Changes in lung epithelial cell volatile metabolite profile induced by pro-fibrotic stimulation with TGF-ß1.

Volatile organic compounds (VOCs) have shown promise as potential biomarkers in idiopathic pulmonary fibrosis. Measuring VOCs in the headspace ofin vitromodels of lung fibrosis may offer a method of determining the origin of those detected in exhaled breath. The aim of this study was to determine the VOCs associated with two lung cell lines (A549 and MRC-5 cells) and changes associated with stimulation of cells with the pro-fibrotic cytokine, transforming growth factor (TGF)-ß1. A dynamic headspace sampling method was used to sample the headspace of A549 cells and MRC-5 cells. These were compared to media control samples and to each other to identify VOCs which discriminated between cell lines. Cells were then stimulated with the TGF-ß1 and samples were compared between stimulated and unstimulated cells. Samples were analysed using thermal desorption-gas chromatography-mass spectrometry and supervised analysis was performed using sparse partial least squares-discriminant analysis (sPLS-DA). Supervised analysis revealed differential VOC profiles unique to each of the cell lines and from the media control samples. Significant changes in VOC profiles were induced by stimulation of cell lines with TGF-ß1. In particular, several terpenoids (isopinocarveol, sativene and 3-carene) were increased in stimulated cells compared to unstimulated cells. VOC profiles differ between lung cell lines and alter in response to pro-fibrotic stimulation. Increased abundance of terpenoids in the headspace of stimulated cells may reflect TGF-ß1 cell signalling activity and metabolic reprogramming. This may offer a potential biomarker target in exhaled breath in IPF.

Circadian regulation of pulmonary disease: the importance of timing.

Circadian regulation causes the activity of biological processes to vary over a 24-h cycle. The pathological effects of this variation are predominantly studied using two different approaches: pre-clinical models or observational clinical studies. Both these approaches have provided useful insights into how underlying circadian mechanisms operate and specifically which are regulated by the molecular oscillator, a key time-keeping mechanism in the body. This review compares and contrasts findings from these two approaches in the context of four common respiratory diseases (asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and respiratory infection). Potential methods used to identify and measure human circadian oscillations are also discussed as these will be useful outcome measures in future interventional human trials that target circadian mechanisms.

Morphologically intact airways in lung fibrosis have an abnormal proteome.

Honeycombing is a histological pattern consistent with Usual Interstitial Pneumonia (UIP). Honeycombing refers to cystic airways located at sites of dense fibrosis with marked mucus accumulation. Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the fibrotic honeycomb airway cells and fibrotic uninvolved airway cells (distant from honeycomb airways and morphologically intact) in specimens from 10 patients with UIP. Non-fibrotic airway cell specimens from 6 patients served as controls. Furthermore, we performed LCM-MS on the mucus plugs found in 6 patients with UIP and 6 patients with mucinous adenocarcinoma. The mass spectrometry data were subject to both qualitative and quantitative analysis and validated by immunohistochemistry. Surprisingly, fibrotic uninvolved airway cells share a similar protein profile to honeycomb airway cells, showing deregulation of the slit and roundabout receptor (Slit and Robo) pathway as the strongest category. We find that (BPI) fold-containing family B member 1 (BPIFB1) is the most significantly increased secretome-associated protein in UIP, whereas Mucin-5AC (MUC5AC) is the most significantly increased in mucinous adenocarcinoma. We conclude that fibrotic uninvolved airway cells share pathological features with fibrotic honeycomb airway cells. In addition, fibrotic honeycomb airway cells are enriched in mucin biogenesis proteins with a marked derangement in proteins essential for ciliogenesis. This unbiased spatial proteomic approach generates novel and testable hypotheses to decipher fibrosis progression.

ClinCirc identifies alterations of the circadian peripheral oscillator in critical care patients.

BackgroundAssessing circadian rhythmicity from infrequently sampled data is challenging; however, these types of data are often encountered when measuring circadian transcripts in hospitalized patients.MethodsWe present ClinCirc. This method combines 2 existing mathematical methods (Lomb-Scargle periodogram and cosinor) sequentially and is designed to measure circadian oscillations from infrequently sampled clinical data. The accuracy of this method was compared against 9 other methods using simulated and frequently sampled biological data. ClinCirc was then evaluated in 13 intensive care unit (ICU) patients as well as in a separate cohort of 29 kidney-transplant recipients. Finally, the consequences of circadian alterations were investigated in a retrospective cohort of 726 kidney-transplant recipients.ResultsClinCirc had comparable performance to existing methods for analyzing simulated data or clock transcript expression of healthy volunteers. It had improved accuracy compared with the cosinor method in evaluating circadian parameters in PER2:luc cell lines. In ICU patients, it was the only method investigated to suggest that loss of circadian oscillations in the peripheral oscillator was associated with inflammation, a feature widely reported in animal models. Additionally, ClinCirc was able to detect other circadian alterations, including a phase shift following kidney transplantation that was associated with the administration of glucocorticoids. This phase shift could explain why a significant complication of kidney transplantation (delayed graft dysfunction) oscillates according to the time of day kidney transplantation is performed.ConclusionClinCirc analysis of the peripheral oscillator reveals important clinical associations in hospitalized patients.FundingUK Research and Innovation (UKRI), National Institute of Health Research (NIHR), Engineering and Physical Sciences Research Council (EPSRC), National Institute on Academic Anaesthesia (NIAA), Asthma+Lung UK, Kidneys for Life.

Residual Lung Abnormalities after COVID-19 Hospitalization: Interim Analysis of the UKILD Post-COVID-19 Study.

Rationale: Shared symptoms and genetic architecture between coronavirus disease (COVID-19) and lung fibrosis suggest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may lead to progressive lung damage. Objectives: The UK Interstitial Lung Disease Consortium (UKILD) post-COVID-19 study interim analysis was planned to estimate the prevalence of residual lung abnormalities in people hospitalized with COVID-19 on the basis of risk strata. Methods: The PHOSP-COVID-19 (Post-Hospitalization COVID-19) study was used to capture routine and research follow-up within 240 days from discharge. Thoracic computed tomography linked by PHOSP-COVID-19 identifiers was scored for the percentage of residual lung abnormalities (ground-glass opacities and reticulations). Risk factors in linked computed tomography were estimated with Bayesian binomial regression, and risk strata were generated. Numbers within strata were used to estimate posthospitalization prevalence using Bayesian binomial distributions. Sensitivity analysis was restricted to participants with protocol-driven research follow-up. Measurements and Main Results: The interim cohort comprised 3,700 people. Of 209 subjects with linked computed tomography (median, 119 d; interquartile range, 83-155), 166 people (79.4%) had more than 10% involvement of residual lung abnormalities. Risk factors included abnormal chest X-ray (risk ratio [RR], 1.21; 95% credible interval [CrI], 1.05-1.40), percent predicted DlCO less than 80% (RR, 1.25; 95% CrI, 1.00-1.56), and severe admission requiring ventilation support (RR, 1.27; 95% CrI, 1.07-1.55). In the remaining 3,491 people, moderate to very high risk of residual lung abnormalities was classified at 7.8%, and posthospitalization prevalence was estimated at 8.5% (95% CrI, 7.6-9.5), rising to 11.7% (95% CrI, 10.3-13.1) in the sensitivity analysis. Conclusions: Residual lung abnormalities were estimated in up to 11% of people discharged after COVID-19-related hospitalization. Health services should monitor at-risk individuals to elucidate long-term functional implications.

The UIP/IPF fibroblastic focus is a collagen biosynthesis factory embedded in a distinct extracellular matrix.

Usual interstitial pneumonia (UIP) is a histological pattern characteristic of idiopathic pulmonary fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection-coupled mass spectrometry, we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 nonfibrotic alveolar specimens as controls. The data were subjected to qualitative and quantitative analysis and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared with the nonfibrotic control. Furthermore, FF were positive for both TGFB1 and TGFB3, whereas the aberrant basaloid cell lining of FF was predominantly positive for TGFB2. In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that FF are the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This essential information will inform future mechanistic studies on fibrosis progression.

Understanding the burden of interstitial lung disease post-COVID-19: the UK Interstitial Lung Disease-Long COVID Study (UKILD-Long COVID).

INTRODUCTION: The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD). METHODS AND ANALYSIS: The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment. ETHICS AND DISSEMINATION: All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals. CONCLUSION: This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD.

Shift work is associated with positive COVID-19 status in hospitalised patients.

INTRODUCTION: Shift work is associated with lung disease and infections. We therefore investigated the impact of shift work on significant COVID-19 illness. METHODS: 501 000 UK Biobank participants were linked to secondary care SARS-CoV-2 PCR results from Public Health England. Healthcare worker occupational testing and those without an occupational history were excluded from analysis. RESULTS: Multivariate logistic regression (age, sex, ethnicity and deprivation index) revealed that irregular shift work (OR 2.42, 95% CI 1.92 to 3.05), permanent shift work (OR 2.5, 95% CI 1.95 to 3.19), day shift work (OR 2.01, 95% CI 1.55 to 2.6), irregular night shift work (OR 3.04, 95% CI 2.37 to 3.9) and permanent night shift work (OR 2.49, 95% CI 1.67 to 3.7) were all associated with positive COVID-19 tests compared with participants that did not perform shift work. This relationship persisted after adding sleep duration, chronotype, premorbid disease, body mass index, alcohol and smoking to the model. The effects of workplace were controlled for in three ways: (1) by adding in work factors (proximity to a colleague combined with estimated disease exposure) to the multivariate model or (2) comparing participants within each job sector (non-essential, essential and healthcare) and (3) comparing shift work and non-shift working colleagues. In all cases, shift work was significantly associated with COVID-19. In 2017, 120 307 UK Biobank participants had their occupational history reprofiled. Using this updated occupational data shift work remained associated with COVID-19 (OR 4.48 (95% CI 1.8 to 11.18). CONCLUSIONS: Shift work is associated with a higher likelihood of in-hospital COVID-19 positivity. This risk could potentially be mitigated via additional workplace precautions or vaccination.

Night shift work is associated with an increased risk of asthma.

INTRODUCTION: Shift work causes misalignment between internal circadian time and the external light/dark cycle and is associated with metabolic disorders and cancer. Approximately 20% of the working population in industrialised countries work permanent or rotating night shifts, exposing this large population to the risk of circadian misalignment-driven disease. Analysis of the impact of shift work on chronic inflammatory diseases is lacking. We investigated the association between shift work and asthma. METHODS: We describe the cross-sectional relationship between shift work and prevalent asthma in >280000 UK Biobank participants, making adjustments for major confounding factors (smoking history, ethnicity, socioeconomic status, physical activity, body mass index). We also investigated chronotype. RESULTS: Compared with day workers, 'permanent' night shift workers had a higher likelihood of moderate-severe asthma (OR 1.36 (95% CI 1.03 to 1.8)) and all asthma (OR 1.23 (95% CI 1.03 to 1.46)). Individuals doing any type of shift work had higher adjusted odds of wheeze/whistling in the chest. Shift workers who never or rarely worked on nights and people working permanent nights had a higher adjusted likelihood of having reduced lung function (FEV1 <80% predicted). We found an increase in the risk of moderate-severe asthma in morning chronotypes working irregular shifts, including nights (OR 1.55 (95% CI 1.06 to 2.27)). CONCLUSIONS: The public health implications of these findings are far-reaching due to the high prevalence and co-occurrence of both asthma and shift work. Future longitudinal follow-up studies are needed to determine if modifying shift work schedules to take into account chronotype might present a public health measure to reduce the risk of developing inflammatory diseases such as asthma.

Circadian asthma airway responses are gated by REV-ERBα.

BACKGROUND: The circadian clock powerfully regulates inflammation and the clock protein REV-ERBα is known to play a key role as a repressor of the inflammatory response. Asthma is an inflammatory disease of the airways with a strong time of day rhythm. Airway hyper-responsiveness (AHR) is a dominant feature of asthma; however, it is not known if this is under clock control. OBJECTIVES: To determine if allergy-mediated AHR is gated by the clock protein REV-ERBα. METHODS: After exposure to the intra-nasal house dust mite (HDM) allergen challenge model at either dawn or dusk, AHR to methacholine was measured invasively in mice. MAIN RESULTS: Wild-type (WT) mice show markedly different time of day AHR responses (maximal at dusk/start of the active phase), both in vivo and ex vivo, in precision cut lung slices. Time of day effects on AHR were abolished in mice lacking the clock gene Rev-erbα, indicating that such effects on asthma response are likely to be mediated via the circadian clock. We suggest that muscarinic receptors one (Chrm 1) and three (Chrm 3) may play a role in this pathway. CONCLUSIONS: We identify a novel circuit regulating a core process in asthma, potentially involving circadian control of muscarinic receptor expression, in a REV-ERBα dependent fashion. CLINICAL IMPLICATION: These insights suggest the importance of considering the timing of drug administration in clinic trials and in clinical practice (chronotherapy).

The clock gene Bmal1 inhibits macrophage motility, phagocytosis, and impairs defense against pneumonia.

The circadian clock regulates many aspects of immunity. Bacterial infections are affected by time of day, but the mechanisms involved remain undefined. Here we show that loss of the core clock protein BMAL1 in macrophages confers protection against pneumococcal pneumonia. Infected mice show both reduced weight loss and lower bacterial burden in circulating blood. In vivo studies of macrophage phagocytosis reveal increased bacterial ingestion following Bmal1 deletion, which was also seen in vitro. BMAL1-/- macrophages exhibited marked differences in actin cytoskeletal organization, a phosphoproteome enriched for cytoskeletal changes, with reduced phosphocofilin and increased active RhoA. Further analysis of the BMAL1-/- macrophages identified altered cell morphology and increased motility. Mechanistically, BMAL1 regulated a network of cell movement genes, 148 of which were within 100 kb of high-confidence BMAL1 binding sites. Links to RhoA function were identified, with 29 genes impacting RhoA expression or activation. RhoA inhibition restored the phagocytic phenotype to that seen in control macrophages. In summary, we identify a surprising gain of antibacterial function due to loss of BMAL1 in macrophages, associated with a RhoA-dependent cytoskeletal change, an increase in cell motility, and gain of phagocytic function.

The circadian clock protein REVERBα inhibits pulmonary fibrosis development.

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinß1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Pulmonary airway epithelial cells (AECs) form a critical interface between host and environment. We investigated the role of the circadian clock using mice bearing targeted deletion of the circadian gene brain and muscle ARNT-like 1 (Bmal1) in AECs. Pulmonary neutrophil infiltration, biomechanical function, and responses to influenza infection were all disrupted. A circadian time-series RNA sequencing study of laser-captured AECs revealed widespread disruption in genes of the core circadian clock and output pathways regulating cell metabolism (lipids and xenobiotics), extracellular matrix, and chemokine signaling, but strikingly also the gain of a novel rhythmic transcriptome in Bmal1-targeted cells. Many of the rhythmic components were replicated in primary AECs cultured in air-liquid interface, indicating significant cell autonomy for control of pulmonary circadian physiology. Finally, we found that metabolic cues dictate phasing of the pulmonary clock and circadian responses to immunologic challenges. Thus, the local circadian clock in AECs is vital in lung health by coordinating major cell processes such as metabolism and immunity.-Zhang, Z. Hunter, L., Wu, G., Maidstone, R., Mizoro, Y., Vonslow, R., Fife, M., Hopwood, T., Begley, N., Saer, B., Wang, P., Cunningham, P., Baxter, M., Durrington, H., Blaikley, J. F., Hussell, T., Rattray, M., Hogenesch, J. B., Gibbs, J., Ray, D. W., Loudon, A. S. I. Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Incidence of primary graft dysfunction after lung transplantation is altered by timing of allograft implantation.

The importance of circadian factors in managing patients is poorly understood. We present two retrospective cohort studies showing that lungs reperfused between 4 and 8 AM have a higher incidence (OR 1.12; 95% CI 1.03 to 1.21; p=0.01) of primary graft dysfunction (PGD) in the first 72 hours after transplantation. Cooling of the donor lung, occurring during organ preservation, shifts the donor circadian clock causing desynchrony with the recipient. The clock protein REV-ERBα directly regulates PGD biomarkers explaining this circadian regulation while also allowing them to be manipulated with synthetic REV-ERB ligands.