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Autophagy (or macroautophagy) is a key cellular process that removes damaged molecules (particularly proteins) and subcellular organelles to maintain cellular homeostasis. There is growing evidence that abnormalities in autophagy may contribute to the pathogenesis of many chronic diseases, including asthma and chronic obstructive pulmonary disease (COPD). In asthma, increased autophagy plays a role in promoting type 2 immune responses and eosinophilic inflammation, whereas decreased autophagy may be important in neutrophilic asthma. Acute exposure to cigarette smoke may activate autophagy, resulting in ciliary dysfunction and death of airway epithelial cells, whereas in stable COPD most studies have demonstrated an impairment in autophagy, with reduced autophagic flux and accumulation of abnormal mitochondria (defective mitophagy) and linked to cellular senescence. Autophagy may be increased or decreased in different cell types and depending on the cellular environment, making it difficult to target autophagy therapeutically. Several existing drugs may activate autophagy, including rapamycin, metformin, carbamazepine, cardiac glycosides and statins, whereas others, such as chloroquine, inhibit this process. However, these drugs are nonspecific and more selective drugs are now in development, which may prove useful as novel agents to treat asthma and COPD in the future.

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating novel therapies. Here, we show that COPD patients have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared to control subjects. IL-36γ is derived mainly from small airway epithelial cells (SAEC) and further induced by a viral mimetic, whereas IL-36RA is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, that was inhibited by exogenous IL-36RA. The use of a therapeutic antibody that inhibits binding to the IL-36 receptor (IL-36R) attenuated IL-36γ driven inflammation and cellular cross talk. We have demonstrated a novel mechanism for the amplification and propagation of neutrophilic inflammation in COPD and that blocking this cytokine family via a IL-36R neutralising antibody could be a promising new therapeutic strategy in the treatment of COPD.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow–derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

There is increasing evidence that COPD is a disease of accelerated lung aging, with the accumulation of senescent cells that lose their ability to repair and secrete multiple inflammatory proteins known as the senescence-associated secretory phenotype (SASP), which mimic the profile of inflammatory mediators secreted in COPD. This review discusses novel drugs (senotherapies) that target cellular senescence and which may be a promising therapeutic approach to prevent currently unaddressed disease progression and mortality in COPD. A major pathway leading to senescence is via the activation of phosphoinositide-3-kinase/mammalian target of rapamycin signaling. Existing drugs, such as rapamycin and metformin, target this pathway. Mitochondrial oxidative stress is a key driving mechanism for this pathway, and mitochondria-targeted antioxidants are promising. A key finding in COPD is loss of antiaging molecules such as sirtuin-1 and sirtuin-6, which are reduced by phosphoinositide-3-kinase/mammalian target of rapamycin signaling through microRNA-34a. Sirtuin activators are in development, and inhibiting microRNA-34a restores sirtuin expression experimentally in COPD cells. Senolytic therapies induce apoptosis and removal of senescent cells and reduce the senescence-associated secretory phenotype response in animal models of aging and in pilot clinical studies of other age-related diseases. A combination of senolytics and senostatics (drugs that inhibit cellular senescence) may be a valuable new approach to COPD, especially if started early in the disease process. Furthermore, COPD is associated with several comorbidities that share the same aging pathways which may be spread by extracellular vesicles, and thus a single treatment for all these diseases is feasible in the future to extend health span.

Community-based clinical trials of the inhaled corticosteroid budesonide in early COVID-19 have shown improved patient outcomes. We aimed to understand the inflammatory mechanism of budesonide in the treatment of early COVID-19.The STOIC trial was a randomised, open label, parallel group, phase 2 clinical intervention trial where patients were randomly assigned (1:1) to receive usual care (as needed antipyretics were only available treatment) or inhaled budesonide at a dose of 800 μg twice a day plus usual care. For this experimental analysis, we investigated the nasal mucosal inflammatory response in patients recruited to the STOIC trial and in a cohort of SARS-CoV-2-negative healthy controls, recruited from a long-term observational data collection study at the University of Oxford. In patients with SARS-CoV-2 who entered the STOIC study, nasal epithelial lining fluid was sampled at day of randomisation (day 0) and at day 14 following randomisation, blood samples were also collected at day 28 after randomisation. Nasal epithelial lining fluid and blood samples were collected from the SARS-CoV-2 negative control cohort. Inflammatory mediators in the nasal epithelial lining fluid and blood were assessed for a range of viral response proteins, and innate and adaptive response markers using Meso Scale Discovery enzyme linked immunoassay panels. These samples were used to investigate the evolution of inflammation in the early COVID-19 disease course and assess the effect of budesonide on inflammation.146 participants were recruited in the STOIC trial (n=73 in the usual care group; n=73 in the budesonide group). 140 nasal mucosal samples were available at day 0 (randomisation) and 122 samples at day 14. At day 28, whole blood was collected from 123 participants (62 in the budesonide group and 61 in the usual care group). 20 blood or nasal samples were collected from healthy controls. In early COVID-19 disease, there was an enhanced inflammatory airway response with the induction of an anti-viral and T-helper 1 and 2 (Th1/2) inflammatory response compared with healthy individuals. Individuals with COVID-19 who clinically deteriorated (ie, who met the primary outcome) showed an early blunted respiratory interferon response and pronounced and persistent Th2 inflammation, mediated by CC chemokine ligand (CCL)-24, compared with those with COVID-19 who did not clinically deteriorate. Over time, the natural course of COVID-19 showed persistently high respiratory interferon concentrations and elevated concentrations of the eosinophil chemokine, CCL-11, despite clinical symptom improvement. There was persistent systemic inflammation after 28 days following COVID-19, including elevated concentrations of interleukin (IL)-6, tumour necrosis factor-α, and CCL-11. Budesonide treatment modulated inflammation in the nose and blood and was shown to decrease IL-33 and increase CCL17. The STOIC trial was registered with ClinicalTrials.gov, NCT04416399.An initial blunted interferon response and heightened T-helper 2 inflammatory response in the respiratory tract following SARS-CoV-2 infection could be a biomarker for predicting the development of severe COVID-19 disease. The clinical benefit of inhaled budesonide in early COVID-19 is likely to be as a consequence of its inflammatory modulatory effect, suggesting efficacy by reducing epithelial damage and an improved T-cell response.Oxford National Institute of Health Research Biomedical Research Centre and AstraZeneca.

This editorial describes the ERS CRC programme, which addresses research areas across all major respiratory disease domains. It explains how the ERS Research Agency supports the CRCs to obtain external funding and ensures links with other ERS activities.https://bit.ly/2WHIQPa

SummaryVaccinations against SARS-CoV-2 are effective in COVID-19. However, with limited vaccine access, vaccine hesitancy and variant breakthroughs, there is still a need for effective and safe early treatments. Two community-based clinical trials of the inhaled corticosteroid, budesonide, have recently been published showing and improvement in patients with COVID-19 treated early with budesonide1,2. To understand mechanistically how budesonide was beneficial, inflammatory mediators were assessed in the nasal mucosa of patients recruited to the Steroids in COVID (STOIC1) trial and a cohort of SARS-CoV-2 negative individuals. Here we show that in early COVID-19, elevation in viral response proteins and Th1 and Th2 inflammation occurs. Longitudinal sampling in the natural course of COVID-19 showed persistently high interferon levels and elevated concentrations of the eosinophil chemokine, CCL11. In patients who deteriorate, the initial nasal mucosal signal is characterised by a marked suppression of the early inflammatory response, with reduced concentrations of interferon and inflammatory cytokines, but elevated eosinophil chemokines. Systemic inflammation remained altered in COVID-19 patients, implying that even after symptom resolution, changes in immunological mediators do not resolve. Budesonide treatment decreased IL-33 and IFN-γ, implying a reduction in epithelial damage and dampening of the interferon response. Budesonide treatment also increased CCL17 concentrations, suggesting an improved T-cell response; and significantly alters inflammatory pathways giving further insight into how this treatment can accelerate patient recovery.Abstract Figure

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared with control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and is further induced by a viral mimetic, whereas IL-36Ra is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ, thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, which was inhibited by exogenous IL-36Ra. The use of a therapeutic antibody that inhibits binding to the IL-36R attenuated IL-36γ-driven inflammation and cellular crosstalk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and have shown that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising therapeutic strategy in the treatment of COPD.

Introduction: Chronic obstructive pulmonary disease (COPD) is associated with accelerated lung aging. In the COPD lung, there is an accumulation of senescent cells that may, in part, be due to a lack of clearance. To investigate this, a robust source of well-characterised senescent cells is required. Doxorubicin (DOXO) and etoposide (Etop) at sub-cytotoxic concentrations activate the DNA-damage response (DDR) pathway, leading to the induction of cellular senescence and could be used to generate a senescent cell population for study. Aim: To induce senescence in BEAS-2B cells using DOXO and Etop. Methods: Senescent BEAS-2B cells were generated by repeated exposure to 50nM DOXO or 1mM Etop for a total of 6 days. Cell cycle checkpoint inhibitors, anti-aging molecule sirtuin-1 and senescence associated secretory phenotype (SASP) marker expression were measured using western blotting and ELISA. Senescent cells were identified by senescence-associated-b-galactosidase (SA-β-gal) expression. Results: In BEAS-2B cells, 50nM DOXO treatment induced senescence in ~60% of cells (n=7, p Conclusion: Sub-cytotoxic double-hit DOXO and Etop treatments are required to induce senescence in BEAS-2B cells via p21 induction of cell cycle arrest and may provide a model that can be used in future experiments.

Background: Multiple early hospital cohorts of coronavirus disease 2019 (COVID-19) showed that patients with chronic respiratory disease were significantly under-represented. We hypothesised that the widespread use of inhaled glucocorticoids was responsible for this finding and tested if inhaled glucorticoids would be an effective treatment for early COVID-19 illness. Methods: We conducted a randomised, open label trial of inhaled budesonide, compared to usual care, in adults within 7 days of the onset of mild Covid-19 symptoms. The primary end point was COVID-19-related urgent care visit, emergency department assessment or hospitalisation. The trial was stopped early after independent statistical review concluded that study outcome would not change with further participant enrolment. Results: 146 patients underwent randomisation. For the per protocol population (n=139), the primary outcome occurred in 10 participants and 1 participant in the usual care and budesonide arms respectively (difference in proportion 0.131, p=0.004). The number needed to treat with inhaled budesonide to reduce COVID-19 deterioration was 8. Clinical recovery was 1 day shorter in the budesonide arm compared to the usual care arm (median of 7 days versus 8 days respectively, logrank test p=0.007). Proportion of days with a fever and proportion of participants with at least 1 day of fever was lower in the budesonide arm. Fewer participants randomised to budesonide had persistent symptoms at day 14 and day 28 compared to participants receiving usual care. Conclusion: Early administration of inhaled budesonide reduced the likelihood of needing urgent medical care and reduced time to recovery following early COVID-19 infection. Trial Registration: ClinicalTrials.gov number, NCT04416399 Funding: Oxford NIHR Biomedical Research Centre and AstraZeneca Declaration of Interests: Dr. Ramakrishnan reports grants and non-financial support from Oxford Respiratory NIHR BRC, during the conduct of the study; non-financial support from AstraZeneca, personal fees from Australian Government Research Training Program, outside the submitted work; . Dr. Nicolau has nothing to disclose. Mrs Langford has nothing to disclose. Mr. Mahdi has nothing to disclose. Mrs Helen Jeffers reports personal fees from AstraZeneca, outside the submitted work; . Miss Mwasuku has nothing to disclose. Mrs Krassowska has nothing to disclose. Dr Fox has nothing to disclose. Dr Binnian has nothing to disclose. Dr Glover has nothing to disclose. Dr Bright has nothing to disclose. Dr. Butler reports grants from National Institute for Health Research (NIHR), Roche Molecular Diagnostics, Janssen Pharmaceuticals, and various public funding bodies for research related to diagnostics and infections. He has revcied personal fees from Pfizer INC, Roche Diagnostics, and Janssen Pharmaceuticals, outside the submitted work. Dr. Cane has nothing to disclose. Mr. Halner has nothing to disclose. Dr. Matthews has nothing to disclose. Dr. Donnelly reports grants from AstraZeneca, from Boehringer-Ingelheim, outside the submitted work; . Dr. Simpson has nothing to disclose. Dr Baker has nothing to disclose. Dr. Fadai has nothing to disclose. Dr. Peterson reports personal fees from AstraZeneca, outside the submitted work; . Mr. Bengtsson reports personal fees from AstraZeneca, outside the submitted work; Dr. Barnes reports grants and personal fees from AstraZeneca, grants and personal fees from Boehringer Ingelheim, personal fees from Teva, personal fees from Covis, during the conduct of the study; Dr. Russell reports grants from AstraZeneca, personal fees from Boehringer Ingelheim, personal fees from Chiesi UK, personal fees from Glaxo-SmithKline, during the conduct of the study; . Dr. Bafadhel reports grants from AstraZeneca, personal fees from AstraZeneca, Chiesi, GSK, other from Albus Health, ProAxsis, outside the submitted work; . Ethics Approval Statement: The trial was sponsored by the University of Oxford, and was approved by the Fulham London Research Ethics Committee (20/HRA/2531) and the National Health Research Authority.The ethical approval number is 20/HRA/2531.

Chronic obstructive pulmonary disease (COPD) is a progressive lung condition affecting 10% of the global population over 45 years. Currently, there are no disease-modifying treatments, with current therapies treating only the symptoms of the disease. COPD is an inflammatory disease, with a high infiltration of leukocytes being found within the lung of COPD patients. These leukocytes, if not kept in check, damage the lung, leading to the pathophysiology associated with the disease. In this review, we focus on the main leukocytes found within the COPD lung, describing how the release of chemokines from the damaged epithelial lining recruits these cells into the lung. Once present, these cells become active and may be driven towards a more pro-inflammatory phenotype. These cells release their own subtypes of inflammatory mediators, growth factors and proteases which can all lead to airway remodeling, mucus hypersecretion and emphysema. Finally, we describe some of the current therapies and potential new targets that could be utilized to target aberrant leukocyte function in the COPD lung. Here, we focus on old therapies such as statins and corticosteroids, but also look at the emerging field of biologics describing those which have been tested in COPD already and potential new monoclonal antibodies which are under review.

Cellular senescence is now considered an important driving mechanism for chronic lung diseases, particularly chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Cellular senescence is due to replicative and stress-related senescence with activation of p53 and p16INK4a, respectively, leading to activation of p21CIP1 and cell cycle arrest. Senescent cells secrete multiple inflammatory proteins known as the senescence-associated secretory phenotype, leading to low-grade chronic inflammation, which further drives senescence. Loss of key antiaging molecules sirtuin-1 and sirtuin-6 may be important in acceleration of aging and arises from oxidative stress reducing phosphatase PTEN (phosphatase tensin homolog), thereby activating PI3K (phosphoinositide-3-kinase) and mTOR (mammalian target of rapamycin). MicroRNA-34a (miR-34a), which is regulated by PI3K-mTOR signaling, plays a pivotal role in reducing sirtuin-1/6, and its inhibition with an antagomir results in their restoration, reducing markers of senescence, reducing senescence-associated secretory phenotype, and reversing cell cycle arrest in epithelial cells from peripheral airways of patients with COPD. miR-570 is also involved in reduction of sirtuin-1 and cellular senescence and is activated by p38 mitogen-activated protein kinase. These miRNAs may be released from cells in extracellular vesicles that are taken up by other cells, thereby spreading senescence locally within the lung but also outside the lung through the circulation; this may account for comorbidities of COPD and other lung diseases. Understanding the mechanisms of cellular senescence may result in new treatments for chronic lung disease, either by inhibiting PI3K-mTOR signaling, by inhibiting specific miRNAs, or by deletion of senescent cells with senolytic therapies, already shown to be effective in experimental lung fibrosis.

Neutrophilic inflammation is characteristic of chronic obstructive pulmonary disease (COPD); yet, there are no effective antiinflammatory therapies. The PDE4 inhibitor roflumilast is approved for use in COPD and suppresses sputum neutrophilia. The mechanism underlying this observation is unclear; therefore, this study addressed whether roflumilast directly affected neutrophil migration. Blood-derived neutrophils were isolated from nonsmokers, smokers, and patients with COPD, and chemotaxis was measured using Boyden chambers. Intracellular calcium ion concentration was measured by fluorimetry, and shape change and CD11b expression were measured by flow cytometry. Neutrophils from patients with COPD showed enhanced chemotactic responses toward both CXCL1 and leukotriene B4 compared with control cells. Chemotaxis was inhibited by both the active metabolite roflumilast N-oxide and rolipram in a concentration-dependent manner with no difference in responsiveness between subjects. Roflumilast N-oxide and rolipram were less efficacious against CXCL1 and leukotriene B4-mediated intracellular calcium ion concentration, suggesting that inhibition was not via this pathway. Both PDE4 inhibitors attenuated chemoattractant-mediated shape change and CD11b upregulation, suggesting common mechanisms. The stable cAMP analog 8-bromoadenosine 3',5'-cAMP inhibited chemotaxis, as did the direct Epac1 (exchange protein directly activated by cAMP 1) activator 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cAMP but not the direct protein kinase A activator N6-benzoyladenosine-3',5'-cAMP. These data suggest that roflumilast inhibits neutrophil chemotaxis directly via a cAMP-mediated mechanism requiring activation of Epac1 and that Epac1 activators could reduce COPD neutrophilic inflammation.

We presenta robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, for example due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilize a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade-off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/-100 μm. We characterize the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localization microscopy.Many microscopy experiments involve extended imaging of samples over timescales from minutes to days, during which the microscope can ‘drift’ out of focus. When imaging at high magnification, the depth of field is of the order of one micron and so the imaging system should keep the sample in the focal plane of the microscope objective lens to this precision. Unfortunately, temperature changes in the laboratory can cause thermal expansion of microscope components that can move the focal plane by more than a micron and such changes can occur on a timescale of minutes. This is a particular issue for super-resolved microscopy experiments using single molecule localization microscopy (SMLM) techniques, for which 1000s of images are acquired, and for automated imaging of multiple samples in multiwell plates. It is possible to maintain the sample in the focal plane focus position by either automatically moving the sample or adjusting the imaging system, for example by moving the objective lens. This is called ‘autofocus’ and is frequently achieved by reflecting a light beam from the microscope coverslip and measuring its position of beam profile as a function of defocus of the microscope. The correcting adjustment is then usually calculated analytically but there is recent interest in using machine learning techniques to determine the required focussing adjustment. Here, we present a system that uses a neural network to determine the required defocus correcting adjustment from camera images of a laser beam that is reflected from the coverslip. Unfortunately, this approach will only work when the microscope is in the same condition as it was when the neural network was trained - and this can be compromised by the same drift of the optical system that causes the defocus needing to be corrected. We show, however, that by training a neural network over an extended period, for example 10 days, this approach can ‘learn’ about the optical system drifts and provide the required autofocus function. We also show that an optical system utilizing a rectangular slit can make two measurements of the defocus simultaneously, with one measurement being optimized for high accuracy over a limited range (±10 μm) near focus and the other providing lower accuracy but over a much longer range (±100 μm). This robust autofocus system is suitable for automated super-resolved microscopy of arrays of samples in a multiwell plate using SMLM, for which an experiment routinely lasts more than 5 h.

We present a robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, e.g. due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilise a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade-off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/− 100 μm. We characterise the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localisation microscopy.

Summary Background Multiple early reports of patients admitted to hospital with COVID-19 showed that patients with chronic respiratory disease were significantly under-represented in these cohorts. We hypothesised that the widespread use of inhaled glucocorticoids among these patients was responsible for this finding, and tested if inhaled glucocorticoids would be an effective treatment for early COVID-19. Methods We performed an open-label, parallel-group, phase 2, randomised controlled trial (Steroids in COVID-19; STOIC) of inhaled budesonide, compared with usual care, in adults within 7 days of the onset of mild COVID-19 symptoms. The trial was done in the community in Oxfordshire, UK. Participants were randomly assigned to inhaled budsonide or usual care stratified for age (≤40 years or >40 years), sex (male or female), and number of comorbidities (≤1 and ≥2). Randomisation was done using random sequence generation in block randomisation in a 1:1 ratio. Budesonide dry powder was delivered using a turbohaler at a dose of 400 μg per actuation. Participants were asked to take two inhalations twice a day until symptom resolution. The primary endpoint was COVID-19-related urgent care visit, including emergency department assessment or hospitalisation, analysed for both the per-protocol and intention-to-treat (ITT) populations. The secondary outcomes were self-reported clinical recovery (symptom resolution), viral symptoms measured using the Common Cold Questionnare (CCQ) and the InFLUenza Patient Reported Outcome Questionnaire (FLUPro), body temperature, blood oxygen saturations, and SARS-CoV-2 viral load. The trial was stopped early after independent statistical review concluded that study outcome would not change with further participant enrolment. This trial is registered with ClinicalTrials.gov, NCT04416399. Findings From July 16 to Dec 9, 2020, 167 participants were recruited and assessed for eligibility. 21 did not meet eligibility criteria and were excluded. 146 participants were randomly assigned—73 to usual care and 73 to budesonide. For the per-protocol population (n=139), the primary outcome occurred in ten (14%) of 70 participants in the usual care group and one (1%) of 69 participants in the budesonide group (difference in proportions 0·131, 95% CI 0·043 to 0·218; p=0·004). For the ITT population, the primary outcome occurred in 11 (15%) participants in the usual care group and two (3%) participants in the budesonide group (difference in proportions 0·123, 95% CI 0·033 to 0·213; p=0·009). The number needed to treat with inhaled budesonide to reduce COVID-19 deterioration was eight. Clinical recovery was 1 day shorter in the budesonide group compared with the usual care group (median 7 days [95% CI 6 to 9] in the budesonide group vs 8 days [7 to 11] in the usual care group; log-rank test p=0·007). The mean proportion of days with a fever in the first 14 days was lower in the budesonide group (2%, SD 6) than the usual care group (8%, SD 18; Wilcoxon test p=0·051) and the proportion of participants with at least 1 day of fever was lower in the budesonide group when compared with the usual care group. As-needed antipyretic medication was required for fewer proportion of days in the budesonide group compared with the usual care group (27% [IQR 0–50] vs 50% [15–71]; p=0·025) Fewer participants randomly assigned to budesonide had persistent symptoms at days 14 and 28 compared with participants receiving usual care (difference in proportions 0·204, 95% CI 0·075 to 0·334; p=0·003). The mean total score change in the CCQ and FLUPro over 14 days was significantly better in the budesonide group compared with the usual care group (CCQ mean difference −0·12, 95% CI −0·21 to −0·02 [p=0·016]; FLUPro mean difference −0·10, 95% CI −0·21 to −0·00 [p=0·044]). Blood oxygen saturations and SARS-CoV-2 load, measured by cycle threshold, were not different between the groups. Budesonide was safe, with only five (7%) participants reporting self-limiting adverse events. Interpretation Early administration of inhaled budesonide reduced the likelihood of needing urgent medical care and reduced time to recovery after early COVID-19. Funding National Institute for Health Research Biomedical Research Centre and AstraZeneca.

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases., Carolin Koss et al. show that IL-36 produced from neutrophils is a key amplifier of lung inflammation in mice by activating neutrophils, macrophages and fibroblasts in cooperation with GM-CSF and the viral mimic poly(I:C). This study suggests that targeting IL-36 may improve the treatment of neutrophilic lung diseases.

BackgroundMultiple early hospital cohorts of coronavirus disease 2019 (COVID-19) showed that patients with chronic respiratory disease were significantly under-represented. We hypothesised that the widespread use of inhaled glucocorticoids was responsible for this finding and tested if inhaled glucorticoids would be an effective treatment for early COVID-19 illness.MethodsWe conducted a randomised, open label trial of inhaled budesonide, compared to usual care, in adults within 7 days of the onset of mild Covid-19 symptoms. The primary end point was COVID-19-related urgent care visit, emergency department assessment or hospitalisation. The trial was stopped early after independent statistical review concluded that study outcome would not change with further participant enrolment.Results146 patients underwent randomisation. For the per protocol population (n=139), the primary outcome occurred in 10 participants and 1 participant in the usual care and budesonide arms respectively (difference in proportion 0.131, p=0.004). The number needed to treat with inhaled budesonide to reduce COVID-19 deterioration was 8. Clinical recovery was 1 day shorter in the budesonide arm compared to the usual care arm (median of 7 days versus 8 days respectively, logrank test p=0.007). Proportion of days with a fever and proportion of participants with at least 1 day of fever was lower in the budesonide arm. Fewer participants randomised to budesonide had persistent symptoms at day 14 and day 28 compared to participants receiving usual care.ConclusionEarly administration of inhaled budesonide reduced the likelihood of needing urgent medical care and reduced time to recovery following early COVID-19 infection.(Funded by Oxford NIHR Biomedical Research Centre and AstraZeneca;ClinicalTrials.govnumber,NCT04416399)Research in contextEvidence before this studyThe majority of interventions studied for the COVID-19 pandemic are focused on hospitalised patients. Widely available and broadly relevant interventions for mild COVID-19 are urgently needed.Added value of this studyIn this open label randomised controlled trial, inhaled budesonide, when given to adults with early COVID-19 illness, reduces the likelihood of requiring urgent care, emergency department consultation or hospitalisation. There was also a quicker resolution of fever, a known poor prognostic marker in COVID-19 and a faster self-reported and questionnaire reported symptom resolution. There were fewer participants with persistent COVID-19 symptoms at 14 and 28 days after budesonide therapy compared to usual care.Implications of all the available evidenceThe STOIC trial potentially provides the first easily accessible effective intervention in early COVID-19. By assessing health care resource utilisation, the study provides an exciting option to help with the worldwide pressure on health care systems due to the COVID-19 pandemic. Data from this study also suggests a potentially effective treatment to prevent the long term morbidity from persistent COVID-19 symptoms.

Background: Cellular senescence is one of the hallmarks of COPD. The miR-34a and mir-570 are involved in the induction of cellular senescence by inhibiting anti-ageing sirtuins. Extracellular vesicles (EVs) play a key role in intercellular communication. Senescent cells produce high amounts of EVs and contain miRNA that can regulate cellular senescence. The role of EVs in spreading cell senescence remains unclear. Aim: To characterize extracellular vesicles released by epithelial cells in response to oxidative stress and to assess their effect on epithelial cells senescence. Methods: BEAS-2B bronchial epithelial cells were stimulated with H2O2 for 24 or 48h. Microvesicles and exosomes were isolated by differential ultracentrifugation of cell supernatant and characterised by Western blot and labelling membrane with the dye PKH67. The RNA content of EVs was determined by fluorescence and by qRT-PCR. Results: After 48h treatment, EVs are released by cells stimulated with medium or H2O2. The fluorescence intensity of pkh67 increased in response to H2O2 (n=2, from 2.6 fold for microvesicles, and 1.4 fold for exosomes) suggesting that epithelial cells release more EVs in response to oxidative stress. In response to H2O2, miR-34a and miR-570 expression is augmented only in exosomes. Conclusion: These data suggest that in response to oxidative stress, epithelial cells produced exosomes enriched in miRNA involved in cellular senescence. This may contribute to COPD pathophysiology by spreading senescence in adjacent epithelial and other cells.

Background: Altered iron metabolism is associated with COPD pathogenesis, and elevated iron levels are observed in the COPD lung. COPD is also associated with elevated levels of senescent cells that contain increased intracellular iron. Whether this is a cause or consequence of senescence is unknown. Aim: To examine the relationship between excess intracellular iron and senescence in COPD. Methods: Total intracellular iron was detected in small airway epithelial cells (SAEC) from non-smokers (NS) and COPD patients by atomic absorbance spectrometry. Heme iron was measured using absorbance at 400 nM. SAEC were treated with ferric ammonium iron citrate (FAC) or the iron chelator, deferoxamine. Iron and senescence markers were detected by western blot. Results: Intracellular iron was increased 1.7-fold (p=0.002) and heme iron 2.1-fold in COPD SAEC compared NS cells (n=6 p=0.008). Treatment of NS and COPD SAEC with FAC induced a 3.65 (p=0.4) and 4.8-fold (p=0.02) increase in iron respectively, with COPD SAEC accumulating significantly more iron than NS cells (n=6 p=0.04). Iron chelation significantly reduced intracellular iron in COPD, but not NS SAEC (n=4 p=0.02). Elevated expression of transferrin heavy chain and transferrin receptor were detected in COPD SAEC compared to NS, correlating with alterations in senescence markers p21 and Sirtuin-1/-6 (n=6). Conclusion: Elevated levels of intracellular iron were observed in COPD SAEC compared to NS, with COPD SAEC having increased capacity to accumulate extracellular iron, possibly via increased transferrin receptor. Altered senescence markers were also seen in COPD, but whether altered iron metabolism regulates this requires further investigation.

Background: In COPD, senescence of small airway fibroblasts (SAF) is closely related to the oxidative imbalance. The SAF secretome is characterised by a pro-inflammatory pattern that may drive small airway fibrosis. Molecular events leading to senescence are not clearly defined but in vitro studies suggest endoplasmic reticulum (ER) stress may be a key event in COPD pathogenesis. Aim: Assess the role of ER stress in SAF senescence. Methods: SAF were isolated from COPD lung tissue and age-matched non-smokers (NS) (n=6). Senescence (p21, γ-H2AX) and ER stress markers (p-IRE1, p-PERK, p-eIF2α, XBP1) were evaluated by Western blot. CXCL-8 and PAI-1 release were measured by ELISA. Co-expression of XBP1 and p21 was studied by immunochemistry. To link ER stress and senescence, SAF were exposed to ISRIB, a selective inhibitor of P-eIF2α and modification of senescence markers as well as the level of antioxidant markers (NRF2 stabilisation, SOD3 expression, catalase activity) were evaluated. Results: At baseline, the expression of p21, γ-H2AX and CXCL-8 were increased in COPD SAF compared to NS (2.6, 3.2 and 2.7-fold-change). Phosphorylation of PERK and eIF2α was only observed in COPD SAF. XBP1 and p21 were co-expressed in the nucleus of ~35% of COPD SAF. ISRIB reduced expression of p21 and γ-H2AX (47% and 63% decrease, p Conclusion: These data imply a role for ER stress in SAF senescence in COPD and suggest p-eIF2α as a new target for therapeutic strategy.

Background: Senescence is a critical cellular process involved in COPD progression. The molecular mechanisms that trigger senescence in small airways fibroblasts (SAF) are not clearly identified. These cells secrete proinflammatory cytokines and resist to apoptosis. In vitro studies suggest endoplasmic reticulum (ER) stress is important in COPD pathogenesis but whether this is linked to senescence is unknown. Aim: Assess the role of ER stress and the related unfolded protein response (UPR) pathways in fibroblast senescence in COPD. Methods: SAF were isolated from peripheral lung tissue from COPD patients and age-matched non-smokers (NS). Expression of senescence markers p21, p16, p-γH2AX and UPR pathway actors p-IRE1, p-PERK, p-eIF2α, ATF4, ATF6α were evaluated by Western blotting or immunocytochemistry. To link ER stress to senescence, protein expression of senescence markers was evaluated in SAF treated with ISRIB, a specific inhibitor of p-eiF2α. Apoptosis was measured using a caspase 3 activity assay in SAF exposed to cigarette smoke extract (CSE) (5%) treated with or without ISRIB. Results: p21, p16 and p-γH2AX were increased in COPD SAF (n=6, 57%, 27% and 62%) as well as UPR markers p-IRE1, p-PERK and p-eIF2α (n=6, 53%, 65% and 63%). COPD SAF showed also an elevated nuclear localization of the UPR transcription factors ATF4 and ATF6α (n=4). ISRIB treatment decreased expression of p21, p16 and p-γH2AX in COPD SAF (n=6). CSE induced more active caspase 3 positive cells in NS (n=2, 43%) than in COPD (n=2, 14%), ISRIB treatment restored the apoptotic effect of CSE in COPD SAF (n=2, 35%). Conclusion: ER stress, especially the p-eIF2α pathway, may play a role in primary SAF senescence in COPD suggesting that this molecular pathway could be targeted for new therapeutic strategies

Introduction: COPD is associated with cellular senescence, hallmarks of which include increased cell size, autofluorescence, senescence associated-β-galactosidase (SA-β-Gal) staining, mitochondrial dysfunction, reduced proliferation and cell cycle inhibitor expression. Dilution of these markers due to proliferation of non-senescent cells makes studying senescence in proliferative cells, such as fibroblasts, challenging. Aim: Assess whether senescent fibroblasts can be isolated from a mixed COPD fibroblast population. Method: Fibroblasts were isolated from COPD patient lung resections (n=6). Cells were sorted using a FACSAria Fusion into a “senescence-enriched population” (largest and most autofluorescence) and a “non-senescent population” (smallest, least autofluorescence). Senescence was investigated in these populations using SA-β-Gal for senescent cell identification, iCELLigence technology to assess proliferation, Seahorse assay to investigate mitochondrial function and RT-qPCR to measure cell cycle inhibitor expression. Results: Senescence-enriched fibroblasts were larger and more autofluorescence and displayed greater SA-β-Gal staining. These cells trended towards reduced proliferation over the first 48h of growth compared to the non-senescent population. Senescence-enriched fibroblasts showed increased basal mitochondrial function (p Conclusion: These data suggest FACS isolate senescent-enriched fibroblasts. This technique can be used to study fibroblast senescence mechanisms and provide therapeutic targets for COPD.

Background: Cellular senescence is one of the hallmarks of COPD. The miR-34a and mir-570 are involved in the induction of cellular senescence by inhibiting anti-ageing sirtuins. Extracellular vesicles (EVs) play a key role in intercellular communication. Senescent cells produce high amounts of EVs and contain miRNA that can regulate cellular senescence. The role of EVs in spreading cell senescence remains unclear. Aim: To characterize extracellular vesicles released by epithelial cells in response to oxidative stress and to assess their effect on epithelial cells senescence. Methods: BEAS-2B bronchial epithelial cells were stimulated with H2O2 for 24 or 48h. Microvesicles and exosomes were isolated by differential ultracentrifugation of cell supernatant and characterised by Western blot and labelling membrane with the dye PKH67. The RNA content of EVs was determined by fluorescence and by qRT-PCR. Results: After 48h treatment, EVs are released by cells stimulated with medium or H2O2. The fluorescence intensity of pkh67 increased in response to H2O2 (n=2, from 2.6 fold for microvesicles, and 1.4 fold for exosomes) suggesting that epithelial cells release more EVs in response to oxidative stress. In response to H2O2, miR-34a and miR-570 expression is augmented only in exosomes. Conclusion: These data suggest that in response to oxidative stress, epithelial cells produced exosomes enriched in miRNA involved in cellular senescence. This may contribute to COPD pathophysiology by spreading senescence in adjacent epithelial and other cells.

Background: Accelerated ageing is associated with COPD, with more senescent cells found within the COPD lung. Elevated oxidative stress is observed in the COPD lung, as well as increased deposition of iron. Whether increased oxidative stress and increased intracellular iron are linked to senescence is unknown. Methods: Immunohistochemistry was performed on human lung sections from controls and COPD patients. Senescent cells were detected by senescence-associated-β-galactosidase (SA-β-Gal) staining. Iron and senescence markers were detected by western blot. Total intracellular iron was detected by atomic absorption spectroscopy (AAS). Results: Elevated levels of p16 and p21 staining were detected in COPD (N=12) lung sections compared to controls (p Conclusion: Increased levels of senescence markers and iron uptake proteins, along with total intracellular iron are seen in cells exposed to oxidative stress. These data are the first linking oxidative stress-induced senescence and iron uptake in airway epithelial cells. However, more work is needed to elucidate whether the uptake of iron induces senescence or is a consequence of senescence.

Super-resolved microscopy techniques have overcome the diffraction limit to provide image resolutions approaching the scale of fluorescent labels. However, many of these techniques require significant experimental resources and expertise and impose long image data acquisition times, making it difficult to acquire super-resolved data from sufficiently large sample numbers to overcome intrinsic biological variation. We have worked to make stimulated emission depletion (STED) microscopy and single molecule localisation microscopy (SMLM) more straightforward to implement and more practical to image larger numbers of cells. Here we present work in progress developing easySLM STED and easySTORM, including a new modular microscope frame that we believe can make it easier to prototype microscopy techniques and to implement and maintain them in lower resourced settings.

Background and Purpose Chronic obstructive pulmonary disease (COPD) is a corticosteroid-resistant airway inflammatory condition. Resveratrol exhibits anti-inflammatory activities in COPD but has weak potency and poor pharmacokinetics. This study aimed to evaluate the potential of isorhapontigenin, another dietary polyphenol, as a novel anti-inflammatory agent for COPD by examining its effects in vitro and pharmacokinetics in vivo. Experimental Approach Primary human airway epithelial cells derived from healthy and COPD subjects, and A549 epithelial cells were incubated with isorhapontigenin or resveratrol and stimulated with IL-1β in the presence or absence of cigarette smoke extract. Effects of isorhapontigenin and resveratrol on the release of IL-6 and chemokine (C-X-C motif) ligand 8 (CXCL8), and the activation of NF-κB, activator protein-1 (AP-1), MAPKs and PI3K/Akt/FoxO3A pathways were determined and compared with those of dexamethasone. The pharmacokinetic profiles of isorhapontigenin, after i.v. or oral administration, were assessed in Sprague–Dawley rats. Key Results Isorhapontigenin concentration-dependently inhibited IL-6 and CXCL8 release, with IC50 values at least twofold lower than those of resveratrol. These were associated with reduced activation of NF-κB and AP-1 and, notably, the PI3K/Akt/FoxO3A pathway, that was relatively insensitive to dexamethasone. In vivo, isorhapontigenin was rapidly absorbed with abundant plasma levels after oral dosing. Its oral bioavailability was approximately 50% higher than resveratrol. Conclusions and Implications Isorhapontigenin, an orally bioavailable dietary polyphenol, displayed superior anti-inflammatory effects compared with resveratrol. Furthermore, it suppressed the PI3K/Akt pathway that is insensitive to corticosteroids. These favourable efficacy and pharmacokinetic properties support its further development as a novel anti-inflammatory agent for COPD.