RESUMEN
BACKGROUND: Natural immunity against Mycobacterium tuberculosis exists, and > 90% of those infected remain disease-free. Innate and adaptive immune responses required to mediate such protection against tuberculosis (TB) are, however, poorly understood. METHODS: This is an analytical study exploring protective and non-protective pathways of immunity against Mycobacterium tuberculosis. Adults without HIV infection are recruited at community healthcare clinics in high TB incidence areas of the Western Cape Province, South Africa. Data regarding participants' medical, social and medication usage will be collected, and clinical examinations and point-of-care tests documented. Reference tests for TB (chest radiographs and sputum tests for GeneXpert MTB/RIF Ultra®, Auramine smear and liquid cultures) and investigations to classify infection states [interferon-gamma release assay (IGRA) and SARS-CoV-2 polymerase chain reaction (PCR) nasopharyngeal swab and IgG], are done on all participants who meet the inclusion criteria. 18F-Fluorodeoxyglucose positron emission tomography combined with computerized tomography will be done on all close contacts (contacts) and healthy control (controls) participants. Participants are divided into 12 study groups representing a spectrum of TB clinical phenotypes and prior SARS-CoV-2 infection based on their TB status, exposure history, results of IGRA test at baseline and 3 months, SARS-CoV-2 serology, and PCR results, and for contacts and controls, PET-CT imaging findings indicative of sub-clinical TB lesions. Samples for experimental assays include whole blood for isolation of peripheral blood mononuclear cells and blood in PAXgene® tubes for RNA isolation. All SARS-CoV-2 PCR negative study participants undergo bronchoscopy for collecting bronchoalveolar lavage samples. DISCUSSION: The paired blood and BAL samples will be used for comprehensive analyses of the tissue-specific and systemic immunity that will include e.g., cytometry by time-of-flight analyses, RNA-sequencing, multiplex immunoassays, epigenetic analysis, and mechanistic studies of control of infection by Mycobacterium tuberculosis. Results will be integrated with those from mice and non-human primate studies to provide a comprehensive analysis of protective pathways in natural and vaccine-induced immunity against Mycobacterium tuberculosis.
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COVID-19 , Infecciones por VIH , Mycobacterium tuberculosis , Tuberculosis Ganglionar , Animales , Infecciones por VIH/epidemiología , Humanos , Leucocitos Mononucleares , Ratones , Tomografía Computarizada por Tomografía de Emisión de Positrones , ARN , SARS-CoV-2 , Sudáfrica/epidemiologíaRESUMEN
OBJECTIVES: The bacille Calmette-Guérin (BCG) vaccine is usually administered at birth to protect against severe forms of tuberculosis in children. BCG also confers some protection against other infections, possibly mediated by innate immune training. We investigated whether newborn BCG vaccination modulates myeloid and natural killer (NK) cell responses to mycobacteria. METHODS: BCG vaccination was either administered at birth or delayed to 6 or 10 weeks of age in 130 South African infants. Whole blood was stimulated with BCG and clusters of differentiation (CD)4+ T, myeloid, and NK cell responses were measured by flow cytometry; the levels of secreted cytokines were measured by a multiplex bead array. RESULTS: Newborn BCG vaccination was associated with significantly higher frequencies of BCG-reactive, cytokine-expressing CD4+ T cells, and interferon (IFN)-γ-expressing NK cells than in unvaccinated infants but no differences in cytokine-expressing CD33+ myeloid cells were observed. The induction of BCG-reactive IFN-γ-expressing NK cells was not associated with the markers of NK cell maturation, differentiation, or cytokine receptor expression. BCG-reactive NK cell responses correlated directly with the levels of secreted interleukin (IL)-2 and IFN-γ and the innate pro-inflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor (TNF) in BCG-vaccinated infants only. CONCLUSION: We showed that BCG-reactive IFN-γ-expressing NK cells are strongly induced by BCG vaccination in infants and are likely amplified through bystander cytokines.
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Interferón gamma , Mycobacterium , Recién Nacido , Niño , Lactante , Humanos , Vacuna BCG , Células Asesinas Naturales , Citocinas , VacunaciónRESUMEN
Tuberculosis (TB) disease remains a major health crisis. Infection with Mycobacterium tuberculosis (M.tb) cause a range of diseases ranging from latent infection to active TB disease. This active state of the disease is characterised by the formation of granulomas (a physical barrier in the lung), a structure thought to protect the host by controlling the infection through preventing the growth of the bacilli. Subsequently, the surviving bacteria become inactive and in most cases, TB reactivation is prevented by the immune response of the host. B-cells perform numerous immunological functions beyond antibody production to positively regulate the response to pathogenic assault. A subgroup of B-cells with regulatory functions express death-inducing ligands, such as Fas ligand (FasL). Expression and interaction of the Fas receptor-ligand promotes the induction of apoptosis and the induction of T-cell tolerance. Here, we focus on the significance of B-cells by addressing their FasL phenotype and regulatory functions during TB, with reference to disease in humans, non-human primates and mice.
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Linfocitos B Reguladores/metabolismo , Proteína Ligando Fas/metabolismo , Mycobacterium tuberculosis/fisiología , Tuberculosis/inmunología , Tuberculosis/microbiología , Animales , Citocinas/biosíntesis , Humanos , Modelos BiológicosRESUMEN
In tuberculosis, T cell-mediated immunity is extensively studied whilst B cells received limited attention in human and mice. Of interest, Mycobacterium tuberculosis (Mtb) does increase IL-4 Receptor-alpha (IL4Rα) expression in murine B cells. To better understand the role of IL4Rα signalling in B cells, we compared wild type mice with B cell-specific IL4Rα deficient mice (mb1creIL-4Rα-/lox mice). Chronic Mtb aerosol infection in mb1creIL-4Rα-/lox mice reduced lung and spleen bacterial burdens, compared to littermate (IL-4Rα-/lox) control animals. Consequently, lung pathology, inflammation and inducible nitric oxide synthase (iNOS) expression were reduced in the lungs of mb1creIL-4Rα-/lox mice, which was also accompanied by increased lung IgA and decreased IgG1 levels. Furthermore, intratracheal adoptive transfer of wild-type B cells into B cell-specific IL4Rα deficient mice reversed the protective phenotype. Moreover, constitutively mCherry expressing Mtb showed decreased association with B cells from mb1creIL-4Rα-/lox mice ex vivo. In addition, supernatants from Mtb-exposed B cells of mb1creIL-4Rα-/lox mice also increased the ability of macrophages to produce nitric oxide, IL-1ß, IL-6 and TNF. Together, this demonstrates that IL-4-responsive B cells are detrimental during the chronic phase of tuberculosis in mice with perturbed antibody profiles, inflammatory cytokines and tnf and stat1 levels in the lungs.
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Linfocitos B/inmunología , Inmunoglobulina A/metabolismo , Interleucina-4/metabolismo , Pulmón/metabolismo , Macrófagos/patología , Mycobacterium tuberculosis/fisiología , Tuberculosis/inmunología , Animales , Enfermedad Crónica , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Superficie Celular/genética , Transducción de SeñalRESUMEN
INTRODUCTION: B-cells are essential in the defense against Mycobacterium tuberculosis. Studies on isolated cells may not accurately reflect the responses that occur in vivo due to the presence of other cells. This study elucidated the influence of microenvironment complexity on B-cell polarization and function in the context of tuberculosis disease. METHODS: B-cell function was tested in whole blood, peripheral blood mononuclear cells (PBMCs), and as isolated cells. The different fractions were stimulated and the B-cell phenotype and immunoglobulin profiles analyzed. RESULTS: The immunoglobulin profile and developmental B-cell frequencies varied for each of the investigated sample types, while in an isolated cellular environment, secretion of immunoglobulin isotypes immunoglobulin A (IgA), IgG2, and IgG3 was hampered. The differences in the immunoglobulin profile highlight the importance of cell-cell communication for B-cell activation. Furthermore, a decrease in marginal zone B-cell frequencies and an increase in T1 B-cells was observed following cell isolation, indicating impaired B-cell development in response to in vitro antigenic stimulation in isolation. CONCLUSION: Our results suggest that humoral B-cell function and development was impaired likely due to a lack of costimulatory signals from other cell types. Thus, B-cell function should ideally be studied in a PBMC or whole blood fraction.
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Linfocitos B , Leucocitos Mononucleares , Microambiente Celular , Humanos , Proyectos Piloto , SudáfricaRESUMEN
Regulatory B cells (Bregs) have been shown to be present during several disease states. The phenotype of the cells is not completely defined and the function of these cells differ between disease. The presence of FASL expressing (killer) B cells during latent and successfully treated TB disease have been shown but whether these cells are similar to regulatory B cells remain unclear. We assessed the receptor expression of FASL/IL5 (killer B cells), CD24/CD38 (regulatory B cells) on whole peripheral blood of participants with untreated active TB and healthy controls. We then isolated B cells from a second cohort of M.tb exposed (Quantiferon (QFN) positive) and unexposed (Quantiferon negative) HIV negative participants, and evaluated the frequency of killer B cells induced following stimulation with BCG and/or CD40 and IL5. Our data reveal no difference in the expression on CD24 and CD38 between participants with active TB and the controls. There was also no difference in the frequency of regulatory B cells measured in the peripheral blood mononuclear cells (PBMC) fraction between latent TB and uninfected controls. We did however notice that regulatory B cells (CD24hiCD38hi) population express the FASL receptor. The expression of killer B cell phenotype (CD178+IL5RA+) was significantly higher in controls compared to those with active TB disease (1,06% vs 0,455%). Furthermore, we found that BCG restimulation significantly induced the FASL/IL5RA B cells but this was only evident in the QFN positive group. Our data suggest that both regulatory and killer B cells are present during latent and active TB disease but that the frequency of these populations are increased during latent disease. We also show that the FASL+IL5RA+ B killer B cells are induced in latent TB infection following BCG restimulation but whether these cells are indicative of protection remains unclear.
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Linfocitos B Reguladores/inmunología , Proteína Ligando Fas/inmunología , Células Asesinas Naturales/inmunología , Tuberculosis Latente/inmunología , Activación de Linfocitos , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , ADP-Ribosil Ciclasa 1/sangre , ADP-Ribosil Ciclasa 1/inmunología , Linfocitos B Reguladores/metabolismo , Linfocitos B Reguladores/microbiología , Antígeno CD24/sangre , Antígeno CD24/inmunología , Estudios de Casos y Controles , Proliferación Celular , Proteína Ligando Fas/sangre , Interacciones Huésped-Patógeno , Humanos , Subunidad alfa del Receptor de Interleucina-5/sangre , Subunidad alfa del Receptor de Interleucina-5/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/microbiología , Tuberculosis Latente/sangre , Tuberculosis Latente/microbiología , Recuento de Linfocitos , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/inmunología , FenotipoRESUMEN
Activated B-cells increase T-cell behaviour during autoimmune disease and other infections by means of cytokine production and antigen-presentation. Functional studies in experimental autoimmune encephalomyelitis (EAE) indicate that B-cell deficiencies, and a lack of IL10 and IL35 leads to a poor prognosis. We hypothesised that B-cells play a role during tuberculosis. We evaluated B-cell mRNA expression using real-time PCR from healthy community controls, individuals with other lung diseases and newly diagnosed untreated pulmonary TB patients at three different time points (diagnosis, month 2 and 6 of treatment).We show that FASLG, IL5RA, CD38 and IL4 expression was lower in B-cells from TB cases compared to healthy controls. The changes in expression levels of CD38 may be due to a reduced activation of B-cells from TB cases at diagnosis. By month 2 of treatment, there was a significant increase in the expression of APRIL and IL5RA in TB cases. Furthermore, after 6 months of treatment, APRIL, FASLG, IL5RA and CD19 were upregulated in B-cells from TB cases. The increase in the expression of APRIL and CD19 suggests that there may be restored activation of B-cells following anti-TB treatment. The upregulation of FASLG and IL5RA indicates that B-cells expressing regulatory genes may play an important role in the protective immunity against M.tb infection. Our results show that increased activation of B-cells is present following successful TB treatment, and that the expression of FASLG and IL5RA could potentially be utilised as a signature to monitor treatment response.
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Antituberculosos/uso terapéutico , Linfocitos B/efectos de los fármacos , Proteína Ligando Fas/genética , Subunidad alfa del Receptor de Interleucina-5/genética , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores/metabolismo , Biomarcadores Farmacológicos , Estudios de Casos y Controles , Monitoreo de Drogas/métodos , Proteína Ligando Fas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Receptor de Interleucina-5/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Masculino , Proyectos Piloto , Pronóstico , ARN Mensajero/efectos de los fármacos , Resultado del Tratamiento , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/inmunologíaRESUMEN
INTRODUCTION: Studies show that B-cells, in addition to producing antibodies and antigen-presentation, are able to produce cytokines as well. These include regulatory cytokines such as IL-10 by regulatory B-cells. Furthermore, a rare regulatory subset of B-cells have the potential to express FasL, which is a death-inducing ligand. This subset of B-cells have a positive role during autoimmune disease, but has not yet been studied during tuberculosis. These FasL-expressing B-cells are induced by bacterial LPS and CpG, thus we hypothesized that this phenotype might be induced during tuberculosis as well. METHODS: B-cells from participants with TB (at diagnosis and during treatment) and controls were collected, and analyzed by means of real-time PCR and flow cytometry. In addition to this, BAL was collected from TB participants as well and analyzed by means of MAGPix (multi-cytokine) technology. RESULTS: Gene expression analysis show that FASL transcript levels increase by the end of treatment. Similarly, phenotypic analysis show that there is a higher frequency of FasL-expressing B-cells by the end of treatment. CONCLUSION: Collectively, these results indicate that these FasL-expressing B-cells are being induced during anti-TB treatment, and thus may play a positive role. Further studies are required to elucidate this.
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Antituberculosos/farmacología , Linfocitos B/efectos de los fármacos , Proteína Ligando Fas/genética , Tuberculosis Pulmonar/genética , Antituberculosos/uso terapéutico , Linfocitos B/inmunología , Linfocitos B/metabolismo , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/sangre , Citocinas/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Receptor de Interleucina-5/genética , Fenotipo , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/inmunologíaRESUMEN
Tuberculosis is a global threat affecting millions of people and requires more efficient methods of diagnosis, monitoring treatment response and the development of more efficacious drug therapies and new vaccines. The use of transcriptomic approaches and gene expression techniques have contributed to the elucidation of these aspects concerning the study of tuberculosis, and more specifically, the utilization of transcriptional profiles to identify biomarkers. These markers are the key to developing tools required to improve diagnosis and treatment of tuberculosis. Several studies have led to the identification of markers able to distinguish between different infection states, as well as other pulmonary diseases. Utilizing a systems biology approach will assist in obtaining more reliable results, leading to the implementation of significant findings.