Effects of Moderate- versus Vigorous-Intensity Exercise Training on Asthma Outcomes in Adults.

BACKGROUND: The therapeutic effects of exercise has prompted calls for it to be embedded into standard asthma care, but evidence informing the optimal exercise intensity is lacking. OBJECTIVE: This study aimed to compare the effects of moderate- and vigorous-intensity aerobic exercise training on asthma outcomes and inflammation. METHODS: This was a 12-week randomised controlled trial in 46 adults with asthma randomised to either 1) 45min moderate-intensity exercise training three times/week, 2) 30min vigorous-intensity exercise training three times/week, or 3) control group. Asthma-related quality of life (AQLQ), asthma control (ACQ), cardiorespiratory fitness, body composition, and airway and systemic inflammation were assessed pre- and post-intervention. RESULTS: Forty-one participants completed the study (89% retention). The moderate-intensity group had a statistically and clinically significant improvement in AQLQ [0.63 (0.33 to 0.93) p<0.001) and ACQ [-0.51 (-0.83 to -0.19), p=0.003] relative to control. The vigorous-intensity group had a statistically, but not clinically, significant improvement in AQLQ [0.46 (0.14 to 0.80, p=0.007] and ACQ [-0.36 (-0.69 to -0.02, p=0.040] relative to control. Following moderate-intensity training, there was a reduction in sputum macrophage [-1341 (-2491 to -191)x104/mL, p=0.024] and lymphocyte [-114 (-220 to -8)x104/mL, p=0.036] counts relative to control. A reduction in android fat mass, but not a change in fitness, was associated with improved AQLQ (rs=-0.341, p=0.030) and reduced sputum IL-6 (rs=0.422, p=0.013). CONCLUSION: Our findings suggest that both moderate-intensity and vigorous-intensity aerobic exercise training are associated with improvements in clinical asthma outcomes, and therefore both intensities could be recommended as an adjuvant asthma therapy.

Airway remodelling in asthma and the epithelium: on the edge of a new era.

Asthma is a chronic, heterogeneous disease of the airways, often characterised by structural changes known collectively as airway remodelling. In response to environmental insults, including pathogens, allergens and pollutants, the epithelium can initiate remodelling via an inflammatory cascade involving a variety of mediators that have downstream effects on both structural and immune cells. These mediators include the epithelial cytokines thymic stromal lymphopoietin, interleukin (IL)-33 and IL-25, which facilitate airway remodelling through cross-talk between epithelial cells and fibroblasts, and between mast cells and airway smooth muscle cells, as well as through signalling with immune cells such as macrophages. The epithelium can also initiate airway remodelling independently of inflammation in response to the mechanical stress present during bronchoconstriction. Furthermore, genetic and epigenetic alterations to epithelial components are believed to influence remodelling. Here, we review recent advances in our understanding of the roles of the epithelium and epithelial cytokines in driving airway remodelling, facilitated by developments in genetic sequencing and imaging techniques. We also explore how new and existing therapeutics that target the epithelium and epithelial cytokines could modify airway remodelling.

Alveolar epithelial cells of lung fibrosis patients are susceptible to severe virus-induced injury.

Patients with pulmonary fibrosis (PF) often experience exacerbations of their disease, characterised by a rapid, severe deterioration in lung function that is associated with high mortality. Whilst the pathobiology of such exacerbations is poorly understood, virus infection is a trigger. The present study investigated virus-induced injury responses of alveolar and bronchial epithelial cells (AECs and BECs, respectively) from patients with PF and age-matched controls (Ctrls). Air-liquid interface (ALI) cultures of AECs, comprising type I and II pneumocytes or BECs were inoculated with influenza A virus (H1N1) at 0.1 multiplicity of infection (MOI). Levels of interleukin-6 (IL-6), IL-36γ and IL-1ß were elevated in cultures of AECs from PF patients (PF-AECs, n = 8-11), being markedly higher than Ctrl-AECs (n = 5-6), 48 h post inoculation (pi) (P<0.05); despite no difference in H1N1 RNA copy numbers 24 h pi. Furthermore, the virus-induced inflammatory responses of PF-AECs were greater than BECs (from either PF patients or controls), even though viral loads in the BECs were overall 2- to 3-fold higher than AECs. Baseline levels of the senescence and DNA damage markers, nuclear p21, p16 and H2AXγ were also significantly higher in PF-AECs than Ctrl-AECs and further elevated post-infection. Senescence induction using etoposide augmented virus-induced injuries in AECs (but not viral load), whereas selected senotherapeutics (rapamycin and mitoTEMPO) were protective. The present study provides evidence that senescence increases the susceptibility of AECs from PF patients to severe virus-induced injury and suggests targeting senescence may provide an alternative option to prevent or treat the exacerbations that worsen the underlying disease.

Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) Samples from Advanced Non-Small Cell Lung Cancer for Whole Genome, Whole Exome and Comprehensive Panel Sequencing.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often the only source of tumor tissue from patients with advanced, inoperable lung cancer. EBUS-TBNA aspirates are used for the diagnosis, staging, and genomic testing to inform therapy options. Here we extracted DNA and RNA from 220 EBUS-TBNA aspirates to evaluate their suitability for whole genome (WGS), whole exome (WES), and comprehensive panel sequencing. For a subset of 40 cases, the same nucleic acid extraction was sequenced using WGS, WES, and the TruSight Oncology 500 assay. Genomic features were compared between sequencing platforms and compared with those reported by clinical testing. A total of 204 aspirates (92.7%) had sufficient DNA (100 ng) for comprehensive panel sequencing, and 109 aspirates (49.5%) had sufficient material for WGS. Comprehensive sequencing platforms detected all seven clinically reported tier 1 actionable mutations, an additional three (7%) tier 1 mutations, six (15%) tier 2-3 mutations, and biomarkers of potential immunotherapy benefit (tumor mutation burden and microsatellite instability). As expected, WGS was more suited for the detection and discovery of emerging novel biomarkers of treatment response. WGS could be performed in half of all EBUS-TBNA aspirates, which points to the enormous potential of EBUS-TBNA as source material for large, well-curated discovery-based studies for novel and more effective predictors of treatment response. Comprehensive panel sequencing is possible in the vast majority of fresh EBUS-TBNA aspirates and enhances the detection of actionable mutations over current clinical testing.

Treatment of idiopathic pulmonary fibrosis and progressive pulmonary fibrosis: A position statement from the Thoracic Society of Australia and New Zealand 2023 revision.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease leading to significant morbidity and mortality. In 2017 the Thoracic Society of Australia and New Zealand (TSANZ) and Lung Foundation Australia (LFA) published a position statement on the treatment of IPF. Since that time, subsidized anti-fibrotic therapy in the form of pirfenidone and nintedanib is now available in both Australia and New Zealand. More recently, evidence has been published in support of nintedanib for non-IPF progressive pulmonary fibrosis (PPF). Additionally, there have been numerous publications relating to the non-pharmacologic management of IPF and PPF. This 2023 update to the position statement for treatment of IPF summarizes developments since 2017 and reaffirms the importance of a multi-faceted approach to the management of IPF and progressive pulmonary fibrosis.

Ventilation Heterogeneity Is a Treatable Trait in Severe Asthma.

BACKGROUND: Ventilation heterogeneity (VH) is a feature of asthma and indicates small airway disease. Nuclear imaging methods assess VH, which can facilitate clinical diagnosis and further our understanding of disease aetiology. OBJECTIVE: We sought to assess VH in severe eosinophilic asthma (SEA) using ventilation/perfusion single-photon emission computed tomography (V/P SPECT), and to assess its use as an objective test of the effect of biologic treatment for ventilation defects in SEA. METHODS: Adults (≥18 y) with severe asthma were recruited to participate in a cross-sectional observational study. Participants underwent a clinical assessment and V/P SPECT CT using Technegas as the ventilation agent. Measures were repeated for a nested before-after treatment study in people with SEA commencing biologics. RESULTS: A total of 62 participants with severe asthma were recruited. From this, 38 participants with SEA were included in the before-after study. The VH was associated with clinical variables such as lung function impairment and significantly improved after monoclonal antibody treatment in the severe asthma group. The changes in VH correlated with change in post bronchodilator forced expiratory volume in 1 second (FEV1) %predicted (r = -0.503; P = .001) and post bronchodilator FEV1/FVC (forced vital capacity) (r = -0.415; P = .01). CONCLUSIONS: The VH is clinically significant, measurable, and treatable, which establishes VH as a treatable trait in severe asthma.

Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells.

Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)-(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions.

Mechanical forces suppress antiviral innate immune responses from asthmatic airway epithelial cells following rhinovirus infection.

Bronchoconstriction is the main physiological event in asthma, which leads to worsened clinical symptoms and generates mechanical stress within the airways. Virus infection is the primary cause of exacerbations in people with asthma, however, the impact that bronchoconstriction itself on host antiviral responses and viral replication is currently not well understood. Here we demonstrate how mechanical forces generated during bronchoconstriction may suppress antiviral responses at the airway epithelium without any difference in viral replication. Primary bronchial epithelial cells from donors with asthma were differentiated at the air-liquid interface. Differentiated cells were apically compressed (30 cmH2O) for 10 min every hour for 4 days to mimic bronchoconstriction. Two asthma disease models were developed with the application of compression, either before ("poor asthma control model," n = 7) or following ("exacerbation model," n = 4) rhinovirus (RV) infection. Samples were collected at 0, 24, 48, 72, and 96 h postinfection (hpi). Viral RNA, interferon (IFN)-ß, IFN-λ, and host defense antiviral peptide gene expressions were measured along with IFN-ß, IFN-λ, TGF-ß2, interleukin-6 (IL-6), and IL-8 protein expression. Apical compression significantly suppressed RV-induced IFN-ß protein from 48 hpi and IFN-λ from 72 hpi in the poor asthma control model. There was a nonsignificant reduction of both IFN-ß and IFN-λ proteins from 48 hpi in the exacerbation model. Despite reductions in antiviral proteins, there was no significant change in viral replication in either model. Compressive stress mimicking bronchoconstriction inhibits antiviral innate immune responses from asthmatic airway epithelial cells when applied before RV infection.NEW & NOTEWORTHY Bronchoconstriction is the main physiological event in asthma, which leads to worsened clinical symptoms and generates mechanical stress within the airways. Virus infection is the primary cause of exacerbations in people with asthma, however, the impact of bronchoconstriction on host antiviral responses and viral replication is unknown. We developed two disease models, in vitro, and found suppressed IFN response from cells following the application of compression and RV-A1 infection. This explains why people with asthma have deficient IFN response.

Inhaled pirfenidone solution (AP01) for IPF: a randomised, open-label, dose-response trial.

INTRODUCTION: Oral pirfenidone reduces lung function decline and mortality in patients with idiopathic pulmonary fibrosis (IPF). Systemic exposure can have significant side effects, including nausea, rash, photosensitivity, weight loss and fatigue. Reduced doses may be suboptimal in slowing disease progression. METHODS: This phase 1b, randomised, open-label, dose-response trial at 25 sites in six countries (Australian New Zealand Clinical Trials Registry (ANZCTR) registration number ACTRN12618001838202) assessed safety, tolerability and efficacy of inhaled pirfenidone (AP01) in IPF. Patients diagnosed within 5 years, with forced vital capacity (FVC) 40%-90% predicted, and intolerant, unwilling or ineligible for oral pirfenidone or nintedanib were randomly assigned 1:1 to nebulised AP01 50 mg once per day or 100 mg two times per day for up to 72 weeks. RESULTS: We present results for week 24, the primary endpoint and week 48 for comparability with published trials of antifibrotics. Week 72 data will be reported as a separate analysis pooled with the ongoing open-label extension study. Ninety-one patients (50 mg once per day: n=46, 100 mg two times per day: n=45) were enrolled from May 2019 to April 2020. The most common treatment-related adverse events (frequency, % of patients) were all mild or moderate and included cough (14, 15.4%), rash (11, 12.1%), nausea (8, 8.8%), throat irritation (5, 5.5%), fatigue (4, 4.4%) and taste disorder, dizziness and dyspnoea (three each, 3.3%). Changes in FVC % predicted over 24 and 48 weeks, respectively, were -2.5 (95% CI -5.3 to 0.4, -88 mL) and -4.9 (-7.5 to -2.3,-188 mL) in the 50 mg once per day and 0.6 (-2.2 to 3.4, 10 mL) and -0.4 (-3.2 to 2.3, -34 mL) in the 100 mg two times per day group. DISCUSSION: Side effects commonly associated with oral pirfenidone in other clinical trials were less frequent with AP01. Mean FVC % predicted remained stable in the 100 mg two times per day group. Further study of AP01 is warranted. TRIAL REGISTRATION NUMBER: ACTRN12618001838202 Australian New Zealand Clinical Trials Registry.

Randomised controlled trial for the titration of oral corticosteroids using markers of inflammation in severe asthma.

INTRODUCTION: Biomarkers are used to select biologic therapies for patients with severe asthma, but not to regularly adjust therapy, especially oral corticosteroids (OCS). OBJECTIVE: Our goal was to test the efficacy of an algorithm to guide the titration of OCS using blood eosinophil count and fraction of exhaled nitric oxide (FeNO) levels. DESIGN, PARTICIPANTS, INTERVENTIONS AND SETTING: This proof-of-concept prospective randomised controlled trial assigned adult participants with severe uncontrolled asthma (n=32) to biomarker-based management (BBM) where OCS dose was adjusted based on a composite biomarker score comprised of blood eosinophil count and FeNO, or a standard best practice (SBP) arm. The study was conducted at the Hunter Medical Research Institute, Newcastle, Australia. Participants were recruited from the local Severe Asthma Clinic and were blinded to their study allocation. MAIN OUTCOME: The coprimary outcomes were number of severe exacerbations and time to first severe exacerbation assessed over 12 months. RESULTS: There was a longer median time to first severe exacerbation with BBM, although not significant (295 vs 123 days, Adj. HR: 0.714; 95% CI: 0.25 to 2.06; p=0.533). The relative risk of a severe exacerbation in BBM (n=17) vs SBP (n=15) was 0.88 (Adj.; 95% CI: 0.47 to 1.62; p=0.675) with a mean exacerbation rate per year of 1.2 and 2.0, respectively. There was a significant reduction in the proportion of patients requiring an emergency department (ED) visit using BBM (OR 0.09, 95% CI: 0.01 to 0.91; p=0.041). There was no difference in the cumulative OCS dose used between the two groups. CONCLUSION: A treatment algorithm to adjust OCS using blood eosinophil count and FeNO is feasible in a clinical setting and resulted in a reduced odds of an ED visit. This warrants further study to optimise the use of OCS in the future. TRIAL REGISTRATION NUMBER: This trial was registered with the Australia and New Zealand Clinical Trials Registry (ACTRN12616001015437).

Conditionally reprogrammed asthmatic bronchial epithelial cells express lower FOXJ1 at terminal differentiation and lower IFNs following RV-A1 infection.

Primary bronchial epithelial cells (pBECs) obtained from donors have limited proliferation capacity. Recently, conditional reprogramming (CR) technique has overcome this and has provided the potential for extended passaging and subsequent differentiation of cells at air-liquid interface (ALI). However, there has been no donor-specific comparison of cell morphology, baseline gene expression, barrier function, and antiviral responses compared with their "parent" pBECs, especially cells obtained from donors with asthma. We, therefore, collected and differentiated pBECs at ALI from mild donors with asthma (n = 6) for the parent group. The same cells were conditionally reprogrammed and later differentiated at ALI. Barrier function was measured during the differentiation phase. Morphology and baseline gene expression were compared at terminal differentiation. Viral replication kinetics and antiviral responses were assessed following rhinovirus (RV) infection over 96 h. Barrier function during the differentiation phase and cell structural morphology at terminal differentiation appear similar in both parent and CR groups, however, there were elongated cell structures superficial to basal cells and significantly lower FOXJ1 expression in CR group. IFN gene expression was also significantly lower in CR group compared with parent asthma group following RV infection. The CR technique is a beneficial tool to proliferate pBECs over extended passages. Considering lower FOXJ1 expression, viral replication kinetics and antiviral responses, a cautious approach should be taken while choosing CR cells for experiments. In addition, as lab-to-lab cell culture techniques vary, the most appropriate technique must be utilized to best match individual cell functions and morphologies to address specific research questions and experimental reproducibility across the labs.

Quantitative computed tomography predicts outcomes in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Prediction of disease course in patients with progressive pulmonary fibrosis remains challenging. The purpose of this study was to assess the prognostic value of lung fibrosis extent quantified at computed tomography (CT) using data-driven texture analysis (DTA) in a large cohort of well-characterized patients with idiopathic pulmonary fibrosis (IPF) enrolled in a national registry. METHODS: This retrospective analysis included participants in the Australian IPF Registry with available CT between 2007 and 2016. CT scans were analysed using the DTA method to quantify the extent of lung fibrosis. Demographics, longitudinal pulmonary function and quantitative CT metrics were compared using descriptive statistics. Linear mixed models, and Cox analyses adjusted for age, gender, BMI, smoking history and treatment with anti-fibrotics were performed to assess the relationships between baseline DTA, pulmonary function metrics and outcomes. RESULTS: CT scans of 393 participants were analysed, 221 of which had available pulmonary function testing obtained within 90 days of CT. Linear mixed-effect modelling showed that baseline DTA score was significantly associated with annual rate of decline in forced vital capacity and diffusing capacity of carbon monoxide. In multivariable Cox proportional hazard models, greater extent of lung fibrosis was associated with poorer transplant-free survival (hazard ratio [HR] 1.20, p < 0.0001) and progression-free survival (HR 1.14, p < 0.0001). CONCLUSION: In a multi-centre observational registry of patients with IPF, the extent of fibrotic abnormality on baseline CT quantified using DTA is associated with outcomes independent of pulmonary function.

Deep Learning-based Outcome Prediction in Progressive Fibrotic Lung Disease Using High-Resolution Computed Tomography.

Rationale: Reliable outcome prediction in patients with fibrotic lung disease using baseline high-resolution computed tomography (HRCT) data remains challenging. Objectives: To evaluate the prognostic accuracy of a deep learning algorithm (SOFIA [Systematic Objective Fibrotic Imaging Analysis Algorithm]), trained and validated in the identification of usual interstitial pneumonia (UIP)-like features on HRCT (UIP probability), in a large cohort of well-characterized patients with progressive fibrotic lung disease drawn from a national registry. Methods: SOFIA and radiologist UIP probabilities were converted to Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED)-based UIP probability categories (UIP not included in the differential, 0-4%; low probability of UIP, 5-29%; intermediate probability of UIP, 30-69%; high probability of UIP, 70-94%; and pathognomonic for UIP, 95-100%), and their prognostic utility was assessed using Cox proportional hazards modeling. Measurements and Main Results: In multivariable analysis adjusting for age, sex, guideline-based radiologic diagnosis, anddisease severity (using total interstitial lung disease [ILD] extent on HRCT, percent predicted FVC, DlCO, or the composite physiologic index), only SOFIA UIP probability PIOPED categories predicted survival. SOFIA-PIOPED UIP probability categories remained prognostically significant in patients considered indeterminate (n = 83) by expert radiologist consensus (hazard ratio, 1.73; P < 0.0001; 95% confidence interval, 1.40-2.14). In patients undergoing surgical lung biopsy (n = 86), after adjusting for guideline-based histologic pattern and total ILD extent on HRCT, only SOFIA-PIOPED probabilities were predictive of mortality (hazard ratio, 1.75; P < 0.0001; 95% confidence interval, 1.37-2.25). Conclusions: Deep learning-based UIP probability on HRCT provides enhanced outcome prediction in patients with progressive fibrotic lung disease when compared with expert radiologist evaluation or guideline-based histologic pattern. In principle, this tool may be useful in multidisciplinary characterization of fibrotic lung disease. The utility of this technology as a decision support system when ILD expertise is unavailable requires further investigation.

IL-25 blockade augments antiviral immunity during respiratory virus infection.

IL-25 is implicated in the pathogenesis of viral asthma exacerbations. However, the effect of IL-25 on antiviral immunity has yet to be elucidated. We observed abundant expression and colocalization of IL-25 and IL-25 receptor at the apical surface of uninfected airway epithelial cells and rhinovirus infection increased IL-25 expression. Analysis of immune transcriptome of rhinovirus-infected differentiated asthmatic bronchial epithelial cells (BECs) treated with an anti-IL-25 monoclonal antibody (LNR125) revealed a re-calibrated response defined by increased type I/III IFN and reduced expression of type-2 immune genes CCL26, IL1RL1 and IL-25 receptor. LNR125 treatment also increased type I/III IFN expression by coronavirus infected BECs. Exogenous IL-25 treatment increased viral load with suppressed innate immunity. In vivo LNR125 treatment reduced IL-25/type 2 cytokine expression and increased IFN-ß expression and reduced lung viral load. We define a new immune-regulatory role for IL-25 that directly inhibits virus induced airway epithelial cell innate anti-viral immunity.

TLR7 agonist loaded airway epithelial targeting nanoparticles stimulate innate immunity and suppress viral replication in human bronchial epithelial cells.

Nanoparticle-based delivery is a strategy for increasing the therapeutic window of inhaled immunomodulatory drugs that have inflammatory activity. TLR7 agonists are a class of immunomodulators that have been considered for the treatment of virus-induced respiratory diseases. However, due to high immune-stimulatory activity, TLR7 agonists, delivered via direct exposure, generally have a narrow therapeutic window. To address this, we have developed lipid/polymer hybrid nanoparticles (NPs) conjugated with anti-EpCAM monoclonal antibody for targeted delivery of TLR7 agonist (CL264) to airway epithelial cells (AECs)2 - the primary site of respiratory virus infection. These airway epithelial targeting nanoparticles (AEC-NPs)3 showed safety and biocompatibility, and approximately two-fold increased cellular uptake compared to non-targeting NPs. Upon cell entry, AEC-NPs were able to deliver CL264 to cytoplasm and endosomes where TLR7 is located. CL264 delivered by AEC-NPs significantly increased innate immune response through expression of IFN-ß, IFN-λ 2/3 and IFN-stimulated genes and suppressed more than 92% of viral load at 48 h post-infection compared to the drug alone and non-targeting NPs. In conclusion, AEC-NPs exhibited increased cellular uptake leading to enhanced innate immune activation and suppression of viral replication. These findings support the use of AEC-targeting approach for delivering drugs with a narrow therapeutic window.

Biomarker signatures for progressive idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease in which circulatory biomarkers have the potential for guiding management in clinical practice. We assessed the prognostic role of serum biomarkers in three independent IPF cohorts: Australian Idiopathic Pulmonary Fibrosis Registry (AIPFR), Trent Lung Fibrosis (TLF) and Prospective Observation of Fibrosis in the Lung Clinical Endpoints (PROFILE). METHODS: In the AIPFR cohort, candidate proteins were assessed by ELISA as well as in an unbiased proteomic approach. LASSO (least absolute shrinkage and selection operator) regression was used to restrict the selection of markers that best accounted for the progressor phenotype at 1 year in the AIPFR cohort, and subsequently prospectively selected for replication in the validation TLF cohort and assessed retrospectively in the PROFILE cohort. Four significantly replicating biomarkers were aggregated into a progression index model based on tertiles of circulating concentrations. RESULTS: 189 participants were included in the AIPFR cohort, 205 participants from the TLF cohort and 122 participants from the PROFILE cohort. Differential biomarker expression was observed by ELISA and replicated for osteopontin, matrix metallopeptidase-7, intercellular adhesion molecule-1 and periostin for those with a progressor phenotype at 1 year. Proteomic data did not replicate. The progression index in the AIPFR, TLF and PROFILE cohorts predicted risk of progression, mortality and progression-free survival. A statistical model incorporating the progression index demonstrated the capacity to distinguish disease progression at 12 months, which was increased beyond the clinical GAP (gender, age and physiology) score model alone in all cohorts, and significantly so within the incidence-based TLF and PROFILE cohorts. CONCLUSION: A panel of circulatory biomarkers can provide potentially valuable clinical assistance in the prognosis of IPF patients.

TELO-SCOPE study: a randomised, double-blind, placebo-controlled, phase 2 trial of danazol for short telomere related pulmonary fibrosis.

INTRODUCTION: Recent discoveries have identified shortened telomeres and related mutations in people with pulmonary fibrosis (PF). There is evidence to suggest that androgens, including danazol, may be effective in lengthening telomeres in peripheral blood cells. This study aims to assess the safety and efficacy of danazol in adults and children with PF associated with telomere shortening. METHODS AND ANALYSIS: A multi-centre, double-blind, placebo-controlled, randomised trial of danazol will be conducted in subjects aged >5 years with PF associated with age-adjusted telomere length ≤10th centile measured by flow fluorescence in situ hybridisation; or in children, a diagnosis of dyskeratosis congenita. Adult participants will receive danazol 800 mg daily in two divided doses or identical placebo capsules orally for 12 months, in addition to standard of care (including pirfenidone or nintedanib). Paediatric participants will receive danazol 2 mg/kg/day orally in two divided doses or identical placebo for 6 months. If no side effects are encountered, the dose will be escalated to 4 mg/kg/day (maximum 800 mg daily) orally in two divided doses for a further 6 months. The primary outcome is change in absolute telomere length in base pairs, measured using the telomere shortest length assay (TeSLA), at 12 months in the intention to treat population. ETHICS AND DISSEMINATION: Ethics approval has been granted in Australia by the Metro South Human Research Ethics Committee (HREC/2020/QMS/66385). The study will be conducted and reported according to Standard Protocol Items: Recommendations for Interventional Trials guidelines. Results will be published in peer-reviewed journals and presented at international and national conferences. TRIAL REGISTRATION NUMBERS: NCT04638517; Australian New Zealand Clinical Trials Registry (ACTRN12620001363976p).

A Senescence Bystander Effect in Human Lung Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a dense fibrosing of the lung parenchyma. An association between IPF and cellular senescence is well established and several studies now describe a higher abundance of senescent fibroblasts and epithelial cells in the lungs of IPF patients compared with age-matched controls. The cause of this abnormal accumulation of senescent cells is unknown but evidence suggests that, once established, senescence can be transferred from senescent to non-senescent cells. In this study, we investigated whether senescent human lung fibroblasts (LFs) and alveolar epithelial cells (AECs) could induce a senescent-like phenotype in "naïve" non-senescent LFs in vitro. Primary cultures of LFs from adult control donors (Ctrl-LFs) with a low baseline of senescence were exposed to conditioned medium (CM) from: (i) Ctrl-LFs induced to become senescent using H2O2 or etoposide; (ii) LFs derived from IPF patients (IPF-LFs) with a high baseline of senescence; or (iii) senescence-induced A549 cells, an AEC line. Additionally, ratios of non-senescent Ctrl-LFs and senescence-induced Ctrl-LFs (100:0, 0:100, 50:50, 90:10, 99:1) were co-cultured and their effect on induction of senescence measured. We demonstrated that exposure of naïve non-senescent Ctrl-LFs to CM from senescence-induced Ctrl-LFs and AECs and IPF-LFs increased the markers of senescence including nuclear localisation of phosphorylated-H2A histone family member X (H2AXγ) and expression of p21, IL-6 and IL-8 in Ctrl-LFs. Additionally, co-cultures of non-senescent and senescence-induced Ctrl-LFs induced a senescent-like phenotype in the non-senescent cells. These data suggest that the phenomenon of "senescence-induced senescence" can occur in vitro in primary cultures of human LFs, and provides a possible explanation for the abnormal abundance of senescent cells in the lungs of IPF patients.

A cGAS-dependent response links DNA damage and senescence in alveolar epithelial cells: a potential drug target in IPF.

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5-7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF.