RESUMEN
Tuberculosis pathogenesis was earlier thought to be mainly related to the host but now it appears to be clear that bacterial factors are also involved. Genetic variability of Mycobacterium tuberculosis (Mtb) could be slight but it may lead to sharp phenotypic differences. We have previously reported that nonopsonized Mtb H37Rv induce apoptosis of polymorphonuclear neutrophils (PMNs) by a mechanism that involves the p38 pathway. Here we evaluated the capability to induce PMN apoptosis of two prevalent Mtb lineages in Argentina, the Latin America and Mediterranean (LAM), and Haarlem, using the H37Rv as a reference strain. Results showed that LAM strains strongly induced apoptosis of PMN which correlated with the induction of reactive oxygen species (ROS) production and p38 activation. Interestingly, the highly prosperous multidrug-resistant M strain, belonging to the Haarlem lineage, lacked the ability to activate and to induce PMN apoptosis as a consequence of (1) a weak ROS production and (2) the contribution of antiapoptotic mechanisms mediated at least by ERK. Although with less skill, M is able to enter the PMN so that phenotypic differences could lead PMN to be a reservoir allowing some pathogens to prevail and persist over other strains in the community.
Asunto(s)
Apoptosis/inmunología , Mycobacterium tuberculosis/inmunología , Neutrófilos/inmunología , Estallido Respiratorio/inmunología , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Moléculas de Adhesión Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Ligadas a GPI/metabolismo , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , Neutrófilos/metabolismo , Neutrófilos/microbiología , Fagocitosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: The proinflammatory cytokine interleukin 17 (IL-17) plays an important role in immune responses but it is also associated with tissue-damaging inflammation. So, we evaluated the ability of Mycobacterium tuberculosis clinical isolates to induce IL-17 in tuberculosis (TB) patients and in healthy human tuberculin reactors (PPD(+)HD). METHODS: IL-17, interferon γ (IFN-γ), and interleukin 23 (IL-23) receptor expression were evaluated ex vivo and cultured peripheral blood mononuclear cells from TB and PPD(+)HD stimulated with irradiated clinical isolates from multidrug resistant (MDR) outbreaks M (Haarlem family) and Ra (Latin American-Mediterranean family), as well as drug-susceptible isolates belonging to the same families and laboratory strain H37Rv for 48 hours in T-cell subsets by flow cytometry. RESULTS: We observed that: (1) MDR strains M and Ra are stronger IL-17 inducers than drug-susceptible Mtb strains of the Haarlem and Latin American-Mediterranean families, (2) MDR-TB patients show the highest IL-17 expression that is independent on the strain, (3) IL-17 expression is dependent on CD4(+) and CD8(+) T cells associates with persistently high antigen load. CONCLUSIONS: IL-17--producing T cells could play an immunopathological role in MDR-TB promoting severe tissue damage, which may be associated with the low effectiveness of the second-line drugs employed in the treatment.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Adulto , Células Cultivadas , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de Interleucina/metabolismo , Subgrupos de Linfocitos T/metabolismo , Adulto JovenRESUMEN
Tuberculous pleurisy allows the study of specific cells at the site of Mycobacterium tuberculosis infection. Among pleural lymphocytes, natural killer (NK) cells are a major source of interferon gamma (IFN-gamma), and their functions are regulated by activating and inhibitory receptors. Programmed death-1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) are recognized inhibitory receptors in adaptive immunity, but their role during innate immunity remains poorly understood. We investigated the PD-1:PD-L1/PD-L2 pathway on NK cell effector functions in peripheral blood and pleural fluid from patients with tuberculosis. M. tuberculosis stimulation significantly up-regulated PD-1, PD-L1, and PD-L2 levels on NK cells. Interestingly, a direct correlation between PD-1 and IFN-gamma expression on NK cells was observed. Moreover, blockade of the PD-1 pathway markedly augmented lytic degranulation and IFN-gamma production of NK cells against M. tuberculosis. Furthermore, PD-1(+) NK cells displayed a diminished IFN-gamma mean fluorescence intensity, denoting the relevance of PD-1 on IFN-gamma regulation. Together, we described a novel inhibitory role played by PD-1:PD-L interactions in innate immunity in tuberculosis.
Asunto(s)
Antígenos CD/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Apoptosis , Inmunidad Innata , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Tuberculosis/patología , Adulto , Antígeno B7-H1 , Sangre/inmunología , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Pleura/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1 , Receptor de Muerte Celular Programada 1 , Regulación hacia ArribaRESUMEN
Tuberculous pleurisy, one of the most common manifestations of extrapulmonary tuberculosis, is characterized by a T-cell-mediated hypersensitivity reaction along with a Th1 immune profile. In this study, we investigated functional cross-talk among T and NK cells in human tuberculous pleurisy. We found that endogenously activated pleural fluid-derived NK cells express high ICAM-1 levels and induce T-cell activation ex vivo through ICAM-1. Besides, upon in vitro stimulation with monokines and PAMP, resting peripheral blood NK cells increased ICAM-1 expression leading to cellular activation and Th1 polarization of autologous T cells. Furthermore, these effects were abolished by anti-ICAM-1 Ab. Hence, NK cells may contribute to the adaptive immune response by a direct cell-contact-dependent mechanism in the context of Mycobacterium tuberculosis infection.
Asunto(s)
Molécula 1 de Adhesión Intercelular/metabolismo , Células Asesinas Naturales/inmunología , Mycobacterium tuberculosis , Linfocitos T/inmunología , Tuberculosis Pleural/inmunología , Adulto , Antígeno CD11a/inmunología , Antígeno CD56/inmunología , Comunicación Celular/inmunología , Cisteína/análogos & derivados , Cisteína/farmacología , Humanos , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/inmunología , Interleucina-12/farmacología , Interleucina-15/farmacología , Interleucina-18/farmacología , Lipopolisacáridos/farmacología , Lipoproteínas/farmacología , Persona de Mediana Edad , Fosfotransferasas (Aceptor del Grupo Fosfato)RESUMEN
Tuberculous pleurisy allows the study of human cells at the site of active Mycobacterium tuberculosis infection. In this study, we found that among pleural fluid (PF) lymphocytes, natural killer (NK) cells are a major source of early gamma interferon (IFN-gamma) upon M. tuberculosis stimulation, leading us to investigate the mechanisms and molecules involved in this process. We show that the whole bacterium is the best inducer of IFN-gamma, although a high-molecular-weight fraction of culture filtrate proteins from M. tuberculosis H37Rv and the whole-cell lysate also induce its expression. The mannose receptor seems to mediate the inhibitory effect of mannosylated lipoarabinomannan, and Toll-like receptor 2 and 4 agonists activate NK cells but do not induce IFN-gamma like M. tuberculosis does. Antigen-presenting cells (APC) and NK cells bind M. tuberculosis, and although interleukin-12 is required, it is not sufficient to induce IFN-gamma expression, indicating that NK cell-APC contact takes place. Indeed, major histocompatibility complex class I, adhesion, and costimulatory molecules as well as NK receptors regulate IFN-gamma induction. The signaling pathway is partially inhibited by dexamethasone and sensitive to Ca2+ flux and cyclosporine. Inhibition of p38 and extracellular-regulated kinase mitogen-activated protein kinase pathways reduces the number of IFN-gamma+ NK cells. Phosphorylated p38 (p-p38) is detected in ex vivo PF-NK cells, and M. tuberculosis triggers p-p38 in PF-NK cells at the same time that binding between NK and M. tuberculosis reaches its maximum value. Thus, interplay between M. tuberculosis and NK cells/APC triggering IFN-gamma would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a type 1 profile.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Lectinas Tipo C/inmunología , Lectinas de Unión a Manosa/inmunología , Mycobacterium tuberculosis/inmunología , Receptores de Superficie Celular/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Tuberculosis Pleural/inmunología , Adulto , Anciano , Células Presentadoras de Antígenos/microbiología , Antígenos de Superficie/inmunología , Adhesión Bacteriana , Proteínas Bacterianas/inmunología , Mezclas Complejas/inmunología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Asesinas Naturales/microbiología , Lipopolisacáridos/inmunología , Receptor de Manosa , Persona de Mediana Edad , Derrame Pleural/inmunología , Derrame Pleural/microbiología , Tuberculosis Pleural/microbiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16- subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-gamma production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.
Asunto(s)
Apoptosis/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Tuberculosis Pleural/inmunología , Tuberculosis Pleural/patología , Adulto , Anciano , Antígenos CD/metabolismo , Antígenos CD19/metabolismo , Complejo CD3/metabolismo , Antígeno CD56/metabolismo , Proteínas Ligadas a GPI , Humanos , Técnicas In Vitro , Interferón gamma/biosíntesis , Células Asesinas Naturales/clasificación , Receptores de Lipopolisacáridos/metabolismo , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/patología , Persona de Mediana Edad , Fenotipo , Derrame Pleural/inmunología , Derrame Pleural/patología , Receptores de IgG/metabolismoRESUMEN
Tuberculous pleuritis usually shows lymphocytic preponderance, but neutrophils are also present. Therefore, pleuritis is a good model for the study of neutrophil fate at sites of active Mycobacterium tuberculosis infection. We have previously demonstrated in vitro that M. tuberculosis-induced neutrophil apoptosis involves p38 mitogen protein kinase activation through Toll-like receptor 2. Herein, we demonstrate that, in tuberculous pleuritis, neutrophil apoptosis increases together with the expression of Toll-like receptor 2 and phosphorylated p38 (p-p38) kinase. In addition, receptors associated with activation/apoptotis (CD11b, CD64, tumor necrosis factor receptor, and Fas ligand) are up-regulated, together with a loss of CD16 expression. However, neutrophils express CD86, CD83, and major histocompatibility complex class II antigens, acquiring dendritic cell (DC) characteristics. Therefore, the cytokine milieu in the pleural space may influence signaling pathways on activated neutrophils, thereby inducing apoptosis and inhibiting their proinflammatory capacity, as well as allowing them acquire DC characteristics that influence the immune response.
Asunto(s)
Apoptosis/fisiología , Diferenciación Celular/fisiología , Células Dendríticas/patología , Neutrófilos/patología , Derrame Pleural/sangre , Tuberculosis Pulmonar/sangre , Antígenos CD/sangre , Células Dendríticas/fisiología , Citometría de Flujo , Humanos , Neutrófilos/fisiología , Fenotipo , Valores de ReferenciaRESUMEN
Polymorphonuclear neutrophils (PMN) exposed to Mycobacterium tuberculosis display bactericidal responses and produce inflammatory proteins. This PMN-mediated inflammatory response is regulated by an activation of the apoptotic program, which collaborates to avoid tissue injury. In vitro, circulating PMN from patients with tuberculosis (TB) show an increased spontaneous apoptosis, and M. tuberculosis-induced activation accelerates the PMN apoptosis. In this study, we evaluated the mechanisms involved in spontaneous and M. tuberculosis-induced apoptosis. We demonstrate that apoptosis of PMN is not induced by lipoarabinomannan or by a whole-cell lysate of M. tuberculosis and that neither tumor necrosis factor alpha nor CD11b, CD14, and Fcgamma receptors are involved. Apoptosis of PMN from patients with active TB (TB-PMN) is induced by the interaction with the whole M. tuberculosis via Toll-like receptor 2 (TLR2), and, in contrast to spontaneous apoptosis, it involves the p38 mitogen-activated protein kinase (MAPK) pathway. These results correlate with a high expression of phosphorylated p38 (p-p38) in circulating TB-PMN and with the ability of M. tuberculosis to induce in vitro the expression of p-p38 in PMN. Therefore, when the bacterial burden is low, TB-PMN could be detecting nonopsonized M. tuberculosis via TLR2, leading to the activation of the p38 MAPK pathway, which in turn would induce PMN activation and apoptosis. This mechanism needs further confirmation at the site of infection.
Asunto(s)
Apoptosis/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mycobacterium tuberculosis/patogenicidad , Neutrófilos/fisiología , Receptores de Superficie Celular/metabolismo , Células Cultivadas , Activación Enzimática , Humanos , Mycobacterium tuberculosis/metabolismo , Receptor Toll-Like 2 , Receptores Toll-Like , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The activation of circulating polymorphonuclear neutrophils (PMN) from patients with active tuberculosis (TB-PMN) may be associated with induction of apoptosis. Spontaneous or Mycobacterium tuberculosis (MTB)-induced apoptosis of PMN were evaluated by microscopy, DNA content, and their binding to Annexin V at 0, 3, and 18 h. In addition, the expression of CD11b and of CD16 were evaluated as parameters of activation and apoptosis, respectively. Recently isolated TB-PMN showed a higher CD11b expression than normal PMN (N-PMN), but there were no features of apoptosis, even though an enhancement of Fas expression was observed. Spontaneous apoptosis was accelerated in TB-PMN at 3 h, but no differences were observed in TB- and N-PMN at 18 h of culture. When stimulated with MTB, both TB- and N-PMN steadily increased CD11b expression along the culture period. MTB induced apoptosis of N-PMN at 3 h with loss of CD16 expression. By contrast, MTB delayed the apoptotic rate of TB-PMN, preserving the CD16 receptor at 3 h, whereas it accelerated apoptosis at 18 h, increasing at the same time the expression of CD11b. Taken together, these data suggest that the acceleration of apoptosis observed in TB-PMN could be associated with the MTB-induced activation.