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Samuel James Brake,1 Mathew Suji Eapen,1 Kielan Darcy McAlinden,1 James Markos,1,2 Greg Haug,1,2 Josie Larby,1,2 Collin Chia,1,2 Ashutosh Hardikar,1,3 Gurpreet Kaur Singhera,4,5 Tillie L Hackett,4,5 Wenying Lu,1 Sukhwinder Singh Sohal1 1Respiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, TAS, 7248, Australia; 2Department of Respiratory Medicine, Launceston General Hospital, Launceston, TAS, 7250, Australia; 3Department of Cardiothoracic Surgery, Royal Hobart Hospital, Hobart, TAS, 7000, Australia; 4Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, BC, Canada; 5Department of Medicine, University of British Columbia Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, BC, CanadaCorrespondence: Sukhwinder Singh SohalRespiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Locked Bag – 1322, Newnham Drive, Launceston, TAS, 7248, AustraliaTel +61 3 6324 5434Email sssohal@utas.edu.auBackground: Smokers and patients with COPD are highly susceptible to SARS-CoV-2 infection, leading to severe COVID-19.Methods: This cross-sectional study involved resected lung tissues from 16 patients with GOLD stage I or II COPD; of which 8 were current smokers COPD (COPD-CS), and 8 ex-smokers COPD (COPD-ES), 7 normal lung function smokers (NLFS), 9 patients with small airways disease (SAD), and 10 were never-smoking normal controls (NC). Immunostaining for ACE2, Furin, and TMPRSS2 was performed and analysed for percent expression in small airway epithelium (SAE) and counts for positively and negatively stained type 2 pneumocytes and alveolar macrophages (AMs) were done using Image ProPlus V7.0. Furthermore, primary small airway epithelial cells (pSAEC) were analysed by immunofluorescence after exposure to cigarette smoke extract (CSE).Results: ACE2, Furin, and TMPRSS2 expression significantly increased in SAE and type 2 pneumocytes in all the subjects (except Furin for NLFS) compared to NC (p < 0.001). Similar significance was observed for ACE2 positive AM (p < 0.002), except COPD-ES, which decreased in ACE2 positive AMs (p < 0.003). Total type 2 pneumocytes and AMs significantly increased in the pathological groups compared to NC (p < 0.01), except SAD (p = 0.08). However, AMs are significantly reduced in COPD-ES (p < 0.003). Significant changes were observed for tissue co-expression of Furin and TMPRSS2 with ACE2 in SAE, type 2 pneumocytes and AMs. These markers also negatively correlated with lung function parameters, such as FEV1/FVC % predicted, FEF25-75%, DLCO% predicted. A strong co-localisation and expression for ACE2 (p < 0.0001), Furin (p < 0.01), and TMPRSS2 (p < 0.0001) was observed in pSAEC treated with 1% CSE than controls.Discussion: The increased expression of ACE2, TMPRSS2 and Furin, in the SAE, type 2 pneumocytes and AMs of smokers and COPD are detrimental to lung function and proves that these patient groups could be more susceptible to severe COVID-19 infection. Increased type 2 pneumocytes suggest that these patients are vulnerable to developing post-COVID-19 interstitial pulmonary fibrosis or fibrosis in general. There could be a silently developing interstitial pathology in smokers and patients with COPD. This is the first comprehensive study to report such changes.Keywords: COVID-19, SARS-CoV-2, smoking, COPD, ACE2, type 2 pneumocytes, alveolar macrophages, epithelium

Purva Asrani,1 Afzal Hussain,2 Khalida Nasreen,3 Mohamed Fahad AlAjmi,2 Samira Amir,4 Sukhwinder Singh Sohal,5 Md Imtaiyaz Hassan3 1Molecular Biology and Biotechnology, ICAR- National Institute for Plant Biotechnology, New Delhi, 110012, India; 2Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia; 3Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India; 4Department of Chemistry, College of Science and General Studies, Alfaisal University, Riyadh, Kingdom of Saudi Arabia; 5Respiratory Translational Research Group, Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Tasmania, AustraliaCorrespondence: Md Imtaiyaz HassanCentre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, IndiaEmail mihassan@jmi.ac.inAbstract: Coronavirus disease 2019 (COVID-19) is an emerging challenging area for the researchers to buckle up against the spread and control of the virus. Since earlier times, the diagnosis has been an important procedure in estimating the fate of epidemics by indicating the extent to which disease has been spread and to the extent, further disease prognosis would occur. The absence of anti-viral therapies and vaccines for COVID-19 at present suggests early diagnosis and isolation of the patients as the only smart approach available as of now. Presently, the increasing death rates, faster rates of transmission, non-availability of vaccines, and treatment have over-pressurized the researchers, health professionals, and government officials to develop effective clinical strategies in diagnosis and to come up with guidelines to be followed during conduction of each diagnostic procedure for maintaining healthcare systems. Since the incubation period of this virus is 2– 14 days, a patient can transmit the infection without showing symptoms. Therefore, early diagnosis and isolation of susceptible individuals are the only way to limit the spread of the virus. Significance of diagnosis and triaging, information on specimen collection, safety considerations while handling, transport, and storage of samples have been highlighted in this paper to make people more aware and develop better clinical strategies in the future.Keywords: COVID-19, diagnosis guidelines, specimen collection, safety consideration, SARS-COV-2

Mathew Suji Eapen,1 Pawan Sharma,1–3 Archana Vijay Gaikwad,1 Wenying Lu,1 Stephen Myers,1 Philip M Hansbro4,5, Sukhwinder Singh Sohal11Respiratory Translational Research Group, Department of Laboratory Medicine, College of Health and Medicine, University of Tasmania, Launceston, TAS 7248, Australia; 2Medical Sciences, University of Technology Sydney, Sydney, NSW 2007, Australia; 3Woolcock Emphysema Centre, Woolcock Institute of Medical Research, University of Sydney, Sydney, NSW 2037, Australia; 4Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute and the University of Newcastle, Newcastle, NSW 2308, Australia; 5Centre for Inflammation, Centenary Institute and University of Technology Sydney, Sydney, NSW 2007, AustraliaAbstract: COPD is a common and highly destructive disease with huge impacts on people and health services throughout the world. It is mainly caused by cigarette smoking though environmental pollution is also significant. There are no current treatments that affect the overall course of COPD; current drugs focus on symptomatic relief and to some extent reducing exacerbation rates. There is an urgent need for in-depth studies of the fundamental pathogenic mechanisms that underpin COPD. This is vital, given the fact that nearly 40%–60% of the small airway and alveolar damage occurs in COPD well before the first measurable changes in lung function are detected. These individuals are also at a high risk of lung cancer. Current COPD research is mostly centered around late disease and/or innate immune activation within the airway lumen, but the actual damage to the airway wall has early onset. COPD is the end result of complex mechanisms, possibly triggered through initial epithelial activation. To change the disease trajectory, it is crucial to understand the mechanisms in the epithelium that are switched on early in smokers. One such mechanism we believe is the process of epithelial to mesenchymal transition. This article highlights the importance of this profound epithelial cell plasticity in COPD and also its regulation. We consider that understanding early changes in COPD will open new windows for therapy.Keywords: epithelial-to-mesenchymal transition, EMT, cancer, fibrosis, inflammation, HuR, EGFR, MMP, TGFβ

Sukhwinder Singh SohalDiscipline of Laboratory Medicine, School of Health Sciences, Faculty of Health, University of Tasmania, Launceston, AustraliaIt was with great interest that I read the recent comprehensive review by Christer Janson et al1 published in the International Journal of COPD, where authors discussed the possible mechanisms behind the increased risk of pneumonia in COPD patients using inhaled corticosteroids (ICSs), especially with fluticasone propionate (FP), where the risk was highest.1 It is an important area, and it is encouraging and reassuring that leading clinical journals are recognizing this. Understanding the fundamental mechanisms behind pneumococcal infections is critical.2 I would like to suggest that a broader discussion of new insights into the potential mechanisms contributing to the increased adherence of pneumococcus to airway wall and particularly in response to FP might has been appropriate with this opportunity.Authors’ replyChrister Janson,1 Georgios Stratelis,1,2 Anna Miller-Larsson,3 Tim W Harrison,4 Kjell Larsson51Respiratory, Allergy and Sleep Research Unit, Department of Medical Sciences, Uppsala University, Uppsala, 2Respiratory, Inflammation and Autoimmunity, AstraZeneca Nordic, Södertälje, 3Respiratory GMed, AstraZeneca Gothenburg, Mölndal, Sweden; 4Nottingham Respiratory Research Unit, City Hospital Campus, University of Nottingham, Nottingham, UK; 5Lung and Airway Research, National Institute of Environmental Medicine, Karolinska Institutet, Stockholm, SwedenWe thank Dr Sohal for his interest in our recently published manuscript discussing the scientific rationale for the possible inhaled corticosteroid (ICS) intraclass difference in the risk of pneumonia in COPD. His own work on platelet-activating factor receptor (PAFR) expression on airway epithelial cells in COPD patients shows a trend toward increased expression of PAFR by 6-month treatment with fluticasone propionate (FP), suggesting an increased rate of pneumococcal adhesion with FP, and hence the possible development of infection or pneumonia.1 This supports in part the work by Heijink et al2 that we cited in our paper.2View the original article by Janson et al

Amir Soltani,1 Eugene Haydn Walters,1,* David W Reid,1,2 Shakti Dhar Shukla,1 Kaosia Nowrin,1 Chris Ward,3 H Konrad Muller,1 Sukhwinder Singh Sohal1,4,* 1NHMRC Center of Research Excellence for Chronic Respiratory Disease, School of Medicine, University of Tasmania, Hobart, TAS, Australia; 2Iron Metabolism Laboratory, Queensland Institute of Medical Research, Brisbane, QLD, Australia; 3Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK; 4School of Health Sciences, University of Tasmania, Launceston, TAS, Australia *These authors contributed equally to this work Background: This study assessed the effects of inhaled corticosteroid (ICS) on airway vascular remodeling in chronic obstructive pulmonary disease (COPD). Methods: Thirty-four subjects with mild-to-moderate COPD were randomly allocated 2:1 to ICS or placebo treatment in a double-blinded clinical trial over 6 months. Available tissue was compared before and after treatment for vessel density, and expression of VEGF, TGF-β1, and TGF-β1-related phosphorylated transcription factors p-SMAD 2/3. This clinical trial has been registered and allocated with the Australian New Zealand Clinical Trials Registry (ANZCTR) on 17/10/2012 with reference number ACTRN12612001111864. Results: There were no significant baseline differences between treatment groups. With ICS, vessels and angiogenic factors did not change in hypervascular reticular basement membrane, but in the hypovascular lamina propria (LP), vessels increased and this had a proportionate effect on lung air trapping. There was modest evidence for a reduction in LP vessels staining for VEGF with ICS treatment, but a marked and significant reduction in p-SMAD 2/3 expression. Conclusion: Six-month high-dose ICS treatment had little effect on hypervascularity or angiogenic growth factors in the reticular basement membrane in COPD, but normalized hypovascularity in the LP, and this was physiologically relevant, though accompanied by a paradoxical reduction in growth factor expression. Keywords: airway remodeling, bronchial biopsy, COPD, inhaled corticosteroid, vascular remodeling

Shakti D Shukla,1,* Rory L Fairbairn,1,* David A Gell,1 Roger D Latham,1 Sukhwinder S Sohal,1,2 Eugene H Walters,1 Ronan F O’Toole11Breathe Well Centre, School of Medicine, Faculty of Health, University of Tasmania, Hobart, TAS, Australia; 2School of Health Sciences, Faculty of Health, University of Tasmania, Launceston, TAS, Australia*These authors contributed equally to this workBackground: COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells.Objective: To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae.Methods: Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken.Results: PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure.Conclusion: WEB-2086 represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD. Keywords: airway epithelium, NTHi, pneumococci, WEB-2086, platelet-activating factor receptor, PAFr antagonist

Sukhwinder Singh Sohal, Eugene Haydn Walters School of Health Sciences, University of Tasmania, Launceston, TAS, Australia We read with interest the recent comprehensive review by Sowmya P Lakshmi et al on potential new therapies for COPD in the International Journal of Chronic Obstructive Pulmonary Disease.1 The review says that only by understanding the core pathological processes, new therapeutics emerge, and it encourages that leading respiratory journals are recognizing this. However, we would suggest that, in this review, the overall view of the pathology of COPD airway disease does not reflect the current literature. In particular, the airway wall in at least mild to moderate COPD is hypo-cellular and hypo-vascular, with markedly active epithelial–mesenchymal transition (EMT)2 as part of epithelial activation and reprogramming.3 This process is closely related to small airway fibrosis and airflow obstruction. Inhaled corticosteroids (ICSs) affect these key epithelial cellular activation and vascular aspects of COPD,2 and more research on alternatives to corticosteroid on these aspects is urgently needed. It is of interest that the peroxisome proliferator-activated receptor system that the reviewers mention has implication for EMT induction,1 as has the TGF/Activin family and the Wnt system; these pathways are replete with potential drug targets. View the original paper by Lakshimi and colleagues.

Malik Quasir Mahmood,1,* Sukhwinder Singh Sohal,1,2,* Shakti Dhar Shukla,1 Chris Ward,3 Ashutosh Hardikar,4 Wan Danial Noor,1 Hans Konrad Muller,1 Darryl A Knight,5 Eugene Haydn Walters1 1NHMRC Centre of Research Excellence for Chronic Respiratory Disease and Lung Ageing, School of Medicine, University of Tasmania, Hobart, TAS, Australia; 2School of Health Sciences, Faculty of Health, University of Tasmania, Launceston, TAS, Australia; 3Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK; 4Royal Hobart Hospital, Hobart, TAS, Australia; 5School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia *These authors contributed equally to this work Background: Small airway fibrosis is the main contributor in airflow obstruction in chronic obstructive pulmonary disease. Epithelial mesenchymal transition (EMT) has been implicated in this process, and in large airways, is associated with angiogenesis, ie, Type-3, which is classically promalignant. Objective: In this study we have investigated whether EMT biomarkers are expressed in small airways compared to large airways in subjects with chronic airflow limitation (CAL) and what type of EMT is present on the basis of vascularity. Methods: We evaluated epithelial activation, reticular basement membrane fragmentation (core structural EMT marker) and EMT-related mesenchymal biomarkers in small and large airways from resected lung tissue from 18 lung cancer patients with CAL and 9 normal controls. Tissues were immunostained for epidermal growth factor receptor (EGFR; epithelial activation marker), vimentin (mesenchymal marker), and S100A4 (fibroblast epitope). Type-IV collagen was stained to demonstrate vessels. Results: There was increased expression of EMT-related markers in CAL small airways compared to controls: EGFR (P

Shakti Dhar Shukla,1,* Sukhwinder Singh Sohal,1,* Malik Quasir Mahmood,1 David Reid,2 Hans Konrad Muller,1 Eugene Haydn Walters1 1NHMRC Centre for Research Excellence for Chronic Respiratory Disease and Lung Ageing, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia; 2Queensland Institute of Medical Research, Iron Metabolism Laboratory, Brisbane, Queensland, Australia *Shakti Dhar Shukla and Sukhwinder Singh Sohal are joint first authors Background: We recently published that platelet-activating factor receptor (PAFr) is upregulated on the epithelium of the proximal airways of current smokers and also in bronchial epithelial cells exposed to cigarette smoke extract. These treated cells also showed upregulation of Streptococcus pneumoniae adhesion. Bacterial wall phosphorylcholine specifically binds to PAFr expressed on airway epithelium, thus facilitating adherence and tissue invasion, which may be relevant to chronic obstructive pulmonary disease (COPD). Moreover, the use of inhaled corticosteroids (ICS) in COPD patients is associated with an increased risk of invasive respiratory pneumococcal infections. Objective: In this study, we have investigated whether PAFr expression is especially upregulated in airway epithelium in COPD patients and whether this expression may be modulated by ICS therapy. Methods: We cross-sectionally evaluated PAFr expression in bronchial biopsies from 15 COPD patients who were current smokers (COPD-smokers) and 12 COPD-ex-smokers, and we compared these to biopsies from 16 smokers with normal lung function. We assessed immunostaining with anti-PAFr monoclonal antibody. We also used material from a previous double-blinded randomized placebo-controlled 6-month ICS intervention study in COPD patients to explore the effect of ICS on PAFr expression. We employed computer-aided image analysis to quantify the percentage of epithelium stained for PAFr. Results: Markedly enhanced expression of PAFr was found in both COPD-smokers (P

Sukhwinder Singh Sohal,1,* Amir Soltani,1,* David Reid,1,2 Chris Ward,1,3 Karen E Wills,1,4 H Konrad Muller,1 Eugene Haydn Walters1 1National Health and Medical Research Council Centre of Research Excellence for Chronic Respiratory Disease, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia; 2Iron Metabolism Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia; 3Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, Tyne and Wear, UK; 4Department of Biostatistics, Menzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia *These authors contributed equally to this workBackground: We recently reported that epithelial–mesenchymal transition (EMT) is active in the airways in chronic obstructive pulmonary disease (COPD), suggesting presence of an active profibrotic and promalignant stroma. With no data available on potential treatment effects, we undertook a blinded analysis of inhaled corticosteroids (ICS) effects versus placebo on EMT markers in previously obtained endobronchial biopsies in COPD patients, as a “proof of concept” study.Methods: Assessment of the effects of inhaled fluticasone propionate (FP; 500 µg twice daily for 6 months) versus placebo in 34 COPD patients (23 on fluticasone propionate and eleven on placebo). The end points were epidermal growth factor receptor (EGFR; marker of epithelial activation) and the biomarkers of EMT: reticular basement membrane (Rbm) fragmentation (“hallmark” structural marker), matrix metalloproteinase-9 (MMP-9) cell expression, and S100A4 expression in basal epithelial and Rbm cells (mesenchymal transition markers).Results: Epithelial activation, “clefts/fragmentation” in the Rbm, and changes in the other biomarkers all regressed on ICS, at or close to conventional levels of statistical significance. From these data, we have been able to nominate primary and secondary end points and develop power calculations that would be applicable to a definitive prospective study.Conclusion: Although only a pilot “proof of concept” study, this trial provided strong suggestive support for an anti-EMT effect of ICS in COPD airways. A larger and fully powered prospective study is now indicated as this issue is likely to be extremely important. Such studies may clarify the links between ICS use and better clinical outcomes and protection against lung cancer in COPD.Keywords: pilot trial, reticular basement membrane, S100A4, EGFR, MMP-9, lung cancer