Real time analysis of β2-adrenoceptor-mediated signaling kinetics in Human Primary Airway Smooth Muscle Cells reveals both ligand and dose dependent differences

BACKGROUND: β(2)-adrenoceptor agonists elicit bronchodilator responses by binding to β(2)-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β(2)-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP. METHODS: Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β(2)-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used. RESULTS: Temporal analysis revealed that in hASM cells the β(2)-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response. CONCLUSIONS: We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β(2)-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β(2)-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo

Signaling and regulation of G protein-coupled receptors in airway smooth muscle

Signaling through G protein-coupled receptors (GPCRs) mediates numerous airway smooth muscle (ASM) functions including contraction, growth, and "synthetic" functions that orchestrate airway inflammation and promote remodeling of airway architecture. In this review we provide a comprehensive overview of the GPCRs that have been identified in ASM cells, and discuss the extent to which signaling via these GPCRs has been characterized and linked to distinct ASM functions. In addition, we examine the role of GPCR signaling and its regulation in asthma and asthma treatment, and suggest an integrative model whereby an imbalance of GPCR-derived signals in ASM cells contributes to the asthmatic state

Clonally expanded human airway smooth muscle cells exhibit morphological and functional heterogeneity

Background: Mesenchyme-derived airway cell populations including airway smooth muscle (ASM) cells, fibroblasts and myofibroblasts play key roles in the pathogenesis of airway inflammation and remodeling. Phenotypic and functional characterisation of these cell populations are confounded by their heterogeneity in vitro. It is unclear which mechanisms underlie the creation of these different sub-populations. The study objectives were to investigate whether ASM cells are capable of clonal expansion and if so (i) what proportion possess this capability and (ii) do clonal populations exhibit variation in terms of morphology, phenotype, proliferation rates and pro-relaxant or pro-contractile signaling pathways. Methods: Early passage human ASM cells were subjected to single-cell cloning and their doubling time was recorded. Immunocytochemistry was performed to assess localization and levels of markers previously reported to be specifically associated with smooth muscle or fibroblasts. Finally functional assays were used to reveal differences between clonal populations specifically assessing mitogen-induced proliferation and pro-relaxant and pro-contractile signaling pathways. Results: Our studies provide evidence that a high proportion (58%) of single cells present within early passage human ASM cell cultures have the potential to create expanded cell populations. Despite being clonally-originated, morphological heterogeneity was still evident within these clonal populations as assessed by the range in expression of markers associated with smooth muscle cells. Functional diversity was observed between clonal populations with 10 μM isoproterenol-induced cyclic AMP responses ranging from 1.4 - 5.4 fold cf basal and bradykinin-induced inositol phosphate from 1.8 - 5.2 fold cf basal. Conclusion: In summary we show for the first time that primary human ASM cells are capable of clonal expansion and that the resulting clonal populations themselves exhibit phenotypic plasticity

Defining the inflammatory signature of human lung explant tissue in the presence and absence of glucocorticoid

Background: Airway inflammation is a feature of many respiratory diseases and there is a need for newer, more effective anti-inflammatory compounds. The aim of this study was to develop an ex vivo human lung explant model which can be used to help study the mechanisms underlying inflammatory responses and which can provide a tool to aid drug discovery for inflammatory respiratory diseases such as asthma and COPD. Method: Parenchymal lung tissue from 6 individual donors was dissected and cultured with two pro-inflammatory stimuli, lipopolysaccharide (LPS) (1 µg/ml) and interleukin-1 beta (IL-1β) (10 ng/ml) in the presence or absence of dexamethasone (1 µM). Inflammatory responses were assessed using Luminex analysis of tissue culture supernatants to measure levels of 21 chemokines, growth factors and cytokines. Results: A robust and reproducible inflammatory signal was detected across all donors for 12 of the analytes measured following LPS stimulation with a modest fold increase (4-fold in CCL3, CCL4, GM-CSF, IL-10, TNF-α and IL-1β. The inflammatory signal induced by IL-1β stimulation was less than that observed with LPS but resulted in elevated levels of 7 analytes (CXCL8, CCL3, CCL4, GM-CSF, IL-6, IL-10 and TNF-α). The inflammatory responses induced by both stimulations was supressed by dexamethasone for the majority of analytes. Conclusions: These data provide proof of concept that this ex vivo human lung explant model is responsive to inflammatory signals and could be used to investigate the anti-inflammatory effects of existing and novel compounds. In addition this model could be used to help define the mechanisms and pathways involved in development of inflammatory airway disease

Novel polymorphisms influencing transcription of the human CHRM2 gene in airway smooth muscle

Muscarinic receptors are a functionally important family of G-protein-coupled receptors. Using a combination of rapid amplification of 5′ cDNA ends and reporter gene assays, we characterized the 5′ untranslated region of the CHRM2 gene as expressed in human airway smooth muscle (HASM) cells. A splice site is present 46 bp upstream from the ATG start codon. Five exons with alternative splicing patterns are present upstream of this splice site, separated by introns ranging from 87 bp to > 145 kb. There is evidence for the gene being under the control of a TATA-less promoter with Sp1, GATA, and activator protein-2 binding sites. Multiple transcription start sites (TSSs) were identified. We identified a novel 0.5-kb hypervariable region located 648 bp upstream of the most 5′ TSS, a multiallelic (CA) tandem repeat 96 bp downstream of the most 5′ TSS, and a common C→A SNP located 136 bp upstream of the most 5′ TSS. Functional studies in primary HASM cells and the BEAS-2B cell line demonstrated highest promoter activity to be upstream of the most 3′ TSS, with potential repressor elements operating in a cell type-dependent manner, located upstream of the most 5′ TSS. We present functional data to show that the CA repeat may influence the transcription of the gene in HASM and BEAS-2B cells.peer-reviewe

Functional genomics of GPR126 in airway smooth muscle and bronchial epithelial cells

GPR126 is an adhesion G protein-coupled receptor which lies on chromosome 6q24. Genetic variants in this region are reproducibly associated with lung function and COPD in genome wide association studies (GWAS). The aims of this study were to define the role of GPR126 in the human lung and in pulmonary disease and identify possible casual variants. Online tools (GTEx and LDlink) identified SNPs which may have effects on GPR126 function/ expression, including missense variant Ser123Gly and an intronic variant that shows eQTL effects on GPR126 expression. GPR126 signaling via cAMP-mediated pathways was identified in human structural airway cells when activated with the tethered agonist, stachel. RNA-seq was used to identify downstream genes/ pathways affected by stachel-mediated GPR126 activation in human airway smooth muscle cells. We identified ~350 differentially expressed genes at 4 and 24 hours post stimulation with ~20% overlap. We identified that genes regulated by GPR126 activation include IL33, CTGF, and SERPINE1, which already have known roles in lung biology. Pathways altered by GPR126 included those involved in cell cycle progression and cell proliferation. Here, we suggest a role for GPR126 in airway remodeling

Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP-activated whole cell chloride currents in human bronchial epithelial cells

Chloride channels are known to play critical physiological roles in many cell types. Here we describe the expression of anion channels using RNA Seq in primary cultures of human Bronchial Epithelial Cells (hBECs). Chloride intracellular channel (CLIC) family members were the most abundant chloride channel transcripts, and CLIC1 showed the highest level of expression. In addition, we characterise the chloride currents in hBECs and determine how inhibition of CLIC1 via pharmacological and molecular approaches impacts these. We demonstrate that CLIC1 is able to modulate cyclic AMP-induced chloride currents and suggest that CLIC1 modulation could be important for chloride homeostasis in this cell type

MMP-1 activation contributes to airway smooth muscle growth and asthma severity

Introduction: Matrix metalloproteinase-1 and mast cells are present in the airways of people with asthma. We hypothesised that matrix metalloproteinase-1 could be activated by mast cells and increase asthma severity. Methods: Patients with stable asthma and healthy controls underwent spirometry, methacholine challenge, bronchoscopy and their airway smooth muscle cells were grown in culture. A second asthma group and controls had symptom scores, spirometry and bronchoalveolar lavage before and after rhinovirus-induced asthma exacerbations. Extra-cellular matrix was prepared from decellularised airway smooth muscle cultures. Matrix metalloproteinase-1 protein and activity were assessed. Results: Airway smooth muscle cells generated pro-matrix metalloproteinase-1 which was proteolytically activated by mast cell tryptase. Airway smooth muscle treated with activated mast cell supernatants produced extra-cellular matrix which enhanced subsequent airway smooth muscle growth by 1.5 fold (p<0.05) which was dependent on matrix metalloproteinase-1 activation. In asthma, airway pro-matrix metalloproteinase-1 was 5.4 fold higher than control subjects (p=0.002). Mast cell numbers were associated with airway smooth muscle proliferation and matrix metalloproteinase-1 protein associated with bronchial hyper-responsiveness. During exacerbations, matrix metalloproteinase-1 activity increased and was associated with fall in FEV1 and worsening asthma symptoms. Conclusions: Matrix metalloproteinase-1 is activated by mast cell tryptase resulting in a pro-proliferative extra-cellular matrix. In asthma, mast cells are associated with airway smooth muscle growth, matrix metalloproteinase-1 levels are associated with bronchial hyper-responsiveness and matrix metalloproteinase-1 activation with exacerbation severity. Our findings suggest that airway smooth muscle/mast cell interactions contribute to asthma severity by transiently increasing matrix metalloproteinase activation, airway smooth muscle growth and airway responsiveness

The Ser82 RAGE variant affects lung function and serum RAGE in smokers and sRAGE production in vitro

Introduction: Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. Methods: Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. Results: Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. Conclusions: This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism

Determining the contribution of IL33 and IL1RL1 polymorphisms to clinical and immunological features of asthma

Rationale: IL33 (9p24.1) and the IL33 receptor (IL1RL, 2q12) have been reproducibly identified as asthma susceptibility genes. However, the variants driving genetic associations are not yet fully defined. Using a population based birth cohort of 1059 children (Manchester Asthma and Allergy Study-(MAAS)) and 2536 adults with asthma (Genetics of Asthma Severity and Phenotypes- (GASP)) cohort we aimed to define genetic variants associated with clinical and immunological features of asthma. Methods: MAAS samples were genotyped using the Illumina 610 Quad array and imputed using 1000G reference panel. GASP samples were genotyped using two custom designed Affymetrix arrays (UK BiLEVE/UK Biobank array). Datasets were quality controlled for gender mismatches, outliers and relatedness. Data was generated for the IL33/IL1RL1 regions consisting of the genes and surrounding regions (chr9:5715785−6757983 & chr2:102427961−103468497) on the following traits: asthma diagnosis (MAAS), atopy, FEV1 (GASP) and FEV1/FVC (MAAS and GASP) as well as total blood eosinophil counts and serum total IgE levels (GASP). Variables for blood eosinophils and total IgE were log10 transformed. Analysis was carried out in PLINK using linear or logistic regression modelling including appropriate covariates for each trait. Results: In the MAAS cohort, we replicated the association of the IL33 locus with asthma diagnosis, identifying potentially two independent novel signals in that locus (rs10975398; P=1.70E-05; B= -1.519; MAF=0.32 and rs2890697; P=1.10E-04; B= -1.573; MAF=0.43). This association survived a Bonferroni correction for multiple testing. Although not surviving correction, an association was also identified for atopy in the IL1RL1 locus for MAAS (P=1.08E-04; MAF=0.48). In GASP we identified modest associations not in known LD with published loci (P-value range: 5.00E-02 – 7.60E-04) for FEV1, FEV1/FVC, atopy, blood eosinophils and total IgE in both the IL33 and IL1RL1 loci. Multiple SNPs presented nominal association (P<0.01) with more than one trait such as atopy & total IgE, providing supporting evidence for association. Conclusion: We replicated the association of IL33 region SNPs with asthma diagnosis in MAAS, highlighting the role of this locus in childhood asthma. Although trait association signals did not survive correction for multiple testing, nominal association across multiple phenotypes in GASP provides suggestive evidence of the role of the IL33/IL1RL1 genetic polymorphisms in determining clinical and immunological features of asthma