RESUMEN
Airway resistance measurements using oscillometry provide a potential alternative to spirometry in assessing airway obstruction and dynamics due to measurements taken during tidal breathing. Oscillometry typically requires participants to form a tight seal around a mouthpiece that can prove challenging for some people. To address this challenge, we conducted a prospective study to evaluate the effect of different interfaces like mouthpiece, mouth mask, and nasal mask on respiratory impedance results from oscillometry in a cohort of healthy adults. Ten healthy adults [7 females; mean age: 38.9 yr (SD ±15.5)] underwent oscillometry using each of the three interfaces. We measured resistance at 5 Hz (Rrs5), frequency dependence of resistance at 5-20 Hz (Rrs5-20), and reactance area (Ax). Rrs5 was not different when using the mouthpiece compared with the mouth mask [mean 2.98 cmH2O/L/s (SD ±0.68) vs. mean 3.2 cmH2O/L/s (SD ±0.81); P = 0.92; 95% CI -0.82 to +0.38], respectively. Nasal mask Rrs5 measurements were significantly higher than mouthpiece measurements (mean 7.31 cmH2O/L/s; SD ±2.62; P < 0.01; 95%CI -6.91 to -1.75). With Ax5, we found a mean of 4.01 cmH2O/L (SD ±2.04) with the mouth mask compared with a mean of 4.02 cmH2O/L (SD ±1.87; P = 1.0 95% CI -1.86 to +1.87) for the mouthpiece, however, we found a significant difference between the mouthpiece and nasal mask for Ax (mean = 10.71; SD ±7.0 H2O/L; P = 0.04, 95% CI -12.96 to -0.43). Our findings show that oscillometry using a mouth mask may be just as effective as using a mouthpiece in assessing airway dynamics and resistance.NEW & NOTEWORTHY This is the first study to compare the use of different interfaces: mouthpiece, mouth mask, and nasal mask, for oscillometry in an adult population. We report that using a mouth mask in oscillometry may provide a valid alternative to a mouthpiece in cohorts who may struggle to form the required tight seal that is typically required in oscillometry or spirometry.
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Resistencia de las Vías Respiratorias , Pulmón , Femenino , Humanos , Adulto , Oscilometría/métodos , Estudios Prospectivos , Espirometría , BocaRESUMEN
BACKGROUND: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2. METHODS: We used house dust mite (HDM) or interleukin-1ß in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1ß and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database. RESULTS: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1ß-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1ß-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma. CONCLUSIONS: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease.
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Asma , Proteínas Portadoras/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Proteínas de la Membrana/metabolismo , Piruvato Quinasa , Hormonas Tiroideas/metabolismo , Animales , Proteínas Portadoras/genética , Glutatión/metabolismo , Gutatión-S-Transferasa pi/genética , Glutatión Transferasa , Glucólisis , Humanos , Pulmón/metabolismo , Proteínas de la Membrana/genética , Ratones , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Hormonas Tiroideas/genética , Proteínas de Unión a Hormona TiroideRESUMEN
Aging is associated with a gradual loss of lung function due to increased cellular senescence, decreased regenerative capacity, and impaired innate host defense. One important aspect of innate airway epithelial host defense to nonmicrobial triggers is the secretion of alarmins such as IL-33 and activation of type 2 inflammation, which were previously found to depend on activation of the NADPH oxidase (NOX) homolog DUOX1, and redox-dependent signaling pathways that promote alarmin secretion. Here, we demonstrate that normal aging of C57BL/6J mice resulted in markedly decreased lung innate epithelial type 2 responses to exogenous triggers such as the airborne allergen Dermatophagoides pteronyssinus, which was associated with marked downregulation of DUOX1, as well as DUOX1-mediated redox-dependent signaling. DUOX1 deficiency was also found to accelerate age-related airspace enlargement and decline in lung function but did not consistently affect other features of lung aging such as senescence-associated inflammation. Intriguingly, observations of age-related DUOX1 downregulation and enhanced airspace enlargement due to DUOX1 deficiency in C57BL/6J mice, which lack a functional mitochondrial nicotinamide nucleotide transhydrogenase (NNT), were much less dramatic in C57BL/6NJ mice with normal NNT function, although the latter mice also displayed impaired innate epithelial injury responses with advancing age. Overall, our findings indicate a marked aging-dependent decline in (DUOX1-dependent) innate airway injury responses to external nonmicrobial triggers, but the impact of aging on DUOX1 downregulation and its significance for age-related senile emphysema development was variable between different C57BL6 substrains, possibly related to metabolic alterations due to differences in NNT function.
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Lesión Pulmonar Aguda/patología , Envejecimiento/patología , Oxidasas Duales/fisiología , Inflamación/patología , Enfisema Pulmonar/patología , Mucosa Respiratoria/patología , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Animales , Femenino , Inflamación/etiología , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfisema Pulmonar/etiología , Enfisema Pulmonar/metabolismo , Mucosa Respiratoria/metabolismoRESUMEN
Airway oscillometry has become the de facto standard for quality assessment of lung physiology in laboratory animals and has demonstrated its usefulness in understanding diseases of small airways. Nowadays, it is seeing extensive use in daily clinical practice and research; however, a question that remains unanswered is how well physiological findings in animals and humans correlate? Methodological and device differences are obvious between animal and human studies. However, all devices deliver an oscillated airflow test signal and output respiratory impedance. In addition, despite analysis differences, there are ways to interpret animal and human oscillometry data to allow suitable comparisons. The potential with oscillometry is its ability to reveal universal features of the respiratory system across species, making translational extrapolation likely to be predictive. This means that oscillometry can thus help determine if an animal model displays the same physiological characteristics as the human disease. Perhaps more importantly, it can also be useful to determine whether an intervention is effective as well as to understand if it affects the desired region of the respiratory system, e.g., the periphery of the lung. Finally, findings in humans can also inform preclinical scientists and give indications as to what type of physiological changes should be observed in animal models to make them relevant as models of human disease. The present article will attempt to demonstrate the potential of oscillometry in respiratory research, an area where the development of novel therapies is plagued with a failure rate higher than in other disease areas.
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Asma/tratamiento farmacológico , Asma/fisiopatología , Impedancia Eléctrica/uso terapéutico , Pulmón/fisiopatología , Oscilometría , Animales , Humanos , Pulmón/efectos de los fármacos , Pulmón/fisiología , Oscilometría/métodos , Pruebas de Función Respiratoria/métodos , Fenómenos Fisiológicos Respiratorios/efectos de los fármacosRESUMEN
In the community setting, assessing spirometry in school-aged children is often limited by the unavailability of respirology technicians at the point-of-care. We developed a new technique called the Rapid Expiratory Occlusion Method (REOM) that measures respiratory resistance during normal breathing, without specialized training. The aim was to examine the concordance between respiratory resistance measured with the REOM and respiratory resistance measured by oscillometry on the tremoflo. Children aged 6-17 yr, with or without asthma, received respiratory resistance testing on the tremoflo, then on the REOM. Three to five replicates with a coefficient of variation ≤15% were obtained on each instrument; the primary outcome was the concordance between the average respiratory resistance on the REOM and that measured at 5 Hz (R5) on the tremoflo. Thirty-two children (11 girls; 21 boys) were enrolled with a mean age of 11.2 (range 6-17) yr; after excluding two children not meeting reproducibility criteria, 9 healthy controls, 15 controlled asthmatics, and 6 poorly controlled asthmatics were included. Resistance measured on the REOM showed a strong correlation with R5 measured on the tremoflo (P < 0.0001) with no significant differences on the Bland-Altman analyses. Children and their parents found the REOM easy to use and would consider for home use if recommended by their doctor. With the high concordance between resistance values measured on the REOM and that on the tremoflo combined with perceived ease of use, the REOM appears as a promising means for measuring lung function, thus supporting further testing of other psychometric properties.NEW & NOTEWORTHY We have developed a novel version of the interrupter technique to measure respiratory resistance. The Rapid Expiratory Occlusion Method (REOM) is a small handheld device that measures respiratory resistance and demonstrates excellent correlation with airway oscillometry. With its ease of use, REOM may be promising for use in community practice, patient's homes, and, if paired with a telemedicine application, could enable the healthcare provider to monitor patients in their homes.
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Resistencia de las Vías Respiratorias , Pulmón , Niño , Femenino , Humanos , Masculino , Oscilometría , Reproducibilidad de los Resultados , Pruebas de Función Respiratoria , EspirometríaRESUMEN
Research using animal models of asthma is currently dominated by mouse models. This has been driven by the comprehensive knowledge on inflammatory and immune reactions in mice, as well as tools to produce genetically modified mice. Many of the identified therapeutic targets influencing airway hyper-responsiveness and inflammation in mouse models, have however been disappointing when tested clinically in asthma. It is therefore a great need for new animal models that more closely resemble human asthma. The guinea pig has for decades been used in asthma research and a comprehensive table of different protocols for asthma models is presented. The studies have primarily been focused on the pharmacological aspects of the disease, where the guinea pig undoubtedly is superior to mice. Further reasons are the anatomical and physiological similarities between human and guinea pig airways compared with that of the mouse, especially with respect to airway branching, neurophysiology, pulmonary circulation and smooth muscle distribution, as well as mast cell localization and mediator secretion. Lack of reagents and specific molecular tools to study inflammatory and immunological reactions in the guinea pig has however greatly diminished its use in asthma research. The aim in this position paper is to review and summarize what we know about different aspects of the use of guinea pig in vivo models for asthma research. The associated aim is to highlight the unmet needs that have to be addressed in the future.
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Asma/patología , Modelos Animales de Enfermedad , Cobayas/fisiología , Animales , Desarrollo de Medicamentos , Edición Génica , Cobayas/genética , Pulmón/patología , Pulmón/fisiopatologíaRESUMEN
The stress-induced kinase, c-Jun-N-terminal kinase 1 (JNK1) has previously been implicated in the pathogenesis of lung fibrosis. However, the exact cell type(s) wherein JNK1 exerts its pro-fibrotic role(s) remained enigmatic. Herein we demonstrate prominent activation of JNK in bronchial epithelia using the mouse models of bleomycin- or AdTGFß1-induced fibrosis. Furthermore, in lung tissues of patients with idiopathic pulmonary fibrosis (IPF), active JNK was observed in various regions including type I and type II pneumocytes and fibroblasts. No JNK activity was observed in adjacent normal tissue or in normal control tissue. To address the role of epithelial JNK1, we ablated Jnk1 form bronchiolar and alveolar type II epithelial cells using CCSP-directed Cre recombinase-mediated ablation of LoxP-flanked Jnk1 alleles. Our results demonstrate that ablation of Jnk1 from airway epithelia resulted in a strong protection from bleomycin- or adenovirus expressing active transforming growth factor beta-1 (AdTGFß1)-induced fibrosis. Ablation of the Jnk1 allele at a time when collagen increases were already present showed a reversal of existing increases in collagen content. Epithelial Jnk1 ablation resulted in attenuation of mesenchymal genes and proteins in lung tissue and preserved expression of epithelial genes. Collectively, these data suggest that epithelial JNK1 contributes to the pathogenesis of pulmonary fibrosis. Given the presence of active JNK in lungs from patients with IPF, targeting JNK1 in airway epithelia may represent a potential treatment strategy to combat this devastating disease.
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Eliminación de Gen , Fibrosis Pulmonar Idiopática/terapia , Pulmón/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Animales , Bleomicina/efectos adversos , Dependovirus/genética , Modelos Animales de Enfermedad , Células Epiteliales/química , Femenino , Humanos , Fibrosis Pulmonar Idiopática/etiología , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/citología , Masculino , Ratones , Fosforilación , Factor de Crecimiento Transformador beta1/administración & dosificación , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Impedance, or oscillometry, measurements of the respiratory system can generate information about the function of the respiratory system not possible with traditional spirometry. There are currently several instruments on the market using different perturbations. We have compared a new respiratory oscillometry instrument, the tremoflo, with Impulse Oscillometry (IOS). Patients with a physician's diagnosis of chronic obstructive lung disease (COPD) and healthy subjects were recruited. They underwent assessment of respiratory function with oscillometry using the IOS and tremoflo devices and the resulting impedance data from the two methods were compared. The two devices were also tested against a reference respiratory phantom with variable resistances. Whereas both devices detected impairments in the patients' lung function commensurate with small airways pathology, the tremoflo appeared to be more sensitive than the IOS. We found systematic differences between the two instruments especially for reactance measurements where the area over the reactance curve (AX) was significantly lower with the IOS compared with the tremoflo (p < 0.001). Moreover, the agreement between the two devices was reduced with increasing severity of the disease as determined with a Bland-Altman test. Testing both instruments against a respiratory phantom unit confirmed that the resistance measured by the tremoflo compares closely with the known resistance of test loads, whereas the IOS' resistance correlated with a test load of 0.19, kPa.s.L-1 at higher loads it deviated significantly from the known resistance (p < 0.0028). We conclude that the absolute values measured with the two devices may not be directly comparable and suggest that differences in the calibration procedures might account for the differences.
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Oscilometría/métodos , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oscilometría/instrumentación , Pruebas de Función RespiratoriaRESUMEN
INTRODUCTION: The measurement of specific volatile organic compounds in breath has been proposed as a potential diagnostic for a variety of diseases. The most well-studied bacterial lung infection in the breath field is that caused by Pseudomonas aeruginosa. OBJECTIVES: To determine a discriminatory core of molecules in the "breath-print" of mice during a lung infection with four strains of P. aeruginosa (PAO1, PA14, PAK, PA7). Furthermore, we attempted to extrapolate a strain-specific "breath-print" signature to investigate the possibility of recapitulating the genetic phylogenetic groups (Stewart et al. Pathog Dis 71(1), 20-25, 2014. https://doi.org/10.1111/2049-632X.12107 ). METHODS: Breath was collected into a Tedlar bag and shortly after drawn into a thermal desorption tube. The latter was then analyzed into a comprehensive multidimensional gas chromatography coupled with a time-of-flight mass spectrometer. Random forest algorithm was used for selecting the most discriminatory features and creating a prediction model. RESULTS: Three hundred and one molecules were significantly different between animals infected with P. aeruginosa, and those given a sham infection (PBS) or inoculated with UV-killed P. aeruginosa. Of those, nine metabolites could be used to discriminate between the three groups with an accuracy of 81%. Hierarchical clustering showed that the signature from breath was due to a specific response to live bacteria instead of a generic infection response. Furthermore, we identified ten additional volatile metabolites that could differentiate mice infected with different strains of P. aeruginosa. A phylogram generated from the ten metabolites showed that PAO1 and PA7 were the most distinct group, while PAK and PA14 were interspersed between the former two groups. CONCLUSIONS: To the best of our knowledge, this is the first study to report on a 'core' murine breath print, as well as, strain level differences between the compounds in breath. We provide identifications (by running commercially available analytical standards) to five breath compounds that are predictive of P. aeruginosa infection.
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Pruebas Respiratorias/métodos , Metabolómica/métodos , Compuestos Orgánicos Volátiles/análisis , Animales , Femenino , Cromatografía de Gases y Espectrometría de Masas/métodos , Espectrometría de Masas/métodos , Metaboloma/fisiología , Ratones , Ratones Endogámicos C57BL , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/metabolismoRESUMEN
BACKGROUND: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma. OBJECTIVES: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma. METHODS: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis. RESULTS: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1ß into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1ß or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1ß- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1ß on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1ß and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1ß levels, and lactate content correlated negatively with lung function. CONCLUSIONS: Collectively, these findings demonstrate that IL-1ß/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease.
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Asma/metabolismo , Hipersensibilidad/metabolismo , Interleucina-1beta/metabolismo , Pulmón/metabolismo , Nariz/patología , Mucosa Respiratoria/metabolismo , Esputo/metabolismo , Animales , Antígenos Dermatofagoides/inmunología , Células Cultivadas , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Glucólisis , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-1beta/genética , Ácido Láctico/metabolismo , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Neutrófilos/patología , Proteínas Proto-Oncogénicas/metabolismo , Pyroglyphidae , ARN Interferente Pequeño/genética , Mucosa Respiratoria/patología , Transducción de SeñalRESUMEN
In the present research, the potential of breath analysis by comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC×GC-MS) was investigated for the discrimination between healthy and infected mice. A pilot study employing a total of 16 animals was used to develop a method for breath analysis in a murine model for studying Mycobacterium tuberculosis complex (MTBC) using the M. bovis bacillus Calmette-Guérin. Breath was collected in Tedlar bags and concentrated onto thermal desorption tubes for subsequent analysis by GC×GC-MS. Immunological test and bacterial cell count in bronchoalveolar lavage fluid and mice lung homogenate confirmed the presence of bacteria in the infected group. From the GC×GC-MS analysis, 23 molecules were found to mainly drive the separation between control and infected mice and their tentative identification is provided.This study shows that the overall used methodology is able to differentiate breath between healthy and infected animals, and the information herein can be used to further develop the mouse breath model to study MTBC pathogenesis, evaluate pre-clinical drug regimen efficacy, and to further develop the concept of breath-based diagnostics.
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Pruebas Respiratorias/métodos , Infecciones por Mycobacterium/diagnóstico , Mycobacterium bovis/aislamiento & purificación , Animales , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Cromatografía de Gases y Espectrometría de Masas , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Infecciones por Mycobacterium/microbiología , Infecciones por Mycobacterium/patología , Neutrófilos/patología , Proyectos Piloto , Análisis de Componente PrincipalRESUMEN
The use of computed tomography (CT scans) has increased dramatically in recent decades, raising questions about the long-term safety of CT-emitted x-rays especially in infants who are more sensitive to radiation-induced effects. Cancer risk estimates for CT scans typically are extrapolated from models; therefore, new approaches measuring actual DNA damage are needed for improved estimations. Hence, changes in a dosimeter of DNA double-strand breaks, micronucleated reticulocytes (MN-RETs) measured by flow cytometry, were investigated in mice and infants exposed to CT scans. In male C57BL/6N mice (6-8 weeks-of-age), there was a dose-related increase in MN-RETs in blood samples collected 48h after CT scans delivering targeted exposures of 1-130 cGy x-rays (n=5-10/group, r=0.994, p=0.01), with significant increases occurring at exposure levels as low as 0.83 cGy x-rays compared to control mice (p=0.002). In paired blood specimens from infants with no history of a prior CT scan, there was no difference in MN-RET frequencies found 2h before (mean, 0.10±0.07%) versus 48h after (mean, 0.11±0.05%) a scheduled CT scan/cardiac catheterization. However, in infants having prior CT scan(s), MN-RET frequencies measured at 48h after a scheduled CT scan (mean=0.22±0.12%) were significantly higher than paired baseline values (mean, 0.17±0.07%; p=0.032). Increases in baseline (r=0.722, p<0.001) and 48-h post exposure (r=0.682, p<0.001) levels of MN-RETs in infants with a history of prior CT scans were significantly correlated with the number of previous CT scans. These preliminary findings suggest that prior CT scans increase the cellular responses to subsequent CT exposures. Thus, further investigation is needed to characterize the potential cancer risk from single versus repeated CT scans or cardiac catheterizations in infants.
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Cateterismo Cardíaco/efectos adversos , Roturas del ADN de Doble Cadena , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Reticulocitos/patología , Tomografía Computarizada por Rayos X/efectos adversos , Animales , Relación Dosis-Respuesta en la Radiación , Femenino , Citometría de Flujo , Inestabilidad Genómica , Humanos , Lactante , Recién Nacido , Masculino , Ratones Endogámicos C57BL , Micronúcleos con Defecto Cromosómico/estadística & datos numéricos , Proyectos Piloto , Embarazo , Estudios Prospectivos , Dosis de Radiación , Irradiación Corporal TotalRESUMEN
iNKT cells and mast cells have both been implicated in the syndrome of allergic asthma through their activation-induced release of Th2 type cytokines and secretion of histamine and other mediators, respectively, which can promote airways hyperresponsiveness (AHR) to agents such as methacholine. However, a mechanistic link between iNKT cells and mast cell recruitment or activation has never been explored. Our objective was to determine whether iNKT cells are necessary for the recruitment of mast cells and if iNKT cells can influence the acute allergen induced bronchoconstriction (AIB) caused by mast cell mediator release. To do so, we pharmacologically eliminated iNKT cells using a specific antibody (NKT-14) and examined its impact on airway inflammation and physiological phenotype. In mice treated with NKT-14, the elimination of iNKT cells was sufficient to prevent AHR and pulmonary eosinophilic inflammation elicited by administration of the iNKT cell agonist αGalCer. In mice treated with NKT-14 and then sensitized and challenged with house dust mite extract (HDM), eliminating the iNKT cells significantly reduced both AHR and AIB but did not affect pulmonary inflammation, the mast cell population, nor the release of the mast cell mediators mast cell protease-1 and prostaglandin D2. We conclude that while iNKT cells contribute to the phenotype of allergic airways disease through the manifestation of AIB and AHR, their presence is not required for mast cell recruitment and activation, or to generate the characteristic inflammatory response subsequent to allergen challenge.
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Broncoconstricción/inmunología , Mastocitos/metabolismo , Células T Asesinas Naturales/metabolismo , Hipersensibilidad Respiratoria/inmunología , Alérgenos/inmunología , Animales , Quimasas/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Femenino , Hipersensibilidad/inmunología , Inflamación/inmunología , Pulmón/inmunología , Pulmón/patología , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Células T Asesinas Naturales/inmunología , Fenotipo , Prostaglandina D2/metabolismo , Pyroglyphidae/inmunologíaRESUMEN
BACKGROUND: The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-α levels are high in rheumatoid arthritis; however, it is unknown if TNF-α can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-α-induced arthritis, but a role for PAD4 in lung citrullination and TNF-α-induced lung inflammation has not been explored. Our aim was to use TNF-α-overexpressing mice to clarify the intersection of TNF-α, citrullination, PAD4, arthritis, and lung inflammation. METHODS: Lung protein citrullination in wild-type mice, mice that overexpress TNF-α systemically (TNF(+)), TNF(+)PAD4(+/+), and TNF(+)PAD4(-/-) mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (H&E)-stained lung sections from TNF(+)PAD4(+/+) and TNF(+)PAD4(-/-) mice were scored for lung inflammation. H&E-stained ankle joint sections from mice that overexpress TNF-α only in the lungs were assessed for arthritis. RESULTS: TNF(+) mice have increased lung protein citrullination. TNF(+)PAD4(-/-) mice do not have significantly reduced lung protein citrullination, but do have decreased lung inflammation compared to TNF(+)PAD4(+/+) mice. Mice that overexpress TNF-α only in the lungs do not develop arthritis. CONCLUSIONS: PAD4 exacerbates lung inflammation downstream of TNF-α without having a major role in generalized protein citrullination in inflamed lungs. Also, TNF-α-induced lung inflammation is not sufficient to drive murine arthritis.
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Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Hidrolasas/metabolismo , Neumonía/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Western Blotting , Citrulina , Humanos , Ratones , Ratones Transgénicos , Neumonía/patología , Procesamiento Proteico-Postraduccional/fisiología , Arginina Deiminasa Proteína-Tipo 4RESUMEN
Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.
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Glutarredoxinas/metabolismo , Hipersensibilidad/patología , Hipersensibilidad/parasitología , Pulmón/patología , Pulmón/parasitología , Pyroglyphidae/fisiología , Animales , Citocinas/genética , Citocinas/metabolismo , Glutatión/metabolismo , Hiperplasia , Hipersensibilidad/sangre , Hipersensibilidad/complicaciones , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Moco/metabolismo , Neumonía/sangre , Neumonía/complicaciones , Neumonía/parasitología , Neumonía/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Hipersensibilidad Respiratoria/sangre , Hipersensibilidad Respiratoria/parasitología , Hipersensibilidad Respiratoria/patología , Hipersensibilidad Respiratoria/fisiopatología , Mecánica Respiratoria , Células Th2/inmunologíaRESUMEN
We have previously advanced the hypothesis that the allergic inflammatory response in the lungs occurs as a self-limited sequence of events that begins with the onset of inflammation and then resolves back to baseline over a predetermined time course (Pothen JJ, Poynter ME, Bates JH. J Immunol 190: 3510-3516, 2013). In the present study we tested a key prediction of this hypothesis, which is that the instigation of the allergic inflammatory response should be accompanied by a later refractory period during which the response cannot be reinitiated. We challenged groups of ovalbumin-sensitized BALB/c mice for 3, 14, 21 and 31 consecutive days with aerosolized ovalbumin. We measured airways responsiveness as well as cell counts and cytokines in bronchoalveolar lavage fluid after the final challenge in subgroups from each group. In other subgroups we performed the same measurements following rest periods and after a final single recall challenge with antigen. We determined that the refractory periods for GM-CSF, KC, and IL-5 are no longer than 10 days, while those for IFNγ and IL-10 are no longer than 28 days. The refractory periods for total leukocytes and neutrophils were no greater than 28 days, while that for eosinophils was more than 28 days. The refractory period for airways resistance was less than 17, while for lung elastance it was longer than 28 days. Our results thus demonstrate that the components of the allergic inflammatory response in the lung have finite refractory periods, with the refractory period of the entire response being in the order of a month.
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Citocinas/metabolismo , Pulmón/inmunología , Neumonía/inmunología , Animales , Asma/inmunología , Asma/fisiopatología , Femenino , Recuento de Leucocitos , Pulmón/metabolismo , Pulmón/fisiopatología , Ratones Endogámicos BALB C , Neumonía/patología , Neumonía/fisiopatologíaRESUMEN
S-glutathionylation has emerged as an oxidant-induced post-translational modification of protein cysteines that affects structure and function. The oxidoreductase glutaredoxin-1 (Glrx1), under physiological conditions, catalyzes deglutathionylation and restores the protein thiol group. The involvement of Grx1/S-glutathionylation in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 for the pathogenesis of house dust mite (HDM)-induced allergic airway disease in mice. Wild-type (WT) or Glrx1(-/-) mice in the BALB/c background were instilled intranasally with 50 µg of HDM 5 consecutive days for 3 weeks and killed 72 hours post final exposure. As expected, overall protein S-glutathionylation was increased in Glrx1(-/-) mice exposed to HDM as compared with WT animals. Total cells in the bronchoalveolar lavage fluid were similarly increased in WT and Glrx1(-/-) HDM-treated mice compared with phosphate-buffered saline-treated control mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils but fewer eosinophils than HDM-exposed WT mice. mRNA expression of the Th2-associated cytokine IL-13, as well as MUC5ac, was significantly attenuated in Glrx1(-/-) HDM-treated mice compared with WT mice. Conversely, expression of IL-17A was increased in Glrx1(-/-) HDM mice compared with WT mice. Last, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Grx1/S-glutathionylation redox status plays a pivotal role in HDM-induced allergic inflammation and airway hyperresponsiveness and suggest a potential role of Glrx1/S-glutathionylation in controlling the nature of the HDM-induced adaptive immune responses by promoting Type-2-driven inflammation and restricting IL-17A.
RESUMEN
Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.
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Anticuerpos Monoclonales de Origen Murino/farmacología , Asma/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Asma/genética , Asma/patología , Asma/terapia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Depleción Linfocítica/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Células T Asesinas Naturales/patología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Mechanical ventilation is a crucial component of the supportive care provided to patients with acute respiratory distress syndrome. Current practice stipulates the use of a low tidal volume (VT) of 6 ml/kg ideal body weight, the presumptive notion being that this limits overdistension of the tissues and thus reduces volutrauma. We have recently found, however, that airway pressure release ventilation (APRV) is efficacious at preventing ventilator-induced lung injury, yet APRV has a very different mechanical breath profile compared with conventional low-VT ventilation. To gain insight into the relative merits of these two ventilation modes, we measured lung mechanics and derecruitability in rats before and following Tween lavage. We fit to these lung mechanics measurements a computational model of the lung that accounts for both the degree of tissue distension of the open lung and the amount of lung derecruitment that takes place as a function of time. Using this model, we predicted how tissue distension, open lung fraction, and intratidal recruitment vary as a function of ventilator settings both for conventional low-VT ventilation and for APRV. Our predictions indicate that APRV is more effective at recruiting the lung than low-VT ventilation, but without causing more overdistension of the tissues. On the other hand, low-VT ventilation generally produces less intratidal recruitment than APRV. Predictions such as these may be useful for deciding on the relative benefits of different ventilation modes and thus may serve as a means for determining how to ventilate a given lung in the least injurious fashion.