Introduction:Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality worldwide and currently lacks effective disease-modifying therapies. Extracellular vesicles (EVs) are key mediators of intercellular communication and transport biologically active cargo, including microRNAs (miRNAs). We previously identified 8 differentially expressed EV miRNA (miR-223-3p, miR-2110, miR-182-5p, miR-200b-5p, miR-625-3p, miR-204-5p, miR-138-5p and miR-338-3p) that were differentially expressed in individuals with COPD compared with healthy volunteer ex-smoker controls (HV-ES). This study aimed to identify miRNA-mRNA interactions in diseased lung epithelium that may contribute to COPD pathogenesis. Methods:Gene expression was quantified by RNA sequencing of epithelial brushings obtained from 24 subjects with COPD and 20 HV-ES. In silico analyses were performed to identify target genes of the previously identified EV-derived miRNAs isolated from the same individuals. MiRNA-mRNA interactions were examined using negative correlation analysis and network-based approaches, and enrichment of biological processes was assessed using Cytoscape. Associations between computer tomography (CT) disease probability measures (DPM) and gene expression were assessed using Spearman correlation across the pooled cohort. Results:A total of 191 genes were differentially expressed in epithelial brushings from subjects with COPD compared with HV-ES. In silico analysis identified 121 miRNA-mRNA interactions involving these genes and the EV-associated miRNAs. Network analysis revealed miR-182-5p as a central hub, targeting multiple highly differentially expressed genes (DEGs). Expression of these DEGs correlated with CT DPM of emphysema and small airways disease (SAD). Exploratory pathway analyses suggested potential trends toward coordinated network regulation involving metabolic and immune -related processes; however, no biological processes remained significant after correction for multiple testing. Discussion:These findings highlight a potential role for EV-derived miRNA-mRNA regulatory networks in COPD pathogenesis, with miR-182-5p emerging as a putative regulator within this network. While exploratory analyses suggested possible associations with metabolic and immune-related pathways, these did not withstand multiple testing correction and should therefore be interpreted as hypothesis-generating. These findings support further mechanistic investigation of EV-derived miRNA-mRNA regulatory networks in COPD and may help inform future translational studies exploring their biological relevance.

Extracellular matrix (ECM) dysregulation is a key process in the pathology of COPD. However, an inability to characterise ECM remodelling in vivo has limited our understanding of its relationship with functional decline and disease mechanisms. We aimed to quantify in vivo ECM remodelling using probe-based confocal laser endomicroscopy (pCLE) and determine associations with physiological, radiological, histological, and serological markers of COPD. 16 patients with COPD and 20 controls underwent pulmonary function testing, CT imaging, bronchoscopy, and pCLE. Alveolar morphometrics and elastin linearity scores (ELS) were quantified using a novel automated algorithm. Bronchial biopsies were analysed for elastin and collagen content. Serum biomarkers of elastin and collagen turnover were measured in a combined cohort of 54 COPD patients and 61 controls. Compared with never-smoking controls, current smokers without airflow obstruction demonstrated larger alveolar dimensions including increased alveolar opening area (AOA) (46,282 ± 16,805 vs. 33,549 ± 2,595 μm², p = 0.003). Alveolar dimensions were further increased in COPD, with larger AOA (56,468 ± 11,079 vs. 46,282 ± 16,805 μm², p < 0.001) compared with all controls. COPD was also associated with greater elastin fibre disorganisation (ELS 54.9 ± 6.0 vs. 47.5 ± 10.7, p = 0.032). Across the cohort, ELS correlated with airflow obstruction and surrogate markers of small airway disease. Airway collagen content was increased in COPD and correlated with ELS (r = 0.665, p = 0.005). COPD was associated with higher circulating elastin and collagen degradation biomarkers, including ELP-3, C1M, C6M, and EL-CG (all p < 0.05), which correlated with pCLE morphometrics. Using an innovative lung imaging technique, we provide the first objective quantification of in vivo airway elastic fibre disorganisation and demonstrate quantifiable lung microstructural changes that may precede abnormalities detected by established techniques. These quantifiable signals relate to biomarkers of lung ECM turnover, offering a new platform for early disease detection and mechanistic understanding of COPD.

Background Coronavirus disease 2019 (COVID-19) is a viral pneumonia that can result in serious respiratory illness. It is associated with extensive systemic inflammation, changes to the lung extracellular matrix, and long-term lung impairment such as interstitial lung disease (ILD). In this study, the aim was to investigate whether tissue remodelling, wound healing, and neutrophil activity is altered in patients with COVID-19 and how these relate to the development of post-COVID ILD. Method Serum samples were collected from 63 patients three months after discharge as part of the Research Evaluation Alongside Clinical Treatment study in COVID-19 (REACT COVID-19), 10 of whom developed ILD, and 16 healthy controls. Samples were quantified using neo-epitope specific biomarkers reflecting tissue stiffness and formation (PC3X, PRO-C3, and PRO-C6), tissue degradation (C1M, C3M, and C6M), wound healing (PRO-FIB and X-FIB), and neutrophil activity (CPa9-HNE and ELP-3). Results Mean serum levels of PC3X (p < 0.0001), PRO-C3 (p = 0.002), C3M (p = 0.009), PRO-FIB (p < 0.0001), CPa9-HNE (p < 0.0001), and ELP-3 (p < 0.0001) were significantly elevated in patients with COVID-19 compared to healthy controls. Moreover, PC3X (p = 0.023) and PRO-C3 (p = 0.032) were significantly elevated in post-COVID ILD as compared to COVID-19. Conclusion Serological biomarkers reflecting type III collagen remodelling, clot formation, and neutrophil activity were significantly elevated in COVID-19 and type III collagen formation markers were further elevated in post-COVID ILD. The findings suggest an increased type III collagen remodelling in COVID-19 and warrants further investigations to assess the potential of tissue remodelling biomarkers as a tool to identify COVID-19 patients at high risk of developing ILD.

Objectives COVID-19 is a heterogeneous disease, and many reports have described variations in demographic, biochemical and clinical features at presentation influencing overall hospital mortality. However, there is little information regarding longitudinal changes in laboratory prognostic variables in relation to disease progression in hospitalised patients with COVID-19. Design and setting This retrospective observational report describes disease progression from symptom onset, to admission to hospital, clinical response and discharge/death among patients with COVID-19 at a tertiary centre in South East England. Participants Six hundred and fifty-one patients treated for SARS-CoV-2 between March and September 2020 were included in this analysis. Ethical approval was obtained from the HRA Specific Review Board (REC 20/HRA/2986) for waiver of informed consent. Results The majority of patients presented within 1 week of symptom onset. The lowest risk patients had low mortality (1/45, 2%), and most were discharged within 1 week after admission (30/45, 67%). The highest risk patients, as determined by the 4C mortality score predictor, had high mortality (27/29, 93%), with most dying within 1 week after admission (22/29, 76%). Consistent with previous reports, most patients presented with high levels of C reactive protein (CRP) (67% of patients >50 mg/L), D-dimer (98%>upper limit of normal (ULN)), ferritin (65%>ULN), lactate dehydrogenase (90%>ULN) and low lymphocyte counts (81%<lower limit of normal (LLN)). Increases in platelet counts and decreases in CRP, neutrophil:lymphocyte ratio (p<0.001), lactate dehydrogenase, neutrophil counts, urea and white cell counts (all p<0.01) were each associated with discharge. Conclusions Serial measurement of routine blood tests may be a useful prognostic tool for monitoring treatment response in hospitalised patients with COVID-19. Changes in other biochemical parameters often included in a 'COVID-19 bundle' did not show significant association with outcome, suggesting there may be limited clinical benefit of serial sampling. This may have direct clinical utility in the context of escalating healthcare costs of the pandemic.

Objectives: Dexamethasone has now been incorporated into the standard of care for COVID-19 hospital patients. However, larger intensive care unit studies have failed to show discernible improvements in mortality in the recent wave. We aimed to investigate the impacts of these factors on disease outcomes in a UK hospital study. Methods: This retrospective observational study reports patient characteristics, interventions and outcomes in COVID-19 patients from a UK teaching hospital; cohort 1, pre 16th June-2020 (pre-dexamethasone); cohort 2, 17th June to 30th November-2020 (post-dexamethasone, pre-VOC 202,012/01 as dominant strain); cohort 3, 1st December-2020 to 3rd March-2021 (during establishment of VOC202012/01 as the dominant strain). Results: Dexamethasone treatment was more common in cohorts 2 and 3 (42.7% and 51.6%) compared with cohort 1 (2.5%). After adjusting for risk, odds of death within 28 days were 2-fold lower in cohort 2 vs 1 (OR:0.47, [0.27,0.79],p = 0.006). Mortality was higher cohort 3 vs 2 (20% vs 14%); but not significantly different to cohort 1 (OR: 0.86, [0.64, 1.15],p = 0.308). Conclusions: The real world finding of lower mortality following dexamethasone supports the published trial evidence and highlights ongoing need for research with introduction of new treatments and ongoing concern over new COVID-19 variants.

Background Routine follow-up of patients hospitalised with COVID-19 is recommended, however due to the ongoing high number of infections this is not without significant health resource and economic burden. In a previous study we investigated the prevalence of, and risk factors for, persistent chest radiograph (CXR) abnormalities post-hospitalisation with COVID-19 and identified a 5-point composite score that strongly predicted risk of persistent CXR abnormality at 12-weeks. Here we sought to validate and refine our findings in an independent cohort of patients. Methodology A single-centre prospective study of consecutive patients attending a virtual post-hospitalisation COVID-19 clinic and CXR as part of their standard clinical care between 2nd March – 22nd June 2021. Inpatient and follow-up CXRs were scored by the assessing clinician for extent of pulmonary infiltrates (0–4 in each lung) with complete resolution defined as a follow-up score of zero. Results 182 consecutive patients were identified of which 31% had persistent CXR abnormality at 12-weeks. Patients with persistent CXR abnormality were significantly older (p < 0.001), had a longer hospital length of stay (p = 0.005), and had a higher incidence of both level 2 or 3 facility admission (level 2/3 care) (p = 0.003) and ever-smoking history (p = 0.038). Testing our composite score in the present cohort we found it predicted persistent CXR abnormality with reasonable accuracy (area under the receiver operator curve [AUROC 0.64]). Refining this score replacing obesity with Age ≥ 50 years, we identify the SHADE-750 score (1-point each for; Smoking history, Higher-level care (level 2/3 admission), Age ≥ 50 years, Duration of admission ≥ 15 days and Enzyme-lactate dehydrogenase (LDH ≥ 750U/L), that accurately predicted risk of persistent CXR abnormality, both in the present cohort (AUROC 0.73) and when retrospectively applied to our 1st cohort (AUROC 0.79). Applied to both cohorts combined (n = 213) it again performed strongly (AUROC 0.75) with all patients with a score of zero (n = 18) having complete CXR resolution at 12-weeks. Conclusions In two independent cohorts of patients hospitalised with COVID-19, we identify a 5-point score which accurately predicts patients at risk of persistent CXR abnormality at 12-weeks. This tool could be used by clinicians to identify patients in which radiological follow-up may not be required.

Introduction Chronic obstructive pulmonary disease (COPD) is a heterogeneous condition without effective disease modifying therapies. Identification of novel inflammatory endotype markers such as extracellular vesicles (EVs), which are important intercellular messengers carrying microRNA (miRNA), may enable earlier diagnosis and disease stratification for a targeted treatment approach. Our aim was to identify differentially expressed EV miRNA in the lungs of COPD patients compared with healthy ex-smokers and determine whether they can help define inflammatory COPD endotypes. Methods EV miRNA were isolated and sequenced from ex-smoking COPD patients and healthy ex-smoker bronchoalveolar lavage fluid. Results were validated with RT-qPCR and compared to differential inflammatory cell counts. Results Expression analysis identified five upregulated miRNA in COPD (miR-223-3p, miR-2110, miR-182-5p, miR-200b-5p and miR-625-3p) and three downregulated miRNA (miR-138-5p, miR-338-3p and miR-204-5p), all with a log2 fold change of >1/−1, FDR < 0.05. These miRNAs correlated with disease defining characteristics such as FEF 25–75% (a small airways disease measure) and DLCO% (a surrogate measure of emphysema). Receiver operator curve analysis demonstrated miR-2110, miR-223-3p, and miR-182-5p showed excellent combinatory predictive ability (AUC 0.91, p < 0.0001) in differentiating between health and mild COPD. Furthermore, miR-223-3p and miR-338-3p correlated with airway eosinophilia and were able to distinguish “pure eosinophilic” COPD from other airway inflammatory subtypes (AUC 0.94 and 0.85, respectively). Discussion This is the first study to identify differentially expressed miRNA in COPD bronchoalveolar lavage fluid EVs. These findings suggest specific lung derived EV miRNA are a strong predictor of disease presence even in mild COPD. Furthermore, specific miRNA correlated with inflammatory cell numbers in COPD, and may have a role in defining inflammatory endotypes for future treatment stratification.

Introduction: Probe-based confocal laser endomicroscopy (pCLE) images the elastin structure in the airway walls whilst serum markers of protein fragments (neo-epitopes) of elastin and collagen are associated with disease activity in COPD. This study aims to characterise ECM remodelling in COPD by pCLE imaging and the serological assessment of neo-epitopes. Methods: We completed pCLE-bronchoscopy in 15 patients with COPD and 18 healthy subjects. Alveolar dimensions were measured and the alignment of airway fibres was quantified using our novel image analysis software. We assessed serum levels of neo-epitopes in 61 patients with COPD and 54 healthy controls for the evaluation of elastin (ELP-3, EL-NE, EL-CG) and collagen type I (C1M), type IV (C4M) and type VI (C6M) remodelling. Results: COPD was associated with larger alveolar diameter (352µm +/− 15 vs 293µm +/− 27, p<0.001) and greater disorder of airway elastin fibre alignment related to markers of small airways disease including FEV1/FVC, MEF25%-75% and mean lung density expiratory/inspiratory ratio. COPD was associated with higher serum levels of ELP-3, EL-CG, C1M, C6M whilst ELP-3, EL-CG, C1M, C4M and C6M were related to lung function impairment (see Fig1). Conclusion: Novel imaging and neo-epitope biomarkers identify evidence of ECM remodelling which underpin the development of abnormal lung function and insights into key mechanisms driving the origins of COPD.

Rationale Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms. Methods Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed. Results Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log2 fold change (log2FC) −2.0, −2.2, −1.5, −0.5, −0.7 and −0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= −0.56, r= −0.58, r= −0.45, r= −0.36, r= −0.44, r= −0.37, r= −0.40 and r= −0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= −0.55, r= −0.61, r= −0.48, r= −0.51, r= −0.41, r= −0.31 and r= −0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log2FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85). Conclusions Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease.

Introduction The COVID-19 pandemic caused by SARS-CoV-2 places immense worldwide demand on healthcare services. Earlier identification of patients at risk of severe disease may allow intervention with experimental targeted treatments, mitigating the course of their disease and reducing critical care service demand. Methods and analysis This prospective observational study of patients tested or treated for SARS-CoV-2, who are under the care of the tertiary University Hospital Southampton NHS Foundation Trust (UHSFT), captured data from admission to discharge; data collection commenced on 7 March 2020. Core demographic and clinical information, as well as results of disease-defining characteristics, was captured and recorded electronically from hospital clinical record systems at the point of testing. Manual data were collected and recorded by the clinical research team for assessments which are not part of the structured electronic healthcare record, for example, symptom onset date. Thereafter, participant records were continuously updated during hospital stay and their follow-up period. Participants aged >16 years were given the opportunity to provide consent for excess clinical sample storage with optional further biological sampling. These anonymised samples were linked to the clinical data in the Real-time Analytics for Clinical Trials platform and were stored within a biorepository at UHSFT. Ethics and dissemination Ethical approval was obtained from the HRA Specific Review Board (REC 20/HRA/2986) for waiver of informed consent for the database-only cohort; the procedures conform with the Declaration of Helsinki. The study design, protocol and patient-facing documentation for the biobanking arm of the study have been approved by North West Research Ethics Committee (REC 17/NW/0632) as an amendment to the National Institute for Health Research Southampton Clinical Research Facility-managed Southampton Research Biorepository. This study will be published as peer-reviewed articles and presented at conferences, presentations and workshops.

Presentation, disease severity and outcomes of coronavirus disease 2019 (COVID-19) vary widely from asymptomatic carriage to death. Currently, there are little data on the relationship between the timing of symptom onset at presentation to hospital and disease outcome. Therefore, we aimed to identify the differences between ‘early presenters’ (patients presenting to hospital <7 days from symptom onset) and ‘late presenters’ (≥7 days from symptom onset). This retrospective single-centre observational study collected demographic, clinical and radiological data from electronic patient healthcare records with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the University Hospital Southampton NHS Foundation Trust (Southampton, UK). Research ethics approval was gained as part of the REACT Study (Bromley REC 20/HRA/2986) and data were extracted from this study. There is no patient identifiable data included in this manuscript. Chest radiograph (CXR) on admission was assessed for pulmonary infiltrates by an unblinded hospital clinician and radiological severity scores were assigned using an adapted Radiographic Assessment of Lung Oedema (RALE) score. The composite end point was admission to the intensive care, the use of mechanical ventilation and/or death. Logistical regression models were used to explore the relationship between outcome and independent variables. Between March and July 2020, data were collected for 574 SARS-CoV-2-positive patients. Early presenters were older (P < 0.001), more likely to have significant medical comorbidities (hypertension, thromboembolic and renal diseases; P < 0.001) and less likely to report cardinal symptoms of COVID-19: fever, cough and shortness of breath (SOB) (P < 0.001) (Table 1). In the cohort overall, the presence of CXR infiltrates was not predictive of adverse outcome (P = 0.214). However, although early presenters were less likely to have infiltrates on their CXR (P < 0.001), the presence of infiltrates in this group revealed increased risk of adverse outcome (odds ratio (OR): 1.90, 95% CI: 1.11, 3.25). The presence or lack of infiltrates in late presenters was not predictive of outcome (OR: 0.58, 95% CI: 0.30, 1.16). To our knowledge, we are the first to investigate radiological change in predicting outcome in the context of symptom duration at presentation to hospital. We demonstrate that heterogeneity in SARS-CoV-2 presentation varies with symptom duration, with early presenters being more likely to present atypically. This is notable in the context of national criteria for self-isolation and testing. Furthermore, late presentation is more likely to be associated with radiological change, reflective of current literature, but is not predictive of outcome. Most importantly, early presenters with radiological changes are at increased risk of adverse outcome, suggesting that symptom onset and detection of CXR infiltrates are important for clinical assessment at presentation to hospital. This may reflect a more

Background The long-term consequences of COVID-19 remain unclear. There is concern a proportion of patients will progress to develop pulmonary fibrosis. We aimed to assess the temporal change in CXR infiltrates in a cohort of patients following hospitalisation for COVID-19. Methods We conducted a single-centre prospective cohort study of patients admitted to University Hospital Southampton with confirmed SARS-CoV2 infection between 20th March and 3rd June 2020. Patients were approached for standard-of-care follow-up 12-weeks after hospitalisation. Inpatient and follow-up CXRs were scored by the assessing clinician for extent of pulmonary infiltrates; 0–4 per lung (Nil = 0, < 25% = 1, 25–50% = 2, 51–75% = 3, > 75% = 4). Results 101 patients with paired CXRs were included. Demographics: 53% male with a median (IQR) age 53.0 (45–63) years and length of stay 9 (5–17.5) days. The median CXR follow-up interval was 82 (77–86) days with median baseline and follow-up CXR scores of 4.0 (3–5) and 0.0 (0–1) respectively. 32% of patients had persistent CXR abnormality at 12-weeks. In multivariate analysis length of stay (LOS), smoking-status and obesity were identified as independent risk factors for persistent CXR abnormality. Serum LDH was significantly higher at baseline and at follow-up in patients with CXR abnormalities compared to those with resolution. A 5-point composite risk score (1-point each; LOS ≥ 15 days, Level 2/3 admission, LDH > 750 U/L, obesity and smoking-status) strongly predicted risk of persistent radiograph abnormality (0.81). Conclusion Persistent CXR abnormality 12-weeks post COVID-19 was common in this cohort. LOS, obesity, increased serum LDH, and smoking-status were risk factors for radiograph abnormality. These findings require further prospective validation.

BackgroundThe clinical presentation and disease severity in SARS-Cov-2 infection ranges from asymptomatic carriage to death There is little data regarding the timeframe of symptom onset to presentation to hospital, and disease outcomes Therefore, we aim to investigate differences between ‘early presenters’ (7 days) and their clinical and radiological outcomes MethodsIn this retrospective cohort study, symptom onset, epidemiological, and clinical characteristics were collected from patient electronic medical records at University Hospital Southampton Foundation Trust with laboratory confirmed SARS-Cov-2 infection Logistical regression models were used to explore the relationships between these data and time of presentation to hospital ResultsBetween March and July 2020, symptom onset data was collected for 626 SARS-Cov-2 positive patients, 574 of whom had chest radiographs (CXR) Early presenters comprised 388 (62%) and 238 (38%) were late presenters Early presenters were significantly older (p<0 001), more likely to have significant comorbidities – hypertension, thromboembolic and renal disease (p<0 001) – and also significantly less likely to report cardinal symptoms of Covid-19;fever, cough, SOB, myalgia, fatigue/malaise, headache (p<0 001) In the cohort overall, the presence of infiltrates was not predictive of adverse outcome (ICU admission, ventilation or death) (p=0 214) Although early presenters were less likely to have infiltrates on their CXR (58% vs 76 8%), (p<0 001), the presence of CXR infiltrates in early presenters demonstrated an increased risk of adverse outcome (OR 1 90, 95% CI 1 11, 3 25) ConclusionWe have demonstrated that SARS-Cov-2 infection presents in an heterogenous manner that varies with symptom duration Atypical presentation of SARS-Cov-2 infection is more common earlier on in disease course, where viral shedding is likely to be higher, and this finding is of note in the context of national criteria for self-isolation and testing Late presentation is more likely to be associated with radiological change, but this does not reflect an increased likelihood of adverse outcome Patients who present early in their illness with radiological changes are at increased risk of adverse clinical outcome, suggesting that symptom onset and detection of CXR infiltrates are important for clinical assessment of severity at presentation to hospital in Covid-19

Introduction and Objectives Pulmonary surfactant homeostasis is critical to lung function as it coats the vast air-liquid interface, reducing surface tension and preventing alveolar collapse. surfactant proteins (SP)-A and D play essential anti-pathogenic defence and immunoregulation roles to maintain lung homeostasis. SP-D is reduced in COPD. We hypothesised lipid and protein surfactant dysregulation could contribute to pathological COPD mechanisms, this requires detailed characterisation. Methods We conducted mass spectrometry-based multiomic (proteomic, lipidomic and metabolomic) analysis of bronchoalveolar lavage to comprehensively characterize surfactant dysregulation in a well-characterised ex-smoking mild/moderate COPD cohort (n=31), with median (IQR) age of 70 (9.5) and FEV1% predicted 73 (21), compared with healthy ex-smoking volunteers (n=20), with age 67.5 (6.75) and FEV1% predicted 100.5 (11.75). To identify the multiomic signatures of COPD, we fitted linear mixed-effects models to the data, accounting for the effects of confounding variables, such as age, gender and individual differences simultaneously. We further characterised purified alveolar macrophage gene expression differences. Results SP-A, SP-B, and SP-D levels were lower in COPD vs control with log2fold changes (FC) of -0.7 (p=1.1x10-6), -0.6 (p=4.8x10-6) and -0.6 (p=3.0x10-5), and showed positive correlations of 0.65 (p=1.5x10-12), 0.62 (p=7.7x10-11) and 0.43 (p=2.3x10-4) with lung function (FEV1/FVC), respectively. SP-C was below detection limit. NAPSA and CTSH, responsible for SP-B synthesis, were lower in COPD, log2FC of -0.1 (p=2.8x10-6) and -0.4 (p=4.3x10-4), respectively. Fatty acid binding protein 4 (FABP4) and CD44, involved in surfactant regulation, were decreased in COPD, log2FC of -0.4 (p=8.0x10-3) and -0.7 (p=1.1x10-5), respectively. Histamine and hypoxanthine metabolites were increased in COPD, log2FC of 0.8 (p=2.5x10-2) and 1.0 (p=4.0x10-2), respectively, which could indicate immune-dysregulation and inflammation. Surfactant lipid levels were decreased in COPD, specifically, PC, PG, and PI, log2FC of -2.1 (p=2.6×10-7), -2.4 (p=5.3×10-8) and -1.5 (p=9.0×10-6), respectively. Conclusions We used a multiomics approach to comprehensively describe surfactant dysregulation in a well-characterised COPD cohort, with an aim to generate novel insights into key aspects of this biology within the COPD lung. Further mechanistic studies could help to understand the role this plays in pathological mechanisms and allow novel therapeutic target identification. Please refer to page A190 for declarations of interest related to this abstract.

Exosomes are small extracellular vesicles released by all cell types in the lung. They contain miRNAs, small non-coding RNAs, able to regulate vast networks of genes relevant to pathologies such as COPD. The aim of this work was to identify differentially expressed exosomal miRNA in the lungs of COPD that target genes important in the underlying pathogenesis. Exosomes were isolated from bronchoalveolar lavage fluid (BALF) by size exclusion chromatography from 14 healthy ex-smokers and 17 mild to moderate COPD patients, with a mean FEV1 of 78.9% predicted (SD±14.4%). Exosomal miRNA was sequenced using the Illumina NextSeq500, de-multiplexed, trimmed and aligned to hg19. Differentially expressed miRNA were identified and validated by RT-qPCR. A greater proportion of miRNA (p=0.02) and small RNA (p=0.01) were found in COPD exosomes. 54 miRNAs were significantly differentially expressed between COPD and healthy ex-smokers (FDR <0.05). Five had a fold change of >2, with two upregulated and three downregulated in COPD. 3 of these miRNA had a good predictive ability of discriminating between COPD and healthy ex-smokers (area under receiver operating characteristic curve >0.8). Network-based analysis identified these miRNA targeted genes as central to pro-inflammatory pathways (e.g. Jak-STAT & MAPK signalling) important in COPD pathogenesis. These genes and pathways are being investigated using primary ex-vivo cell culture models. Importantly, we observed differential expression of exosomal microRNAs in these relatively mild COPD subjects, which target key proinflammatory genes. These data provide new insights into the persistent inflammation seen in COPD and could identify future targets for disease modifying therapies.

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation, caused by emphysema and small airways disease (SAD). Computed tomography (CT) coupled with image analysis enables the quantification of these abnormalities; however, the optimum method for doing so has not been determined.Objectives: This study aims to compare two CT quantitative analysis techniques, disease probability measure (DPM) and parametric response mapping (PRM), and assess their relationship with specific physiological measures of SAD.Methods: Subjects with mild to moderate COPD, never smokers, and healthy ex-smokers were recruited. Each had airway oscillometry and multiple-breath nitrogen washout, measuring peripheral airway resistance, peripheral airway reactance, and acinar airway inhomogeneity. Subjects also had an inspiratory and expiratory chest CT, with DPM and PRM analysis performed by coregistering images and classifying each voxel as normal, emphysema, or nonemphysematous gas trapping related to SAD.Results: Thirty-eight subjects with COPD, 18 never smokers, and 23 healthy ex-smokers were recruited. There were strong associations between DPM and PRM analysis when measuring gas trapping (ρ = 0.87; P < 0.001) and emphysema (ρ = 0.99; P < 0.001). DPM assigned significantly more voxels as emphysema and gas trapped than PRM (P < 0.001). Both techniques showed significantly greater emphysema and gas trapping in subjects with COPD than in never smokers and ex-smokers (P < 0.001). All CT measures had significant associations with peripheral airway resistance and reactance, with disease probability measure of nonemphysematous gas trapping related to SAD having the strongest independent association with peripheral airway resistance (β = 0.42; P = 0.001) and peripheral airway reactance (β = 0.41; P = 0.001). Emphysema measures had the strongest associations with acinar airway inhomogeneity (β = 0.35-0.38).Conclusions: These results provide further validation for the use of DPM/PRM analysis in COPD by demonstrating significant relationships with specific physiological measures of SAD.

Introduction: Exercise intervention may modulate the inflammatory basis for asthma, offering clinical benefit beyond functional improvement. Interval training is tolerated in asthmatics, and may improve symptom control. Aims and Objectives: This proof of concept study has recruited poorly controlled asthmatics to a 12-week Interval Training Programme to ascertain if it is feasible and safe, improves symptom control, airway and systemic inflammation, and physical fitness. Methods: Participants were sampled at baseline and 3, 6 and 12 weeks of training. Results: Early results (n=7) suggest safety and tolerability, with improvement between baseline and week 3. Wilcoxon signed rank showed that Asthma Control Questionnaire score improved significantly (mdn=2 [IQR=1.8,2.3] vs mdn=1.63 [IQR=1.37,1.96], p=0.028). Asthma Quality of Life Questionnaire score improved significantly (mdn=4.97 [IQR=4.46,5.3] vs median 5.5 [IQR=5.09,5.84] p=0.018), but was not associated with improved physical fitness, assessed by VO2 at anaerobic threshold (mdn=10.1 [IQR=9.25,11.35] vs 9.7ml/kg/min [IQR=8.6,14]; p=0.46) and VO2peak (mdn= 21.30 [IQR=12.80,25.40] vs 22.8ml/kg/min [IQR=13.80,28.80]; p=0.86). There were no statistically significant improvements in FeNO (median=37.75 [IQR=27.63,66] vs median=36 [IQR=20,82], p=0.39) or peripheral blood eosinophilia (mdn=0.2 [IQR=0.2,0.38] vs mdn=0.3 [IQR=0.2,0.4], p=0.57). Conclusion: These interim data suggests even a short exercise intervention in poorly controlled asthma is tolerated and beneficial for symptom control, which may precede improvement in markers of inflammation. Prescribed training programmes may provide a cost-effective treatment adjunct in poorly controlled asthma.

Introduction Many asthmatics remain symptomatic despite optimized medical therapy. New treatments are needed that are scalable at minimal cost. Pulmonary rehabilitation is established in respiratory disease, and emerging data suggests that prescribed exercise intervention may modulate the immune and inflammatory basis for asthma, offering clinical benefit beyond functional capacity improvement. High-intensity interval training (HIIT) seems better tolerated in asthmatics than constant work-rate training, but HIIT with strength training has not been assessed in terms of immunomodulatory potential, and mechanistic work is needed to explain these responses to exercise. Methods This study will randomise ten suboptimally controlled asthma patients to a 12 week HIIT Structured Responsive Exercise Training Programme (SRETP) or SRETP with strength training, which allows adaptation of the programme as fitness improves. Participants will be assessed at 3, 6 and 12 weeks to ascertain if SRETP is feasible, safe and tolerable, improves symptom control, modifies airway and systemic inflammation, and physiology in asthmatic patients. An interim analysis of the first 5 patients is presented. Results Early results suggest safety and tolerability. Wilcoxon signed rank tests between Baseline and Week 3 showed that ACQ score improved significantly (median=2.00 [IQR=1.82–2.38] vs median=1.50 [IQR=1.00–1.83], p=0.043). There were no statistically significant improvements in airways inflammation as assessed by FeNO (median=44.50 [IQR=29.00–87.25] vs median=49 [IQR=21.25–92.50], p=0.686), peripheral blood eosinophilia (n=4, median=0.30 [IQR=0.20–0.50] vs median=0.35 [IQR=0.30–0.48], p=0.102), or aerobic fitness as assessed by VO2 at anaerobic threshold (AT) (n=4, median=10.90 [IQR=9.55–20.25] vs 11.00 ml/kg/min [IQR=9.48–22.35]; p=0.285) and VO2peak (n=4, median=21.80 [IQR=17.15–33.45] vs 23.00 ml/kg/min [IQR=15.73–35.68]; p=0.465) Conclusion This interim data suggests even a very short period of exercise intervention is tolerated and beneficial for symptom control in sub-optimally controlled asthma. Further data will allow assessment of whether this precedes improvement in markers of inflammation, and current results are due to early subclinical cellular improvements in inflammation, or whether symptoms improve independent of any reduction in inflammation. We aim to further investigate this response to exercise training in asthma in a fully powered, observational mechanistic study. Prescribed exercise training programmes may provide a cost-effective treatment adjunct for suboptimally controlled asthmatics.

Introduction and objectives Viral infections are major drivers of exacerbations and clinical burden in patients with asthma and COPD. IFN-β is essential for controlling the infection and spread of viruses such as influenza. However, in recent clinical trials, treatment with IFN-β failed to prevent asthma exacerbations. We aimed to compare the utility of IFN-β as either a treatment or prophylactic for modulating influenza infection in macrophages from health and COPD, with an aim of generating new insights for the optimal design of future clinical trials. Methods Monocyte-derived macrophages (MDM) and primary alveolar macrophages were isolated from healthy and COPD patients. Cells were then infected with influenza virus either prior to or after IFN-β stimulation. Levels of influenza infection were measured by%NP1 +cells using flow cytometry. Viral RNA shedding and anti-viral gene expression (Including OAS1, MX1 and RIG-I) were measured by qPCR. Concentration of inflammatory cytokines (including GM-CSF, TSLP, IL-33, IL-25, TNF-α, IL-1β, IL-6, CCL17, CCL22 and RANTES) in supernatants was measured using MSD. Results Levels of influenza infection in MDMs was similar for both healthy and COPD patients with a%NP1 of 24.4 and 22.0, respectively (p=0.79). Treatment after infection of MDMs with IFN-β was ineffective at modulating influenza infection rates or viral load but IFN-β prophylaxis effectively reduced%NP1+ cells and shedding by 85% (p<0.001) and, 20 fold (p<0.01), respectively. The effect of IFN-β prophylaxis in modulating influenza infection lasted up to 1 week in MDMs from both health and COPD. This was mirrored by the expression of OAS1, MX1 and RIG-I interferon stimulated genes. Exogenous IFN-β did not induce inflammatory cytokine production by MDMs. Conclusions We modelled IFN-β dynamics in vitro and highlight the potential for intermittent prophylactic doses of exogenous IFN-β to modulate viral infection. This generates novel insights to both understand recent clinical trial results in asthma and to aid the optimal design of future clinical trials in both asthma and COPD.