RESUMEN
PGAP3 is a glycosylphosphatidylinositol (GPI) phospholipase gene localized within chromosome 17q12-21, a region highly linked to asthma. Although much is known about the function of other chromosome 17q12-21 genes expressed at increased levels in bronchial epithelium such as ORMDL3 and GSDMB, little is known about the function of increased PGAP3 expression in bronchial epithelium in the context of asthma. The aim of this study was therefore to determine whether increased PGAP3 expression in human bronchial epithelial cells regulated expression of mRNA pathways important to the pathogenesis of asthma by utilizing RNA-sequencing and bioinformatic analysis. We performed RNA-sequencing on normal human bronchial epithelial cells transfected with PGAP3 for 24 and 48 hours. PGAP3 regulated genes were compared to asthma and respiratory virus (influenza A, rhinovirus, respiratory syncytial virus) reference data sets to identify PGAP3 target genes and pathways. Approximately 9% of the upregulated PGAP3-induced genes were found in an asthma reference data set, 41% in a rhinovirus reference data set, 33% in an influenza A reference data set, and 3% in a respiratory syncytial virus reference data set. PGAP3 significantly upregulated the expression of several genes associated with the innate immune response and viral signatures of respiratory viruses associated with asthma exacerbations. Two of the highest expressed genes induced by PGAP3 are RSAD2, OASL, and IFN-λ, which are anti-viral genes associated with asthma. PGAP3 also upregulated the antiviral gene BST2, which like PGAP3 is a GPI-anchored protein. We conclude that PGAP3 expression in human bronchial epithelial cells regulates expression of genes known to be linked to asthma, and also regulates the bronchial epithelial expression of genes pertinent to the pathogenesis of respiratory viral triggered asthma exacerbations.
RESUMEN
BACKGROUND: Dysregulation of airway smooth muscle cells (ASM) is central to the severity of asthma. Which molecules dominantly control ASM in asthma is unclear. High levels of the cytokine LIGHT (aka TNFSF14) have been linked to asthma severity and lower baseline predicted FEV1 percentage, implying that signals through its receptors might directly control ASM dysfunction. OBJECTIVE: Our study sought to determine whether signaling via lymphotoxin beta receptor (LTßR) or herpesvirus entry mediator from LIGHT dominantly drives ASM hyperreactivity induced by allergen. METHODS: Conditional knockout mice deficient for LTßR or herpesvirus entry mediator in smooth muscle cells were used to determine their role in ASM deregulation and airway hyperresponsiveness (AHR) in vivo. Human ASM were used to study signals induced by LTßR. RESULTS: LTßR was strongly expressed in ASM from normal and asthmatic subjects compared to several other receptors implicated in smooth muscle deregulation. Correspondingly, conditional deletion of LTßR only in smooth muscle cells in smMHCCreLTßRfl/fl mice minimized changes in their numbers and mass as well as AHR induced by house dust mite allergen in a model of severe asthma. Intratracheal LIGHT administration independently induced ASM hypertrophy and AHR in vivo dependent on direct LTßR signals to ASM. LIGHT promoted contractility, hypertrophy, and hyperplasia of human ASM in vitro. Distinguishing LTßR from the receptors for IL-13, TNF, and IL-17, which have also been implicated in smooth muscle dysregulation, LIGHT promoted NF-κB-inducing kinase-dependent noncanonical nuclear factor kappa-light-chain enhancer of activated B cells in ASM in vitro, leading to sustained accumulation of F-actin, phosphorylation of myosin light chain kinase, and contractile activity. CONCLUSIONS: LTßR signals directly and dominantly drive airway smooth muscle hyperresponsiveness relevant for pathogenesis of airway remodeling in severe asthma.
Asunto(s)
Asma , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Humanos , Ratones , Animales , Receptor beta de Linfotoxina/genética , Asma/patología , Músculo Liso , Miocitos del Músculo Liso/patología , Ratones Noqueados , Alérgenos , Pulmón/patologíaAsunto(s)
Asma , Hiperreactividad Bronquial , Asma/tratamiento farmacológico , Bronquios , Células Epiteliales , HumanosRESUMEN
BACKGROUND: Genetic association studies have demonstrated that the SNP rs12603332 located on chromosome 17q21 is highly associated with the risk of the development of asthma. METHODS: To determine whether SNP rs1260332 is functional in regulating levels of ORMDL3 expression, we used a Cytosine Base Editor (CBE) plasmid DNA or a CBE mRNA to edit the rs12603332 C risk allele to the T non-risk allele in a human lymphocyte cell line (i.e., Jurkat cells) and in primary human CD4 T cells that carry the C risk alleles. RESULTS: Jurkat cells with the rs12603332 C risk allele expressed significantly higher levels of ORMDL3 mRNA, as well as the ORMDL3 regulated gene ATF6α as assessed by qPCR compared to Jurkat clones with the T non-risk allele. In primary human CD4 T cells, we edited 90 ± 3% of the rs12603332-C risk allele to the T non-risk allele and observed a reduction in ORMDL3 and ATF6α expression. Bioinformatic analysis predicted that the non-risk allele rs12603332-T could be the central element of the E-box binding motif (CANNTG) recognized by the E47 transcription factor. An EMSA assay confirmed the bioinformatics prediction demonstrating that a rs12603332-T containing probe bound to the transcription factor E47 in vitro. CONCLUSIONS: SNP rs12603332 is functional in regulating the expression of ORMDL3 as well as ORMDL3 regulated gene ATF6α expression. In addition, we demonstrate the use of CBE technology in functionally interrogating asthma-associated SNPs using studies of primary human CD4 cells.
Asunto(s)
Asma , Cromosomas Humanos Par 17 , Citosina , Asma/genética , Estudios de Casos y Controles , Cromosomas , Predisposición Genética a la Enfermedad , Humanos , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , ARN Mensajero , Factores de Transcripción/genéticaRESUMEN
ORM1-like 3 (ORMDL3) has strong genetic linkage to childhood onset asthma. To determine whether ORMDL3 selective expression in airway smooth muscle (ASM) influences ASM function, we used Cre-loxP techniques to generate transgenic mice (hORMDL3Myh11eGFP-cre), which express human ORMDL3 selectively in smooth muscle cells. In vitro studies of ASM cells isolated from the bronchi of hORMDL3Myh11eGFP-cre mice demonstrated that they developed hypertrophy (quantitated by FACS and image analysis), developed hyperplasia (assessed by BrdU incorporation), and expressed increased levels of tropomysin proteins TPM1 and TPM4. siRNA knockdown of TPM1 or TPM4 demonstrated their importance to ORMDL3-mediated ASM proliferation but not hypertrophy. In addition, ASM derived from hORMDL3Myh11eGFP-cre mice had increased contractility to histamine in vitro, which was associated with increased levels of intracellular Ca2+; increased cell surface membrane Orai1 Ca2+ channels, which mediate influx of Ca2+ into the cytoplasm; and increased expression of ASM contractile genes sarco/endoplasmic reticulum Ca2+ ATPase 2b and smooth muscle 22. In vivo studies of hORMDL3Myh11eGFP-cre mice demonstrated that they had a spontaneous increase in ASM and airway hyperreactivity (AHR). ORMDL3 expression in ASM thus induces changes in ASM (hypertrophy, hyperplasia, increased contractility), which may explain the contribution of ORMDL3 to the development of AHR in childhood onset asthma, which is highly linked to ORMDL3 on chromosome 17q12-21.
Asunto(s)
Proteínas de la Membrana/genética , Músculo Liso/patología , Tropomiosina/genética , Animales , Asma/genética , Asma/patología , Hiperreactividad Bronquial/etiología , Calcio/metabolismo , Proliferación Celular , Células Cultivadas , Histamina/farmacología , Humanos , Hiperplasia , Hipertrofia , Ratones Transgénicos , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Tropomiosina/metabolismoRESUMEN
Lung fibrosis and tissue remodeling are features of chronic diseases such as severe asthma, idiopathic pulmonary fibrosis, and systemic sclerosis. However, fibrosis-targeted therapies are currently limited. We demonstrate in mouse models of allergen- and bleomycin-driven airway inflammation that neutralization of the TNF family cytokine TL1A through Ab blocking or genetic deletion of its receptor DR3 restricted increases in peribronchial smooth muscle mass and accumulation of lung collagen, primary features of remodeling. TL1A was found as a soluble molecule in the airways and expressed on the surface of alveolar macrophages, dendritic cells, innate lymphoid type 2 cells, and subpopulations of lung structural cells. DR3 was found on CD4 T cells, innate lymphoid type 2 cells, macrophages, fibroblasts, and some epithelial cells. Suggesting in part a direct activity on lung structural cells, administration of recombinant TL1A into the naive mouse airways drove remodeling in the absence of other inflammatory stimuli, innate lymphoid cells, and adaptive immunity. Correspondingly, human lung fibroblasts and bronchial epithelial cells were found to express DR3 and responded to TL1A by proliferating and/or producing fibrotic molecules such as collagen and periostin. Reagents that disrupt the interaction of TL1A with DR3 then have the potential to prevent deregulated tissue cell activity in lung diseases that involve fibrosis and remodeling.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Fibrosis Pulmonar Idiopática/inmunología , Pulmón/inmunología , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Inmunidad Adaptativa/inmunología , Animales , Asma/inmunología , Bleomicina/inmunología , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Células Dendríticas/inmunología , Células Epiteliales/inmunología , Femenino , Humanos , Inmunidad Innata/inmunología , Inflamación/inmunología , Linfocitos/inmunología , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BLAsunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Asma/genética , Cromosomas Humanos Par 17 , Pulmón/patología , Músculo Liso/patología , Polimorfismo de Nucleótido Simple , Adulto , Asma/mortalidad , Asma/patología , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Humanos , Hipertrofia , Masculino , Persona de Mediana Edad , Fenotipo , Medición de Riesgo , Factores de RiesgoRESUMEN
Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.
Asunto(s)
Factores Reguladores del Interferón , Receptores de Reconocimiento de Patrones , Transducción de Señal/inmunología , Células Th17 , Células Th2 , Animales , Línea Celular Tumoral , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Citocinas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Factores Reguladores del Interferón/antagonistas & inhibidores , Factores Reguladores del Interferón/inmunología , Factores Reguladores del Interferón/metabolismo , Factor 4 Similar a Kruppel , Ratones , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Células Th2/inmunología , Células Th2/metabolismoAsunto(s)
Alternaria/fisiología , Alternariosis/inmunología , Hipersensibilidad/inmunología , Subgrupos Linfocitarios/inmunología , Linfocitos/inmunología , Alérgenos/inmunología , Animales , Antígenos Fúngicos/inmunología , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Proteína 1 Similar al Receptor de Interleucina-1/genética , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Ratones , Ratones Noqueados , Células Th2/inmunologíaRESUMEN
Asthma is a common disorder of the airways characterized by airway inflammation and by decline in lung function and airway remodeling in a subset of asthmatics. Airway remodeling is characterized by structural changes which include airway smooth muscle hypertrophy/hyperplasia, subepithelial fibrosis due to thickening of the reticular basement membrane, mucus metaplasia of the epithelium, and angiogenesis. Epidemiologic studies suggest that both genetic and environmental factors may contribute to decline in lung function and airway remodeling in a subset of asthmatics. Environmental factors include respiratory viral infection-triggered asthma exacerbations, and tobacco smoke. There is also evidence that several asthma candidate genes may contribute to decline in lung function, including ADAM33, PLAUR, VEGF, IL13, CHI3L1, TSLP, GSDMB, TGFB1, POSTN, ESR1 and ARG2. In addition, mediators or cytokines, including cysteinyl leukotrienes, matrix metallopeptidase-9, interleukin-33 and eosinophil expression of transforming growth factor-ß, may contribute to airway remodeling in asthma. Although increased airway smooth muscle is associated with reduced lung function (i.e. forced expiratory volume in 1 second) in asthma, there have been few long-term studies to determine how individual pathologic features of airway remodeling contribute to decline in lung function in asthma. Clinical studies with inhibitors of individual gene products, cytokines or mediators are needed in asthmatic patients to identify their individual role in decline in lung function and/or airway remodeling.
RESUMEN
The recent discovery of innate lymphoid cells has revolutionized our understanding of the pathogenesis of immune diseases including allergy and asthma. Innate lymphoid cells (ILCs) are a heterogeneous collection of lymphocytes that lack antigen-specificity (non-T, non-B cells) and potently produce characteristic cytokines of T cell subsets (Th1, Th2, Th17). ILCs are divided into group 1 (ILC1s), group 2 (ILC2s), or group 3 (ILC3s). Similar to Th2 cells, ILC2s produce IL-4, IL-5, and IL-13, among others, and are present in increased numbers in samples from patients with many allergic disorders including asthma and chronic rhinosinusitis (CRS). Animal models have identified that ILC2s contribute to eosinophilic tissue infiltration, airway hyperresponsiveness, mucus production, as well as coordinate adaptive immune responses. Finally, recent studies support regulation of ILC2s by neuro-immune mechanisms as well as demonstrate a significant degree of plasticity between ILC subsets that may impact the immune responses in asthma and allergic airway diseases. Here, we review the current literature on ILC2s in human asthma and allergic airway diseases, as well as highlight some recent mechanistic insights into ILC2 function from in vitro studies and in vivo animal models.
Asunto(s)
Linfocitos/inmunología , Hipersensibilidad Respiratoria/inmunología , Animales , Plasticidad de la Célula , Humanos , Inmunidad InnataAsunto(s)
Asma/metabolismo , Cromosomas Humanos Par 17/genética , Proteínas del Huevo/metabolismo , Pulmón/fisiología , Proteínas de la Membrana/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Zona Pelúcida/metabolismo , Animales , Antígenos Dermatofagoides/inmunología , Asma/genética , Metilación de ADN , Modelos Animales de Enfermedad , Proteínas del Huevo/genética , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pyroglyphidae/inmunología , Hipersensibilidad Respiratoria/genéticaAsunto(s)
Asma Inducida por Aspirina , Dinoprostona , Citocinas , Humanos , Inmunidad Innata , LinfocitosRESUMEN
BACKGROUND: Group 2 innate lymphoid cells (ILC2s) expand in the lungs of mice during type 2 inflammation induced by the fungal allergen Alternaria alternata. The increase in ILC2 numbers in the lung has been largely attributed to local proliferation and whether ILC2s migrate from the circulation to the lung after Alternaria exposure is unknown. OBJECTIVE: We examined whether human (lung, lymph node, and blood) and mouse lung ILC2s express ß1 and ß2 integrin adhesion molecules and whether these integrins are required for trafficking of ILC2s into the lungs of mice. METHODS: Human and mouse ILC2s were assessed for surface expression of ß1 and ß2 integrin adhesion molecules by using flow cytometry. The role of ß1 and ß2 integrins in ILC2 trafficking to the lungs was assessed by in vivo blocking of these integrins before airway exposure to Alternaria in mice. RESULTS: Both human and mouse lung ILC2s express high levels of ß1 and ß2 integrin adhesion receptors. Intranasal administration of Alternaria challenge reduced ILC2 numbers in the bone marrow and concurrently increased blood and lung ILC2 numbers. In vivo blocking of ß2 integrins (CD18) significantly reduced ILC2 numbers in the lungs but did not alter ILC2 proliferation, apoptosis, and function. In contrast, in vivo blocking of ß1 integrins or α4 integrins did not affect lung ILC2 numbers. CONCLUSION: ILC2 numbers increase in the mouse lung not only through local proliferation but also through trafficking from the circulation into the lung using ß2 rather than ß1 or α4 integrins.
Asunto(s)
Alternaria/inmunología , Antígenos CD18/metabolismo , Inmunidad Innata , Integrina beta1/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Animales , Apoptosis/inmunología , Biomarcadores , Médula Ósea/inmunología , Médula Ósea/metabolismo , Antígenos CD18/genética , Citocinas/metabolismo , Citometría de Flujo , Expresión Génica , Humanos , Integrina beta1/genética , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Selectina L/genética , Selectina L/metabolismo , Pulmón/patología , Recuento de Linfocitos , Ratones , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
BACKGROUND: Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. OBJECTIVE: We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). METHODS: Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3Zp3-Cre mice, which do not have a blood supply. RESULTS: Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. CONCLUSION: Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients.