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Severe asthma is a syndromic label assigned to patients based on clinical parameters, yet there are diverse underlying molecular endotypes in severe asthma pathobiology. Immunophenotyping of asthma biospecimens commonly includes a mixture of granulocytes and lymphocytes. Recently, a subset of severe asthma patients was defined as non-type 2 with neutrophil-enriched inflammation associated with increased Th17 CD4+ T cells and IL-17 levels. Here, we used an allergen-driven mouse model of increased IL-17 and mixed granulocyte lung inflammation to determine the impact of upstream regulation by an Anticalin protein that specifically binds IL-23. Airway administration of the IL-23 binding Anticalin protein (AcIL-23) decreased lung neutrophils, eosinophils, macrophages, and lymphocytes, IL-17+ CD4 T cells, mucous cell metaplasia and methacholine-induced airway hyperresponsiveness. Selective targeting of IL-23 with a monoclonal antibody (IL-23p19) (αIL-23) also decreased macrophages, IL-17+ CD4 T cells and airway hyperresponsiveness. In contrast, a monoclonal antibody against IL-17A (αIL-17A) had no significant effect on airway hyperresponsiveness, but did decrease lung neutrophils, macrophages, and IL-17+ CD4 T cells. Targeting the IL-23 pathway did not significantly change IL-5+ or IL-13+ CD4 T cells. Together, these data indicate that airway AcIL-23 mirrored the activity of systemic anti-IL-23 antibody to decrease airway hyperresponsiveness in addition to mixed granulocytic inflammation, and that these protective actions were broader than blocking only IL-17A or IL-5, which selectively decreased airway neutrophils and eosinophils, respectively.
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Specialized proresolving mediators (SPMs) promote local macrophage efferocytosis but excess leukocytes early in inflammation require additional leukocyte clearance mechanism for resolution. Here, neutrophil clearance mechanisms from localized acute inflammation were investigated in mouse dorsal air pouches. 15-HEPE (15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid) levels were increased in the exudates. Activated human neutrophils converted 15-HEPE to lipoxin A5 (5S,6R,15S-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), 15-epi-lipoxin A5 (5S,6R,15R-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), and resolvin E4 (RvE4; 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosapentaenoic acid). Exogenous 15-epi-lipoxin A5, 15-epi-lipoxin A4 and a structural lipoxin mimetic significantly decreased exudate neutrophils and increased local tissue macrophage efferocytosis, with comparison to naproxen. 15-epi-lipoxin A5 also cleared exudate neutrophils faster than the apparent local capacity for stimulated macrophage efferocytosis, so the fate of exudate neutrophils was tracked with CD45.1 variant neutrophils. 15-epi-lipoxin A5 augmented the exit of adoptively transferred neutrophils from the pouch exudate to the spleen, and significantly increased splenic SIRPa+ and MARCO+ macrophage efferocytosis. Together, these findings demonstrate new systemic resolution mechanisms for 15-epi-lipoxin A5 and RvE4 in localized tissue inflammation, which distally engage the spleen to activate macrophage efferocytosis for the clearance of tissue exudate neutrophils.
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Lipoxinas , Macrófagos , Neutrófilos , Baço , Animais , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Humanos , Lipoxinas/metabolismo , Lipoxinas/farmacologia , Baço/metabolismo , Baço/citologia , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacologia , Ácido Eicosapentaenoico/metabolismo , Camundongos Endogâmicos C57BL , Fagocitose , Masculino , Inflamação/metabolismo , Ácidos HeptanoicosRESUMO
Vitamin D possesses immunomodulatory functions and vitamin D deficiency has been associated with the rise in chronic inflammatory diseases, including asthma (Litonjua and Weiss, 2007). Vitamin D supplementation studies do not provide insight into the molecular genetic mechanisms of vitamin D-mediated immunoregulation. Here, we provide evidence for vitamin D regulation of two human chromosomal loci, Chr17q12-21.1 and Chr17q21.2, reliably associated with autoimmune and chronic inflammatory diseases. We demonstrate increased vitamin D receptor (Vdr) expression in mouse lung CD4+ Th2 cells, differential expression of Chr17q12-21.1 and Chr17q21.2 genes in Th2 cells based on vitamin D status and identify the IL-2/Stat5 pathway as a target of vitamin D signaling. Vitamin D deficiency caused severe lung inflammation after allergen challenge in mice that was prevented by long-term prenatal vitamin D supplementation. Mechanistically, vitamin D induced the expression of the Ikzf3-encoded protein Aiolos to suppress IL-2 signaling and ameliorate cytokine production in Th2 cells. These translational findings demonstrate mechanisms for the immune protective effect of vitamin D in allergic lung inflammation with a strong molecular genetic link to the regulation of both Chr17q12-21.1 and Chr17q21.2 genes and suggest further functional studies and interventional strategies for long-term prevention of asthma and other autoimmune disorders.
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Asma , Pneumonia , Deficiência de Vitamina D , Camundongos , Animais , Humanos , Vitamina D/farmacologia , Interleucina-2 , Inflamação , Células Th2 , Deficiência de Vitamina D/metabolismo , VitaminasRESUMO
Chronic airway inflammatory diseases like asthma, chronic obstructive pulmonary disease (COPD), and their associated exacerbations cause significant socioeconomic burden. There are still major obstacles to effective therapy for controlling severe asthma and COPD progression. Advances in understanding the pathogenesis of the two diseases at the cellular and molecular levels are essential for the development of novel therapies. In recent years, significant efforts have been made to identify natural products as potential drug leads for treatment of human diseases and to investigate their efficacy, safety, and underlying mechanisms of action. Many major active components from various natural products have been extracted, isolated, and evaluated for their pharmacological efficacy and safety. For the treatment of asthma and COPD, many promising natural products have been discovered and extensively investigated. In this chapter, we will review a range of natural compounds from different chemical classes, including terpenes, polyphenols, alkaloids, fatty acids, polyketides, and vitamin E, that have been demonstrated effective against asthma and/or COPD and their exacerbations in preclinical models and clinical trials. We will also elaborate in detail their underlying mechanisms of action unraveled by these studies and discuss new opportunities and potential challenges for these natural products in managing asthma and COPD.
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Eosinophils (Eos) reside in multiple organs during homeostasis and respond rapidly to an inflammatory challenge. Although Eos share chemical staining properties, they also demonstrate phenotypic and functional plasticity that is not fully understood. Here, we used a murine model of allergic lung inflammation to characterize Eos subsets and determine their spatiotemporal and functional regulation during inflammation and its resolution in response to resolvin D2 (RvD2), a potent specialized proresolving mediator. Two Eos subsets were identified by CD101 expression with distinct anatomic localization and transcriptional signatures at baseline and during inflammation. CD101low Eos were predominantly located in a lung vascular niche and responded to allergen challenge by moving into the lung interstitium. CD101high Eos were predominantly located in bronchoalveolar lavage (BAL) and extravascular lung, only present during inflammation, and had transcriptional evidence for cell activation. RvD2 reduced total Eos numbers and changed their phenotype and activation by at least two distinct mechanisms: decreasing interleukin 5-dependent recruitment of CD101low Eos and decreasing conversion of CD101low Eos to CD101high Eos. Collectively, these findings indicate that Eos are a heterogeneous pool of cells with distinct activation states and spatiotemporal regulation during resolution of inflammation and that RvD2 is a potent proresolving mediator for Eos recruitment and activation.
Assuntos
Alveolite Alérgica Extrínseca , Pneumonia , Eosinofilia Pulmonar , Camundongos , Animais , Eosinófilos/metabolismo , Líquido da Lavagem Broncoalveolar , Eosinofilia Pulmonar/metabolismo , Inflamação/metabolismo , Pneumonia/metabolismo , FenótipoRESUMO
Lipid phosphate phosphatases are a family of enzymes with diverse cellular metabolic functions. Phospholipid phosphatase 6 (PLPP6) is a regulator of cellular polyisoprenyl phosphates; however, its in vivo functions remain to be determined. Here, mouse PLPP6 was characterized to possess similar catalytic properties as the human enzyme. Plpp6 knockout mice (Plpp6 -/- ) were generated and displayed decreased airway allergen sensitization, pointing to a role for PLPP6 in the early events of lung allergic responses. Dendritic cell (DC) responses were investigated and endocytosis of allergen via macropinocytosis was decreased in Plpp6 -/- DCs that had lower cholesterol content. When reversed by cholesterol loading, the DC macropinocytosis defect is corrected. Adoptive transfer of Plpp6 -/- DCs to wild-type mice during sensitization was sufficient to decrease allergen-induced responses. Together, our findings have identified PLPP6 as a pivotal regulator of DC cholesterol content and macropinocytosis, cellular mechanisms that are important for pathologic responses in allergen-induced lung inflammation.
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Alveolar macrophages (AMs) are critical for lung immune defense and homeostasis. They are orchestrators of chronic obstructive pulmonary disease (COPD), with their number significantly increased and functions altered in COPD. However, it is unclear how AM number and function are controlled in a healthy lung and if changes in AMs without environmental assault are sufficient to trigger lung inflammation and COPD. We report here that absence of isthmin 1 (ISM1) in mice (Ism1-/- ) leads to increase in both AM number and functional heterogeneity, with enduring lung inflammation, progressive emphysema, and significant lung function decline, phenotypes similar to human COPD. We reveal that ISM1 is a lung resident anti-inflammatory protein that selectively triggers the apoptosis of AMs that harbor high levels of its receptor cell-surface GRP78 (csGRP78). csGRP78 is present at a heterogeneous level in the AMs of a healthy lung, but csGRP78high AMs are expanded in Ism1-/- mice, cigarette smoke (CS)-induced COPD mice, and human COPD lung, making these cells the prime targets of ISM1-mediated apoptosis. We show that csGRP78high AMs mostly express MMP-12, hence proinflammatory. Intratracheal delivery of recombinant ISM1 (rISM1) depleted csGRP78high AMs in both Ism1-/- and CS-induced COPD mice, blocked emphysema development, and preserved lung function. Consistently, ISM1 expression in human lungs positively correlates with AM apoptosis, suggesting similar function of ISM1-csGRP78 in human lungs. Our findings reveal that AM apoptosis regulation is an important physiological mechanism for maintaining lung homeostasis and demonstrate the potential of pulmonary-delivered rISM1 to target csGRP78 as a therapeutic strategy for COPD.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Apoptose/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático/fisiologia , Feminino , Homeostase , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Pulmão/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/fisiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/metabolismo , Fumaça/efeitos adversos , Fumar/efeitos adversos , Nicotiana/efeitos adversosRESUMO
A disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by cells implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its contributions to COPD are unknown. To address this gap, ADAM15 levels were measured in samples from cigarette smoke (CS)-versus air-exposed wild-type (WT) mice. CS-induced COPD-like disease was compared in CS-exposed WT, Adam15-/-, and Adam15 bone marrow chimeric mice. CS exposure increased Adam15 expression in lung macrophages and CD8+ T cells and to a lesser extent in airway epithelial cells in WT mice. CS-exposed Adam15-/- mice had greater emphysema, small airway fibrosis, and lung inflammation (macrophages and CD8+ T cells) than WT mice. Adam15 bone marrow chimera studies revealed that Adam15 deficiency in leukocytes led to exaggerated pulmonary inflammation and COPD-like disease in mice. Adam15 deficiency in CD8+ T cells was required for the exaggerated pulmonary inflammation and COPD-like disease in CS-exposed Adam15-/- mice (as assessed by genetically deleting CD8+ T cells in Adam15-/- mice). Adam15 deficiency increased pulmonary inflammation by rendering CD8+ T cells and macrophages resistant to CS-induced activation of the mitochondrial apoptosis pathway by preserving mTOR signaling and intracellular Mcl-1 levels in these cells. These results strongly link ADAM15 deficiency to the pathogenesis of COPD.
Assuntos
Proteínas ADAM/metabolismo , Linfócitos T CD8-Positivos/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Pneumonia/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Proteínas ADAM/genética , Animais , Células Cultivadas , Fumar Cigarros , Modelos Animais de Doenças , Humanos , Depleção Linfocítica , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pneumonia/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The average respiration rate for an adult is 12-20 breaths per minute, which constantly exposes the lungs to allergens and harmful particles. As a result, respiratory diseases, which includes asthma, chronic obstructive pulmonary disease (COPD) and acute lower respiratory tract infections (LTRI), are a major cause of death worldwide. Although asthma, COPD and LTRI are distinctly different diseases with separate mechanisms of disease progression, they do share a common feature - airway inflammation with intense recruitment and activation of granulocytes and mast cells. Neutrophils, eosinophils, basophils, and mast cells are crucial players in host defense against pathogens and maintenance of lung homeostasis. Upon contact with harmful particles, part of the pulmonary defense mechanism is to recruit these cells into the airways. Despite their protective nature, overactivation or accumulation of granulocytes and mast cells in the lungs results in unwanted chronic airway inflammation and damage. As such, understanding the bright and the dark side of these leukocytes in lung physiology paves the way for the development of therapies targeting this important mechanism of disease. Here we discuss the role of granulocytes in respiratory diseases and summarize therapeutic strategies focused on granulocyte recruitment and activation in the lungs.
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Granulócitos/efeitos dos fármacos , Medicamentos para o Sistema Respiratório/uso terapêutico , Sistema Respiratório/efeitos dos fármacos , Doenças Respiratórias/tratamento farmacológico , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Granulócitos/imunologia , Granulócitos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Terapia de Alvo Molecular , Fenótipo , Sistema Respiratório/imunologia , Sistema Respiratório/metabolismo , Sistema Respiratório/fisiopatologia , Medicamentos para o Sistema Respiratório/efeitos adversos , Doenças Respiratórias/imunologia , Doenças Respiratórias/metabolismo , Doenças Respiratórias/fisiopatologia , Transdução de SinaisRESUMO
In this study, we developed a water-in-oil microemulsion containing vitamin A (retinol) and vitamin E (α-tocopherol), which serves as a multifunctional nanosystem that co-delivers antioxidants and displayed additive effect against acute skin inflammation. Microemulsion (ME) was prepared by mixing a surfactant blend (Tween 80 and propylene glycol, 5:1) with isopropyl myristate and water (ratio of 50:40:10, respectively). Vitamin A (0.05% w/w concentration) and/or vitamin E (0.1% w/w concentration) were incorporated into the surfactant mixture of ME by stirring with a magnetic stirrer for 30 min. This multifunctional ME displayed physical stability, with low cytotoxicity in 3T3 cell line, as well as cellular internalization into the cytosol. In vivo treatments using ME delivering α-tocopherol reduced dermal expression of TNF-α by 1.3-fold (pâ¯<â¯0.01), when compared to unloaded ME treatment group. When retinol was added into the ME containing α-tocopherol, it further reduced TNF-α expression by 2-fold (pâ¯<â¯0.001), suggesting the additive effect of vitamin E and vitamin A in the treatment against skin inflammation. In conclusion, we successfully developed the use of water-in-oil ME to pack both vitamin E and vitamin A, and demonstrated for the first time its anti-inflammatory potential when applied topically to TPA-induced inflamed skin.
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Sistemas de Liberação de Medicamentos , Inflamação/tratamento farmacológico , Inflamação/patologia , Pele/patologia , Vitamina A/administração & dosagem , Vitamina E/administração & dosagem , Doença Aguda , Administração Tópica , Animais , Varredura Diferencial de Calorimetria , Sobrevivência Celular/efeitos dos fármacos , Emulsões , Células HaCaT , Humanos , Camundongos , Camundongos Pelados , Células NIH 3T3 , Pele/efeitos dos fármacos , Suínos , Vitamina A/farmacologia , Vitamina A/uso terapêutico , Vitamina E/farmacologia , Vitamina E/uso terapêuticoRESUMO
Cigarette smoking is the leading cause of chronic obstructive pulmonary disease (COPD). Cigarette smoke heightens oxidative stress and impairs autophagy, advancing COPD progression. Andrographolide is a bioactive diterpenoid lactone isolated from the plant Andrographis paniculata which has been a traditional medicinal herb for respiratory diseases. As airway epithelial cells form the first interface to be exposed to cigarette smoke, this study aimed to explore the modulatory effects of andrographolide on oxidative stress and autophagy in human bronchial epithelial BEAS-2B cells exposed to cigarette smoke extract (CSE). CSE (2%) exposure increased autophagic markers p62 and LC3B-II levels in BEAS-2B cells. Andrographolide alone increased p62 and p-p62 (S349) but not LC3B-II in BEAS-2B cells. However, in the presence of CSE, andrographolide was able to simultaneously increase LC3B-II level and enhance antioxidant defense by decreasing oxidative stress and increasing total antioxidant capacity, through upregulation of nuclear Nrf2 via the p62-Nrf2 positive feedback loop. Using RFP-GFP-LC3B transfected BEAS-2B cells exposed to CSE, andrographolide was found to impair autophagosome fusion with lysosome, which may account for the moderate increase in activated caspase 3/7 and annexin V levels. Our findings revealed for the first time that andrographolide simultaneously upregulated antioxidant defense through the p62-Nrf2 loop and moderately induced apoptosis through impairment of autophagic flux in CSE-exposed bronchial epithelium. Andrographolide facilitated cigarette smoke-induced apoptosis may be a potential toxicological outcome or may protect against chronic inflammation and aberrant DNA repair. Validation of these in-vitro findings in an experimental COPD model by andrographolide is warranted.
Assuntos
Antioxidantes/metabolismo , Autofagia/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Diterpenos/farmacologia , Células Epiteliais/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fumaça/efeitos adversos , Apoptose/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Fumar/efeitos adversos , Nicotiana/efeitos adversos , Regulação para Cima/efeitos dos fármacosRESUMO
Chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death by 2030. Transcription factor NF-κB may play a critical role in COPD pathogenesis. Ribosomal protein S3 (RPS3), a 40S ribosomal protein essential for executing protein translation, has recently been found to interact with the NF-κB p65 subunit and promote p65 DNA-binding activity. We sought to study whether RPS3 gene silencing could protect against cigarette-smoke (CS)-induced acute lung injury in a mouse model. Effects of an intratracheal RPS3 siRNA in CS-induced lung injury were determined by measuring bronchoalveolar lavage (BAL) fluid cell counts, levels of inflammatory and oxidative damage markers, and NF-κB translocation. Lung RPS3 level was found to be upregulated for the first time with CS exposure, and RPS3 siRNA blocked CS-induced neutrophil counts in BAL fluid. RPS3 siRNA suppressed CS-induced lung inflammatory mediator and oxidative damage marker levels, as well as nuclear p65 accumulation and transcriptional activation. RPS3 siRNA was able to disrupt CS extract (CSE)-induced NF-κB activation in an NF-κB reporter gene assay. We report for the first time that RPS3 gene silencing ameliorated CS-induced acute lung injury, probably via interruption of the NF-κB activity, postulating that RPS3 is a novel therapeutic target for COPD.
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Inflammation and oxidative stress contribute to emphysema in COPD. Although corticosteroids are the standard of care for COPD, they do not reduce oxidative stress, and a subset of patients is steroid-resistant. Vitamin E isoform γ-tocotrienol possesses both anti-inflammatory and anti-oxidative properties that may protect against emphysema. We aimed to establish the therapeutic potential of γ-tocotrienol in cigarette smoke-induced COPD models in comparison with prednisolone. BALB/c mice were exposed to cigarette smoke for 2 weeks or 2 months. γ-Tocotrienol and prednisolone were given orally. Bronchoalveolar lavage (BAL) fluid and lung tissues were assessed for inflammation, oxidative damage, and regulation of transcription factor activities. Emphysema and lung function were also evaluated. γ-Tocotrienol dose-dependently reduced cigarette smoke-induced BAL fluid neutrophil counts and levels of cytokines, chemokines and oxidative damage biomarkers, and pulmonary pro-inflammatory and pro-oxidant gene expression, but restored lung endogenous antioxidant activities. γ-Tocotrienol acted by inhibiting nuclear translocation of STAT3 and NF-κB, and up-regulating Nrf2 activation in the lungs. In mice exposed to 2-month cigarette smoke, γ-tocotrienol ameliorated bronchial epithelium thickening and destruction of alveolar sacs in lungs, and improved lung functions. In comparison with prednisolone, γ-tocotrienol demonstrated better anti-oxidative efficacy, and protection against emphysema and lung function in COPD. We revealed for the first time the anti-inflammatory and antioxidant efficacies of γ-tocotrienol in cigarette smoke-induced COPD models. In addition, γ-tocotrienol was able to attenuate emphysematous lesions and improve lung function in COPD. γ-Tocotrienol may have therapeutic potential for the treatment of COPD.
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Anti-Inflamatórios/farmacologia , Nicotiana/efeitos adversos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Enfisema Pulmonar/prevenção & controle , Espécies Reativas de Oxigênio/antagonistas & inibidores , Fumaça/efeitos adversos , Tocotrienóis/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Prednisolona/farmacologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Exposure to environmental allergens is a major risk factor for asthma development. Allergens possess proteolytic activity that is capable of disrupting the airway epithelium. Although there is increasing evidence pointing to asthma as an epithelial disease, the underlying mechanism that drives asthma has not been fully elucidated. In this study, we investigated the direct DNA damage potential of aeroallergens on human bronchial epithelial cells and elucidated the mechanisms mediating the damage. Human bronchial epithelial cells, BEAS-2B, directly exposed to house dust mites (HDM) resulted in enhanced DNA damage, as measured by the CometChip and the staining of DNA double-strand break marker, γH2AX. HDM stimulated cellular reactive oxygen species production, increased mitochondrial oxidative stress, and promoted nitrosative stress. Notably, expression of nuclear factor erythroid 2-related factor 2-dependent antioxidant genes was reduced immediately after HDM exposure, suggesting that HDM altered antioxidant responses. HDM exposure also reduced cell proliferation and induced cell death. Importantly, HDM-induced DNA damage can be prevented by the antioxidants glutathione and catalase, suggesting that HDM-induced reactive oxygen and nitrogen species can be neutralized by antioxidants. Mechanistic studies revealed that HDM-induced cellular injury is NADPH oxidase (NOX)-dependent, and apocynin, a NOX inhibitor, protected cells from double-strand breaks induced by HDM. Our results show that direct exposure of bronchial epithelial cells to HDM leads to the production of reactive oxygen and nitrogen species that damage DNA and induce cytotoxicity. Antioxidants and NOX inhibitors can prevent HDM-induced DNA damage, revealing a novel role for antioxidants and NOX inhibitors in mitigating allergic airway disease.
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Alérgenos/imunologia , Antioxidantes/fisiologia , Brônquios/imunologia , Dano ao DNA , Células Epiteliais/imunologia , Pyroglyphidae/imunologia , Espécies Reativas de Oxigênio/metabolismo , Acetofenonas/imunologia , Acetofenonas/farmacologia , Ar , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Asma/etiologia , Asma/imunologia , Asma/fisiopatologia , Brônquios/citologia , Brônquios/metabolismo , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutationa/farmacologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismoRESUMO
Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD) and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF). The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO), small interfering RNA (siRNA), and microRNA (miRNA) are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir) has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases.
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Asma/tratamento farmacológico , Fibrose Pulmonar Idiopática/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Ensaios Clínicos como Assunto , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , RNA Interferente Pequeno/uso terapêuticoRESUMO
BACKGROUND AND PURPOSE: Ribosomal protein S3 (RPS3) is a 40S ribosomal protein of the S3P family essential for implementing protein translation. RPS3 has recently been found to interact with the p65 subunit of the NF-κB complex and promote p65 DNA-binding activity. Persistent activation of the NF-κB pathway is evident in allergic asthma. We hypothesized that gene silencing of lung RPS3 can ameliorate allergic airway inflammation. EXPERIMENTAL APPROACH: The gene silencing efficacy of RPS3 siRNA was screened in three different mouse cell lines by real-time PCR and immunoblotting. Protective effects of intratracheal RPS3 siRNA in a house dust mite (HDM) mouse asthma model were determined by measuring cell counts in lung lavage fluid and lung sections, lung cytokine profiles and airway hyperresponsiveness (AHR). KEY RESULTS: RPS3 siRNA markedly knocked down RPS3 levels in all mouse cell lines tested, and in mouse lung tissues, blocked TNF-α- or HDM-induced release of mediators by the cultured cells and reduced eosinophil counts in lung lavage fluid from the HDM mouse asthma model. RPS3 siRNA lessened HDM-induced airway mucus hypersecretion, cytokine production and serum IgE elevation. Moreover, RPS3 knockdown significantly suppressed methacholine-induced AHR in experimental asthma. RPS3 siRNA disrupted TNF-α-induced NF-κB activation in a NF-κB reporter gene assay in vitro and prevented the nuclear accumulation of p65 subunit and p65 transcriptional activation in HDM-challenged lungs and cells. CONCLUSIONS AND IMPLICATIONS: RPS3 gene silencing ameliorates experimental asthma, probably by disrupting NF-κB activity. RPS3 could be a novel therapeutic target for allergic airway inflammation.
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Asma/genética , Asma/prevenção & controle , Inativação Gênica , Proteínas Ribossômicas/genética , Animais , Asma/terapia , Feminino , Terapia Genética , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Células RAW 264.7Assuntos
Alérgenos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Asma/terapia , Hipersensibilidade/terapia , Epitopos Imunodominantes/imunologia , Imunoglobulina G/genética , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Asma/imunologia , Células Cultivadas , Humanos , Hipersensibilidade/imunologia , Imunização , Imunoglobulina E/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ácaros/imunologia , Modelos Moleculares , Biblioteca de PeptídeosRESUMO
Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13-dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma.