RESUMEN
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor involved in redox homeostasis and in the response induced by oxidative injury. Nrf2 is present in an inactive state in the cytoplasm of cells. Its activation by internal or external stimuli, such as infections or pollution, leads to the transcription of more than 500 elements through its binding to the antioxidant response element. The lungs are particularly susceptible to factors that generate oxidative stress such as infections, allergens and hyperoxia. Nrf2 has a crucial protective role against these ROS. Oxidative stress and subsequent activation of Nrf2 have been demonstrated in many human respiratory diseases affecting the airways, including asthma and chronic obstructive pulmonary disease (COPD), or the pulmonary parenchyma such as acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. Several compounds, both naturally occurring and synthetic, have been identified as Nrf2 inducers and enhance the activation of Nrf2 and expression of Nrf2-dependent genes. These inducers have proven particularly effective at reducing the severity of the oxidative stress-driven lung injury in various animal models. In humans, these compounds offer promise as potential therapeutic strategies for the management of respiratory pathologies associated with oxidative stress but there is thus far little evidence of efficacy through human trials. The purpose of this review is to summarize the involvement of Nrf2 and its inducers in ARDS, COPD, asthma and lung fibrosis in both human and in experimental models.
RESUMEN
Exposure of mice to high concentrations of chlorine leads to the synthesis of cysteinyl leukotrienes (cysLTs). CysLTs contribute to chlorine-induced airway hyperresponsiveness. The aim of the current study was to determine the cellular source of the cysLTs. To achieve this aim, we exposed mice to 100 ppm of chlorine for 5 minutes. Intranasal instillation of clodronate in liposomes and of diphtheria toxin in CD11c-DTR mice was used to deplete macrophages. CCR2-/- mice were used to assess the contribution of recruited macrophages. Eosinophils and neutrophils were depleted with specific antibodies. Platelet-neutrophil aggregation was prevented with an antibody against P-selectin. The potential roles of phagocytosis of neutrophils by macrophages and of transcellular metabolism between epithelial cells and neutrophils were explored in coculture systems. We found that depletion of neutrophils was the only intervention that inhibited the synthesis of cysLTs at 24 hours after chlorine exposure. Although macrophages did synthesize cysLTs in response to phagocytosis of neutrophils, depletion of macrophages did not reduce the increment in cysLTs triggered by chlorine exposure. However, coculture of airway epithelial cells with neutrophils resulted in a significant increase in the synthesis of cysLTs, dependent on the expression of 5-lipoxygenase by neutrophils. We conclude that cysLT synthesis following chlorine exposure may be dependent on transcellular metabolism by neutrophil-epithelial interactions.
Asunto(s)
Cloro/toxicidad , Cisteína/metabolismo , Leucotrienos/metabolismo , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Líquido del Lavado Bronquioalveolar , Técnicas de Cocultivo , Cisteína/biosíntesis , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interleucina-5/antagonistas & inhibidores , Interleucina-5/metabolismo , Leucotrienos/biosíntesis , Liposomas , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Neumonía/metabolismo , Neumonía/patologíaRESUMEN
BACKGROUND: Inhaled oxidative toxicants present in ambient air cause airway epithelial injury, inflammation, and airway hyperresponsiveness. Effective adaptation to such environmental insults is essential for the preservation of pulmonary function, whereas failure or incomplete adaptation to oxidative injury can render the host susceptible to the development of airway disease. OBJECTIVE: We sought to explore the mechanisms of airway adaptation to oxidative injury. METHODS: For a model to study pulmonary adaptation to oxidative stress-induced lung injury, we exposed mice to repeated nose-only chlorine gas exposures. Outcome measures were evaluated 24 hours after the last chlorine exposure. Lung mechanics and airway responsiveness to methacholine were assessed by using the flexiVent. Inflammation and antioxidant responses were assessed in both bronchoalveolar lavage fluid and lung tissue. Using both loss or gain of function and genomic approaches, we further dissected the cellular and molecular mechanisms involved in pulmonary adaptation. RESULTS: Repeated exposures to oxidative stress resulted in pulmonary adaptation evidenced by abrogation of neutrophilic inflammation and airway hyperresponsiveness. This adaptation was independent of antioxidant mechanisms and regulatory T cells but dependent on residential alveolar macrophages (AMs). Interestingly, 5% of AMs expressed forkhead box P3, and depletion of these cells abolished adaptation. Results from transcriptomic profiling and loss and gain of function suggest that adaptation might be dependent on TGF-ß and prostaglandin E2. CONCLUSION: Pulmonary adaptation during oxidative stress-induced lung injury is mediated by a novel subset of forkhead box P3-positive AMs that limits inflammation, favoring airway adaptation and host fitness through TGF-ß and prostaglandin E2.
Asunto(s)
Adaptación Fisiológica/fisiología , Macrófagos Alveolares/metabolismo , Estrés Oxidativo/inmunología , Hipersensibilidad Respiratoria/metabolismo , Animales , Cloro/toxicidad , Dinoprostona/metabolismo , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/metabolismo , Irritantes/toxicidad , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/inmunología , Lesión Pulmonar/metabolismo , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Inhalation of organic dust (OD) from swine confinement facilities leads to pulmonary inflammation, airway hyperresponsiveness, and oxidative stress. In mice, pretreatment with a hydroxyl radical scavenger prevents airway inflammation and airway hyperresponsiveness (AHR) induced by OD exposure. We sought to determine a mechanism by which OD could induce oxidative stress in bronchial epithelial cells. Human bronchial epithelial cells (BEAS-2B or NHBE) were treated with various concentrations of OD, followed by evaluation of intracellular oxidative stress using 2',7'-dichlorofluorescein diacetate (DCFDA). After stimulation with OD, gene expression of antioxidant genes was assessed by real-time quantitative PCR followed by quantification of Nrf2 nuclear translocation using a luciferase reporter assay. Phagocytic markers (CD36 and CD68) were analyzed by FACS. Cells were treated with an actin inhibitor, cytochalasin D, before OD exposure and evaluated for Nrf2 nuclear translocation and DCFDA. Mice were pretreated with sulforaphane, the Nrf2 activator, before OD exposure and evaluated for pulmonary inflammation and airway reactivity. OD induced a time- and concentration-dependent increase in DCFDA. mRNA expression levels of Nrf2-dependent genes and Nrf2 nuclear translocation were increased after OD exposure. OD exposure increased the expression of CD68 and CD36. Cytochalasin D prevented oxidative stress and Nrf2 nuclear translocation after OD. Pretreatment with sulforaphane prevented OD-induced inflammation and AHR while increasing the uptake of OD in bronchial epithelial cells. Bronchial epithelial cells can phagocytose OD, resulting in an increase in endogenous oxidative stress. Nrf2-dependent mechanisms mediate the antioxidant response to OD.
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Bronquios/metabolismo , Polvo , Células Epiteliales/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Células Epiteliales/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Isotiocianatos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Fagocitos , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/metabolismo , SulfóxidosRESUMEN
CD4 T cells express the epidermal growth factor (EGF) receptor ligand, heparin-binding EGF (HB-EGF), with no defined immuno-pathophysiological function. Therefore, we wished to elucidate the function of HB-EGF synthesized by CD4 T cells in the context of allergic pulmonary inflammation and the asthma surrogate, airway hyperresponsiveness, in a murine acute model of asthma. In this study, we show how knocking out HB-EGF expression in CD4 T cells in vivo attenuates IL-5 synthesis in the lung that is accompanied by diminished eosinophilic inflammation and airway hyperresponsiveness. HB-EGF coimmunoprecipitates with the transcriptional repressor B cell lymphoma 6 (Bcl-6) in CD4 T cells. Knocking out HB-EGF in CD4 T cells resulted in increased Bcl-6 binding to the IL-5 gene and decreased IL-5 mRNA expression. Thus, these findings suggest an immunoregulatory function for intrinsic HB-EGF expressed by CD4 T cells in TH2 inflammation and airway dysfunction by modulating IL-5 expression via binding to and inhibiting the repressive function of Bcl-6.
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Asma/inmunología , Eosinofilia/inmunología , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Hipersensibilidad Respiratoria/inmunología , Células Th2/inmunología , Animales , Antígenos CD4/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Humanos , Interleucina-5/genética , Interleucina-5/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismoRESUMEN
Membrane-associated RING-CH-1 (March1) is a member of the March family of E3 ubiquitin ligases. March1 downregulates cell surface expression of MHC II and CD86 by targeting them to lysosomal degradation. Given the key roles of MHC class II and CD86 in T cell activation and to get further insights into the development of allergic inflammation, we asked whether March1 deficiency exacerbates or attenuates features of allergic asthma in mice. Herein, we used an acute model of allergy to compare the asthmatic phenotype of March1-deficient and -sufficient mice immunized with ovalbumin (OVA) and later challenged by intranasal instillation of OVA in the lungs. We found that eosinophilic inflammation in airways and lung tissue was similar between WT and March1-/- allergic mice, whereas neutrophilic inflammation was significant only in March1-/- mice. Airway hyperresponsiveness as well as levels of IFN-γ, IL-13, IL-6, and IL-10 was lower in the lungs of asthmatic March1-/- mice compared to WT, whereas lung levels of TNF-α, IL-4, and IL-5 were not significantly different. Interestingly, in the serum, levels of total and ova-specific IgE were reduced in March1-deficient mice as compared to WT mice. Taken together, our results demonstrate a role of March1 E3 ubiquitin ligase in modulating allergic responses.
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Asma/inmunología , Pulmón/inmunología , Neumonía/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Alérgenos/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina E/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila , Ovalbúmina/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Activated CD4 T cells connect to airway smooth muscle cells (ASMCs) in vitro via lymphocyte-derived membrane conduits (LMCs) structurally similar to membrane nanotubes with unknown intercellular signals triggering their formation. We examined the structure and function of CD4 T cell-derived LMCs, and we established a role for ASMC-derived basic fibroblast growth factor 2 (FGF2b) and FGF receptor (FGFR)1 in LMC formation. Blocking FGF2b's synthesis and FGFR1 function reduced LMC formation. Mitochondrial flux from ASMCs to T cells was partially FGF2b and FGFR1 dependent. LMC formation by CD4 T cells and mitochondrial transfer from ASMCs was increased in the presence of asthmatic ASMCs that expressed more mRNA for FGF2b compared with normal ASMCs. These observations identify ASMC-derived FGF2b as a factor needed for LMC formation by CD4 T cells, affecting intercellular communication.
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Linfocitos T CD4-Positivos/inmunología , Comunicación Celular/inmunología , Extensiones de la Superficie Celular/inmunología , Factor 2 de Crecimiento de Fibroblastos/inmunología , Miocitos del Músculo Liso/inmunología , Linfocitos T CD4-Positivos/citología , Humanos , Mitocondrias/inmunología , Miocitos del Músculo Liso/citología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/inmunología , Sistema Respiratorio/citología , Sistema Respiratorio/inmunologíaRESUMEN
BACKGROUND AND PURPOSE: Cysteinyl leukotrienes (CysLTs) are pro-inflammatory lipid mediators that exacerbate disease state in several asthma phenotypes including asthma induced by allergen, virus and exercise. However, the role of CysLTs in irritant-induced airway disease is not well characterized. The purpose of the current study was to investigate the effect of montelukast, a CysLT1 receptor antagonist, on parameters of irritant-induced asthma induced by inhalation of chlorine in the mouse. EXPERIMENTAL APPROACH: BALB/c mice were exposed to chlorine in air (100 ppm, for 5 min). Montelukast (3 mg·kg-1 ) or the vehicle (1% methylcellulose) was administered 24 and 1 h prior to chlorine exposure and 1 h prior to outcome measurements. Twenty-four hours after exposure, responses to inhaled aerosolized methacholine, cell composition and an array of cytokines/chemokines in bronchoalveolar lavage (BAL) fluid were measured. Neutralizing antibodies against IL-6 and VEGF were administered prior to exposures. KEY RESULTS: Montelukast reduced chlorine -induced airway hyperresponsiveness (AHR) to methacholine in the peripheral lung compartment as estimated from dynamic elastance, but not in large conducting airways. Montelukast treatment attenuated chlorine-induced macrophage influx, neutrophilia and eosinophilia in BAL fluid. Chlorine exposure increased VEGF, IL-6, the chemokines KC and CCL3 in BAL fluid. Montelukast treatment prevented chlorine-induced increases in VEGF and IL-6. Anti-IL-6 antibody inhibited chlorine-induced neutrophilia and reduced AHR. CONCLUSIONS AND IMPLICATIONS: Pre-treatment with montelukast attenuated chlorine-induced neutrophilia and AHR in mice. These effects are mediated, in part, via IL-6.
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Acetatos/uso terapéutico , Antiinflamatorios/uso terapéutico , Cloro/toxicidad , Irritantes/toxicidad , Antagonistas de Leucotrieno/uso terapéutico , Quinolinas/uso terapéutico , Hipersensibilidad Respiratoria/tratamiento farmacológico , Acetatos/farmacología , Animales , Antiinflamatorios/farmacología , Anticuerpos Monoclonales/farmacología , Líquido del Lavado Bronquioalveolar/inmunología , Línea Celular , Ciclopropanos , Citocinas/inmunología , Antagonistas de Leucotrieno/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones Endogámicos BALB C , Factor 2 Relacionado con NF-E2/metabolismo , Quinolinas/farmacología , Receptores de Interleucina-6/inmunología , Hipersensibilidad Respiratoria/inducido químicamente , SulfurosRESUMEN
The Toll-like receptor (TLR) adaptor proteins myeloid differentiating factor 88 (MyD88) and Toll, interleukin-1 receptor and resistance protein (TIR) domain-containing adaptor inducing interferon-ß (TRIF) comprise the two principal limbs of the TLR signalling network. We studied the role of these adaptors in the TLR4-dependent inhibition of allergic airway disease and induction of CD4+ ICOS+ T cells by nasal application of Protollin™, a mucosal adjuvant composed of TLR2 and TLR4 agonists. Wild-type (WT), Trif-/- or Myd88-/- mice were sensitized to birch pollen extract (BPEx), then received intranasal Protollin followed by consecutive BPEx challenges. Protollin's protection against allergic airway disease was TRIF-dependent and MyD88-independent. TRIF deficiency diminished the CD4+ ICOS+ T-cell subsets in the lymph nodes draining the nasal mucosa, as well as their recruitment to the lungs. Overall, TRIF deficiency reduced the proportion of cervical lymph node and lung CD4+ ICOS+ Foxp3- cells, in particular. Adoptive transfer of cervical lymph node cells supported a role for Protollin-induced CD4+ ICOS+ cells in the TRIF-dependent inhibition of airway hyper-responsiveness. Hence, our data demonstrate that stimulation of the TLR4-TRIF pathway can protect against the development of allergic airway disease and that a TRIF-dependent adjuvant effect on CD4+ ICOS+ T-cell responses may be a contributing mechanism.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Asma/prevención & control , Linfocitos T CD4-Positivos/metabolismo , Pulmón/metabolismo , Rinitis Alérgica Estacional/prevención & control , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Traslado Adoptivo , Animales , Antígenos de Plantas/inmunología , Asma/inmunología , Asma/metabolismo , Asma/fisiopatología , Betula/inmunología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/fisiopatología , Hiperreactividad Bronquial/prevención & control , Broncoconstricción , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Proliferación Celular , Quimiotaxis de Leucocito , Cisteína Endopeptidasas/inmunología , Modelos Animales de Enfermedad , Combinación de Medicamentos , Femenino , Predisposición Genética a la Enfermedad , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Lipopolisacáridos/inmunología , Pulmón/inmunología , Pulmón/fisiopatología , Activación de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Fenotipo , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/metabolismo , Rinitis Alérgica Estacional/fisiopatología , Transducción de Señal , Factores de Tiempo , Receptor Toll-Like 4/inmunologíaRESUMEN
RATIONALE: Chlorine gas (Cl2) is a potent oxidant and trigger of irritant induced asthma. We explored NF-E2-related factor 2 (Nrf2)-dependent mechanisms in the asthmatic response to Cl2, using Nrf2-deficient mice, buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and sulforaphane (SFN), a phytochemical regulator of Nrf2. METHODS: Airway inflammation and airway hyperresponsiveness (AHR) were assessed 24 and 48h after a 5-min nose-only exposure to 100ppm Cl2 of Nrf2-deficient and wild type Balb/C mice treated with BSO or SFN. Animals were anesthetized, paralyzed and mechanically ventilated (FlexiVent™) and challenged with aerosolized methacholine. Bronchoalveolar lavage (BAL) was performed and lung tissues were harvested for assessment of gene expression. RESULTS: Cl2 exposure induced a robust AHR and an intense neutrophilic inflammation that, although similar in Nrf2-deficient mice and wild-type mice at 24h after Cl2 exposure, were significantly greater at 48h post exposure in Nrf2-deficient mice. Lung GSH and mRNA for Nrf2-dependent phase II enzymes (NQO-1 and GPX2) were significantly lower in Nrf2-deficient than wild-type mice after Cl2 exposure. BSO reduced GSH levels and promoted Cl2-induced airway inflammation in wild-type mice, but not in Nrf2-deficient mice, whereas SFN suppressed Cl2-induced airway inflammation in wild-type but not in Nrf2-deficient mice. AHR was not affected by either BSO or SFN at 48h post Cl2 exposure. CONCLUSIONS: Nrf2-dependent phase II enzymes play a role in the resolution of airway inflammation and AHR after Cl2 exposure. Moderate deficiency of GSH affects the magnitude of acute inflammation but not AHR.
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Inflamación/metabolismo , Pulmón/metabolismo , Factor 2 Relacionado con NF-E2/genética , Hipersensibilidad Respiratoria/metabolismo , Animales , Lavado Broncoalveolar , Butionina Sulfoximina/metabolismo , Cloro/toxicidad , Regulación de la Expresión Génica/genética , Glutatión/antagonistas & inhibidores , Glutatión/biosíntesis , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/fisiopatología , Isotiocianatos/metabolismo , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Cloruro de Metacolina/metabolismo , Ratones , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , ARN Mensajero/genética , Hipersensibilidad Respiratoria/fisiopatología , SulfóxidosRESUMEN
The bronchoconstrictive and proinflammatory properties of cysteinyl leukotrienes (cysLTs) in allergic asthma mediate their effects predominantly through the cysLT1 receptor (cysLT1R). However, the role of cysLTs and cysLT1R in innate immune-triggered asthma is largely unexplored. We explored the synthesis of cysLTs and cysLT1R as determinants of airway responses in an oxidative stress-induced model of irritant asthma. Wild-type (WT) mice exposed to 100 ppm Cl2 for 5 min had airway neutrophilia, increased cysLT production, and pulmonary expression of cysLT-related biosynthetic genes. CysLT1R-deficient (CysLTr1(-/-)) mice that were exposed to Cl2 demonstrated airway hyperresponsiveness to inhaled methacholine significantly greater than in WT BALB/c mice. Compared to WT mice, airway neutrophilia and keratinocyte chemoattractant production levels were higher in CysLTr1(-/-) mice and airway hyperresponsiveness was ameliorated using a granulocyte depletion Ab. CysLTr1(-/-) mice also demonstrated prolonged bronchial epithelial cell apoptosis following Cl2 WT mice showed increased antioxidant and NF erythroid 2-related factor 2 (Nrf2) gene expression, Nrf2 nuclear translocation in bronchial epithelial cells, and increased reduced glutathione/oxidized glutathione following Cl2 exposure whereas CysLTr1(-/-) mice did not. Furthermore, CysLTr1(-/-) mice demonstrated increased pulmonary E-cadherin expression and soluble E-cadherin shedding compared with WT mice. Loss of a functional cysLT1R results in aberrant antioxidant response and increased susceptibility to oxidative injury, apparently via a cysLT1R-dependent impairment of Nrf2 function.
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Asma/inmunología , Queratinocitos/inmunología , Neutrófilos/inmunología , Estrés Oxidativo , Receptores de Leucotrienos/metabolismo , Alérgenos/inmunología , Animales , Asma/inducido químicamente , Cadherinas/metabolismo , Células Cultivadas , Cloro/inmunología , Humanos , Inmunidad Innata , Irritantes/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Receptores de Leucotrienos/genéticaRESUMEN
Airway exposure to organic dust (OD) from swine confinement facilities induces airway inflammation dominated by neutrophils and airway hyperresponsiveness (AHR). One important neutrophilic innate defense mechanism is the induction of oxidative stress. Therefore, we hypothesized that neutrophils exacerbate airway dysfunction following OD exposure by increasing oxidant burden. BALB/C mice were given intranasal challenges with OD or PBS (1/day for 3 days). Mice were untreated or treated with a neutrophil-depleting antibody, anti-Ly6G, or the antioxidant dimethylthiourea (DMTU) prior to OD exposure. Twenty-four hours after the final exposure, we measured airway responsiveness in response to methacholine (MCh) and collected bronchoalveolar lavage fluid to assess pulmonary inflammation and total antioxidant capacity. Lung tissue was harvested to examine the effect of OD-induced antioxidant gene expression and the effect of anti-Ly6G or DMTU. OD exposure induced a dose-dependent increase of airway responsiveness, a neutrophilic pulmonary inflammation, and secretion of keratinocyte cytokine. Depletion of neutrophils reduced OD-induced AHR. DMTU prevented pulmonary inflammation involving macrophages and neutrophils. Neutrophil depletion and DMTU were highly effective in preventing OD-induced AHR affecting large, conducting airways and tissue elastance. OD induced an increase in total antioxidant capacity and mRNA levels of NRF-2-dependent antioxidant genes, effects that are prevented by administration of DMTU and neutrophil depletion. We conclude that an increase in oxidative stress and neutrophilia is critical in the induction of OD-induced AHR. Prevention of oxidative stress diminishes neutrophil influx and AHR, suggesting that mechanisms driving OD-induced AHR may be dependent on neutrophil-mediated oxidant pathways.
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Hiperreactividad Bronquial/metabolismo , Neutrófilos/inmunología , Estrés Oxidativo/inmunología , Neumonía/inmunología , Hipersensibilidad Respiratoria/inmunología , Animales , Hiperreactividad Bronquial/inmunología , Citocinas/metabolismo , Masculino , Cloruro de Metacolina/farmacología , Ratones Endogámicos BALB C , Infiltración Neutrófila/inmunología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neumonía/metabolismoRESUMEN
The effect of remodeling on airway function is uncertain. It may affect airway compressibility during forced expirations differently than airflow resistance, providing a tool for its assessment. The aim of the current study was to compare the effects of acute and chronic antigen challenge on methacholine-induced bronchoconstriction assessed from resistance and maximal tidal expiratory flow. Balb/C mice were sensitized with ovalbumin (OVA) and challenged either daily for three days with intra-nasal OVA or daily for 5 days and three times a week for 5 subsequent weeks. Acute and chronic allergen challenge induced airway hyperresponsiveness (AHR) to methacholine. However the relationship between maximal tidal expiratory flow and resistance during methacholine challenge was different between the two conditions, suggesting that the determinants of AHR are not identical following acute and chronic allergen exposure. We conclude that the contrast of changes in maximal tidal expiratory flow and respiratory resistance during methacholine-induced bronchoconstriction may allow the detection of the mechanical consequences of airway remodeling.
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Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Resistencia de las Vías Respiratorias/fisiología , Hipersensibilidad Respiratoria/fisiopatología , Enfermedad Aguda , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Broncoconstrictores/farmacología , Enfermedad Crónica , Modelos Animales de Enfermedad , Elasticidad , Femenino , Células Caliciformes/patología , Cloruro de Metacolina/farmacología , Ratones Endogámicos BALB C , Músculo Liso Vascular/patología , Ovalbúmina , Ventilación Pulmonar/efectos de los fármacos , Ventilación Pulmonar/fisiología , Distribución Aleatoria , Hipersensibilidad Respiratoria/patología , Volumen de Ventilación Pulmonar/efectos de los fármacos , Volumen de Ventilación Pulmonar/fisiologíaRESUMEN
Airway colonization by the mold Aspergillus fumigatus is common in patients with underlying lung disease and is associated with chronic airway inflammation. Studies probing the inflammatory response to colonization with A. fumigatus hyphae have been hampered by the lack of a model of chronic colonization in immunocompetent mice. By infecting mice intratracheally with conidia embedded in agar beads (Af beads), we have established an in vivo model to study the natural history of airway colonization with live A. fumigatus hyphae. Histopathological examination and galactomannan assay of lung homogenates demonstrated that hyphae exited beads and persisted in the lungs of mice up to 28 days postinfection without invasive disease. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammatory reaction and peribronchial infiltration of lymphocytes. Whole-lung cytokine analysis from Af bead-infected mice revealed an increase in proinflammatory cytokines and chemokines early in infection. Evidence of a Th2 type response was observed only early in the course of colonization, including increased levels of interleukin-4 (IL-4), elevated IgE levels in serum, and a mild increase in airway responsiveness. Pulmonary T cell subset analysis during infection mirrored these results with an initial transient increase in IL-4-producing CD4(+) T cells, followed by a rise in IL-17 and Foxp3(+) cells by day 14. These results provide the first report of the evolution of the immune response to A. fumigatus hyphal colonization.
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Hifa/inmunología , Aspergilosis Pulmonar/inmunología , Aspergilosis Pulmonar/patología , Animales , Aspergillus fumigatus/inmunología , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Contact between airway smooth muscle (ASM) cells and activated CD4(+) T cells, a key interaction in diseases such as asthma, triggers ASM cell proliferation and enhances T cell survival. We hypothesized that direct contact between ASM and CD4(+) T cells facilitated the transfer of anti-apoptotic proteins via nanotubes, resulting in increased survival of activated CD4(+) T cells. CD4(+) T cells, isolated from PBMCs of healthy subjects, when activated and cocultured with ASM cells for 24 h, formed nanotubes that were visualized by immunofluorescence and atomic force microscopy. Cell-to-cell transfer of the fluorescent dye calcein-AM confirmed cytoplasmic communication via nanotubes. Immunoreactive B cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1), two major anti-apoptotic proteins, were present within the nanotubes. Downregulation of Mcl-1 by small interfering RNA in ASM cells significantly increased T cell apoptosis, whereas downregulation of Bcl-2 had no effect. Transfer of GFP-tagged Mcl-1 from ASM cells to CD4(+) T cells via the nanotubes confirmed directionality of transfer. In conclusion, activated T cells communicate with ASM cells via nanotube formation. Direct transfer of Mcl-1 from ASM to CD(+) T cells via nanotubes is involved in T cell survival. This study provides a novel mechanism of survival of CD4(+) T cells that is dependent on interaction with a structural cell.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Miocitos del Músculo Liso/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Transporte Biológico , Linfocitos T CD4-Positivos/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio , Adhesión Celular/inmunología , Comunicación Celular/inmunología , Supervivencia Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Humanos , Receptores de Hialuranos/inmunología , Activación de Linfocitos/inmunologíaRESUMEN
Chlorine gas (Cl2) inhalation causes oxidative stress, airway epithelial damage, airway hyperresponsiveness (AHR), and neutrophilia. We evaluated the effect of neutrophil depletion on Cl2-induced AHR and its effect on the endogenous antioxidant response, and if eosinophils or macrophages influence Cl2-induced AHR. We exposed male Balb/C mice to 100 ppm Cl2 for 5 minutes. We quantified inflammatory cell populations in bronchoalveolar lavage (BAL), the antioxidant response in lung tissue by quantitative PCR, and nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation by immunofluorescence. In vitro, NRF2 nuclear translocation in response to exogenous hypochlorite was assessed using a luciferase assay. Anti-granulocyte receptor-1 antibody or anti-Ly6G was used to deplete neutrophils. The effects of neutrophil depletion on IL-13 and IL-17 were measured by ELISA. Eosinophils and macrophages were depleted using TRFK5 or clodronate-loaded liposomes, respectively. AHR was evaluated with the constant-phase model in response to inhaled aerosolized methacholine. Our results show that Cl2 exposure induced neutrophilia and increased expression of NRF2 mRNA, superoxide dismutase-1, and heme-oxygenase 1. Neutrophil depletion abolished Cl2-induced AHR in large conducting airways and prevented increases in antioxidant gene expression and NRF2 nuclear translocation. Exogenous hypochlorite administration resulted in increased NRF2 nuclear translocation in vitro. After Cl2 exposure, neutrophils occupied 22 ± 7% of the luminal space in large airways. IL-17 in BAL was increased after Cl2, although this effect was not prevented by neutrophil depletion. Neither depletion of eosinophils nor macrophages prevented Cl2-induced AHR. Our data suggest the ability of neutrophils to promote Cl2-induced AHR is dependent on increases in oxidative stress and occupation of luminal space in large airways.
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Asma Ocupacional/inmunología , Cloro/toxicidad , Neutrófilos/inmunología , Transporte Activo de Núcleo Celular , Animales , Asma Ocupacional/inducido químicamente , Ácido Clodrónico/administración & dosificación , Citocinas/metabolismo , Granulocitos/inmunología , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones Endogámicos BALB C , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Regulación hacia ArribaRESUMEN
The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in naïve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.
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Pruebas de Función Respiratoria/métodos , Mecánica Respiratoria/fisiología , Animales , Broncoconstrictores/administración & dosificación , Volumen Espiratorio Forzado , Cloruro de Metacolina/administración & dosificación , Ratones , Modelos Animales , Pruebas de Función Respiratoria/instrumentación , Mecánica Respiratoria/efectos de los fármacosRESUMEN
Asthma is a chronic inflammatory disease that is associated with airway remodeling, including hyperplasia of airway epithelial cells and airway smooth muscle cells, and goblet cell differentiation. We wished to address the potential role of histamine, a key biogenic amine involved in allergic reactions, in airway remodeling through the epidermal growth factor receptor (EGFR) pathway. Here, we demonstrate that histamine releases 2 EGFR ligands, amphiregulin and heparin-binding epidermal growth factor-like growth factor (HB-EGF), from airway epithelial cells. Amphiregulin and HB-EGF were expressed in airway epithelium of patients with asthma. Histamine up-regulated their mRNA expression (amphiregulin 3.2-fold, P<0.001; HB-EGF 2.3-fold, P<0.05) and triggered their release (amphiregulin EC(50) 0.50 µM, 31.2 ± 2.7 pg/ml with 10 µM histamine, P<0.01; HB-EGF EC(50) 0.54 µM, 78.5 ± 1.8 pg/ml with 10 µM histamine, P<0.001) compared to vehicle control (amphiregulin 19.3 ± 0.9 pg/ml; HB-EGF 60.2 ± 1.0 pg/ml), in airway epithelial cells. Histamine increased EGFR phosphorylation (2.1-fold by Western blot analysis) and induced goblet cell differentiation (CLCA1 up-regulation by real-time qPCR) in normal human bronchial epithelial (NHBE) cells. Moreover, amphiregulin and HB-EGF caused proliferation and migration of both NHBE cells and human airway smooth muscle cells. These results suggest that histamine may induce airway remodeling via the epithelial-derived EGFR ligands amphiregulin and HB-EGF.
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Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Bronquios/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Histamina/farmacología , Adulto , Anfirregulina , Asma/metabolismo , Asma/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular , Proliferación Celular , Familia de Proteínas EGF , Células Epiteliales/citología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Persona de Mediana Edad , Receptores Histamínicos H1/metabolismo , Adulto JovenRESUMEN
Asthma is a chronic disorder of the airways associated in many instances with structural changes of the airways, termed airway remodelling. Irritant and allergen-induced murine models have been used to further understand the mechanisms of airway remodelling. The infiltration of the airways by inflammatory cells, such as T lymphocytes, mast cells, eosinophils, neutrophils and macrophages after repeated allergen challenges may be important effectors in the initiation and perpetuation of airway remodelling through the release of inflammatory mediators and growth factors. Interleukins-4 and -13 have been widely studied in experimental models, and have been shown to play a significant role in airway remodelling. Recently, a role for Th17 cells has been established. Other mediators involved in this process are ligands of the epidermal growth factor receptor, matrix metalloproteases and cysteinyl leukotrienes. A better understanding of the mechanisms leading to airway remodelling in allergic diseases may lead to the identification of novel therapeutic strategies but validation in human subjects is required for potential targets.
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Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Asma/fisiopatología , Modelos Animales de Enfermedad , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Alérgenos/inmunología , Animales , Asma/enzimología , Asma/inmunología , Eosinófilos/inmunología , Humanos , Mediadores de Inflamación/inmunología , Interleucinas/inmunología , Macrófagos/inmunología , Mastocitos/inmunología , Metaloproteinasas de la Matriz/inmunología , Ratones , Neutrófilos/inmunología , Ratas , Linfocitos T/inmunología , Células Th17/inmunologíaRESUMEN
New therapeutics designed as rescue treatments after toxic gas injury such as from chlorine (Cl(2)) are an emerging area of interest. We tested the effects of the metalloporphyrin catalytic antioxidant AEOL10150, a compound that scavenges peroxynitrite, inhibits lipid peroxidation, and has SOD and catalase-like activities, on Cl(2)-induced airway injury. Balb/C mice received 100ppm Cl(2) gas for 5 min. Four groups were studied: Cl(2) only, Cl(2) followed by AEOL10150 1 and 9 h after exposure, AEOL10150 only, and control. Twenty-four hours after Cl(2) gas exposure airway responsiveness to aerosolized methacholine (6.25-50mg/ml) was measured using a small-animal ventilator. Bronchoalveolar lavage (BAL) was performed to assess airway inflammation and protein. Whole lung tissue was assayed for 4-hydroxynonenal. In separate groups, lungs were collected at 72 h after Cl(2) injury to evaluate epithelial cell proliferation. Mice exposed to Cl(2) showed a significantly higher airway resistance compared to control, Cl(2)/AEOL10150, or AEOL10150-only treated animals in response to methacholine challenge. Eosinophils, neutrophils, and macrophages were elevated in BAL of Cl(2)-exposed mice. AEOL10150 attenuated the increases in neutrophils and macrophages. AEOL10150 prevented Cl(2)-induced increase in BAL fluid protein. Chlorine induced an increase in the number of proliferating airway epithelial cells, an effect AEOL10150 attenuated. 4-Hydroxynonenal levels in the lung were increased after Cl(2) and this effect was prevented with AEOL10150. AEOL10150 is an effective rescue treatment for Cl(2)-induced airway hyperresponsiveness, airway inflammation, injury-induced airway epithelial cell regeneration, and oxidative stress.