RESUMEN
Severe defects in human IFNγ immunity predispose individuals to both Bacillus Calmette-Guérin disease and tuberculosis, whereas milder defects predispose only to tuberculosis1. Here we report two adults with recurrent pulmonary tuberculosis who are homozygous for a private loss-of-function TNF variant. Neither has any other clinical phenotype and both mount normal clinical and biological inflammatory responses. Their leukocytes, including monocytes and monocyte-derived macrophages (MDMs) do not produce TNF, even after stimulation with IFNγ. Blood leukocyte subset development is normal in these patients. However, an impairment in the respiratory burst was observed in granulocyte-macrophage colony-stimulating factor (GM-CSF)-matured MDMs and alveolar macrophage-like (AML) cells2 from both patients with TNF deficiency, TNF- or TNFR1-deficient induced pluripotent stem (iPS)-cell-derived GM-CSF-matured macrophages, and healthy control MDMs and AML cells differentiated with TNF blockers in vitro, and in lung macrophages treated with TNF blockers ex vivo. The stimulation of TNF-deficient iPS-cell-derived macrophages with TNF rescued the respiratory burst. These findings contrast with those for patients with inherited complete deficiency of the respiratory burst across all phagocytes, who are prone to multiple infections, including both Bacillus Calmette-Guérin disease and tuberculosis3. Human TNF is required for respiratory-burst-dependent immunity to Mycobacterium tuberculosis in macrophages but is surprisingly redundant otherwise, including for inflammation and immunity to weakly virulent mycobacteria and many other infectious agents.
Asunto(s)
Macrófagos , Tuberculosis Pulmonar , Factores de Necrosis Tumoral , Adulto , Femenino , Humanos , Masculino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Homocigoto , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/citología , Inflamación/inmunología , Interferón gamma/inmunología , Mutación con Pérdida de Función , Pulmón/citología , Pulmón/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos Alveolares/citología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/patología , Mycobacterium tuberculosis/inmunología , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Estallido Respiratorio , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/genética , Inhibidores del Factor de Necrosis Tumoral/farmacología , Factores de Necrosis Tumoral/deficiencia , Factores de Necrosis Tumoral/genética , Adolescente , Adulto JovenAsunto(s)
VIH-1/fisiología , Macrófagos Alveolares/virología , Mycobacterium tuberculosis/fisiología , Nanotubos , Animales , Diferenciación Celular , Microambiente Celular , Coinfección/microbiología , Coinfección/virología , Progresión de la Enfermedad , Infecciones por VIH/virología , Humanos , Interleucina-10/fisiología , Macaca , Activación de Macrófagos/fisiología , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/ultraestructura , Monocitos/fisiología , Receptores de Superficie Celular/metabolismo , Tuberculosis/microbiología , Replicación ViralAsunto(s)
Antiinfecciosos/farmacología , Factores Inmunológicos/farmacología , Loperamida/farmacología , Monocitos/efectos de los fármacos , Monocitos/microbiología , Tuberculosis/tratamiento farmacológico , Células Cultivadas , Citocinas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Monocitos/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/metabolismo , Tuberculosis/microbiología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Prostaglandin E2 (PGE2) suppresses macrophage effector mechanisms; however, little is known about the function of PGD2 in infected alveolar macrophages (AMs). Using serum-opsonized Histoplasma capsulatum (Ops-H. capsulatum) in vitro, we demonstrated that AMs produced PGE2 and PGD2 in a time-dependent manner, with PGE2 levels exceeding those of PGD2 by 48 h postinfection. Comparison of the effects of both exogenous PGs on AMs revealed that PGD2 increased phagocytosis and killing through the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes receptor, whereas PGE2 had opposite effects, through E prostanoid (EP) receptor 2 (EP2)/EP4-dependent mechanisms. Moreover, PGD2 inhibited phospholipase C-γ (PLC-γ) phosphorylation, reduced IL-10 production, and increased leukotriene B4 receptor expression. In contrast, exogenous PGE2 treatment reduced PLC-γ phosphorylation, p38 and nuclear factor κB activation, TNF-α, H2O2, and leukotriene B4, but increased IL-1ß production. Using specific compounds to inhibit the synthesis of each PG in vitro and in vivo, we found that endogenous PGD2 contributed to fungicidal mechanisms and controlled inflammation, whereas endogenous PGE2 decreased phagocytosis and killing of the fungus and induced inflammation. These findings demonstrate that, although PGD2 acts as an immunostimulatory mediator to control H. capsulatum infection, PGE2 has immunosuppressive effects, and the balance between these two PGs may limit collateral immune damage at the expense of microbial containment.
Asunto(s)
Dinoprostona/farmacología , Histoplasma/efectos de los fármacos , Histoplasmosis/tratamiento farmacológico , Macrófagos Alveolares/efectos de los fármacos , Prostaglandina D2/farmacología , Animales , Células Cultivadas , Macrófagos Alveolares/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Ratas , Ratas WistarRESUMEN
Alveolar macrophages (AMs) are major targets of Mycobacterium tuberculosis (Mtb) infection, critical during the progression of active tuberculosis (TB). The complex immunopathology of TB generates diverse microenvironments in the lung, which shape immune responses by AMs. In the current study, we perform whole genome microarray transcriptional profiling on RNA isolated from AMs from TB patients (AMsTB) compared to AMs from control subjects (AMsCT) using bronchoalveolar lavage (BAL). Our hypothesis was that systemic effects on the local lung microenvironment during TB affect the transcriptional response of AMsTB. We found a unique gene expression profile of 51 genes, including up-regulated CHIT1, CHI3L1, CCL5, CCL22, CCL8, CXCL9, MMP9, MMP7 and MMP12, associated with a robust pro-inflammatory response, cell recruitment and tissue damage, and genes of the cyclin family (CCND1, CCND2, and CCNA1) associated with cell proliferation. These expression profiles may account for the inflammatory condition in the lungs of TB patients. CXCL5, IL1B, CAMP, and TGFB1 were down-regulated, suggesting an altered control of Mtb infection. Also, MARCO and COLEC12, affecting phagocytosis, and CES1, associated with an increase in free cholesterol, were down-regulated. The observed changes in mRNA expression profiles may partially account for the inability of AMsTB to effectively control Mtb infection, suggesting that a balanced control of pro- and anti-inflammatory immune responses is crucial for infection control.
Asunto(s)
Citocinas/genética , Perfilación de la Expresión Génica/métodos , Mediadores de Inflamación , Macrófagos Alveolares/metabolismo , Mycobacterium tuberculosis/patogenicidad , Transcriptoma , Tuberculosis Pulmonar/genética , Adulto , Estudios de Casos y Controles , Células Cultivadas , Citocinas/metabolismo , Femenino , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Masculino , Mycobacterium tuberculosis/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal , Transcripción Genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/metabolismo , Tuberculosis Pulmonar/microbiologíaRESUMEN
Alveolar macrophages (AMs) are multitasking cells that maintain lung homeostasis by clearing apoptotic cells (efferocytosis) and performing antimicrobial effector functions. Different PRRs have been described to be involved in the binding and capture of non-opsonized Streptococcus pneumoniae, such as TLR-2, mannose receptor (MR) and scavenger receptors (SRs). However, the mechanism by which the ingestion of apoptotic cells negatively influences the clearance of non-opsonized S. pneumoniae remains to be determined. In this study, we evaluated whether the prostaglandin E2 (PGE2) produced during efferocytosis by AMs inhibits the ingestion and killing of non-opsonized S. pneumoniae. Resident AMs were pre-treated with an E prostanoid (EP) receptor antagonist, inhibitors of cyclooxygenase and protein kinase A (PKA), incubated with apoptotic Jurkat T cells, and then challenged with S. pneumoniae. Efferocytosis slightly decreased the phagocytosis of S. pneumoniae but greatly inhibited bacterial killing by AMs in a manner dependent on PGE2 production, activation of the EP2-EP4/cAMP/PKA pathway and inhibition of H2O2 production. Our data suggest that the PGE2 produced by AMs during efferocytosis inhibits H2O2 production and impairs the efficient clearance non-opsonized S. pneumoniae by EP2-EP4/cAMP/PKA pathway.
Asunto(s)
Dinoprostona/metabolismo , Macrófagos Alveolares/inmunología , Fagocitosis , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/inmunología , Animales , Apoptosis , Bacteriólisis , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Homeostasis , Humanos , Peróxido de Hidrógeno/metabolismo , Células Jurkat , Macrófagos Alveolares/microbiología , Ratas , Ratas Wistar , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de SeñalRESUMEN
PURPOSE: To investigate the effects of neonatal malnutrition followed by nutritional replacement on the signaling mechanisms developed by the inflammasome complex by analyzing the expression of the targeted TLR2, TLR4, NLRP3, caspase-1 and release of IL-1ß and IL-18 by alveolar macrophages infected in vitro with Candida albicans. METHODS: Male Wistar rats (n = 24), 90-120 days, were suckled by mothers whose diet during lactation contained 17 % protein in the nourish group and 8 % protein in the malnourished group. After weaning, both groups were fed a normal protein diet. Macrophages were obtained after tracheostomy, through the collection of bronchoalveolar lavage fluid. The quantification of the expression levels of targets (TLR2, TLR4, NLRP3 and caspase-1) was performed by real-time RT-PCR. Production of cytokines was performed by ELISA. RESULTS: The malnourished animals during lactation showed reduced body weight from the fifth day of life, remaining until adulthood. Further, the model applied malnutrition induced a lower expression of TLR4 and caspase-1. The quantification of the TLR2 and NLRP3, as well as the release of IL-1ß and IL-18, was not different between groups of animals nourished and malnourished. The system challenged with Candida albicans showed high expression levels of all targets in the study. CONCLUSIONS: The tests demonstrate nutritional restriction during critical periods of development, although nutritional supplementation may compromise defense patterns in adulthood in a timely manner, preserving distinct signaling mechanism, so that the individual does not become widely vulnerable to infections by opportunistic pathogens.
Asunto(s)
Candidiasis/metabolismo , Dieta con Restricción de Proteínas/efectos adversos , Regulación del Desarrollo de la Expresión Génica , Inflamasomas/metabolismo , Macrófagos Alveolares/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Infecciones Oportunistas/metabolismo , Animales , Animales Recién Nacidos , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/microbiología , Candida albicans/inmunología , Candidiasis/inmunología , Candidiasis/microbiología , Candidiasis/patología , Caspasa 1/genética , Caspasa 1/metabolismo , Células Cultivadas , Regulación hacia Abajo , Femenino , Inmunidad Innata , Inflamasomas/inmunología , Lactancia , Activación de Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/patología , Masculino , Infecciones Oportunistas/inmunología , Infecciones Oportunistas/microbiología , Infecciones Oportunistas/patología , Ratas Wistar , Delgadez/etiología , Delgadez/inmunología , Delgadez/microbiología , Delgadez/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Mycobacterium tuberculosis (Mtb) is known to be responsible for tuberculosis (TB), but the pathogenesis of this disease and the host defense mechanisms involved are, for the most part, poorly understood. In this study, we divided 30 male C57BL/6 mice into control and infection groups, and following injection with physiological saline or Mtb, respectively, euthanized five mice from each group on days 1, 3, and 7. TNF-α and IL-10 levels were measured by enzyme-linked immunosorbent assay and flow cytometry, with the latter also being performed to assess apoptosis rates. Protein expression of STAT3 and its phosphorylated form (p-STAT3) was analyzed by western blotting. After Mtb infection, TNF-α and IL-10 levels, alveolar macrophage apoptosis, and STAT3 and p-STAT3 expression increased significantly on days 1, 3, and 7 (P < 0.05), with maximum values on day 3. Furthermore, the Pearson correlation test showed that production of the cytokines TNF-α and IL-10 correlated strongly with expression of STAT3 and p-STAT3 proteins (P < 0.05). Taken together, our results suggest that the STAT3 signaling pathway might play a key role in the regulation of cell proliferation and alveolar macrophage apoptosis in response to Mtb. This provides a theoretical mechanism behind TB pathogenesis and host defense against Mtb, and contributes towards development of an effective treatment.
Asunto(s)
Interleucina-10/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Mycobacterium tuberculosis/fisiología , Tuberculosis/metabolismo , Tuberculosis/microbiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Macrófagos Alveolares/patología , Masculino , Ratones Endogámicos C57BL , Fosforilación , Factor de Transcripción STAT3/metabolismo , Tuberculosis/patologíaRESUMEN
Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. Q fever is an atypical pneumonia transmitted through inhalation of contaminated aerosols. In mammalian lungs, C. burnetii infects and replicates in several cell types, including alveolar macrophages (AMs). The innate immunity and signaling pathways operating during infection are still poorly understood, in part because of the lack of relevant host cell models for infection in vitro In the study described here, we investigated and characterized the infection of primary murine AMs by C. burnetii phase II in vitro Our data reveal that AMs show a pronounced M2 polarization and are highly permissive to C. burnetii multiplication in vitro Murine AMs present an increased susceptibility to infection in comparison to primary bone marrow-derived macrophages. AMs support more than 2 logs of bacterial replication during 12 days of infection in culture, similar to highly susceptible host cells, such as Vero and THP-1 cells. As a proof of principle that AMs are useful for investigation of C. burnetii replication, we performed experiments with AMs from Nos2(-/-) or Ifng(-/-) mice. In the absence of gamma interferon and nitric oxide synthase 2 (NOS2), AMs were significantly more permissive than wild-type cells. In contrast, AMs from Il4(-/-) mice were more restrictive to C. burnetii replication, supporting the importance of M2 polarization for the permissiveness of AMs to C. burnetii replication. Collectively, our data account for understanding the high susceptibility of alveolar macrophages to bacterial replication and support the use of AMs as a relevant model of C. burnetii growth in primary macrophages.
Asunto(s)
Coxiella burnetii/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/microbiología , Animales , Células Cultivadas , Inmunidad Innata/inmunología , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/inmunología , Fiebre Q/inmunología , Fiebre Q/microbiología , Transducción de Señal/inmunologíaRESUMEN
Rhodococcus equi preferentially infects macrophages causing pyogranulomatous pneumonia in young foals. Both the vapA and rhbC genes are up-regulated in an iron (Fe)-deprived environment, such as that found within macrophages. Chloroquine (CQ) is a drug widely used against malaria that suppresses the intracellular availability of Fe in eukaryotic cells. The main objective of this study was to evaluate the ability of CQ to inhibit replication of virulent R. equi within murine (J774A.1) and foal alveolar macrophages (AMs) and to verify whether the mechanism of inhibition could be Fe-deprivation-dependent. CQ effect on R. equi extracellular survival and toxicity to J774A.1 were evaluated. R. equi survival within J774A.1 and foal AMs was evaluated under CQ (10 and 20µM), bovine saturated transferrin (bHTF), and bovine unsaturated transferrin (bATF) exposure. To explore the action mechanism of CQ, the superoxide anion production, the lysozyme activity, as well as the relative mRNA expression of vapA and rhbC were examined. CQ at≤20µM had no effect on R. equi extracellular multiplication and J774A.1 viability. Exposure to CQ significantly and markedly reduced survival of R. equi within J774A.1 and foal AMs. Treatment with bHTF did not reverse CQ effect on R. equi. Exposure to CQ did not affected superoxide anion production or lysozyme activity, however vapA and rhbC expression was significantly increased. Our results reinforce the hypothesis that intracellular availability of Fe is required for R. equi survival, and our initial hypothesis that CQ can limit replication of R. equi in J774A.1 and foal AMs, most likely by Fe starvation.
Asunto(s)
Cloroquina/farmacología , Macrófagos Alveolares/microbiología , Rhodococcus equi/efectos de los fármacos , Animales , Proteínas Bacterianas/genética , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Caballos , Hierro/metabolismo , Macrófagos Alveolares/citología , Ratones , Viabilidad Microbiana/efectos de los fármacos , Muramidasa/metabolismo , Rhodococcus equi/citologíaRESUMEN
OBJECTIVE: Evaluate the effects of neonatal malnutrition on the microbicidal response and viability of in vitro macrophages infected with Staphylococcus aureus sensitive/resistant to methicillin. METHODS: Male Wistar rats (n = 24) were divided into two distinct groups: nourished (rats breast-fed by mothers undergoing diet with 17% casein) and malnourished (rats breast-fed by mothers undergoing diet with 8% casein). Macrophages were recovered after surgical tracheostomy procedure by collecting bronchoalveolar lavage. Four systems were established: negative control, composed only by phagocytes; positive control, macrophages plus lipopolysaccharide; and two test systems, macrophages plus Staphylococcus aureus sensitive and resistant to methicillin. Plates were incubated at 37 °C for 24 h. After this period, tests for the analysis of cell viability and microbicidal response were performed. In the statistical analysis, the Student's t and ANOVA tests were used, accepting p < 0.05. RESULTS: The neonatal malnutrition impaired the animals' body weight. There was a lower expression of the inducible nitric oxide enzyme (iNOS), nitric oxide production, and viability of macrophages infected with methicillin-resistant Staphylococcus aureus. However, increased production of superoxide anion in the malnourished group was detected. CONCLUSION: Neonatal malnutrition focusing on critical periods of development promoted lower expression of iNOS, nitric oxide production, cell viability, and exacerbated reactive oxygen species production. The high levels of reactive oxygen species may favor the onset of serious and systemic infections with fatal outcome if associated with methicillin-resistant Staphylococcus aureus.
Asunto(s)
Radicales Libres/metabolismo , Macrófagos Alveolares/microbiología , Desnutrición/microbiología , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Óxido Nítrico Sintasa de Tipo II/metabolismo , Staphylococcus aureus/aislamiento & purificación , Animales , Animales Recién Nacidos , Peso Corporal , Supervivencia Celular , Dieta , Lipopolisacáridos/efectos adversos , Macrófagos Alveolares/citología , Masculino , Desnutrición/fisiopatología , Meticilina/farmacología , Viabilidad Microbiana/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Fagocitos/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
Cysteinyl leukotrienes (CysLTs) and lipoxins (LXs) are lipid mediators that control inflammation, with the former inducing and the latter inhibiting this process. Because the role played by these mediators in paracoccidioidomycosis was not investigated, we aimed to characterize the role of CysLT in the pulmonary infection developed by resistant (A/J) and susceptible (B10.A) mice. 48 h after infection, elevated levels of pulmonary LTC4 and LXA4 were produced by both mouse strains, but higher levels were found in the lungs of susceptible mice. Blocking the CysLTs receptor by MTL reduced fungal loads in B10.A, but not in A/J mice. In susceptible mice, MLT treatment led to reduced influx of PMN leukocytes, increased recruitment of monocytes, predominant synthesis of anti-inflammatory cytokines, and augmented expression of 5- and 15-lipoxygenase mRNA, suggesting a prevalent LXA4 activity. In agreement, MTL-treated macrophages showed reduced fungal burdens associated with decreased ingestion of fungal cells. Furthermore, the addition of exogenous LX reduced, and the specific blockade of the LX receptor increased the fungal loads of B10.A macrophages. This study showed for the first time that inhibition of CysLTs signaling results in less severe pulmonary paracoccidioidomycosis that occurs in parallel with elevated LX activity and reduced infection of macrophages.
Asunto(s)
Lipoxinas/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/etiología , Acetatos/farmacología , Animales , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Ciclopropanos , Dinoprostona/biosíntesis , Mediadores de Inflamación/metabolismo , Antagonistas de Leucotrieno/farmacología , Leucotrieno C4/biosíntesis , Lipoxinas/biosíntesis , Lipoxinas/inmunología , Macrófagos Alveolares/efectos de los fármacos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos A , Ratones Noqueados , Paracoccidioidomicosis/tratamiento farmacológico , Paracoccidioidomicosis/inmunología , Quinolinas/farmacología , Receptores de Leucotrienos/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , SulfurosRESUMEN
Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-α), CXCL1 or keratinocyte chemoattractant (KC), interleukin-1ß (IL-1ß), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and TLR9 revealed that TNF-α and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class II (MHC-II) molecules induced by gamma interferon (IFN-γ) in AM. The same phenomenon was induced by incubation with heat-killed B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to such survival.
Asunto(s)
Brucella abortus/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Animales , Citocinas/metabolismo , Regulación hacia Abajo , Femenino , Evasión Inmune , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Viabilidad Microbiana , Receptor Toll-Like 2/genéticaRESUMEN
Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.
Asunto(s)
Macrófagos Alveolares/microbiología , Paracoccidioides , Paracoccidioidomicosis , Fosfolipasas/genética , Factores de Virulencia/genética , Animales , Expresión Génica , Masculino , Ratones Endogámicos BALB C , Paracoccidioides/citología , Paracoccidioides/enzimología , Paracoccidioides/patogenicidad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1(-/-) mice at day 3 after bacillus Calmette-Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner.
Asunto(s)
Apoptosis , Macrófagos Alveolares/microbiología , Mycobacterium tuberculosis/patogenicidad , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Tuberculosis/microbiología , Animales , Línea Celular , Membrana Celular/inmunología , Membrana Celular/microbiología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/crecimiento & desarrollo , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Factores de Tiempo , Tuberculosis/inmunología , Tuberculosis/patología , Factor de Necrosis Tumoral alfa/metabolismo , VirulenciaRESUMEN
Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mßCD). Removal of ergosterol by mßCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mßCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mßCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.
Asunto(s)
Proteínas Fúngicas/metabolismo , Histoplasma/metabolismo , Histoplasma/patogenicidad , Histoplasmosis/metabolismo , Macrófagos Alveolares/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Ergosterol/metabolismo , Histoplasmosis/microbiología , Histoplasmosis/patología , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos BALB C , beta-Ciclodextrinas/farmacologíaRESUMEN
BACKGROUND: Phospholipase B (PLB) has been reported to be one of the virulence factors for human pathogenic fungi and has also been described as necessary for the early events in infection. Based on these data, we investigated the role of PLB in virulence and modulation of the alveolar pulmonary immune response during infection using an in-vitro model of host-pathogen interaction, i.e. Paracoccidioides brasiliensis yeast cells infecting alveolar macrophage (MH-S) cells. RESULTS: The effect of PLB was analyzed using the specific inhibitor alexidine dihydrochloride (0.25 µM), and pulmonary surfactant (100 µg mL-1), during 6 hours of co-cultivation of P. brasiliensis and MH-S cells. Alexidine dihydrochloride inhibited PLB activity by 66% and significantly decreased the adhesion and internalization of yeast cells by MH-S cells. Genes involved in phagocytosis (trl2, cd14) and the inflammatory response (nfkb, tnf-α, il-1ß) were down-regulated in the presence of this PLB inhibitor. In contrast, PLB activity and internalization of yeast cells significantly increased in the presence of pulmonary surfactant; under this condition, genes such as clec2 and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of interleukin-10 (IL-10) and increased the levels of IL-12 and tumor necrosis factor-α (TNF-α). In addition, gene expression analysis of plb1, sod3 and icl1 suggests that P. brasiliensis gene re-programming is effective in facilitating adaptation to this inhospitable environment, which mimics the lung-environment interaction. CONCLUSION: P. brasiliensis PLB activity is involved in the process of adhesion and internalization of yeast cells at the MH-S cell surface and may enhance virulence and subsequent down-regulation of macrophage activation.
Asunto(s)
Espacio Extracelular/enzimología , Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno , Lisofosfolipasa/metabolismo , Macrófagos Alveolares/microbiología , Paracoccidioides/enzimología , Paracoccidioidomicosis/microbiología , Animales , Adhesión Bacteriana , Línea Celular , Citocinas/genética , Citocinas/inmunología , Espacio Extracelular/genética , Proteínas Fúngicas/genética , Humanos , Lisofosfolipasa/genética , Macrófagos Alveolares/inmunología , Ratones , Paracoccidioides/genética , Paracoccidioides/inmunología , Paracoccidioides/fisiología , Paracoccidioidomicosis/genética , Paracoccidioidomicosis/inmunologíaRESUMEN
Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guérin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.
Asunto(s)
Apoptosis/inmunología , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Mycobacterium bovis/patogenicidad , Tuberculosis/inmunología , Animales , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Pulmón/metabolismo , Pulmón/microbiología , Macrófagos Alveolares/citología , Macrófagos Alveolares/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tuberculosis/microbiología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Virulencia/inmunologíaRESUMEN
Glucuronoxylomannan (GXM) is the major capsular polysaccharide of Cryptococcus neoformans. It is essential for fungal virulence and causes a number of deleterious effects to host cells. During the last decades, most of the experimental models designed to study the roles of GXM during cryptococcal infection were based on the stimulation of animal cells. This most commonly involved macrophages or other effector cells, with polysaccharide fractions obtained by precipitation with cationic detergents. More recently, it has been demonstrated that GXM interferes with the physiological state of other target cells, such as the epithelium. In addition, recent studies indicate that the structure of the polysaccharide and, consequently, its functions vary according with the method used for its purification. This raises questions as to what is native GXM and the significance of prior studies. In this paper, we discuss some of the aspects of GXM that are still poorly explored in the current literature, including the relevance of the polysaccharide in the interaction of cryptococci with non-phagocytic cells and the relationship between its structure and biological activity.
Asunto(s)
Cryptococcus neoformans/fisiología , Polisacáridos/fisiología , Antígenos Fúngicos/química , Antígenos Fúngicos/metabolismo , Células Cultivadas , Cryptococcus neoformans/química , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Humanos , Interleucina-8/metabolismo , Lisofosfolipasa/metabolismo , Macrófagos Alveolares/microbiología , Metales/metabolismo , Polisacáridos/química , Alveolos Pulmonares/microbiologíaRESUMEN
Os leucotrienos aumentam a fagocitose e a atividade microbicida contra uma série de patógenos. Em macrófagos alveolares, LTB`IND.4´ e LTD`IND.4´ aumentam a fagocitose via Fc`GAMA´R de modo dependente de PKC. Entretanto, o papel das isoformas específicas da PKC, das MAPK, e da Pi3K neste processo, ainda não é conhecido. Além disso, pouco se sabe sobre a importância dos leucotrienos na fagocitose via outros receptores. Os objetivos deste trabalho são: a) ampliar o conhecimento sobre as vias de sinalização ativadas pelos leucotrienos durante a fagocitose de hemácias opsonizadas por IgG; b) avaliar o efeito dos leucotrienos na fagocitose de Candida albicans, por macrófagos alveolares e as vias de sinalização intracelular envolvidas. Observou-se que os leucotrienos endogenamente produzidos ou adicionados aos macrófagos alveolares, aumentam a fagocitose via Fc`GAMA´R e para isso utilizam distintas vias de sinalização intracelular. A ação do LTB`IND.4´ envolveu predominantemente a via ERK1/2 e PKC`ALFA´ e com menor intensidade da PKC`DELTA´...