RESUMEN
BACKGROUND: Two isoforms of Phosphoinositide 3-kinase (PI3K), p110γ and p110δ, are predominantly expressed in leukocytes and represent attractive therapeutic targets for the treatment of allergic asthma. The study aim was to assess the impact of administration of an inhaled PI3Kγδ inhibitor (AZD8154) in a rat model of asthma. METHODS: Firstly, we checked that the tool compound, AZD8154, inhibited rat PI3K γ & δ kinases using rat cell-based assays. Subsequently, a time-course study was conducted in a rat model of asthma to assess PI3K activity in the lung and how it is temporally associated with other key transcription pathways and asthma like features of the model. Finally, the impact on lung dosed AZD8154 on target engagement, pathway specificity, airway inflammation and lung function changes was assessed. RESULTS: Data showed that AZD8154 could inhibit rat PI3K γ & δ isoforms and, in a rat model of allergic asthma the PI3K pathway was activated in the lung. Intratracheal administration of AZD8154 caused a dose related suppression PI3K pathway activation (reduction in pAkt) and unlike after budesonide treatment, STAT and NF-κB pathways were not affected by AZD8154. The suppression of the PI3K pathway led to a marked inhibition of airway inflammation and reduction in changes in lung function. CONCLUSION: These data show that a dual PI3Kγδ inhibitor suppress key features of disease in a rat model of asthma to a similar degree as budesonide and indicate that dual PI3Kγδ inhibition may be an effective treatment for people suffering from allergic asthma.
Asunto(s)
Asma , Modelos Animales de Enfermedad , Animales , Asma/tratamiento farmacológico , Asma/metabolismo , Ratas , Masculino , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Ratas Sprague-Dawley , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Pulmón/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores de Proteínas Quinasas/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Antiasmáticos/farmacología , Ovalbúmina/toxicidadRESUMEN
BACKGROUND: Obesity is a risk factor for asthma, and obese asthmatic individuals are more likely to have severe, steroid-insensitive disease. How obesity affects the pathogenesis and severity of asthma is poorly understood. Roles for increased inflammasome-mediated neutrophilic responses, type 2 immunity, and eosinophilic inflammation have been described. OBJECTIVE: We investigated how obesity affects the pathogenesis and severity of asthma and identified effective therapies for obesity-associated disease. METHODS: We assessed associations between body mass index and inflammasome responses with type 2 (T2) immune responses in the sputum of 25 subjects with asthma. Functional roles for NLR family, pyrin domain-containing (NLRP) 3 inflammasome and T2 cytokine responses in driving key features of disease were examined in experimental high-fat diet-induced obesity and asthma. RESULTS: Body mass index and inflammasome responses positively correlated with increased IL-5 and IL-13 expression as well as C-C chemokine receptor type 3 expression in the sputum of subjects with asthma. High-fat diet-induced obesity resulted in steroid-insensitive airway hyperresponsiveness in both the presence and absence of experimental asthma. High-fat diet-induced obesity was also associated with increased NLRP3 inflammasome responses and eosinophilic inflammation in airway tissue, but not lumen, in experimental asthma. Inhibition of NLRP3 inflammasome responses reduced steroid-insensitive airway hyperresponsiveness but had no effect on IL-5 or IL-13 responses in experimental asthma. Depletion of IL-5 and IL-13 reduced obesity-induced NLRP3 inflammasome responses and steroid-insensitive airway hyperresponsiveness in experimental asthma. CONCLUSION: We found a relationship between T2 cytokine and NLRP3 inflammasome responses in obesity-associated asthma, highlighting the potential utility of T2 cytokine-targeted biologics and inflammasome inhibitors.
Asunto(s)
Asma , Inflamasomas , Citocinas , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Interleucina-13 , Interleucina-1beta , Interleucina-5 , Proteína con Dominio Pirina 3 de la Familia NLR , Obesidad/complicacionesRESUMEN
BACKGROUND: Asthma is an airway inflammatory disease and a major health problem worldwide. Anti-inflammatory steroids and bronchodilators are the gold-standard therapy for asthma. However, they do not prevent the development of the disease, and critically, a subset of asthmatics are resistant to steroid therapy. OBJECTIVE: To elucidate the therapeutic potential of human ß-defensins (hBD), such as hBD2 mild to moderate and severe asthma. METHODS: We investigated the role of hBD2 in a steroid-sensitive, house dust mite-induced allergic airways disease (AAD) model and a steroid-insensitive model combining ovalbumin-induced AAD with C muridarum (Cmu) respiratory infection. RESULTS: In both models, we demonstrated that therapeutic intranasal application of hBD2 significantly reduced the influx of inflammatory cells into the bronchoalveolar lavage fluid. Furthermore, key type 2 asthma-related cytokines IL-9 and IL-13, as well as additional immunomodulating cytokines, were significantly decreased after administration of hBD2 in the steroid-sensitive model. The suppression of inflammation was associated with improvements in airway physiology and treatment also suppressed airway hyper-responsiveness (AHR) in terms of airway resistance and compliance to methacholine challenge. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that hBD2 reduces the hallmark features and has potential as a new therapeutic agent in allergic and especially steroid-resistant asthma.
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Resistencia de las Vías Respiratorias/efectos de los fármacos , Asma/metabolismo , Interleucina-13/metabolismo , Interleucina-9/metabolismo , Rendimiento Pulmonar/efectos de los fármacos , Pulmón/efectos de los fármacos , beta-Defensinas/farmacología , Animales , Asma/fisiopatología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/fisiopatología , Chlamydia muridarum , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/fisiopatología , Pulmón/metabolismo , Pulmón/fisiopatología , Ratones , Ovalbúmina , Pyroglyphidae , Hipersensibilidad Respiratoria/metabolismo , Hipersensibilidad Respiratoria/fisiopatología , Infecciones del Sistema Respiratorio/metabolismo , Infecciones del Sistema Respiratorio/fisiopatologíaRESUMEN
Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild-moderate asthma patients and correlate with lower forced expiratory volume in 1â s (FEV1). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV1/forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 (DMT1) and transferrin receptor 1 (TFR1) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1+ macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo, including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease.
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Asma , Animales , Humanos , Interleucina-13 , Hierro , Pulmón , PyroglyphidaeRESUMEN
Increased iron levels and dysregulated iron homeostasis, or both, occur in several lung diseases. Here, the effects of iron accumulation on the pathogenesis of pulmonary fibrosis and associated lung function decline was investigated using a combination of murine models of iron overload and bleomycin-induced pulmonary fibrosis, primary human lung fibroblasts treated with iron, and histological samples from patients with or without idiopathic pulmonary fibrosis (IPF). Iron levels are significantly increased in iron overloaded transferrin receptor 2 (Tfr2) mutant mice and homeostatic iron regulator (Hfe) gene-deficient mice and this is associated with increases in airway fibrosis and reduced lung function. Furthermore, fibrosis and lung function decline are associated with pulmonary iron accumulation in bleomycin-induced pulmonary fibrosis. In addition, we show that iron accumulation is increased in lung sections from patients with IPF and that human lung fibroblasts show greater proliferation and cytokine and extracellular matrix responses when exposed to increased iron levels. Significantly, we show that intranasal treatment with the iron chelator, deferoxamine (DFO), from the time when pulmonary iron levels accumulate, prevents airway fibrosis and decline in lung function in experimental pulmonary fibrosis. Pulmonary fibrosis is associated with an increase in Tfr1+ macrophages that display altered phenotype in disease, and DFO treatment modified the abundance of these cells. These experimental and clinical data demonstrate that increased accumulation of pulmonary iron plays a key role in the pathogenesis of pulmonary fibrosis and lung function decline. Furthermore, these data highlight the potential for the therapeutic targeting of increased pulmonary iron in the treatment of fibrotic lung diseases such as IPF. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Fibrosis Pulmonar Idiopática/patología , Hierro/metabolismo , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Animales , Bleomicina/farmacología , Proliferación Celular , Células Cultivadas , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones NoqueadosRESUMEN
RATIONALE: Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and concomitant IL-1ß responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma. OBJECTIVES: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL-1ß in severe, steroid-resistant asthma. METHODS: We developed mouse models of Chlamydia and Haemophilus respiratory infection-mediated, ovalbumin-induced severe, steroid-resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1ß responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1ß responses in experimental severe, steroid-resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase-1 inhibitor Ac-YVAD-cho; and neutralizing anti-IL-1ß antibody. Roles for IL-1ß-induced neutrophilic inflammation were examined using IL-1ß and anti-Ly6G. MEASUREMENTS AND MAIN RESULTS: Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1ß responses that drive steroid-resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL-1ß expression. Treatment with anti-IL-1ß, Ac-YVAD-cho, and MCC950 suppressed IL-1ß responses and the important steroid-resistant features of disease in mice, whereas IL-1ß administration recapitulated these features. Neutrophil depletion suppressed IL-1ß-induced steroid-resistant airway hyperresponsiveness. CONCLUSIONS: NLRP3 inflammasome responses drive experimental severe, steroid-resistant asthma and are potential therapeutic targets in this disease.
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Asma/genética , Asma/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Animales , Asma/metabolismo , Modelos Animales de Enfermedad , Resistencia a Medicamentos , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Innate immune responses act as first line defences upon exposure to potentially noxious stimuli. The innate immune system has evolved numerous intracellular and extracellular receptors that undertake surveillance for potentially damaging particulates. Inflammasomes are intracellular innate immune multiprotein complexes that form and are activated following interaction with these stimuli. Inflammasome activation leads to the cleavage of pro-IL-1ß and release of the pro-inflammatory cytokine, IL-1ß, which initiates acute phase pro-inflammatory responses, and other responses are also involved (IL-18, pyroptosis). However, excessive activation of inflammasomes can result in chronic inflammation, which has been implicated in a range of chronic inflammatory diseases. The airways are constantly exposed to a wide variety of stimuli. Inflammasome activation and downstream responses clears these stimuli. However, excessive activation may drive the pathogenesis of chronic respiratory diseases such as severe asthma and chronic obstructive pulmonary disease. Thus, there is currently intense interest in the role of inflammasomes in chronic inflammatory lung diseases and in their potential for therapeutic targeting. Here we review the known associations between inflammasome-mediated responses and the development and exacerbation of chronic lung diseases.
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Inmunidad Innata , Inflamasomas/inmunología , Enfermedades Pulmonares/inmunología , Pulmón/inmunología , Caspasa 1/metabolismo , Enfermedad Crónica , Humanos , Inflamasomas/metabolismo , Inflamación/inmunología , Interleucina-18/biosíntesis , Interleucina-18/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-1beta/metabolismo , Enfermedades Pulmonares/microbiología , Macrófagos Alveolares/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Severe steroid-insensitive asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms of disease pathogenesis. Steroid-insensitive asthma is associated with respiratory tract infections and noneosinophilic endotypes, including neutrophilic forms of disease. However, steroid-insensitive patients with eosinophil-enriched inflammation have also been described. The mechanisms that underpin infection-induced, severe steroid-insensitive asthma can be elucidated by using mouse models of disease. OBJECTIVE: We sought to develop representative mouse models of severe, steroid-insensitive asthma and to use them to identify pathogenic mechanisms and investigate new treatment approaches. METHODS: Novel mouse models of Chlamydia, Haemophilus influenzae, influenza, and respiratory syncytial virus respiratory tract infections and ovalbumin-induced, severe, steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed and interrogated. RESULTS: Infection induced increases in the levels of microRNA (miRNA)-21 (miR-21) expression in the lung during SSIAAD, whereas expression of the miR-21 target phosphatase and tensin homolog was reduced. This was associated with an increase in levels of phosphorylated Akt, an indicator of phosphoinositide 3-kinase (PI3K) activity, and decreased nuclear histone deacetylase (HDAC)2 levels. Treatment with an miR-21-specific antagomir (Ant-21) increased phosphatase and tensin homolog levels. Treatment with Ant-21, or the pan-PI3K inhibitor LY294002, reduced PI3K activity and restored HDAC2 levels. This led to suppression of airway hyperresponsiveness and restored steroid sensitivity to allergic airway disease. These observations were replicated with SSIAAD associated with 4 different pathogens. CONCLUSION: We identify a previously unrecognized role for an miR-21/PI3K/HDAC2 axis in SSIAAD. Our data highlight miR-21 as a novel therapeutic target for the treatment of this form of asthma.
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Asma/genética , Chlamydia muridarum/inmunología , Haemophilus influenzae/inmunología , Histona Desacetilasa 2/metabolismo , Subtipo H1N1 del Virus de la Influenza A/inmunología , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neumonía/genética , Virus Sincitiales Respiratorios/inmunología , Infecciones del Sistema Respiratorio/genética , Animales , Antagomirs/genética , Asma/tratamiento farmacológico , Asma/inmunología , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Resistencia a Medicamentos , Regulación de la Expresión Génica , Histona Desacetilasa 2/genética , Humanos , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Neumonía/tratamiento farmacológico , Neumonía/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/inmunologíaAsunto(s)
Asma/fisiopatología , Inflamasomas/fisiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Asma/genética , Proteínas Portadoras/fisiología , Caspasa 1/fisiología , Citocinas/fisiología , Perfilación de la Expresión Génica , Humanos , Interleucina-1beta/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/fisiología , Fumar/fisiopatologíaRESUMEN
Viral exacerbations of chronic obstructive pulmonary disease (COPD), commonly caused by rhinovirus (RV) infections, are poorly controlled by current therapies. This is due to a lack of understanding of the underlying immunopathological mechanisms. Human studies have identified a number of key immune responses that are associated with RV-induced exacerbations including neutrophilic inflammation, expression of inflammatory cytokines and deficiencies in innate anti-viral interferon. Animal models of COPD exacerbation are required to determine the contribution of these responses to disease pathogenesis. We aimed to develop a short-term mouse model that reproduced the hallmark features of RV-induced exacerbation of COPD. Evaluation of complex protocols involving multiple dose elastase and lipopolysaccharide (LPS) administration combined with RV1B infection showed suppression rather than enhancement of inflammatory parameters compared with control mice infected with RV1B alone. Therefore, these approaches did not accurately model the enhanced inflammation associated with RV infection in patients with COPD compared with healthy subjects. In contrast, a single elastase treatment followed by RV infection led to heightened airway neutrophilic and lymphocytic inflammation, increased expression of tumour necrosis factor (TNF)-α, C-X-C motif chemokine 10 (CXCL10)/IP-10 (interferon γ-induced protein 10) and CCL5 [chemokine (C-C motif) ligand 5]/RANTES (regulated on activation, normal T-cell expressed and secreted), mucus hypersecretion and preliminary evidence for increased airway hyper-responsiveness compared with mice treated with elastase or RV infection alone. In summary, we have developed a new mouse model of RV-induced COPD exacerbation that mimics many of the inflammatory features of human disease. This model, in conjunction with human models of disease, will provide an essential tool for studying disease mechanisms and allow testing of novel therapies with potential to be translated into clinical practice.
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Asma/inmunología , Infecciones por Picornaviridae/inmunología , Enfermedad Pulmonar Obstructiva Crónica/virología , Rhinovirus/inmunología , Animales , Asma/virología , Quimiocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Lipopolisacáridos/inmunología , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Factores de TiempoRESUMEN
BACKGROUND: Steroid-insensitive endotypes of asthma are an important clinical problem and effective therapies are required. They are associated with bacterial infection and non-eosinophilic inflammatory responses in the asthmatic lung. Macrolide therapy is effective in steroid-insensitive endotypes, such as non-eosinophilic asthma. However, whether the effects of macrolides are due to antimicrobial or anti-inflammatory mechanisms is not known. OBJECTIVE: To determine and assess the efficacy of macrolide (ie, clarithromycin) and non-macrolide (ie, amoxicillin) antibiotic treatments in experimental models of infection-induced, severe, steroid-insensitive neutrophilic allergic airways disease (SSIAAD), compared with steroid-sensitive AAD and to delineate the antimicrobial and anti-inflammatory effects of macrolide therapy. METHODS: We developed and used novel mouse models of Chlamydia and Haemophilus lung infection-induced SSIAAD. We used these models to investigate the effects of clarithromycin and amoxicillin treatment on immune responses and airways hyper-responsiveness (AHR) in Ova-induced, T helper lymphocyte (Th) 2 -associated steroid-sensitive AAD and infection-induced Th1/Th17-associated SSIAAD compared with dexamethasone treatment. RESULTS: Clarithromycin and amoxicillin had similar antimicrobial effects on infection. Amoxicillin did attenuate some features, but did not broadly suppress either form of AAD. It did restore steroid sensitivity in SSIAAD by reducing infection. In contrast, clarithromycin alone widely suppressed inflammation and AHR in both steroid-sensitive AAD and SSIAAD. This occurred through reductions in Th2 responses that drive steroid-sensitive eosinophilic AAD and tumour necrosis factor α and interleukin 17 responses that induce SSIAAD. CONCLUSIONS: Macrolides have broad anti-inflammatory effects in AAD that are likely independent of their antimicrobial effects. The specific responses that are suppressed are dependent upon the responses that dominate during AAD.