Genetics of chronic respiratory disease.

Chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD), asthma and interstitial lung diseases are frequently occurring disorders with a polygenic basis that account for a large global burden of morbidity and mortality. Recent large-scale genetic epidemiology studies have identified associations between genetic variation and individual respiratory diseases and linked specific genetic variants to quantitative traits related to lung function. These associations have improved our understanding of the genetic basis and mechanisms underlying common lung diseases. Moreover, examining the overlap between genetic associations of different respiratory conditions, along with evidence for gene-environment interactions, has yielded additional biological insights into affected molecular pathways. This genetic information could inform the assessment of respiratory disease risk and contribute to stratified treatment approaches.

Mendelian randomisation of eosinophils and other cell types in relation to lung function and disease.

RATIONALE: Eosinophils are associated with airway inflammation in respiratory disease. Eosinophil production and survival is controlled partly by interleukin-5: anti-interleukin-5 agents reduce asthma and response correlates with baseline eosinophil counts. However, whether raised eosinophils are causally related to chronic obstructive pulmonary disease (COPD) and other respiratory phenotypes is not well understood. OBJECTIVES: We investigated causality between eosinophils and: lung function, acute exacerbations of COPD, asthma-COPD overlap (ACO), moderate-to-severe asthma and respiratory infections. METHODS: We performed Mendelian randomisation (MR) using 151 variants from genome-wide association studies of blood eosinophils in UK Biobank/INTERVAL, and respiratory traits in UK Biobank/SpiroMeta, using methods relying on different assumptions for validity. We performed multivariable analyses using eight cell types where there was possible evidence of causation by eosinophils. MEASUREMENTS AND MAIN RESULTS: Causal estimates derived from individual variants were highly heterogeneous, which may arise from pleiotropy. The average effect of raising eosinophils was to increase risk of ACO (weighted median OR per SD eosinophils, 1.44 (95%CI 1.19 to 1.74)), and moderate-severe asthma (weighted median OR 1.50 (95%CI 1.23 to 1.83)), and to reduce forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) and FEV1 (weighted median estimator, SD FEV1/FVC: -0.054 (95% CI -0.078 to -0.029), effect only prominent in individuals with asthma). CONCLUSIONS: Broad consistency across MR methods may suggest causation by eosinophils (although of uncertain magnitude), yet heterogeneity necessitates caution: other important mechanisms may be responsible for the impairment of respiratory health by these eosinophil-raising variants. These results could suggest that anti-IL5 agents (designed to lower eosinophils) may be valuable in treating other respiratory conditions, including people with overlapping features of asthma and COPD.

Genome-wide association study of chronic sputum production implicates loci involved in mucus production and infection.

BACKGROUND: Chronic sputum production impacts on quality of life and is a feature of many respiratory diseases. Identification of the genetic variants associated with chronic sputum production in a disease agnostic sample could improve understanding of its causes and identify new molecular targets for treatment. METHODS: We conducted a genome-wide association study (GWAS) of chronic sputum production in UK Biobank. Signals meeting genome-wide significance (p<5×10-8) were investigated in additional independent studies, were fine-mapped and putative causal genes identified by gene expression analysis. GWASs of respiratory traits were interrogated to identify whether the signals were driven by existing respiratory disease among the cases and variants were further investigated for wider pleiotropic effects using phenome-wide association studies (PheWASs). RESULTS: From a GWAS of 9714 cases and 48 471 controls, we identified six novel genome-wide significant signals for chronic sputum production including signals in the human leukocyte antigen (HLA) locus, chromosome 11 mucin locus (containing MUC2, MUC5AC and MUC5B) and FUT2 locus. The four common variant associations were supported by independent studies with a combined sample size of up to 2203 cases and 17 627 controls. The mucin locus signal had previously been reported for association with moderate-to-severe asthma. The HLA signal was fine-mapped to an amino acid change of threonine to arginine (frequency 36.8%) in HLA-DRB1 (HLA-DRB1*03:147). The signal near FUT2 was associated with expression of several genes including FUT2, for which the direction of effect was tissue dependent. Our PheWAS identified a wide range of associations including blood cell traits, liver biomarkers, infections, gastrointestinal and thyroid-associated diseases, and respiratory disease. CONCLUSIONS: Novel signals at the FUT2 and mucin loci suggest that mucin fucosylation may be a driver of chronic sputum production even in the absence of diagnosed respiratory disease and provide genetic support for this pathway as a target for therapeutic intervention.

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.

Phenotypic and functional translation of IL33 genetics in asthma.

BACKGROUND: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. OBJECTIVE: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. METHODS: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. RESULTS: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV1, FEV1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species-capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. CONCLUSIONS: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.

Timing of radiotherapy after radical prostatectomy (RADICALS-RT): a randomised, controlled phase 3 trial.

BACKGROUND: The optimal timing of radiotherapy after radical prostatectomy for prostate cancer is uncertain. We aimed to compare the efficacy and safety of adjuvant radiotherapy versus an observation policy with salvage radiotherapy for prostate-specific antigen (PSA) biochemical progression. METHODS: We did a randomised controlled trial enrolling patients with at least one risk factor (pathological T-stage 3 or 4, Gleason score of 7-10, positive margins, or preoperative PSA ≥10 ng/mL) for biochemical progression after radical prostatectomy (RADICALS-RT). The study took place in trial-accredited centres in Canada, Denmark, Ireland, and the UK. Patients were randomly assigned in a 1:1 ratio to adjuvant radiotherapy or an observation policy with salvage radiotherapy for PSA biochemical progression (PSA ≥0·1 ng/mL or three consecutive rises). Masking was not deemed feasible. Stratification factors were Gleason score, margin status, planned radiotherapy schedule (52·5 Gy in 20 fractions or 66 Gy in 33 fractions), and centre. The primary outcome measure was freedom from distant metastases, designed with 80% power to detect an improvement from 90% with salvage radiotherapy (control) to 95% at 10 years with adjuvant radiotherapy. We report on biochemical progression-free survival, freedom from non-protocol hormone therapy, safety, and patient-reported outcomes. Standard survival analysis methods were used. A hazard ratio (HR) of less than 1 favoured adjuvant radiotherapy. This study is registered with ClinicalTrials.gov, NCT00541047. FINDINGS: Between Nov 22, 2007, and Dec 30, 2016, 1396 patients were randomly assigned, 699 (50%) to salvage radiotherapy and 697 (50%) to adjuvant radiotherapy. Allocated groups were balanced with a median age of 65 years (IQR 60-68). Median follow-up was 4·9 years (IQR 3·0-6·1). 649 (93%) of 697 participants in the adjuvant radiotherapy group reported radiotherapy within 6 months; 228 (33%) of 699 in the salvage radiotherapy group reported radiotherapy within 8 years after randomisation. With 169 events, 5-year biochemical progression-free survival was 85% for those in the adjuvant radiotherapy group and 88% for those in the salvage radiotherapy group (HR 1·10, 95% CI 0·81-1·49; p=0·56). Freedom from non-protocol hormone therapy at 5 years was 93% for those in the adjuvant radiotherapy group versus 92% for those in the salvage radiotherapy group (HR 0·88, 95% CI 0·58-1·33; p=0·53). Self-reported urinary incontinence was worse at 1 year for those in the adjuvant radiotherapy group (mean score 4·8 vs 4·0; p=0·0023). Grade 3-4 urethral stricture within 2 years was reported in 6% of individuals in the adjuvant radiotherapy group versus 4% in the salvage radiotherapy group (p=0·020). INTERPRETATION: These initial results do not support routine administration of adjuvant radiotherapy after radical prostatectomy. Adjuvant radiotherapy increases the risk of urinary morbidity. An observation policy with salvage radiotherapy for PSA biochemical progression should be the current standard after radical prostatectomy. FUNDING: Cancer Research UK, MRC Clinical Trials Unit, and Canadian Cancer Society.

Epithelial cell dysfunction, a major driver of asthma development.

Airway epithelial barrier dysfunction is frequently observed in asthma and may have important implications. The physical barrier function of the airway epithelium is tightly interwoven with its immunomodulatory actions, while abnormal epithelial repair responses may contribute to remodelling of the airway wall. We propose that abnormalities in the airway epithelial barrier play a crucial role in the sensitization to allergens and pathogenesis of asthma. Many of the identified susceptibility genes for asthma are expressed in the airway epithelium, supporting the notion that events at the airway epithelial surface are critical for the development of the disease. However, the exact mechanisms by which the expression of epithelial susceptibility genes translates into a functionally altered response to environmental risk factors of asthma are still unknown. Interactions between genetic factors and epigenetic regulatory mechanisms may be crucial for asthma susceptibility. Understanding these mechanisms may lead to identification of novel targets for asthma intervention by targeting the airway epithelium. Moreover, exciting new insights have come from recent studies using single-cell RNA sequencing (scRNA-Seq) to study the airway epithelium in asthma. This review focuses on the role of airway epithelial barrier function in the susceptibility to develop asthma and novel insights in the modulation of epithelial cell dysfunction in asthma.

Defining a role for lung function associated gene GSTCD in cell homeostasis.

Genome wide association (GWA) studies have reproducibly identified signals on chromosome 4q24 associated with lung function and COPD. GSTCD (Glutathione S-transferase C-terminal domain containing) represents a candidate causal gene in this locus, however little is currently known about the function of this protein. We set out to further our understanding of the role of GSTCD in cell functions and homeostasis using multiple molecular and cellular approaches in airway relevant cells. Recombinant expression of human GSTCD in conjunction with a GST activity assay did not identify any enzymatic activity for two GSTCD isoforms questioning the assignment of this protein to this family of enzymes. Protein structure analyses identified a potential methyltransferase domain contained within GSTCD, with these enzymes linked to cell viability and apoptosis. Targeted knockdown (siRNA) of GSTCD in bronchial epithelial cells identified a role for GSTCD in cell viability as proliferation rates were not altered. To provide greater insight we completed transcriptomic analyses on cells with GSTCD expression knocked down and identified several differentially expressed genes including those implicated in airway biology; fibrosis e.g. TGFBR1 and inflammation e.g. IL6R. Pathway based transcriptomic analyses identified an over-representation of genes related to adipogenesis which may suggest additional functions for GSTCD. These findings identify potential additional functions for GSTCD in the context of airway biology beyond the hypothesised GST activity and warrant further investigation.

Genetic risk factors for the development of pulmonary disease identified by genome-wide association.

Chronic respiratory diseases are a major cause of morbidity and mortality. Asthma and chronic obstructive pulmonary disease (COPD) combined affect over 500 million people worldwide. While environmental factors are important in disease progression, asthma and COPD have long been known to be heritable with genetic components playing an important role in the risk of developing disease. Identification of genetic variation contributing to disease progression is important for a number of reasons including identification of risk alleles, understanding underlying disease mechanisms and development of novel therapies. Genome-wide association studies (GWAS) have been successful in identifying many loci associated with lung function, COPD and asthma. In recent years, meta-analyses and improved imputation have facilitated the growth of GWAS in terms of numbers of subjects and the number of single nucleotide polymorphisms (SNP) that can be interrogated. As a consequence, there has been a significant increase in the number of signals associated with asthma, COPD and lung function. SNP that have shown association with lung function reassuringly show a significant overlap with SNP associated with COPD giving a glimpse at pathways that may be involved in COPD mechanisms including genes in, for example, developmental pathways. In asthma, association signals are often in or near genes involved in both adaptive and innate immune response pathways, epithelial cell homeostasis and airway structural changes. The challenges now are translating these genetic signals into a new understanding of lung biology, understanding how variants impact health and disease and how they may provide opportunities for therapeutic intervention.

Lung function associated gene Integrator Complex subunit 12 regulates protein synthesis pathways.

BACKGROUND: Genetic studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3'end processing of small nuclear RNAs. RESULTS: We found that, in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighbouring GSTCD expression. In contrast to the previous reports in other species, we only observed a minor alteration of snRNA processing following INTS12 depletion. RNAseq analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, ChIPseq experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, we defined the INTS12 regulome which includes genes belonging to the protein synthesis pathways. CONCLUSION: INTS12 has functions beyond the canonical snRNA processing. We show that it regulates translation by regulating the expression of genes belonging to protein synthesis pathways. This study provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the biology behind the genetic association for lung function at 4q24.

Whole exome re-sequencing implicates CCDC38 and cilia structure and function in resistance to smoking related airflow obstruction.

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1) despite many years of heavy smoking. We hypothesised that these "resistant smokers" may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the "resistant smokers" and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34 × 10(-4)) in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.

Exome-wide analysis of rare coding variation identifies novel associations with COPD and airflow limitation in MOCS3, IFIT3 and SERPINA12.

BACKGROUND: Several regions of the genome have shown to be associated with COPD in genome-wide association studies of common variants. OBJECTIVE: To determine rare and potentially functional single nucleotide polymorphisms (SNPs) associated with the risk of COPD and severity of airflow limitation. METHODS: 3226 current or former smokers of European ancestry with lung function measures indicative of Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2 COPD or worse were genotyped using an exome array. An analysis of risk of COPD was carried out using ever smoking controls (n=4784). Associations with %predicted FEV1 were tested in cases. We followed-up signals of interest (p<10(-5)) in independent samples from a subset of the UK Biobank population and also undertook a more powerful discovery study by meta-analysing the exome array data and UK Biobank data for variants represented on both arrays. RESULTS: Among the associated variants were two in regions previously unreported for COPD; a low frequency non-synonymous SNP in MOCS3 (rs7269297, pdiscovery=3.08×10(-6), preplication=0.019) and a rare SNP in IFIT3, which emerged in the meta-analysis (rs140549288, pmeta=8.56×10(-6)). In the meta-analysis of % predicted FEV1 in cases, the strongest association was shown for a splice variant in a previously unreported region, SERPINA12 (rs140198372, pmeta=5.72×10(-6)). We also confirmed previously reported associations with COPD risk at MMP12, HHIP, GPR126 and CHRNA5. No associations in novel regions reached a stringent exome-wide significance threshold (p<3.7×10(-7)). CONCLUSIONS: This study identified several associations with the risk of COPD and severity of airflow limitation, including novel regions MOCS3, IFIT3 and SERPINA12, which warrant further study.

Traditional and emerging indicators of cardiovascular risk in chronic obstructive pulmonary disease.

With the increased cardiovascular (CV) morbidity and mortality in subjects with chronic obstructive pulmonary disease (COPD), there is a priority to identify those patients at increased risk of cardiovascular disease. Stable patients with COPD (n = 185) and controls with a smoking history (n = 106) underwent aortic pulse wave velocity (PWV), blood pressure (BP) and skin autofluorescence (AF) at clinical stability. Blood was sent for fasting lipids, soluble receptor for advanced glycation end products (sRAGE) and CV risk prediction scores were calculated. More patients (18%) had a self-reported history of CV disease than controls (8%), p = 0.02, whilst diabetes was similar (14% and 10%), p = 0.44. Mean (SD) skin AF was greater in patients: 3.1 (0.5) AU than controls 2.8 (0.6) AU, p < 0.001. Aortic PWV was greater in patients: 10.2 (2.3) m/s than controls: 9.6 (2.0) m/s, p = 0.02 despite similar BP. The CV risk prediction scores did not differentiate between patients and controls nor were the individual components of the scores different. The sRAGE levels were not statistically different. We present different indicators of CV risk alongside each other in well-defined subjects with and without COPD. Two non-invasive biomarkers associated with future CV burden: skin AF and aortic PWV are both significantly greater in patients with COPD compared to the controls. The traditional CV prediction scores used in the general population were not statistically different. We provide new data to suggest that alternative approaches for optimal CV risk detection should be employed in COPD management.

Cigarette Smoke and the Induction of Urokinase Plasminogen Activator Receptor In Vivo: Selective Contribution of Isoforms to Bronchial Epithelial Phenotype.

The urokinase plasminogen activator receptor (uPAR) gene (PLAUR) has been identified as an asthma susceptibility gene, with polymorphisms within that gene being associated with baseline lung function, lung function decline, and lung function in a smoking population. Soluble cleaved uPAR (scuPAR), a molecule identified as a marker of increased morbidity and mortality in a number of diseases, has been shown to be elevated in the airways of patients with asthma and in patients with chronic obstructive pulmonary disease. However, the functionality of soluble receptor isoforms and their relationship with an important initiator for obstructive lung disease, cigarette smoke, remains undefined. In this study, we set out to determine the effect of cigarette smoke on soluble uPAR isoforms, its regulatory pathway and the resultant effect on bronchial epithelial cell function. We identified a positive association between cigarette pack-years and uPAR expression in the airway bronchial epithelium of biopsies from patients with asthma (n = 27; P = 0.0485). In vitro, cigarette smoke promoted cleavage of uPAR from the surface of bronchial epithelial cells (1.5× induction; P < 0.0001) and induced the soluble spliced isoform through changes in messenger RNA expression (∼2× change; P < 0.001), driven by loss of endogenous 3' untranslated region suppression. Elevated expression of the soluble isoforms resulted in a proremodeling cell phenotype, characterized by increased proliferation and matrix metalloproteinase-9 expression in primary bronchial epithelial cells. This suggests that cigarette smoke elevates soluble receptor isoforms in bronchial epithelial cells through direct (cleavage) and indirect (messenger RNA expression) means. These findings provide further insight into how cigarette smoke may influence changes in the airways of importance to airway remodeling and obstructive lung disease progression.

Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels.

The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonary disease (COPD; n=219) cohorts when compared to controls (n=96). In these cohorts, a genome-wide association study of serum scuPAR levels identified a human plasma kallikrein gene (KLKB1) promoter polymorphism (rs4253238) associated with serum scuPAR levels in a control/asthma population (P=1.17 × 10(-7)), which was also observed in a COPD population (combined P=5.04 × 10(-12)). Using a fluorescent assay, we demonstrated that serum KLKB1 enzymatic activity was driven by rs4253238 and is inverse to scuPAR levels. Biochemical analysis identified that KLKB1 cleaves scuPAR and negates scuPAR's effects on primary human bronchial epithelial cells (HBECs) in vitro. Chymotrypsin was used as a proproteolytic control, while basal HBECs were used as a control to define scuPAR-driven effects. In summary, we reveal a novel post-translational regulatory mechanism for scuPAR using a hypothesis-free approach with implications for multiple human diseases.

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.

IL-33 is more potent than IL-25 in provoking IL-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction.

BACKGROUND: IL-25 and IL-33 belong to distinct cytokine families, but experimental mouse studies suggest their immunologic functions in type 2 immunity are almost entirely overlapping. However, only polymorphisms in the IL-33 pathway (IL1RL1 and IL33) have been significantly associated with asthma in large-cohort genome-wide association studies. OBJECTIVE: We sought to identify distinct pathways for IL-25 and IL-33 in the lung that might provide insight into their roles in asthma pathogenesis and potential for therapeutic intervention. METHODS: IL-25 receptor-deficient (Il17rb(-/-)), IL-33 receptor-deficient (ST2, Il1rl1(-/-)), and double-deficient (Il17rb(-/-)Il1rl1(-/-)) mice were analyzed in models of allergic asthma. Microarrays, an ex vivo lung slice airway contraction model, and Il13(+/eGFP) mice were then used to identify specific effects of IL-25 and IL-33 administration. RESULTS: Comparison of IL-25 and IL-33 pathway-deficient mice demonstrates that IL-33 signaling plays a more important in vivo role in airways hyperreactivity than IL-25. Furthermore, methacholine-induced airway contraction ex vivo increases after treatment with IL-33 but not IL-25. This is dependent on expression of the IL-33 receptor and type 2 cytokines. Confocal studies with Il13(+/eGFP) mice show that IL-33 more potently induces expansion of IL-13-producing type 2 innate lymphoid cells, correlating with airway contraction. This predominance of IL-33 activity is enforced in vivo because IL-33 is more rapidly expressed and released in comparison with IL-25. CONCLUSION: Our data demonstrate that IL-33 plays a critical role in the rapid induction of airway contraction by stimulating the prompt expansion of IL-13-producing type 2 innate lymphoid cells, whereas IL-25-induced responses are slower and less potent.

Genome-wide SNP-sex interaction analysis of susceptibility to idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition that is more prevalent in males than females. The reasons for this are not fully understood, with differing environmental exposures due to historically sex-biased occupations, or diagnostic bias, being possible explanations. To date, over 20 independent genetic variants have been identified to be associated with IPF susceptibility, but these have been discovered when combining males and females. Our aim was to test for the presence of sex-specific associations with IPF susceptibility and assess whether there is a need to consider sex-specific effects when evaluating genetic risk in clinical prediction models for IPF. Methods: We performed genome-wide single nucleotide polymorphism (SNP)-by-sex interaction studies of IPF risk in six independent IPF case-control studies and combined them using inverse-variance weighted fixed effect meta-analysis. In total, 4,561 cases (1,280 females and 2,281 males) and 23,500 controls (8,360 females and 14,528 males) of European genetic ancestry were analysed. We used polygenic risk scores (PRS) to assess differences in genetic risk prediction between males and females. Findings: Three independent genetic association signals were identified. All showed a consistent direction of effect across all individual IPF studies and an opposite direction of effect in IPF susceptibility between females and males. None had been previously identified in IPF susceptibility genome-wide association studies (GWAS). The predictive accuracy of the PRSs were similar between males and females, regardless of whether using combined or sex-specific GWAS results. Interpretation: We prioritised three genetic variants whose effect on IPF risk may be modified by sex, however these require further study. We found no evidence that the predictive accuracy of common SNP-based PRSs varies significantly between males and females.