells is ineffective at controlling Mtb replication in vivo. We discovered that autophagy-associated proteins function outside of xenophagy to impact Mtb pathogenesis. We reveal a distinct autophagy-independent function of FIP200 in CD11c+ alveolar macrophages (AMs) to control Mtb burden in mice lungs at 7 days post-infection. Intra-tracheal airway labelling shows that despite the abundance of inflammatory cells, including AMs, in airway and interstitium, airway resident AMs precisely exhibit better infection control in Fip200fl/fl-LysM-Cre mice lungs.FIP200, beyond its role in autophagy, is shown to regulate signaling pathways to suppress TNF-induced apoptosis and promote cell survival. Compared to flox control, loss of Fip200 resulted in significantly higher apoptosis in AMs, in the lungs of naïve mice, which is sustained during infection. We also observed higher reactive oxygen species production by the AMs in lungs. This observation extends to Streptococcus pneumoniae infection, where loss of FIP200 in CD11c+ AMs caused similar increase in apoptosis and ROS,with reduced bacterial burden in mouse lungs. Thus, we propose a model where the FIP200 inhibits the apoptosis and ROS production in AMs, which impairs the antimicrobial defense in these cells, thus promoting Mtb infection in the airways following infection.

Adjunctive administration of parabiotic Lactobacillus sakei CVL-001 ameliorates drug-induced toxicity and pulmonary inflammation during antibiotic treatment for tuberculosis

Tuberculosis (TB) treatment requires a long therapeutic duration and induces adverse effects such as hepatotoxicity, causing discontinuation of treatment. Reduced adherence to TB medications elevates the risk of recurrence and the development of drug resistance. Additionally, severe cavitary TB with a high burden of Mycobacterium tuberculosis (Mtb) and inflammation-mediated tissue damage may need an extended treatment duration, resulting in a higher tendency of drug-induced toxicity. We previously reported that the administration of Lactobacillus sakei CVL-001 (L. sakei CVL-001) regulates inflammation and improves mucosal barrier function in a murine colitis model. Since accumulating evidence has reported the functional roles of probiotics in drug-induced liver injury and pulmonary inflammation, we employed a parabiotic form of the L. sakei CVL-001 to investigate whether this supplement may provide beneficial effects on the reduction in drug-induced liver damage and pulmonary inflammation during chemotherapy. Intriguingly, L. sakei CVL-001 administration slightly reduced Mtb burden without affecting lung inflammation and weight loss in both Mtb-resistant and -susceptible mice. Moreover, L. sakei CVL-001 decreased T cell-mediated inflammatory responses and increased regulatory T cells along with an elevated antigen-specific IL-10 production, suggesting that this parabiotic may restrain excessive inflammation during antibiotic treatment. Furthermore, the parabiotic intervention significantly reduced levels of alanine aminotransferase, an indicator of hepatotoxicity, and cell death in liver tissues. Collectively, our data suggest that L. sakei CVL-001 administration has the potential to be an adjunctive therapy by reducing pulmonary inflammation and liver damage during anti-TB drug treatment and may benefit adherence to TB medication in lengthy treatment.

G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival.

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that the coordinated action of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and SIRT6 (H3K9 deacetylase) orchestrate cholesterol build-up in in vitro and in vivo mouse models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while SIRT6 along with G9a represses the genes involved in cholesterol efflux. The accumulated cholesterol in Mtb infected macrophages promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and pharmacological inhibition in vivo; or utilization of BMDMs derived from Sirt6-/- mice or in vivo infection in haplo-insufficient Sirt6-/+ mice; hampered host cholesterol accumulation and restricted Mtb burden. These findings shed light on the novel roles of G9a and SIRT6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

Blood transcriptional correlates of BCG-induced protection against tuberculosis in rhesus macaques

Blood-based correlates of vaccine-induced protection against tuberculosis (TB) are urgently needed. Here, we analyze the blood transcriptome of rhesus macaques immunized with varying doses of intravenous (i.v.) BCG followed by Mycobacterium tuberculosis (Mtb) challenge. We use high-dose i.v. BCG recipients for "discovery" and validate our findings in low-dose recipients and in an independent cohort of macaques receiving BCG via different routes. We identify seven vaccine-induced gene modules, including an innate module (module 1) enriched for type 1 interferon and RIG-I-like receptor signaling pathways. Module 1 on day 2 post-vaccination highly correlates with lung antigen-responsive CD4 Tcells at week 8 and with Mtb and granuloma burden following challenge. Parsimonious signatures within module 1 at day 2 post-vaccination predict protection following challenge with area under the receiver operating characteristic curve (AUROC) ≥0.91. Together, these results indicate that the early innate transcriptional response to i.v. BCG in peripheral blood may provide a robust correlate of protection against TB.

Long-term effects of prenatal arsenic exposure from gestational day 9 to birth on lung, heart, and immune outcomes in the C57BL/6 mouse model

Prenatal arsenic exposure is a major public health concern, associated with altered birth outcomes and increased respiratory disease risk. However, characterization of the long-term effects of mid-pregnancy (second trimester) arsenic exposure on multiple organ systems is scant. This study aimed to characterize the long-term impact of mid-pregnancy inorganic arsenic exposure on the lung, heart, and immune system, including infectious disease response using the C57BL/6 mouse model. Mice were exposed from gestational day 9 till birth to either 0 or 1000 µg/L sodium (meta)arsenite in drinking water. Male and female offspring assessed at adulthood (10–12 weeks of age) did not show significant effects on recovery outcomes after ischemia reperfusion injury but did exhibit increased airway hyperresponsiveness compared to controls. Flow cytometric analysis revealed significantly greater total numbers of cells in arsenic-exposed lungs, lower MHCII expression in natural killer cells, and increased percentages of dendritic cell populations. Activated interstitial (IMs) and alveolar macrophages (AMs) isolated from arsenic-exposed male mice produced significantly less IFN-γ than controls. Conversely, activated AMs from arsenic-exposed females produced significantly more IFN-γ than controls. Although systemic cytokine levels were higher upon Mycobacterium tuberculosis (Mtb) infection in prenatally arsenic-exposed offspring there was no difference in lung Mtb burden compared to controls. This study highlights significant long-term impacts of prenatal arsenic exposure on lung and immune cell function. These effects may contribute to the elevated risk of respiratory diseases associated with prenatal arsenic exposure in epidemiology studies and point to the need for more research into mechanisms driving these maintained responses.

Mycobacterium tuberculosis exploits FIP200 regulated cell-death pathways to promote replication

Abstract Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a leading health concern. The rise in antibacterial resistance has steered focus to developing host directed therapies, which requires an in-depth understanding of Mtb interactions with the host immune system. Xenophagy, the targeting of Mtb to autophagosomes within infected cells, is not effective at controlling Mtb replication in vivo, however, we have discovered that autophagy proteins function outside of xenophagy to impact Mtb pathogenesis. FIP200 is an autophagy pathway protein required for autophagosome formation. In this study, we identified a unique function of FIP200 in promoting Mtb infection in mice that is independent of its role in autophagy. We show that the loss of Fip200, but not the loss of other autophagy genes, in LysM +innate immune cells results in lower Mtb burden in the lungs of mice by 7 dpi. Fip200 fl/fl-LysM-Cre mice lungs harbor higher numbers of neutrophils, macrophages, dendritic cells (DCs), and activated CD4 +T cells relative to Fip200 fl/flmice. Depletion of either neutrophils or T cells did not revert the bacterial burdens, showing that these cells do not contribute to the enhanced control of Mtb. We show that FIP200 is specifically required in CD11c +macrophages and/or DCs during infection. FIP200 has been reported to regulate several signaling pathways to suppress apoptosis and promote cell survival. We observed increased macrophage apoptosis in the airways of Fip200 fl/fl-LysM-Cre mice compared to Fip200 fl/flcontrols in response to Mtb infection. Based on our data, we propose a model where loss of FIP200, but not other autophagy proteins, in CD11c +macrophages leads to increased apoptosis and better control of Mtb replication. NIH NIAID R01 #AI132697 NIH NIAID U19 #AI142784 ND supported with Alexander & Gertrude Berg Fellow

T-cell derived interferon gamma is required for restriction of Mycobacterium tuberculosis in the murine lung: A reappraisal of conflicting models

Abstract An effective vaccine for Mycobacterium tuberculosis (Mtb) is urgently needed, yet its development has been hampered by incomplete understanding of immunity to Mtb. While vaccine candidates are routinely assessed by their ability to induce CD4 +T cell-derived interferon gamma (IFNγ), this marker has been unreliable in predicting vaccine protection against TB. Furthermore, T cell adoptive transfer studies in RAG1 −/−mice have suggested that lung T cell-derived IFNγ only plays a minor role in pulmonary immunity against Mtb. In contrast with prior results, our experiments using IFNγ−/− T cell chimeric mice, and with TCRβ −/−δ −/−mice receiving adoptively transferred IFNγ −/−T cells, demonstrate that control of lung Mtb burden is in fact dependent on T cell-derived IFNγ. We show that this discrepancy is partially due to the confounding effects of RAG −/−vs. the more specific TCRβ −/−δ −/−hosts. Flow cytometry and RNA-seq analysis of lung cells in WT, IFNγ −/−, and TCRβ −/−δ −/−T cell-chimeric mice reveal that absence of T cell-derived IFNγ decreases recruitment of monocyte-derived macrophages to the infected lung, skews infected and bystander macrophages to an alternative M2 phenotype, and promotes neutrophil and eosinophil influx. Together, these results reassert and characterize the role of T cell-derived IFNγ in protection against pulmonary Mtb. Supported by grants from NIH (T32 AI007044, U19 AI135976) and the Firland Foundation (Northrop-Park Fellowship).

A novel self-adjuvanted mRNA vaccine expressing the Antigen-85B-p25 epitope of Mycobacterium tuberculosisprotects mice against tuberculosis

Abstract Mycobacterium tuberculosis (Mtb) is a leading cause of death due to infections. BCG is the most used vaccine despite having partial efficacy against the tuberculosis. We have developed a synthetic mRNA vaccine, SelmRp25-C5, which expresses the p25 epitope of Mtb-derived Antigen-85B and a TLR-2 stimulating C5 adjuvant peptide from CFP-10 protein. We evaluated its efficacy both ex vivo and in mice. Methods: Mouse and human macrophages (MΦ) and mouse dendritic cells (DCs) were treated with SelmRp25-C5 vaccine or EGFP-mRNA control followed by antigen presentation assays and assessment of cytokine secretion. C57Bl/6 mice were vaccinated subcutaneously with two doses (5μg per mouse) of SelmRp25-C5 vaccine, or EGFP-mRNA and one dose of subcutaneous BCG (10 6CFU per mouse) as a control, followed by aerosol infection with Mtb. Four weeks after Mtb challenge, mice were sacrificed, and organs were analyzed for Mtb CFU, RNAseq, Ag85B-p25 tetramer-specific CD4 T cells and memory T cells. Results: SelmRp25-C5 vaccine induced robust antigen presentation to T cells in both mouse MΦs and DCs and in human MΦs, and enhanced Th1 cytokine secretion. Two doses of SelmRp25-C5 vaccine protected mice by reducing Mtb burden by 1log 10. Protection induced by SelmRp25-C5 vaccine was associated with an increase in Ag85B-p25-specific CD4 T cells, increased IFN-γ and IL-2 secreting T cells and CD4 effector memory T cells. Lung and spleens of protected mice showed an upregulation of Spleen tyrosine kinase (Syk) and genes of the C-type lectin receptor-pathway compared to control mice. Conclusions: We propose that the new generation self-adjuvanted mRNA vaccine strengthen both innate and adaptive immunity to provide protection against tuberculosis. Supported by NIAID, NIH AI122070, AI138587; AI161015.

Host Immunity to Mycobacterium tuberculosis Infection Is Similar in Simian Immunodeficiency Virus (SIV)-Infected, Antiretroviral Therapy-Treated and SIV-Naïve Juvenile Macaques.

Pre-existing HIV infection increases tuberculosis (TB) risk in children. Antiretroviral therapy (ART) reduces, but does not abolish, this risk in children with HIV. The immunologic mechanisms involved in TB progression in both HIV-naive and HIV-infected children have not been explored. Much of our current understanding is based on human studies in adults and adult animal models. In this study, we sought to model childhood HIV/Mycobacterium tuberculosis (Mtb) coinfection in the setting of ART and characterize T cells during TB progression. Macaques equivalent to 4 to 8 year-old children were intravenously infected with SIVmac239M, treated with ART 3 months later, and coinfected with Mtb 3 months after initiating ART. SIV-naive macaques were similarly infected with Mtb alone. TB pathology and total Mtb burden did not differ between SIV-infected, ART-treated and SIV-naive macaques, although lung Mtb burden was lower in SIV-infected, ART-treated macaques. No major differences in frequencies of CD4+ and CD8+ T cells and unconventional T cell subsets (Vγ9+ γδ T cells, MAIT cells, and NKT cells) in airways were observed between SIV-infected, ART-treated and SIV-naive macaques over the course of Mtb infection, with the exception of CCR5+ CD4+ and CD8+ T cells which were slightly lower. CD4+ and CD8+ T cell frequencies did not differ in the lung granulomas. Immune checkpoint marker levels were similar, although ki-67 levels in CD8+ T cells were elevated. Thus, ART treatment of juvenile macaques, 3 months after SIV infection, resulted in similar progression of Mtb and T cell responses compared to Mtb in SIV-naive macaques.

Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

Changes of C-reactive protein and Procalcitonin after four weeks of treatment in patients with pulmonary TB

ObjectiveTuberculosis (TB) remains a public health concern worldwide, affecting millions of people every year. Detailed characterization of disease pathophysiology is key to proper diagnosis, disease progression, or treatment follow-up and evaluation. The present study investigated C-reactive protein and Procalcitonin (PCT) as candidate markers of early treatment response and disease activity. MethodsFrom September to December 2019, 21 HIV-negative consecutive TB patients were recruited, within the setting of the Gabonese TB specialized hospital and the National Laboratory of Public Health, in a prospective study. CRP and PCT levels were measured by chemiluminescence at diagnosis and 4 weeks following the initiation of anti-TB treatment. ResultsThe mean concentration of CRP in TB patients was 114.7 mg/L (95 % CI: [83.8–145.6]) at diagnosis and 20.2 mg/L (95 % CI: [14.1–26.4]) 4 weeks following anti-TB treatment. The drop in CRP concentrations between diagnosis, and week 4 following anti-TB treatment showed was significant (p < 0.0001). The average concentration of PCT at the time of diagnosis was 0.3 ng/mL (95 % CI: [0.19–0.41]). PCT Concentration dropped below 0.05 ng/mL 4 weeks following the start of anti-TB treatment (p < 0.01). ConclusionCRP and PCT are potential TB biomarkers, each, carrying important keys. If the drop in both proteins may indicate a significant reduction of the Mtb burden, the maintenance of CRP above the inflammation threshold could indicate the presence of residual bacilli. However, the clinical translation of the present finding will require more investigation.

Mucosal exposure to non-tuberculous mycobacteria elicits B cell-mediated immunity against pulmonary tuberculosis

Bacille Calmette-Guerin (BCG) is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease. However, BCG has limited efficacy, necessitating the development of better vaccines. Non-tuberculous mycobacteria (NTMs) are opportunistic pathogens present ubiquitously in the environment. TB endemic countries experience higher exposure to NTMs, but previous studies have not elucidated the relationship between NTM exposure and BCG efficacy against TB. Therefore, we develop a mouse model (BCG+ NTM) to simulate human BCG immunization regime and continuous NTM exposure. BCG+ NTM mice exhibit superior and prolonged protection against pulmonary TB, with increased B cell influx and anti-Mtb antibodies in serum and airways, compared with BCG alone. Notably, spatial transcriptomics and immunohistochemistry reveal that BCG+ NTM mice formed B cell aggregates with features of germinal center development, which correlate with reduced Mtb burden. Our studies suggest a direct relationship between NTM exposure and TB protection, with B cells playing a crucial role.

An intranasal stringent response vaccine targeting dendritic cells as a novel adjunctive therapy against tuberculosis.

Lengthy tuberculosis (TB) treatment is required to overcome the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine containing a fusion of the relMtb gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3α/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that intramuscular delivery of the MIP-3α/relMtb (fusion) vaccine or intranasal delivery of the relMtb (non-fusion) vaccine potentiate isoniazid activity more than intramuscular delivery of the DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 and 0.5 log10 colony-forming units, respectively; P=0.0002 and P=0.0052), inducing pronounced Mtb-protective immune signatures. The combined approach involving intranasal delivery of the DNA MIP-3α/relMtb fusion vaccine demonstrated the greatest mycobactericidal activity together with isoniazid when compared to each approach alone (absolute reduction of Mtb burden: 1.13 log10, when compared to the intramuscular vaccine targeting relMtb alone; P<0.0001), as well as robust systemic and local Th1 and Th17 responses. This DNA vaccination strategy may be a promising adjunctive approach combined with standard therapy to shorten curative TB treatment, and also serves as proof of concept for treating other chronic bacterial infections.