Emily Hodge

Genetic risk factors for the development of allergic disease identified by genome‐wide association

An increasing proportion of the worldwide population is affected by allergic diseases such as allergic rhinitis (AR), atopic dermatitis (AD) and allergic asthma and improved treatment options are needed particularly for severe, refractory disease. Allergic diseases are complex and development involves both environmental and genetic factors. Although the existence of a genetic component for allergy was first described almost 100 years ago, progress in gene identification has been hindered by lack of high throughput technologies to investigate genetic variation in large numbers of subjects. The development of Genome‐Wide Association Studies (GWAS), a hypothesis‐free method of interrogating large numbers of common variants spanning the entire genome in disease and non‐disease subjects has revolutionised our understanding of the genetics of allergic disease. Susceptibility genes for asthma, AR and AD have now been identified with confidence, suggesting there are common and distinct genetic loci associated with these diseases, providing novel insights into potential disease pathways and mechanisms. Genes involved in both adaptive and innate immune mechanisms have been identified, notably including multiple genes involved in epithelial function/secretion, suggesting that the airway epithelium may be particularly important in asthma. Interestingly, concordance/discordance between the genetic factors driving allergic traits such as IgE levels and disease states such as asthma have further supported the accumulating evidence for heterogeneity in these diseases. While GWAS have been useful and continue to identify novel genes for allergic diseases through increased sample sizes and phenotype refinement, future approaches will integrate analyses of rare variants, epigenetic mechanisms and eQTL approaches, leading to greater insight into the genetic basis of these diseases. Gene identification will improve our understanding of disease mechanisms and generate potential therapeutic opportunities.

Large-Scale Genome-Wide Association Studies and Meta-Analyses of Longitudinal Change in Adult Lung Function

Background Genome-wide association studies (GWAS) have identified numerous loci influencing cross-sectional lung function, but less is known about genes influencing longitudinal change in lung function. Methods We performed GWAS of the rate of change in forced expiratory volume in the first second (FEV1) in 14 longitudinal, population-based cohort studies comprising 27,249 adults of European ancestry using linear mixed effects model and combined cohort-specific results using fixed effect meta-analysis to identify novel genetic loci associated with longitudinal change in lung function. Gene expression analyses were subsequently performed for identified genetic loci. As a secondary aim, we estimated the mean rate of decline in FEV1 by smoking pattern, irrespective of genotypes, across these 14 studies using meta-analysis. Results The overall meta-analysis produced suggestive evidence for association at the novel IL16/STARD5/TMC3 locus on chromosome 15 (P  =  5.71 × 10-7). In addition, meta-analysis using the five cohorts with ≥3 FEV1 measurements per participant identified the novel ME3 locus on chromosome 11 (P  =  2.18 × 10-8) at genome-wide significance. Neither locus was associated with FEV1 decline in two additional cohort studies. We confirmed gene expression of IL16, STARD5, and ME3 in multiple lung tissues. Publicly available microarray data confirmed differential expression of all three genes in lung samples from COPD patients compared with controls. Irrespective of genotypes, the combined estimate for FEV1 decline was 26.9, 29.2 and 35.7 mL/year in never, former, and persistent smokers, respectively. Conclusions In this large-scale GWAS, we identified two novel genetic loci in association with the rate of change in FEV1 that harbor candidate genes with biologically plausible functional links to lung function.

GSTCD and INTS12 regulation and expression in the human lung.

Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C-terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFβ1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD.

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.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFβ1 is considered central to the pathogenesis of IPF. A major mechanism of TGFβ1 activation in the lung involves the epithelially restricted αvβ6 integrin. Expression of the αvβ6 integrin is dramatically increased in IPF. How αvβ6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the β6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvβ6 integrin expression in vitro. We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvβ6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

Effects of atopy and grass pollen season on histamine H4 receptor expression in human leukocytes

BACKGROUND The histamine H4 receptor (H4R) is a novel therapeutic target to treat allergic inflammation. OBJECTIVE To profile messenger RNA (mRNA) expression of H₄R isoforms in human cells and evaluate the effects of atopy and grass pollen season on H₄R expression in peripheral blood leukocytes ex vivo. METHODS H₄R isoform expression was assayed by quantitative polymerase chain reaction in human airway and peripheral RNA. During low and high grass pollen seasons, leukocytes were isolated from venous blood and fractionated into peripheral blood mononuclear cells and polymorphonuclear cells (PMN). H₄R expression was determined and related to atopy, defined by a level of specific IgE to Timothy grass pollen of ≥0.35 kU(A)/L (n = 7 atopic patients and 9 controls). RESULTS Expression of total and full length H₄R was at the limit of detection but predominant in peripheral blood leukocytes, where truncated H₄R was expressed exclusively (≤300-fold less). Suggestive evidence for total H₄R in airway cells and brain indicated an expression ≤260-fold lower than in peripheral blood mononuclear cells. Total H₄R mRNA expression was unaffected by atopy or grass pollen season, but truncated H₄R was significantly reduced during high grass pollen season in total leukocytes, independently of atopy (P < .01). CONCLUSION H₄R mRNA is predominantly expressed in peripheral blood leukocytes, and total H₄R expression levels are unrelated to atopy or grass pollen season. Atopy-independent seasonal variation in truncated H₄R expression might affect putative negative regulation of full length H4R during high grass pollen season. If verified, this should be considered during the design of drugs targeting H₄R to treat allergic inflammation, particularly for seasonal allergic rhinitis.

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 (<2-fold) in levels of CCL22, IL-4, and IL-2; increases of 2-4-fold in levels of CXCL8, VEGF and IL-6 and increases >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. Abbreviations: COPD: Chronic Obstructive Pulmonary Disease; ICS: inhaled corticosteroids; LPS: lipopolysaccharide; IL-1β: interleukin-1 beta; PSF: penicillin, streptomycin and fungizone