RESUMEN
Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen.
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Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Ratones Noqueados , Mycobacterium tuberculosis , Receptor Toll-Like 2 , Tuberculosis , Animales , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD4-Positivos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiología , Ratones Endogámicos C57BL , Pulmón/inmunología , Pulmón/microbiología , Bazo/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Citocinas/metabolismo , Citocinas/inmunologíaRESUMEN
The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.
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Pérdida de Hueso Alveolar , Osteólisis , Osteomielitis , Periodontitis , Ratones , Animales , Osteocitos/metabolismo , Osteólisis/inducido químicamente , Osteólisis/complicaciones , Osteólisis/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Ligando RANK/metabolismo , Porphyromonas gingivalis/metabolismo , Periodontitis/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Osteoclastos/metabolismoRESUMEN
RESUMEN La canaliculitis es una entidad rara, con frecuencia mal diagnosticada por su similitud con otras enfermedades. Se reporta una paciente femenina, de 56 años de edad, remitida a la Consulta de Oculoplastia del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", por secreciones purulentas y epífora del ojo izquierdo. Al examen se observó hiperemia conjuntival, secreción purulenta, punto lagrimal inferior hiperémico, dilatado, y se constató salida de concreciones por este al comprimir el canalículo. Se confirmó el diagnóstico de canaliculitis aguda supurada con concreciones. Se indicó tratamiento quirúrgico, que consistió en la canaliculotomía con remoción de las concreciones. Un examen clínico detallado, con adecuado conocimiento de la vía lagrimal excretora, permitió el diagnóstico certero, con un tratamiento quirúrgico eficaz y una evolución satisfactoria(AU)
ABSTRACT Canaliculitis is an uncommon infectious disease. It is often misdiagnosed due to its overlapping presentation to other common entities. A 56-year-old female patient is reported. She was referred to Ramón Pando Ferrer Cuban Ophthalmologic Institute, Ocular Plastic Surgery consultation, suffering from punctal swelling, discharge, and epiphora. At ocular examination was described conjunctival hyperemia, pouting punctum and mucopurulent discharge. Punctal regurgitation of concretions appears under syringing. It was confirmed acute canaliculitis with concretions in the left eye. A canaliculotomy was performed, and the concretions were removed. Routine clinical examinations helped to get a right diagnosis of canaliculitis and the surgical result was satisfactory(AU)
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Humanos , Femenino , Persona de Mediana Edad , Canaliculitis/diagnóstico , Canaliculitis/terapia , Enfermedades del Aparato Lagrimal/cirugíaRESUMEN
RESUMEN La ubicación anatómica de los párpados los predispone a ser dañados frecuentemente por causas traumáticas. Se informa la evolución de un paciente masculino, de 22 años de edad, remitido a la Consulta de Urgencia del Instituto Cubano de Oftalmología "Ramón Pando Ferrer" por presentar trauma palpebral grave en su ojo izquierdo producido por un alambre. Al examen oftalmológico se constató colgajo extenso del párpado superior, que colgaba fijado de la porción temporal. Se comprobó herida horizontal de la aponeurosis del elevador del párpado superior, de bordes anfractuosos, que se extendía a todo el párpado, así como sección del canalículo superior. Se confirmó el diagnóstico de herida avulsiva grave, con afectación del borde libre palpebral y sección del canalículo superior. Se decidió la conducta quirúrgica: reconstrucción palpebral, que incluyó aponeurosis del músculo elevador del párpado superior, canalículo superior con sonda bicanalicular y borde libre palpebral. Durante el seguimiento se constataron buenos resultados estéticos y funcionales. El conocimiento de la anatomía palpebral y una reconstrucción minuciosa de los tejidos palpebrales permiten resultados posquirúrgicos excelentes en un trauma palpebral grave(AU)
ABSTRACT The anatomical location of the eyelids predisposes them to be frequently damaged by traumatic causes. The evolution of a patient aged 22 years is reported, who was referred at Ramón Pando Ferrer Cuban Institute of Ophthalmology for presenting severe eyelid trauma in his left eye caused by a wire. The ophthalmological examination showed a large flap of the upper eyelid, which hung fixed from the temporal portion. Horizontal wound of the aponeurosis of the upper eyelid elevator, with an abnormal fracture, which extended to the entire eyelid, as well as section of the upper canaliculus, was found. The diagnosis of severe avulsive wound was confirmed, with involvement of the palpebral free edge and section of the upper canaliculus. Surgical behavior was decided: eyelid reconstruction, which included aponeurosis of the upper eyelid lift muscle, upper canaliculus with bicanalicular probe and palpebral free edge. During the follow-up, good aesthetic and functional results were observed. The knowledge of the eyelid anatomy and a thorough reconstruction of the eyelid tissues allow excellent post-surgical results in severe eyelid trauma(AU)
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Humanos , Masculino , Adulto Joven , Procedimientos de Cirugía Plástica/métodos , Párpados/lesiones , Aponeurosis/lesiones , Músculos Oculomotores/lesionesRESUMEN
RESUMEN Objetivo: Evaluar la concordancia entre el diagnóstico clínico inicial y el histopatológico de tumores palpebrales malignos. Métodos: Se realizó un estudio observacional, descriptivo y retrospectivo de pacientes portadores de diferentes tumores palpebrales, atendidos en el Servicio de Cirugía Plástica Ocular del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", en el periodo comprendido de enero a diciembre del año 2016, en el que se describieron las características clínicas y anatomopatológicas, y la concordancia entre ambos diagnósticos. Resultados: Predominó el grupo de 60-79 años de edad (46,1 por ciento), el sexo femenino (51,7 por ciento) y el color de piel blanco (92,6 por ciento) . Las lesiones se presentaron fundamentalmente en el párpado inferior (77,0 por ciento) y las telangectasias constituyeron el signo de malignidad más consistente (56,8 por ciento), con predominio del carcinoma basocelular, seguido del carcinoma espinocelular (81,4 por ciento y 15,2 por ciento, respectivamente). La concordancia entre el diagnóstico clínico y el histopatológico fue de 69,2 por ciento para el carcinoma basocelular; 9,7 por ciento para el espinocelular y 30 por ciento para el sebáceo (índice de Kappa de 0,338; 0,262 y 0,357, respectivamente). Conclusiones: Existe la mayor concordancia entre diagnóstico clínico e histopatológico en el carcinoma basocelular; no así en el resto de los tumores estudiados(AU)
ABSTRACT Objective: To assess the concordance between the initial and histopathological clinical diagnosis of malignant palpebral tumors. Methods: An observational, descriptive and retrospective study of patients with different eyelid tumors was performed. The patients were treated in the Ocular Plastic Surgery Service of Ramón Pando Ferrer Cuban Institute of Ophthalmology, in the period from January to December of 2016, during which the clinical and pathological characteristics were described, together with the concordance between both diagnoses. Results: The age group of 60-79 years predominated (46.1 percent), as it was with the female sex (51.7 percent) and the white skin color (92.6 percent). The lesions occurred mainly in the lower eyelid (77.0 percent) and telangiectasia constituted the most consistent sign of malignancy (56.8 percent), with a predominance of basal cell carcinoma, followed by spinocellular carcinoma (81.4 percent and 15, 2 percent, respectively). The concordance between the clinical and histopathological diagnosis was 69.2 percent for basal cell carcinoma; 9.7 percent for the spinocellular carcinoma; and 30 percent for the sebaceous carcinoma (Kappa index of 0.388, 0.262 and 0.357, respectively). Conclusions: There is the greatest concordance between the clinical and histopathological diagnosis in basal cell carcinoma, but not in the rest of the tumors studied(AU)
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Humanos , Femenino , Anciano , Neoplasias de los Párpados/diagnóstico , Neoplasias/patología , Epidemiología Descriptiva , Estudios Retrospectivos , Estudios Observacionales como AsuntoRESUMEN
Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40+ endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.
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Encéfalo/parasitología , Antígenos CD40/fisiología , Células Endoteliales/inmunología , Retina/parasitología , Toxoplasma/patogenicidad , Animales , Autofagia , Movimiento Celular , Proteínas HSP70 de Choque Térmico/fisiología , Leucocitos/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose-capped lipoarabinomannan (ManLAM) inhibits CD4+ T-cell activation by inhibiting proximal T-cell receptor (TCR) signaling when activated by anti-CD3. To understand the impact of ManLAM on CD4+ T-cell function when both the TCR-CD3 complex and major costimulator CD28 are engaged, we performed label-free quantitative MS and network analysis. Mixed-effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti-CD3- and anti-CD28-activated CD4+ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF-κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x-Akt-mTOR pathway. Thus, ManLAM induced global changes in the CD4+ T-cell proteome by affecting Akt-mTOR signaling, resulting in broad functional impairment of CD4+ T-cell activation beyond inhibition of proximal TCR-CD3 signaling.
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Linfocitos T CD4-Positivos/metabolismo , Redes Reguladoras de Genes , Lipopolisacáridos/farmacología , Mycobacterium tuberculosis/metabolismo , Proteína Oncogénica v-akt/antagonistas & inhibidores , Proteómica/métodos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Ciclo Celular , Femenino , Manosa/química , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Proteína Oncogénica v-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4+ T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4+ T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4+ T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4+ T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4+ T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4+ T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells.
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Linfocitos T CD4-Positivos/inmunología , Interleucina-9/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Receptor Toll-Like 2/metabolismo , Aciltransferasas/inmunología , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Interferón gamma/metabolismo , Interleucina-9/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Receptor Toll-Like 2/inmunología , Transactivadores/metabolismo , TranscriptomaRESUMEN
BACKGROUND: Multiple toll-like receptors (TLRs) are expressed in cells of the monocytic lineage, including microglia, which constitute the major reservoir for human immunodeficiency virus (HIV) infection in the brain. We hypothesized that TLR receptor mediated responses to inflammatory conditions by microglial cells in the central nervous system (CNS) are able to induce latent HIV proviruses, and contribute to the etiology of HIV-associated neurocognitive disorders. RESULTS: Newly developed human microglial cell lines (hµglia), obtained by immortalizing human primary microglia with simian virus-40 (SV40) large T antigen and the human telomerase reverse transcriptase, were used to generate latently infected cells using a single-round HIV virus carrying a green fluorescence protein reporter (hµglia/HIV, clones HC01 and HC69). Treatment of these cells with a panel of TLR ligands showed surprisingly that two potent TLR3 agonists, poly (I:C) and bacterial ribosomal RNA potently reactivated HIV in hµglia/HIV cells. LPS (TLR4 agonist), flagellin (TLR5 agonist), and FSL-1 (TLR6 agonist) reactivated HIV to a lesser extent, while Pam3CSK4 (TLR2/1 agonist) and HKLM (TLR2 agonist) only weakly reversed HIV latency in these cells. While agonists for TLR2/1, 4, 5 and 6 reactivated HIV through transient NF-κB induction, poly (I:C), the TLR3 agonist, did not activate NF-κB, and instead induced the virus by a previously unreported mechanism mediated by IRF3. The selective induction of IRF3 by poly (I:C) was confirmed by chromatin immunoprecipitation (ChIP) analysis. In comparison, in latently infected rat-derived microglial cells (hT-CHME-5/HIV, clone HC14), poly (I:C), LPS and flagellin were only partially active. The TLR response profile in human microglial cells is also distinct from that shown by latently infected monocyte cell lines (THP-1/HIV, clone HA3, U937/HIV, clone HUC5, and SC/HIV, clone HSCC4), where TLR2/1, 4, 5, 6 or 8, but not for TLR3, 7 or 9, reactivated HIV. CONCLUSIONS: TLR signaling, in particular TLR3 activation, can efficiently reactivate HIV transcription in infected microglia, but not in monocytes or T cells. The unique response profile of microglial cells to TLR3 is fundamental to understanding how the virus responds to continuous microbial exposure, especially during inflammatory episodes, that characterizes HIV infection in the CNS.
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VIH-1/fisiología , Microglía/virología , Receptor Toll-Like 3/metabolismo , Latencia del Virus , Animales , Línea Celular , Células Cultivadas , Humanos , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/virología , FN-kappa B/metabolismo , Poli I-C/farmacología , ARN Bacteriano/farmacología , ARN Ribosómico/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/virología , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 4/agonistas , Receptores Toll-Like/agonistas , Activación ViralRESUMEN
Mycobacterium tuberculosis utilizes multiple mechanisms to evade host immune responses, and inhibition of effector CD4+ T cell responses by M. tuberculosis may contribute to immune evasion. TCR signaling is inhibited by M. tuberculosis cell envelope lipoglycans, such as lipoarabinomannan and lipomannan, but a mechanism for lipoglycans to traffic from M. tuberculosis within infected macrophages to reach T cells is unknown. In these studies, we found that membrane vesicles produced by M. tuberculosis and released from infected macrophages inhibited the activation of CD4+ T cells, as indicated by reduced production of IL-2 and reduced T cell proliferation. Flow cytometry and Western blot demonstrated that lipoglycans from M. tuberculosis-derived bacterial vesicles (BVs) are transferred to T cells, where they inhibit T cell responses. Stimulation of CD4+ T cells in the presence of BVs induced expression of GRAIL, a marker of T cell anergy; upon restimulation, these T cells showed reduced ability to proliferate, confirming a state of T cell anergy. Furthermore, lipoarabinomannan was associated with T cells after their incubation with infected macrophages in vitro and when T cells were isolated from lungs of M. tuberculosis-infected mice, confirming the occurrence of lipoarabinomannan trafficking to T cells in vivo. These studies demonstrate a novel mechanism for the direct regulation of CD4+ T cells by M. tuberculosis lipoglycans conveyed by BVs that are produced by M. tuberculosis and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion.
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Linfocitos T CD4-Positivos/inmunología , Evasión Inmune , Pulmón/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Vesículas Secretoras/microbiología , Tuberculosis/inmunología , Animales , Proliferación Celular , Células Cultivadas , Anergia Clonal , Femenino , Humanos , Lipopolisacáridos/inmunología , Pulmón/microbiología , Activación de Linfocitos , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Vesículas Secretoras/inmunologíaRESUMEN
OBJECTIVE: Infection by MTB or exposure to MTB constituents is associated with intense microbial stimulation of the immune system, through both antigenic and TLR components, and induction of a milieu that is rich in pro-inflammatory/anti-inflammatory cytokines. Here, we addressed the basis of induced regulatory T-cell (iT-reg) expansion in response to MTB stimulation, in the absence of prior T cell antigen responsiveness. METHODS: PBMC from HIV-1 un-infected TST negative and TST positive control subjects were stimulated by virulent MTB H37Rv lysate (L), a French press preparation of MTB that includes all bacterial components. Phenotype of MTB H37RvL induced iT-reg was assessed using immunostaining and flow cytometry. Functional capacity of iT-reg was assessed using 3H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in the presence or absence of iT-reg in corresponding culture supernatants in response to TCR stimulation. Realtime PCR was used to assess IDO and FoxP3 mRNA expression. RESULTS: The capacity of MTB H37RvL to induce CD4+CD25hi+ Foxp3+ T-cells in PBMC from TST negative subjects was robust (p<0.001), and in fact comparable to induction of iT-reg in PBMC from TST positive subjects. MTB-induced CD4+CD25hi+ T-reg were TGFß positive (p<0.05). Further, MTB H37RvL induced CD4+CD25hi+ Foxp3+ iT-reg suppressed 3H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in response to TCR stimulation. MTB H37RvL induction of iT-reg was significantly stronger (p<0.01) than that by TLR-2, TLR-4, TLR-9 ligands, or combination of all TLR ligands. MTB H37RvL inducted indoleamine 2,3-dideoxygenase (IDO) mRNA expression in monocytes (p<0.001), and co-culture with the IDO inhibitor, D-1MT, decreased frequencies of T-reg (p<0.05). Inhibition of TGFß by siRNA reduced Foxp3 mRNA expression in CD4 T cells (p<0.05). CONCLUSION: Therefore, MTB and its components expand functional iT-reg in human mononuclear cells from MTB non-sensitized subjects. Also, MTB-induced iT-reg expansion depends on mononuclear phagocyte expression of both TGFß and IDO.
RESUMEN
UNLABELLED: Chemical regulation of macrophage function is one key strategy for developing host-directed adjuvant therapies for tuberculosis (TB). A critical step to develop these therapies is the identification and characterization of specific macrophage molecules and pathways with a high potential to serve as drug targets. Using a barcoded lentivirus-based pooled short-hairpin RNA (shRNA) library combined with next generation sequencing, we identified 205 silenced host genes highly enriched in mycobacteria-resistant macrophages. Twenty-one of these "hits" belonged to the oxidoreductase functional category. NAD(P)H: quinone oxidoreductase 1 (NQO1) was the top oxidoreductase "hit". NQO1 expression was increased after mycobacterial infection, and NQO1 knockdown increased macrophage differentiation, NF-κB activation, and the secretion of pro-inflammatory cytokines TNF-α and IL-1ß in response to infection. This suggests that mycobacteria hijacks NQO1 to down-regulate pro-inflammatory and anti-bacterial functions. The competitive inhibitor of NQO1 dicoumarol synergized with rifampin to promote intracellular killing of mycobacteria. Thus, NQO1 is a new host target in mycobacterial infection that could potentially be exploited to increase antibiotic efficacy in vivo. Our findings also suggest that pooled shRNA libraries could be valuable tools for genome-wide screening in the search for novel druggable host targets for adjunctive TB therapies.
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Antituberculosos/farmacología , Dicumarol/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Macrófagos/inmunología , Mycobacterium tuberculosis/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/genética , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-1beta/agonistas , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/fisiología , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , NAD(P)H Deshidrogenasa (Quinona)/inmunología , FN-kappa B/agonistas , FN-kappa B/genética , FN-kappa B/inmunología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Rifampin/farmacología , Transducción de Señal , Células THP-1 , Factor de Necrosis Tumoral alfa/agonistas , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Mycobacterium tuberculosis cell wall glycolipid, lipoarabinomannan, can inhibit CD4(+) T cell activation by downregulating the phosphorylation of key proximal TCR signaling molecules: Lck, CD3ζ, ZAP70, and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an Ag encounter, yet remain viable and hyporesponsive. We tested whether mannose-capped lipoarabinomannan (LAM)-induced inhibition of CD4(+) T cell activation resulted in CD4(+) T cell anergy. The presence of LAM during primary stimulation of P25 TCR-transgenic murine CD4(+) T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25 TCR-transgenic CD4(+) T cells primed in the presence of LAM also exhibited decreased response upon restimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hyporesponsiveness to restimulation was not due to apoptosis, generation of Foxp3-positive regulatory T cells, or inhibitory cytokines. Acquisition of the anergic phenotype correlated with upregulation of gene related to anergy in lymphocytes (GRAIL) protein in CD4(+) T cells. Inhibition of human CD4(+) T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA-mediated knockdown of GRAIL before LAM treatment abrogated LAM-induced hyporesponsiveness. In addition, exogenous IL-2 reversed defective proliferation by downregulating GRAIL expression. These results demonstrate that LAM upregulates GRAIL to induce anergy in Ag-reactive CD4(+) T cells. Induction of CD4(+) T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition.
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Linfocitos T CD4-Positivos/inmunología , Anergia Clonal/inmunología , Evasión Inmune/inmunología , Lipopolisacáridos/inmunología , Tuberculosis/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Western Blotting , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Femenino , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Humanos , Activación de Linfocitos/inmunología , Manosa/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Mycobacterium tuberculosis/inmunología , ARN Interferente PequeñoRESUMEN
Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.
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Infecciones Bacterianas/inmunología , Gentisatos/inmunología , Hidroxibutirato Deshidrogenasa/inmunología , Inmunidad Innata/inmunología , Sideróforos/inmunología , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/metabolismo , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Candida albicans/inmunología , Candida albicans/fisiología , Candidiasis/genética , Candidiasis/inmunología , Candidiasis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Línea Celular , Enterobactina/inmunología , Enterobactina/metabolismo , Escherichia coli/genética , Escherichia coli/inmunología , Escherichia coli/fisiología , Femenino , Gentisatos/metabolismo , Hidroxibutirato Deshidrogenasa/genética , Hidroxibutirato Deshidrogenasa/metabolismo , Inmunidad Innata/genética , Immunoblotting , Estimación de Kaplan-Meier , Lipocalina 2 , Lipocalinas/genética , Lipocalinas/inmunología , Lipocalinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/inmunología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunología , Proteínas Oncogénicas/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Interferencia de ARN , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sideróforos/metabolismo , Staphylococcus aureus/inmunologíaRESUMEN
We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4(+) T cells and upregulate TCR-triggered IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4(+) T-cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T-cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4(+) T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T-cell TLR2 in priming of CD4(+) T cells in vivo, naive MTB Ag85B-specific TCR transgenic CD4(+) T cells (P25 TCR-Tg) were adoptively transferred into Tlr2(-/-) recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3 Cys-SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN-γ-secreting P25 TCR-Tg T cells 1 week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4(+) T cells increases MTB Ag-specific responses and may contribute to protection against MTB infection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Mycobacterium tuberculosis/inmunología , Receptor Toll-Like 2/inmunología , Tuberculosis/inmunología , Aciltransferasas/biosíntesis , Aciltransferasas/genética , Aciltransferasas/inmunología , Aciltransferasas/farmacología , Animales , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Homólogo de la Proteína Chromobox 5 , Humanos , Inmunización , Interferón gamma/biosíntesis , Interferón gamma/genética , Interferón gamma/inmunología , Ratones , Ratones Noqueados , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/genética , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis/patología , Tuberculosis/prevención & controlRESUMEN
Tuberculosis (TB) is the leading cause of mortality among those infected with human immunodeficiency virus (HIV-1) worldwide. HIV-1 load and heterogeneity are increased both locally and systemically in active TB. Mycobacterium tuberculosis (MTB) infection supports HIV-1 replication through dysregulation of host cytokines, chemokines, and their receptors. However the possibility that mycobacterial molecules released from MTB infected macrophages directly interact with CD4(+) T cells triggering HIV-1 replication has not been fully explored. We studied the direct effect of different MTB molecules on HIV-1 replication (R5-tropic strain Bal) in anti-CD3- stimulated CD4(+) T cells from healthy donors in an antigen presenting cell (APC)-free system. PIM6, a major glycolipid of the mycobacterial cell wall, induced significant increases in the percent of HIV-1 infected T cells and the viral production in culture supernatants. In spite of structural relatedness, none of the other three major MTB cell wall glycolipids had significant impact on HIV-1 replication in T cells. Increased levels of IFN-γ in culture supernatants from cells treated with PIM6 indicate that HIV-1 replication is likely dependent on enhanced T cell activation. In HEK293 cells transfected with TLR2, PIM6 was the strongest TLR2 agonist among the cell wall associated glycolipids tested. PIM6 increased the percentage of HIV infected cells and viral particles in the supernatant in a T-cell-based reporter cell line (JLTRg-R5) transfected with TLR1 and TLR2 but not in the cells transfected with the empty vector (which lack TLR2 expression) confirming that PIM6-induced HIV-1 replication depends at least partially on TLR2 signaling.
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Antígenos Bacterianos/fisiología , Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Mycobacterium tuberculosis/metabolismo , Fosfatidilinositoles/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Replicación Viral/fisiología , Humanos , Células Jurkat , Receptor Toll-Like 2/fisiologíaRESUMEN
Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen-MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide-MHC-II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide-MHC-II complexes in M. tuberculosis-infected human monocyte-derived macrophages (MDMs) using autologous M. tuberculosis-specific CD4(+) T cells. The MDMs were pre-treated with either IFN-γ or IL-10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M. tuberculosis -specific CD4(+) T cells. Our results demonstrated that in MDMs pre-treated with IFN-γ, M. tuberculosis peptide-MHC-II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN-γ, the complexes were detected in the endosomal fractions. In MDMs pre-treated with IL-10, the M. tuberculosis peptide-MHC-II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA-DR at the cell surface only in the IFN-γ-treated MDMs, suggesting that IFN-γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL-10 favours the trafficking of Ag85B to vesicles that do not contain LAMP-1. Therefore, IFN-γ and IL-10 play a role in the formation and trafficking of M. tuberculosis peptide-MHC-II complexes.
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Antígenos Bacterianos/inmunología , Interferón gamma/inmunología , Interleucina-10/inmunología , Fagosomas/inmunología , Línea Celular , Humanos , Mycobacterium tuberculosis/inmunologíaRESUMEN
Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust CD4(+) T cell responses. We have shown previously that M. tuberculosis cell wall glycolipids, including mannose capped lipoarabinomannan (ManLAM), directly inhibit polyclonal murine CD4(+) T cell activation by blocking ZAP-70 phosphorylation. We extended these studies to antigen-specific murine CD4(+) T cells and primary human T cells and found that ManLAM inhibited them as well. Lck and LAT phosphorylation also were inhibited by ManLAM without affecting their localization to lipid rafts. Inhibition of proximal TCR signaling was temperature sensitive, suggesting that ManLAM insertion into T cell membranes was required. Thus, M. tuberculosis ManLAM inhibits antigen-specific CD4(+) T cell activation by interfering with very early events in TCR signaling through ManLAM's insertion in T cell membranes.
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Lipopolisacáridos/inmunología , Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Microdominios de Membrana/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Our recent microarray analysis of infected human alveolar macrophages (AMs) found serine protease inhibitor 9 (PI-9) to be the most prominently expressed of a cluster of apoptosis-associated genes induced by virulent Mycobacterium tuberculosis. In the current study, we show that induction of PI-9 occurs within hours of infection with M. tuberculosis H37Rv and is maintained through 7 days of infection in both AMs and blood monocytes. Inhibition of PI-9 by small inhibitory RNA decreased M. tuberculosis-induced expression of the antiapoptotic molecule Bcl-2 and resulted in a corresponding increase in production of caspase 3, a terminal effector molecule of apoptosis. Further, PI-9 small inhibitory RNA mediated a significant reduction in the subsequent survival of M. tuberculosis within AMs. Thus PI-9 induction within human mononuclear phagocytes by virulent M. tuberculosis serves to protect these primary targets of infection from elimination by apoptosis and thereby promotes intracellular survival of the organism.
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Apoptosis , Macrófagos Alveolares/metabolismo , Mycobacterium tuberculosis/patogenicidad , Serpinas/metabolismo , Caspasa 3/metabolismo , Células Cultivadas , Humanos , Macrófagos Alveolares/microbiología , Monocitos/metabolismo , Monocitos/microbiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5ß1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.