Novel insight into uPAR function in the bronchial epithelium in asthma using functional genomics

The urokinase plasminogen activator receptor (uPAR, PLAUR) is a cell surface receptor actively involved in the regulation of cell homeostasis. Expression is elevated in the bronchial epithelium in vivo and also in serum and sputum in asthma and elevated expression often indicates poor prognosis in a number of human diseases. The relative contribution of uPAR to asthma disease mechanisms is not fully understood and the functional roles of uPAR isoforms remains to be resolved. The key aims of this thesis were to i) investigate how the uPAR pathway may influence bronchial epithelial barrier properties; ii) investigate the gene expression patterns in the bronchial epithelium in asthma; iii) identify functions of different forms of uPAR in human bronchial epithelial cells (HBEC) and to iv) investigate the association between genetic polymorphisms spanning the PLAUR gene with clinical features and the presentation of asthma in moderate to severe asthma. Using two cell based approaches we identified an inverse relationship between soluble-cleaved uPAR expression and epithelial barrier properties. Importantly, we demonstrated that blocking uPAR-integrin interactions provides a potential therapeutic opportunity to improve epithelial barrier function. Using whole transcriptome analysis genes differentially expressed between cultured asthma and control subjects were identified which were related to cell growth, repair and immune regulation. Furthermore, uPAR expression was elevated in epithelial cells in asthma subjects compared to healthy controls, suggesting expression is inherently altered in the bronchial epithelium in asthma. Transcriptomics was used to provide novel insight into the specific and overlapping functions of uPAR isoforms and to determine the effects of elevated uPAR expression on HBEC function. Finally, the contribution of PLAUR genetic variants to clinical and immunological traits within asthma were investigated and found that PLAUR single nucleotide polymorphisms (SNPs) did not show an association with the traits measured in a severe asthma population. Overall this work has provided new insight into the function of uPAR as a regulator of the bronchial epithelium in asthma

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

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

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

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

Novel insight into uPAR function in the bronchial epithelium in asthma using functional genomics

The urokinase plasminogen activator receptor (uPAR, PLAUR) is a cell surface receptor actively involved in the regulation of cell homeostasis. Expression is elevated in the bronchial epithelium in vivo and also in serum and sputum in asthma and elevated expression often indicates poor prognosis in a number of human diseases. The relative contribution of uPAR to asthma disease mechanisms is not fully understood and the functional roles of uPAR isoforms remains to be resolved. The key aims of this thesis were to i) investigate how the uPAR pathway may influence bronchial epithelial barrier properties; ii) investigate the gene expression patterns in the bronchial epithelium in asthma; iii) identify functions of different forms of uPAR in human bronchial epithelial cells (HBEC) and to iv) investigate the association between genetic polymorphisms spanning the PLAUR gene with clinical features and the presentation of asthma in moderate to severe asthma. Using two cell based approaches we identified an inverse relationship between soluble-cleaved uPAR expression and epithelial barrier properties. Importantly, we demonstrated that blocking uPAR-integrin interactions provides a potential therapeutic opportunity to improve epithelial barrier function. Using whole transcriptome analysis genes differentially expressed between cultured asthma and control subjects were identified which were related to cell growth, repair and immune regulation. Furthermore, uPAR expression was elevated in epithelial cells in asthma subjects compared to healthy controls, suggesting expression is inherently altered in the bronchial epithelium in asthma. Transcriptomics was used to provide novel insight into the specific and overlapping functions of uPAR isoforms and to determine the effects of elevated uPAR expression on HBEC function. Finally, the contribution of PLAUR genetic variants to clinical and immunological traits within asthma were investigated and found that PLAUR single nucleotide polymorphisms (SNPs) did not show an association with the traits measured in a severe asthma population. Overall this work has provided new insight into the function of uPAR as a regulator of the bronchial epithelium in asthma

Human bronchial epithelial cells from patients with asthma have an altered gene expression profile

Gene changes observed in asthma bronchial epithelial cells are maintained following repeated culture, presenting with an exaggerated response to viral infection and immune responses as well as having differences in the rate of cell division and replication https://bit.ly/3Cq2xK

Elevated PLAUR is observed in the airway epithelium of asthma patients and blocking improves barrier integrity

Background: Expression of the urokinase plasminogen activator receptor (uPAR) is elevated in the airway epithelium in asthma; however, the contribution of uPAR to asthma pathogenesis and scope for therapeutic targeting remains unknown.Objectives: To determine i) the expression profile of uPAR in cultured human bronchial epithelial cells (HBEC) from asthma patients, ii) the relationship between uPAR and epithelial barrier including blocking uPAR functions and iii) the function of different uPAR isoforms. Methods: uPAR levels in HBEC isolated from asthma patients and cells at air liquid interface (ALI) during differentiation were quantified. Transepithelial electrical resistance or electrical cell impedance sensing was used to relate uPAR levels to barrier properties, including effects of uPAR blocking antibodies. The functional effects of gain of function was determined using transcriptomics, in cells over-expressing: membrane (muPAR), soluble cleaved (scuPAR) or soluble spliced (ssuPAR) isoforms. Results: Elevated expression of uPAR was a feature of cultured HBEC from asthma patients, suggesting intrinsic alterations in asthma patient cells. Soluble uPAR levels inversely correlated with barrier properties of the HBEC layer in 2D and ALI. Blocking uPAR-integrin interactions enhanced barrier formation. Gain of function cells showed limited transcriptomic changes.Conclusion: This study provides a significant advance in our understanding of the relationship between asthma, uPAR and the epithelial barrier, where elevated circulating uPAR results in a reduced cell barrier, a phenotype prevalent in asthm

Dataset for: 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

<i>AGER</i> isoform expression in three HBEC donors using RNA Seq.

<p>Structure and abundance of known <i>AGER</i> isoforms in three human bronchial epithelial cell donors illustrating heterogeneity in expression levels. Percentage abundances (% FPKM) were calculated for each donor. Transcripts for full length and soluble <i>AGER</i> were identified at similar low abundancies. FPKM; fragments per kilobase of transcript per million mapped reads.</p