RATIONALE: Survivors of bronchopulmonary dysplasia (BPD) – a chronic lung disease affecting preterm infants – have a reduced lung function and a greater susceptibility to respiratory viruses, yet the mechanism behind this susceptibility remains poorly understood. Whilst the pathophysiology of BPD is multifactorial, it is underpinned by pulmonary inflammation to which supplemental oxygen therapy contributes. To investigate how BPD affects the host response to influenza A virus (IAV) infection in a preclinical model, we induced BPD by subjecting mice to antenatal inflammation and postnatal hyperoxia before subsequent infection with the IAV strain HKx31 (H3N2). METHODS: Briefly, pregnant dams were injected intraperitoneally on E14 with 150 μg/kg of lipopolysaccharide. Within 24 hours of birth, pups were randomized and exposed to hyperoxia (65% oxygen) continuously for 4 weeks, which induces emphysematous changes in alveolar structure representative of BPD. Control pups were reared in room air (21% oxygen). Subsequently, all pups recovered in room air for 2 weeks, before being inoculated intranasally with 104 plaque forming units (PFU) of HKx31 (H3N2) IAV or saline. Three days post infection, bronchoalveolar lavage was collected for analysis of immune cell infiltrates and the lungs were collected for analysis of alveolar and airway morphology. RESULTS: Early life exposure to BPD altered the innate immune cell subsets within the airways at day 3 following IAV infection, including a 2.6-fold increased abundance of CD3+ T cells compared to air IAV mice (P<0.0001; unpaired t-test). Increased dendritic cells, neutrophils and natural killer cells were also observed. Mice with BPD also had increased airway epithelial thickness following infection compared to infected air controls. CONCLUSION: Early life BPD leads to increased recruitment of inflammatory cells into the lung during following IAV infection, which may contribute to exacerbated pathology and poorer resistance to respiratory viral infections seen in BPD patients.

Acute respiratory distress syndrome (ARDS) results in decreased quality of life, including increased risk of pulmonary hypertension (PH). In animal models, ARDS can be induced by LPS, which can disrupt the pulmonary endothelium and epithelium and induce inflammation. We tested whether in vivo administration or ex vivo treatment with LPS alters the reactivity of intrapulmonary arteries and airways to constrictors relevant to both ARDS and PH, using the precision-cut lung slice (PCLS) technique. Mice were administered LPS (10 μg/50 μl, intranasal) or saline daily for 4 days before collection of BAL fluid or preparation of PCLSs. Alternatively, PCLSs from naive mice were left untreated or treated ex vivo with LPS (10 μg/ml) or TNF (10 ng/ml) for 18 hours. Contraction to endothelin-1, U46619 (a stable mimetic of thromboxane A2), or serotonin was quantified. In vivo LPS administration increased BAL total inflammatory cells 5-fold, neutrophils 125-fold, and protein 2-fold, as well as the thickness of the pulmonary arterial smooth muscle layer. After in vivo LPS, contraction of intrapulmonary arteries in PCLSs to endothelin-1 and U46619, but not serotonin, increased, whereas bronchoconstrictor responses were unchanged. In PCLSs treated with LPS ex vivo, these differential effects on pulmonary artery and airway contraction were maintained. Although LPS increased TNF secretion from PCLSs, TNF treatment only increased U46619-induced vasoconstriction. This study demonstrates the potential contributions of LPS-induced inflammation and vascular remodeling to altered intrapulmonary artery reactivity to specific agonists, with implications for ARDS-associated PH.

RATIONALE Global warming has increased the prevalence of bushfires/wildfires around the world. It is estimated 50% of the population are exposed to air pollution from open fires and wood-burning, causing up to 1.5 million premature deaths globally each year. Particulate Matter (PM) and Polycyclic Aromatic Hydrocarbons (PAHs) are toxins released from bushfire smoke which can be inhaled. The injurious or toxic effects of inhaled PMs and/or PAHs are understudied. We investigated the harmful effects of PMs and/or PAHs from bushfire smoke exposure using precision cut lung slices (PCLS). We aimed to demonstrate size-dependent effects of PMs (1, 2.5, 10 μm) and injurious effects of PM and/or PAHs in human and mouse PCLS, respectively. METHODS PCLS generated from human lung resections (hPCLS, 500μm, N=3) were treated with media (control), PM1, PM2.5, or PM10 (5μg/ml) every 24h for 72h. Mouse PCLS generated from C57/BL6 mice (mPCLS, 300μm, N=5-8) were treated with media, LPS (10ng/mL; positive control), PM1 or PM2.5 (5μg/ml); one or all three PAHs (Fluoranthene, Phenanthrene, Pyrene) at 100, 500, 1000μM; or PM1 or PM2.5 with combination PAHs every 24h for 48h. Viability (MTT assay), senescence (immunohistochemistry for p21), cytokine secretion for IL-6, IL-8/KC, TNF-α, and IL-1β (ELISAs) we assessed. RESULTS PM exposure did not impact viability or induce cytokine secretion in either hPCLS or mPCLS. Exposure to PM2.5 and PM10, but not PM1, increased senescence >2-fold in hPCLSat 72h (p<0.05 cf untreated). Combined PAHs (500 or 1000μM) caused cell death in mPCLS at 48 h (p<0.05 and 0.01 cf untreated, respectively). In mPCLS, KC(IL-8) secretion was induced by PAHs alone and in combination, with phenanthrene more potent than Fluoranthene and Pyrene. Secretion of other cytokines (IL-6, IL-1β, TNF-⍺) from PAH-treated mPCLS was not detectable, and KC(IL-8) secretion in response to PAHs was not further increased by the addition of PMs. CONCLUSION This is the first study to use translational PCLS models to investigate the injurious effects of bushfire-derived PMs and PAHs. PMs induced a size-dependent increase in cellular senescence in hPCLS. Phenanthrene was the most potent inflammatory PAH tested, selectively increasing IL-8 secretion from mPCLS suggestive of PAH-driven neutrophilic inflammation. Future studies should test both lower PAH concentrations and natural PAH sources (ie. bushfire-smoked media) to validate their contributions to bushfire smoke-induced lung injury. Validation of the deleterious effects of PMs and PAHs on lung health would provide a strong public health message for forestry management in bushfire prone areas.

Precision-cut lung slices (PCLS) are gaining traction as a versatile ex vivo tool to study mechanisms and treatments for lung diseases. This preparation, in which the major structural elements of the native lung are preserved, bridges the gap between cell and in vivo models allowing researchers to assess integrated functional responses including smooth muscle reactivity, inflammation and tissue remodelling. To date, the application of PCLS to study outcomes relevant to diseases affecting the pulmonary vasculature, such as pulmonary hypertension, is relatively limited compared to those focussed on chronic airway or interstitial lung diseases. This review explores the specific technical requirements for the preparation of PCLS with viable, patent pulmonary arteries, and their application for investigation of mechanisms and treatments related to pulmonary hypertension. Studies characterising vascular responses to contractile agonists in PCLS, particularly in the context of disease-relevant stimuli and models are described, as well as the use of PCLS for the identification of novel vasodilators. This article also outlines current research to prolong PCLS viability and provides directions for future PCLS studies to investigate inflammation and vascular remodelling, with a view to identify therapeutics that address the current limitations of dilator-only treatment of pulmonary hypertension. Overall, the review highlights the importance of PCLS for mechanistic studies and drug development. While PCLS are currently underutilised in the context of pulmonary hypertension, the evidence provided here of the multifaceted functional outcomes that can be investigated using PCLS supports their wider application for understanding disease pathophysiology and validating novel therapeutics.

Idiopathic pulmonary fibrosis (IPF) is a fatal condition associated with excessive interstitial collagen accumulation and irreversible lung function decline, for which there is no effective cure. Hence, this study evaluated the dose-dependent anti-fibrotic effects of bone marrow mesenchymal stem cell-derived extracellular vesicles (BM-MSC-EVs) in transforming growth factor (TGF)-β1-stimulated human dermal myofibroblasts (1 ×107-1 ×1010 BM-MSC-EVs) and TGF-β1-stimulated lung myofibroblasts isolated from non-IPF versus and IPF patients (1 ×106-1 ×108 BM-MSC-EVs) after 72 h in culture; and when intranasally-administered therapeutically (from days 21-28 post-injury) to bleomycin (BLM)-injured mice (2.5 ×108-2.5 ×1010 BM-MSC-EVs). In each case, changes in myofibroblast differentiation, collagen I deposition, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 levels, and MMP-2:TIMP-2 and MMP-9:TIMP-1 ratios were assessed. BM-MSC-EVs significantly attenuated human dermal and non-IPF patient-derived lung myofibroblast differentiation and collagen I deposition in an inverse dose-dependent manner after 72 h, with the lowest doses evaluated inducing the strongest inhibitory effects. Similarly, BM-MSC-EVs therapeutically reduced the BLM-induced lung TGF-β1 expression and signal transduction, myofibroblast differentiation and collagen I deposition, and restored the BLM-induced loss of dynamic lung compliance in an inverse dose-dependent manner in vivo, after 7-days of treatment. BM-MSC-EVs promoted the MMP-2:TIMP-2 ratio in human dermal myofibroblasts or the MMP-9:TIMP-1 ratio in human lung myofibroblasts and the murine lung as part of their anti-fibrotic effects. Notably, BM-MSC-EVs failed to exert any anti-fibrotic effects in TGF-β1-stimulated lung myofibroblasts isolated from IPF patients. These findings suggested that BM-MSC-EVs may provide an anti-fibrotic treatment option for early-to-moderate IPF, but may not be effective against advanced IPF.

The urgent need for effective treatments for acute and chronic lung diseases underscores the significance of developing innovative preclinical human research tools. The 2023 American Thoracic Society Workshop on Precision-Cut Lung Slices (PCLSs) brought together 35 experts to discuss and address the role of human tissue-derived PCLSs as a unique tool for target and drug discovery and validation in pulmonary medicine. With increasing interest and usage, together with advancements in methods and technology, there is a growing need for consensus on PCLS methodology and readouts. The current document recommends standard reporting criteria and emphasizes the requirement for careful collection and integration of clinical metadata. We further discuss current clinically relevant readouts that can be applied to PCLSs and highlight recent developments and future steps for implementing novel technologies for PCLS modeling and analysis. The collection and correlation of clinical metadata and multiomic analysis will further advance the integration of this preclinical platform into patient endotyping and the development of tailored therapies for patients with lung disease.

Infection by influenza A virus (IAV) and other viruses causes disease exacerbations in chronic obstructive pulmonary disease (COPD). Immune responses are blunted in COPD, a deficit compounded by current standard-of-care glucocorticosteroids (GCS) to further predispose patients to life-threatening infections. The immunosuppressive effects of elevated transforming growth factor-β (TGF-β) in COPD may amplify lung inflammation during infections while advancing fibrosis. In the present study, we investigated potential repurposing of pirfenidone, currently used as an antifibrotic for idiopathic pulmonary fibrosis, as a nonsteroidal treatment for viral exacerbations of COPD. Murine models of lung-specific TGF-β overexpression or chronic cigarette smoke exposure with IAV infection were used. Pirfenidone was administered daily by oral gavage commencing pre- or postinfection, and inhaled pirfenidone and GCS treatment preinfection were also compared. Tissue and BAL were assessed for viral replication, inflammation, and immune responses. Overexpression of TGF-β enhanced the severity of IAV infection, contributing to unrestrained airway inflammation. Mechanistically, TGF-β reduced innate immune responses to IAV by blunting IFN-regulated gene expression and suppressing production of antiviral proteins. Prophylactic pirfenidone administration opposed these actions of TGF-β, curbing IAV infection and airway inflammation associated with TGF-β overexpression and cigarette smoke-induced COPD. Notably, inhaled pirfenidone caused greater inhibition of viral loads and inflammation than inhaled GCS. These proof-of-concept studies demonstrate that repurposing pirfenidone and employing a preventative strategy may yield substantial benefit over antiinflammatory GCS in COPD. Pirfenidone can mitigate damaging viral exacerbations without attendant immunosuppressive actions and merits further investigation, particularly as an inhaled formulation.

OBJECTIVES:To identify the silicosis research priorities of people living with silicosis, workers at risk of silicosis, their partners and caregivers, and of health professionals and researchers. STUDY DESIGN:Research priority setting exercise; modified James Lind Alliance framework for research priority setting partnerships, comprising an online survey followed by two forums in which thematic analysis and nominal group analysis were used to establish a list of research priorities. SETTING, PARTICIPANTS:People with or at risk of silicosis, their partners or caregivers (survey, online forum) and health care professionals, researchers, health and safety professionals (survey, in-person forum), recruited 14 April - 19 December 2023. MAIN OUTCOME MEASURES:Research priorities in four pre-identified areas: prevention, screening and diagnosis, treatment, and living with and managing the impact of silicosis. RESULTS:A total of 164 survey respondents (105 medical or research professionals, 34 workers currently or formerly at risk of silicosis, eleven people with confirmed silicosis, and fourteen partners or caregivers) identified 47 key research topics. Fifty-three health care professionals and thirteen people with or at risk of silicosis and their caregivers then ranked the research topics and developed research questions at the two forums. The highest ranked research priorities were research into assessment and optimisation of the hierarchy of controls, compliance and regulation, establishing minimum standards and developing innovative screening methods, early diagnosis, development of effective treatments, identification of biomarkers for risk of progression, developing an optimal care model that includes mental health care, and estimating the economic impact of silicosis. Both participant groups agreed that research into workplace controls is important, as is improving education and awareness, compliance with preventive measures, and screening and diagnosis, including nationally consistent screening and diagnosis practices. The professional participants rated research into silicosis pathogenesis and biomarkers and technological considerations higher than workers and their carers, who focused more on the barriers for and attitudes of workers, specific treatments, and managing symptoms. CONCLUSIONS:Research into eliminating exposure to silica, early diagnosis of silicosis, preventing disease progression, and reducing the impact of disease were the top research priorities for people with professional or personal interests in silicosis. Our findings should guide research directions and inform policy development.

Background Our simulations previously predicted focal areas of gaseous pollutant dose delivered to the airway mucosa of a patient with idiopathic pulmonary fibrosis (IPF). We hypothesize a relation between these dose predictions and clinically meaningful endpoints in IPF which link toxicant-driven epithelial injury and disrepair to IPF etiology and pathogenesis. Objective To determine associations between patient-specific modeling of tracheal geometry, computer simulations of toxicant dose, and lung histopathology in patients with IPF. Methods The first three conducting airway generations of ten patients diagnosed with IPF were reconstructed from their high-resolution CT chest scans. We quantified geometric abnormalities of the reconstructed tracheas based on their curvature and eccentricity (cross-sectional flattening), and performed three-dimensional computer simulations to predict the average and upper values (i.e. hotspots) of reactive toxicant dose to the underlying mucosa. Distal biopsy tissue samples were characterized by epithelial cell phenotype, extent of fibrosis, and histopathologic severity scores. Non-parametric correlation analysis examined associations between these descriptors. Results Computed values for curvature and eccentricity of IPF-deformed trachea varied widely among patients and correlated with more subjective rankings of tracheal deformation, and with predicted toxicant dose. Overall histopathologic severity was positively correlated with tracheal deformation and upper decile toxicant uptake. Tracheal curvature was significantly correlated with fibroblastic foci. Conclusions These results demonstrate an association of tracheal curvature with predicted toxicant dose and with histopathologic indicators in distal tissue. This suggests that these measures may be predictors of risk for acute IPF exacerbations, subsequent clinical deterioration, and disease progression.

RATIONALE: Current vasodilator therapies for pulmonary arterial hypertension (PAH) reduce elevated pulmonary arterial pressure, but do not target inflammation or features of vascular remodelling such as fibrosis. Preclinical models of PAH are required to validate novel therapeutic targets and screen drug candidates with disease-modifying potential. Our aims were to (1) establish an ex vivo model using mouse and human precision-cut lung slices (mPCLS, hPCLS) treated with PAH-relevant mediators to (2) compare the anti-inflammatory and anti-fibrogenic potential of the formyl peptide receptor (FPR) agonist Compound 17b (Cmpd17b), previously established by us as a novel vasodilator in mPCLS (Studley et al., Br J Pharmacol 2024 181:2287-2301) against current vasodilators used for PAH.METHODS: PCLS prepared from agarose-inflated mouse lung and human lung explants were left untreated or treated with a pulmonary hypertension cocktail (PHC) containing TNF-α, TGF-β, PDGF-AB and ET-1 in the absence or presence of either small-molecule Cmpd17b or sildenafil or iloprost, as these vasodilators are used to treat PAH. hPCLS were additionally treated with nintedanib, an anti-fibrotic that slows disease progression in idiopathic pulmonary fibrosis. PCLS-conditioned media was collected after 1 and 5 days to quantify secretion of the inflammatory cytokine IL-6 and the fibrogenic marker procollagen by ELISA and to measure any effect of treatments on tissue viability using a lactate dehydrogenase activity since this enzyme is released from dying cells. RESULTS: Neither PHC nor drug treatments affected PCLS viability. PHC induced secretion of IL6 and procollagen from both human and mouse PCLS (n=5-7, p<0.05). Cmpd17b (0.1-10µM) inhibited PHC-induced IL-6 secretion by up to 70% from mPCLS (n=5, p<0.05) while neither sildenafil nor iloprost showed significant anti-inflammatory effects. Cmpd17b (0.1-10µM) reduced PHC-mediated procollagen secretion by up to 60% from mPCLS (n=7, p<0.05), while current therapies did not. Cmpd17b (10µM) and nintedanib also significantly reduced procollagen secretion from hPCLS (n=4-6, p<0.05). CONCLUSIONS: We have established a novel PCLS model by mimicking the PAH microenvironment leading to inflammation and fibrogenesis. Cmpd17b exhibited significant anti-inflammatory and anti-fibrotic properties in PHC-treated mPCLS and hPCLS. These findings suggest that FPR agonism may present a more comprehensive therapeutic strategy for PAH than current vasodilators by also modifying the contributions of inflammation and fibrosis to disease progression.