RESUMO
Despite recent improvements in microbial detection, smear-negative TB remains a diagnostic challenge. In this study, we investigated the potential discriminatory role of polychromatic flow cytometry of M. tuberculosis antigen-specific T cells to discriminate smear-negative TB from health controls with or without latent TB infection, and non-TB respiratory illnesses in an endemic setting. A cross-sectional study was conducted on HIV negative, newly diagnosed smear-positive PTB (n = 34), smear-negative/GeneXpert negative PTB (n = 29) patients, non-TB patients with respiratory illness (n = 33) and apparently healthy latent TB infected (n = 30) or non-infected (n = 23) individuals. The expression of activation (HLA-DR, CD-38), proliferation (Ki-67), and functional (IFN-γ, TNF-α) T-cell markers using polychromatic flow cytometry was defined after stimulation with PPD antigens. Sputum samples were collected and processed from all patients for Mtb detection using a concentrated microscopy, LJ/MGIT culture, and RD9 typing by PCR. Our study showed CD4 T cells specific for PPD co-expressed activation/proliferation markers together with induced cytokines IFN-γ or TNF-α were present at substantially higher levels among patients with smear-positive and smear-negative pulmonary TB than among healthy controls and to a lesser extent among patients with non-TB illness. Our study conclude that smear-negative TB can be distinguished from non-TB respiratory illness and healthy controls with a flow cytometric assay for PPD-specific T cells co-expressing activation/proliferation markers and cytokines.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose Pulmonar , Antígenos de Bactérias , Estudos Transversais , Citocinas/metabolismo , Humanos , Tuberculose Latente/diagnóstico , Tuberculose Latente/microbiologia , Escarro/microbiologia , Tuberculina , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Fator de Necrose Tumoral alfaRESUMO
Rationale: Direct evidence for persistence of Mycobacterium tuberculosis (Mtb) during asymptomatic latent tuberculosis infection (LTBI) in humans is currently lacking. Moreover, although a 12-week regimen of once-weekly isoniazid and rifapentine (3HP) is currently recommended by the CDC as treatment for LTBI, experimental evidence for 3HP-mediated clearance of persistent Mtb infection in human lungs has not been established.Objectives: Using a nonhuman primate (NHP) model of TB, we sought to assess 3HP treatment-mediated clearance of Mtb infection in latently infected macaques.Methods: Sixteen NHPs were infected via inhalation with â¼10 cfu of Mtb CDC1551, after which asymptomatic animals were either treated with 3HP or left untreated. Pharmacokinetics of the 3HP regimen were measured. Following treatment, animals were coinfected with simian immunodeficiency virus to assess reactivation of LTBI and development of active TB disease.Measurements and Main Results: Fourteen NHPs remained free of clinical signs or microbiological evidence of active TB following infection with Mtb and were subsequently either treated with 3HP (n = 7) or left untreated (n = 7). Untreated NHPs were asymptomatic for 7 months but harbored persistent Mtb infection, as shown by reactivation of latent infection following simian immunodeficiency virus coinfection. However, none of the treated animals developed TB reactivation disease, and they remained without clinical or microbiological evidence of persistent bacilli, suggesting treatment-mediated clearance of bacteria.Conclusions:Mtb can persist in asymptomatic macaques for at least 7 months. Furthermore, 3HP treatment effectively cleared bacteria and prevented reactivation of TB in latently infected macaques.
Assuntos
Antibióticos Antituberculose/uso terapêutico , Antituberculosos/uso terapêutico , Isoniazida/uso terapêutico , Tuberculose Latente/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/análogos & derivados , Tuberculose/tratamento farmacológico , Animais , Quimioterapia Combinada , Macaca , Modelos Animais , Rifampina/uso terapêutico , Resultado do TratamentoRESUMO
Mycobacterium tuberculosis continues to cause devastating levels of mortality due to tuberculosis (TB). The failure to control TB stems from an incomplete understanding of the highly specialized strategies that M. tuberculosis utilizes to modulate host immunity and thereby persist in host lungs. Here, we show that M. tuberculosis induced the expression of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan catabolism, in macrophages and in the lungs of animals (mice and macaque) with active disease. In a macaque model of inhalation TB, suppression of IDO activity reduced bacterial burden, pathology, and clinical signs of TB disease, leading to increased host survival. This increased protection was accompanied by increased lung T cell proliferation, induction of inducible bronchus-associated lymphoid tissue and correlates of bacterial killing, reduced checkpoint signaling, and the relocation of effector T cells to the center of the granulomata. The enhanced killing of M. tuberculosis in macrophages in vivo by CD4+ T cells was also replicated in vitro, in cocultures of macaque macrophages and CD4+ T cells. Collectively, these results suggest that there exists a potential for using IDO inhibition as an effective and clinically relevant host-directed therapy for TB.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Pulmão/imunologia , Mycobacterium tuberculosis/imunologia , Triptofano/imunologia , Tuberculoma/imunologia , Tuberculose Pulmonar/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Granuloma/imunologia , Granuloma/patologia , Pulmão/patologia , Macaca mulatta , Macrófagos/imunologia , Macrófagos/patologia , Mycobacterium tuberculosis/patogenicidade , Tuberculoma/patologia , Tuberculose Pulmonar/patologiaRESUMO
BACKGROUND: Diagnosis of active tuberculosis (ATB) currently relies on detection of Mycobacterium tuberculosis (Mtb). Identifying patients with extrapulmonary TB (EPTB) remains challenging because microbiological confirmation is often not possible. Highly accurate blood-based tests could improve diagnosis of both EPTB and pulmonary TB (PTB) and timely initiation of anti-TB therapy. METHODS: A case-control study was performed using discriminant analyses to validate an approach using Mtb-specific CD4+T-cell activation markers in blood to discriminate PTB and EPTB from latent TB infection (LTBI) as well as EPTB from PTB in 270 Brazilian individuals. We further tested the effect of human immunodeficiency virus (HIV) coinfection on diagnostic performance. Frequencies of interferon-γâ+CD4+T cells expressing CD38, HLADR, and/or Ki67 were assessed by flow cytometry. RESULTS: EPTB and PTB were associated with higher frequencies of CD4+T cells expressing CD38, HLADR, or Ki67 compared with LTBI (all P values < .001). Moreover, frequencies of HLADR+ (P = .03) or Ki67+ (P < .001) cells accurately distinguished EPTB from PTB. HIV infection did not affect the capacity of these markers to distinguish ATB from LTBI or EPTB from PTB. CONCLUSIONS: Cell activation markers in Mtb-specific CD4+T cells distinguished ATB from LTBI and EPTB from PTB, regardless of HIV infection status. These parameters provide an attractive approach for developing blood-based diagnostic tests for both active and latent TB.
Assuntos
Infecções por HIV , Infecção Latente , Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose Pulmonar , Brasil , Linfócitos T CD4-Positivos , Estudos de Casos e Controles , Testes Diagnósticos de Rotina , Infecções por HIV/diagnóstico , Humanos , Tuberculose Latente/diagnóstico , Tuberculose Pulmonar/diagnósticoRESUMO
Antigen-specific CD4 and CD8 T cells are important components of the immune response to Mycobacterium tuberculosis, yet little information is currently known regarding how the breadth, specificity, phenotype, and function of M. tuberculosis-specific T cells correlate with M. tuberculosis infection outcome in humans. To facilitate evaluation of human M. tuberculosis-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60 M. tuberculosis Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 µl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of M. tuberculosis-unexposed healthy adults, foreign-born adults with latent M. tuberculosis infection residing in the United States, and tuberculosis household contacts with latent M. tuberculosis infection in a tuberculosis-endemic setting in Kenya. The M. tuberculosis-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating M. tuberculosis-specific T cell responses across different states of M. tuberculosis infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of M. tuberculosis-specific T cell responses associated with M. tuberculosis infection outcomes.
Assuntos
Testes Hematológicos/métodos , Ensaios de Triagem em Larga Escala/métodos , Linfócitos T/imunologia , Tuberculose/sangue , Tuberculose/imunologia , Estudos Transversais , Humanos , Técnicas Imunológicas/métodos , Estudos Longitudinais , Reprodutibilidade dos TestesRESUMO
Mycobacterium tuberculosis (Mtb) impairs dendritic cell (DC) functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper cells producing IFN-γ (Th1) and IL-17 (Th17) are important for protecting against tuberculosis (TB), but the mechanisms by which DCs generate antigen-specific T-helper responses during Mtb infection are not well defined. We previously reported that Mtb impairs CD40 expression on DCs and restricts Th1 and Th17 responses. We now demonstrate that CD40-dependent costimulation is required to generate IL-17 responses to Mtb. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced antigen-specific IL-17 responses. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, we show that boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that CD40 costimulation by DCs plays an important role in generating antigen-specific Th17 cells and targeting the CD40-CD40L pathway represents a novel strategy to improve adaptive immunity to TB.
Assuntos
Antígenos CD40/imunologia , Ligante de CD40/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Tuberculose/imunologia , Animais , Técnicas de Cocultura , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/imunologiaRESUMO
Mycobacterium tuberculosis successfully subverts the host immune response to promote disease progression. In addition to its known intracellular niche in macrophages, M. tuberculosis interferes with the functions of dendritic cells (DCs), which are the primary antigen-presenting cells of the immune system. We previously showed that M. tuberculosis dampens proinflammatory responses and impairs DC functions through the cell envelope-associated serine protease Hip1. Here we present data showing that M. tuberculosis GroEL2, a substrate of Hip1, modulates DC functions. The full-length GroEL2 protein elicited robust proinflammatory responses from DCs and promoted DC maturation and antigen presentation to T cells. In contrast, the cleaved form of GroEL2, which predominates in M. tuberculosis, was poorly immunostimulatory and was unable to promote DC maturation and antigen presentation. Moreover, DCs exposed to full-length, but not cleaved, GroEL2 induced strong antigen-specific gamma interferon (IFN-γ), interleukin-2 (IL-2), and IL-17A cytokine responses from CD4+ T cells. Moreover, the expression of cleaved GroEL2 in the hip1 mutant restored the robust T cell responses to wild-type levels, suggesting that proteolytic cleavage of GroEL2 allows M. tuberculosis to prevent optimal DC-T cell cross talk during M. tuberculosis infection.
Assuntos
Chaperonina 60/metabolismo , Células Dendríticas/imunologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Mycobacterium tuberculosis/imunologia , Tuberculose/patologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Proteólise , Tuberculose/microbiologiaRESUMO
The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity.
Assuntos
Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Serina Proteases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Dicroísmo Circular , Cristalografia por Raios X , Estabilidade Enzimática , Metionina/química , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Selenometionina/química , Serina Proteases/química , Serina Proteases/genética , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.
Assuntos
Proteínas de Bactérias/fisiologia , Chaperonina 60/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Mycobacterium tuberculosis/enzimologia , Serina Endopeptidases/fisiologia , Serina Proteases/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Células Cultivadas , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Ligação Proteica , Multimerização Proteica , Proteólise , Serina Endopeptidases/metabolismo , Serina Proteases/metabolismoRESUMO
Mycobacterium tuberculosis is a highly successful human pathogen that primarily resides in host phagocytes, such as macrophages and dendritic cells (DCs), and interferes with their functions. Although multiple strategies used by M. tuberculosis to modulate macrophage responses have been discovered, interactions between M. tuberculosis and DCs are less well understood. DCs are the primary APCs of the immune system and play a central role in linking innate and adaptive immune responses to microbial pathogens. In this study, we show that M. tuberculosis impairs DC cytokine secretion, maturation, and Ag presentation through the cell envelope-associated serine hydrolase, Hip1. Compared to wild-type, a hip1 mutant strain of M. tuberculosis induced enhanced levels of the key Th1-inducing cytokine IL-12, as well as other proinflammatory cytokines (IL-23, IL-6, TNF-α, IL-1ß, and IL-18) in DCs via MyD88- and TLR2/9-dependent pathways, indicating that Hip1 restricts optimal DC inflammatory responses. Infection with the hip1 mutant also induced higher levels of MHC class II and costimulatory molecules CD40 and CD86, indicating that M. tuberculosis impairs DC maturation through Hip1. Further, we show that M. tuberculosis promotes suboptimal Ag presentation, as DCs infected with the hip1 mutant showed increased capacity to present Ag to OT-II- and early secreted antigenic target 6-specific transgenic CD4 T cells and enhanced Th1 and Th17 polarization. Overall, these data show that M. tuberculosis impairs DC functions and modulates the nature of Ag-specific T cell responses, with important implications for vaccination strategies.
Assuntos
Proteínas de Ligação a DNA/imunologia , Células Dendríticas/imunologia , Infecções por Mycobacterium/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Apresentação de Antígeno/imunologia , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/enzimologia , Células Dendríticas/microbiologia , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Human immunodeficiency virus (HIV)-infected individuals with latent Mycobacterium tuberculosis infection have substantially higher rates of progression to active tuberculosis than HIV-uninfected individuals with latent tuberculosis. To explore HIV-induced deficits in M. tuberculosis-specific CD8+ T-cell functions, we compared interferon γ production, degranulation, and proliferation of CD8+ T cells in response to M. tuberculosis peptides (ESAT-6/CFP-10) between HIV-infected (median CD4+ T-cell count, 522 cells/µL; interquartile range, 318-585 cells/µL) and HIV-uninfected individuals with latent tuberculosis from South Africa. We found that M. tuberculosis-specific degranulation and proliferative capacities were impaired in the HIV-infected group. Thus, our results suggest that HIV coinfection compromises CD8+ T-cell functions in latent tuberculosis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/complicações , Infecções por HIV/fisiopatologia , Tuberculose Latente/complicações , Tuberculose Latente/fisiopatologia , Adulto , Linfócitos T CD8-Positivos/fisiologia , Degranulação Celular , Proliferação de Células , Células Cultivadas , Coinfecção/epidemiologia , Coinfecção/imunologia , Coinfecção/fisiopatologia , Feminino , Infecções por HIV/epidemiologia , Humanos , Tuberculose Latente/epidemiologia , Tuberculose Latente/imunologia , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Adulto JovemRESUMO
Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the Δ-dosR, Δ-dosS, and Δ-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although Δ-dosR and Δ-dosT grew comparably to wild-type Mtb, Δ-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. Δ-dosS retained the ability to induce DosR. The Δ-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of Δ-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of Δ-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.
Assuntos
Proteínas de Bactérias/genética , Granuloma do Sistema Respiratório/microbiologia , Mycobacterium tuberculosis/patogenicidade , Protamina Quinase/genética , Tuberculose Pulmonar/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Hipóxia Celular , Células Cultivadas , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Masculino , Camundongos Endogâmicos C3H , Viabilidade Microbiana , Mycobacterium tuberculosis/genética , Protamina Quinase/metabolismo , Regulon , VirulênciaAssuntos
Pesquisa Biomédica/métodos , Erradicação de Doenças/métodos , Tuberculose Pulmonar/prevenção & controle , Antituberculosos/administração & dosagem , Antituberculosos/uso terapêutico , Progressão da Doença , Epidemias/prevenção & controle , Prioridades em Saúde , Humanos , Tuberculose Latente/diagnóstico , Tuberculose Latente/patologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/patologiaRESUMO
CD4 T cells are essential for immunity to M. tuberculosis (Mtb), and emerging evidence indicates that IL-17-producing Th17 cells contribute to immunity to Mtb. While identifying protective T cell effector functions is important for TB vaccine design, T cell antigen specificity is also likely to be important. To identify antigens that induce protective immunity, we reasoned that as in other pathogens, effective immune recognition drives sequence diversity in individual Mtb antigens. We previously identified Mtb genes under evolutionary diversifying selection pressure whose products we term Rare Variable Mtb Antigens (RVMA). Here, in two distinct human cohorts with recent exposure to TB, we found that RVMA preferentially induce CD4 T cells that express RoRγt and produce IL-17, in contrast to 'classical' Mtb antigens that induce T cells that produce IFNγ. Our results suggest that RVMA can be valuable antigens in vaccines for those already infected with Mtb to amplify existing antigen-specific Th17 responses to prevent TB disease.
RESUMO
Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-γ), l-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-γ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.
Assuntos
Macrófagos/metabolismo , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Óxido Nítrico/biossíntese , Arginina/farmacologia , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Linhagem Celular , Humanos , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Nitrogênio/metabolismoRESUMO
BACKGROUND: T cell activation (HLA-DR, CD-38), proliferation (KI-67), and functional (IFN-γ, TNF-α) markers have recently been shown to be useful in predicting and monitoring anti-TB responses in smear positive TB, but previous research did not characterize the activation and proliferation profiles after therapy of smear negative TB. METHODOLOGY: In this study, we used polychromatic flow cytometry to assess selected PPD-specific T cell markers using fresh PBMC of smear negative and positive pulmonary tuberculosis (PTB) patients, recruited from health facilities in Addis Ababa. RESULT: Levels of activation (HLA-DR, CD38) and proliferation (Ki-67) among total unstimulated CD4 T cells decreased significantly after therapy, particularly at month 6. Similarly, levels of PPD-specific T cell activation markers (HLA-DR, CD-38) were significantly lower in smear positive PTB patients following treatment, whereas a consistent decline in these markers was less apparent among smear negative PTB patients at the sixth month. CONCLUSION: After six months of standard anti-TB therapy, persistent levels of activation of HLA-DR and CD-38 from PPD specific CD4+T cells in this study could indicate that those markers have little value in monitoring and predicting anti-TB treatment response in smear negative pulmonary TB patients in Ethiopian context.
Assuntos
Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Tuberculose Pulmonar , Linfócitos T CD4-Positivos , Etiópia , Antígenos HLA-DR/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Leucócitos Mononucleares/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculina/metabolismo , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/metabolismoRESUMO
Early Th17 responses are necessary to provide protection against Mycobacterium tuberculosis (Mtb). Mtb impedes Th17 polarization by restricting CD40 co-stimulatory pathway on dendritic cells (DCs). We previously demonstrated that engaging CD40 on DCs increased Th17 responses. However, the molecular mechanisms that contributed to Th17 polarization were unknown. Here, we identify the Notch ligand DLL4 as necessary for Th17 polarization and demonstrate that Mtb limits DLL4 on DCs to prevent optimal Th17 responses. Although Mtb infection induced only low levels of DLL4, engaging CD40 on DCs increased DLL4 expression. Antibody blockade of DLL4 on DCs reduced Th17 polarization in vitro and in vivo. In addition, we show that the Mtb Hip1 protease attenuates DLL4 expression on lung DCs by impeding CD40 signaling. Overall, our results demonstrate that Mtb impedes CD40-dependent DLL4 expression to restrict Th17 responses and identify the CD40-DLL4 pathways as targets for developing new Th17-inducing vaccines and adjuvants for tuberculosis.
RESUMO
Studies using the nonhuman primate model of Mycobacterium tuberculosis/simian immunodeficiency virus coinfection have revealed protective CD4+ T cell-independent immune responses that suppress latent tuberculosis infection (LTBI) reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV coinfection. Here, we administered combinatorial antiretroviral therapy (cART) 2 weeks after SIV coinfection to study whether restoration of CD4+ T cell immunity occurred more broadly, and whether this prevented reactivation of LTBI compared to cART initiated 4 weeks after SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication, and reduced immune activation in the periphery and lung vasculature, thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed after SIV coinfection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The potential novelty of these findings mainly relates to the development of a robust animal model of human M. tuberculosis/HIV coinfection that allows the testing of underlying mechanisms.
Assuntos
Antirretrovirais/farmacologia , Coinfecção , Tuberculose Latente/metabolismo , Mycobacterium tuberculosis/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia/metabolismo , Animais , Coinfecção/tratamento farmacológico , Coinfecção/metabolismo , Coinfecção/microbiologia , Coinfecção/virologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/microbiologiaRESUMO
A once-weekly oral dose of isoniazid and rifapentine for 3 months (3HP) is recommended by the CDC for treatment of latent tuberculosis infection (LTBI). The aim of this study is to assess 3HP-mediated clearance of M. tuberculosis bacteria in macaques with asymptomatic LTBI. Twelve Indian-origin rhesus macaques were infected with a low dose (~10 CFU) of M. tuberculosis CDC1551 via aerosol. Six animals were treated with 3HP and 6 were left untreated. The animals were imaged via PET/CT at frequent intervals. Upon treatment completion, all animals except 1 were coinfected with SIV to assess reactivation of LTBI to active tuberculosis (ATB). Four of 6 treated macaques showed no evidence of persistent bacilli or extrapulmonary spread until the study end point. PET/CT demonstrated the presence of significantly more granulomas in untreated animals relative to the treated group. The untreated animals harbored persistent bacilli and demonstrated tuberculosis (TB) reactivation following SIV coinfection, while none of the treated animals reactivated to ATB. 3HP treatment effectively reduced persistent infection with M. tuberculosis and prevented reactivation of TB in latently infected macaques.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Animais , Antituberculosos/farmacologia , Isoniazida/farmacologia , Tuberculose Latente/tratamento farmacológico , Tuberculose Latente/microbiologia , Pulmão , Macaca mulatta , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Rifampina/análogos & derivadosRESUMO
New tuberculosis vaccine candidates that are in the development pipeline need to be studied in people with HIV, who are at high risk of acquiring Mycobacterium tuberculosis infection and tuberculosis disease and tend to develop less robust vaccine-induced immune responses. To address the gaps in developing tuberculosis vaccines for people with HIV, a series of symposia was held that posed six framing questions to a panel of international experts: What is the use case or rationale for developing tuberculosis vaccines? What is the landscape of tuberculosis vaccines? Which vaccine candidates should be prioritised? What are the tuberculosis vaccine trial design considerations? What is the role of immunological correlates of protection? What are the gaps in preclinical models for studying tuberculosis vaccines? The international expert panel formulated consensus statements to each of the framing questions, with the intention of informing tuberculosis vaccine development and the prioritisation of clinical trials for inclusion of people with HIV.