Time and phenotype-dependent transcriptome analysis in AAV-TGFβ1 and Bleomycin-induced lung fibrosis models

We have previously established a novel mouse model of lung fibrosis based on Adeno-associated virus (AAV)-mediated pulmonary overexpression of TGFβ1. Here, we provide an in-depth characterization of phenotypic and transcriptomic changes (mRNA and miRNA) in a head-to-head comparison with Bleomycin-induced lung injury over a 4-week disease course. The analyses delineate the temporal state of model-specific and commonly altered pathways, thereby providing detailed insights into the processes underlying disease development. They further guide appropriate model selection as well as interventional study design. Overall, Bleomycin-induced fibrosis resembles a biphasic process of acute inflammation and subsequent transition into fibrosis (with partial resolution), whereas the TGFβ1-driven model is characterized by pronounced and persistent fibrosis with concomitant inflammation and an equally complex disease phenotype as observed upon Bleomycin instillation. Finally, based on an integrative approach combining lung function data, mRNA/miRNA profiles, their correlation and miRNA target predictions, we identify putative drug targets and miRNAs to be explored as therapeutic candidates for fibrotic diseases. Taken together, we provide a comprehensive analysis and rich data resource based on RNA-sequencing, along with a strategy for transcriptome-phenotype coupling. The results will be of value for TGFβ research, drug discovery and biomarker identification in progressive fibrosing interstitial lung diseases

Rheumatic Diseases: Pathophysiology, Targeted Therapy, Focus on Vascular and Pulmonary Manifestations

This book aims to summarize the latest advances in the rheumatic diseases, particularly regarding their pathophysiology and targeted therapy, with a focus on the recent efforts of vascular and pulmonary manifestations in order to anticipate new and future directions of these research topics. Rheumatic diseases represent a heterogeneous group of severe autoimmune disorders. The present Special Issue aims to provide an overview of the diversity and complexity of vascular and pulmonary manifestations of rheumatic diseases and to highlight gaps in our knowledge of how to effectively manage them. Despite their significant morbidity, we have a limited understanding of their pathogenesis. The eleven published articles reported here underline the complexity of rheumatic diseases and the difficulty of managing them. The manuscripts provide an overview of the pathophysiology and current management approach of these disorders, highlighting tools that assist with diagnosis, risk stratification, and therapy. A significant number of articles have reported innovative and effective treatments for the most frequent and debilitating complications of rheumatic diseases. The book emphasizes the importance of multidisciplinary teams using the skills of laboratory researchers, clinicians, radiologists, and pathologists. Furthermore, recent findings are presented and discussed, highlighting strategies to combat worsening symptoms of rheumatic diseases. The research described in this book provide an extremely useful example of the results achieved in the field of anti-rheumatic drug development. Detailed information on new breakthroughs can be found in this book. We strongly encourage a wide group of readers to explore the book that we are presenting for inspiration to develop new approaches to the diagnosis and treatment of rheumatic diseases

Rheumatic Diseases: Pathophysiology, Targeted Therapy, Focus on Vascular and Pulmonary Manifestations

This e-book summarises the latest advances in the rheumatic diseases with a focus on the recent efforts of vascular and pulmonary manifestations and anticipate the new and future directions of these research topic. Rheumatic diseases represent a heterogeneous group of severe autoimmune disorders. The present Special Issue aims to provide an overview of the complexity of vascular and pulmonary manifestations of rheumatologic diseases and helps in knowledge to manage them. The eleven published articles here collected underline the complexity of rheumatic diseases and the difficult to treated them. The manuscripts provide an overview of the pathophysiology and current treatment regimes of these disorders, highlighting tools which assist with diagnosis, risk stratification and therapy. Finally, we underline the importance of a multidisciplinary team working using the skills of clinicians, radiologists, and pathologists

Quantification of Lung Fibrosis in IPF-Like Mouse Model and Pharmacological Response to Treatment by Micro-Computed Tomography

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive degenerative lung disease leading to respiratory failure and death. Although anti-fibrotic drugs are now available for treating IPF, their clinical efficacy is limited and lung transplantation remains the only modality to prolong survival of IPF patients. Despite its limitations, the bleomycin (BLM) animal model remains the best characterized experimental tool for studying disease pathogenesis and assessing efficacy of novel potential drugs. In the present study, the effects of oropharyngeal (OA) and intratracheal (IT) administration of BLM were compared in C57BL/6 mice. The development of lung fibrosis was followed in vivo for 28 days after BLM administration by micro-computed tomography and ex vivo by histological analyses (bronchoalveolar lavage, histology in the left lung to stage fibrosis severity and hydroxyproline determination in the right lung). In a separate study, the antifibrotic effect of Nintedanib was investigated after oral administration (60 mg/kg for two weeks) in the OA BLM model. Lung fibrosis severity and duration after BLM OA and IT administration was comparable. However, a more homogeneous distribution of fibrotic lesions among lung lobes was apparent after OA administration. Quantification of fibrosis by micro-CT based on % of poorly aerated tissue revealed that this readout correlated significantly with the standard histological methods in the OA model. These findings were further confirmed in a second study in the OA model, evaluating Nintedanib anti-fibrotic effects. Indeed, compared to the BLM group, Nintedanib inhibited significantly the increase in % of poorly aerated areas (26%) and reduced ex vivo histological lesions and hydroxyproline levels by 49 and 41%, respectively. This study indicated that micro-computed tomography is a valuable in vivo technology for lung fibrosis quantification, which will be very helpful in the future to better evaluate new anti-fibrotic drug candidates

Transbronchial cryobiopsy and Neutrophil Lymphocyte Ratio - new precision medicine tools and markers in Interstitial Lung Disease

The interstitial lung diseases (ILDs) are a group of over 200 disease that may lead to progressive fibrosis and respiratory failure. ILDs are heterogenous, with varying amounts of inflammation and fibrosis, and differ in response to therapy and outcome. The most severe fibrotic (f) ILD, idiopathic pulmonary fibrosis (IPF), has a median survival of just three years. Progressive fILD may respond to antifibrotic treatments which slow down, but do not reverse, fibrosis albeit often with significant side effects. Better treatments or delivery of treatments are needed. Diagnosis of ILD relies on clinical history, imaging and, in some cases lung biopsy, with associated risks. Better diagnostic and prognostic biomarkers in ILD are urgently needed. This thesis examines the approach to diagnosis, prognostication, and treatment in fILDs, and, in particular IPF. It begins with the finding that Neutrophil Lymphocyte Ratio (NLR), derived from a simple, widely available blood test, is a prognostic biomarker in IPF. The role of lung biopsy in the diagnostic pathway is considered and the use of a relatively new minimally invasive technique of transbronchial cryo lung biopsy (TBCB) as an alternative to surgical lung biopsy (SLB) is described. The value of TBCB to obtain lung tissue for research is demonstrated with evaluation of the distribution of inhaled ipratropium in fILD. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) on samples of lung taken using TBCB, it was demonstrated that inhaled medication was able to reach the fibrotic lung, presenting a new approach to drug delivery in fILD. Further discussion focusses on the current role of SLB in the diagnostic pathway in ILD, the presentation of a systematic literature review, and a discussion of future trials to assess the potential benefits of a wider use of TBCB

Investigating the role of the respiratory microbiome and metabolome in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic lung disease of unknown aetiology. There is growing evidence that the lung microbiota may play a role in IPF. However, no study has investigated the functional impact of the short-chain fatty acids (SCFAs) on disease pathogenesis. The aim of this thesis was to characterise the respiratory microbiome and metabolome in healthy controls and subjects with IPF and to investigate the influence of the SCFAs on human primary bronchial epithelial cells (PBECs) and human lung fibroblasts (HLFs) from these individuals. Subjects diagnosed with IPF (n=201) and healthy controls (n=40) were prospectively recruited and underwent bronchoscopy. Bacterial DNA was isolated and 16S rRNA gene sequencing undertaken to characterise bacterial communities. Untargeted 1H nuclear magnetic resonance spectroscopybased metabolomics and targeted gas chromatography-mass spectrometry captured the metabolic profile of these samples. PBECs, differentiated at air-liquid interface, and HLF from healthy controls and subjects with IPF were either left untreated or exposed to the SCFAs. Compared to healthy controls, the IPF microbiota was less diverse (P<0.01), and had increased proportions of Firmicutes (P<0.01), Streptococcus and Staphylococcus (P<0.05). Relative concentrations of the SCFAs were increased in IPF compared to healthy controls, and in IPF, propionate positively correlated with the bacterial burden (rho=0.47, P=8 x 10-5). Treatment of PBECs from IPF subjects but not healthy controls with the SCFAs led to morphological changes, a dose-dependent release of pro-inflammatory mediators in the cell supernatant, and a decrease in transepithelial electrical resistance (TEER) over time. Compared to baseline, exposure of IPF PBECs to 1 mM of propionate led to a 40% reduction in TEER and a significant increase (P<0.05) in the secretion of IL-6. Treatment of HLFs with 5 mM of propionate or butyrate was found to increase cell proliferation at 72 hours. Subjects with IPF display an altered microbiome which is associated with a distinct metabolic signature in the lower airways. Differences in bacterial genera and an increased bacterial burden in IPF results in changes in the SCFAs in the airways. In vitro work demonstrates the potential of these SCFAs to shape immunological responses in the lung, mediating the pathogenesis of fibrosis.Open Acces

Non-invasive measurement of respiratory mechanics and work of breathing

The mechanical properties of the respiratory system such as resistance, elastance and mechanical work of breathing are rarely measured directly but are inferred from the effect of respiratory disease on maximal lung volumes and flows. Although such tests have proved very useful, they have shortcomings, e.g. changes in lung volumes are poor at detecting progression in interstitial lung disease and correlate only weakly with changes in functional capacity achieved post-bronchodilator in patients with airways obstruction. The direct measurement of mechanical properties is of interest as they have an obvious physical interpretation but their usefulness has as yet not been systematically tested. Resistance aside, their measurement is rarely performed as it is invasive, requiring either a sedated patient on controlled ventilation to abolish spontaneous respiratory muscle activity or measurement of oesophageal and gastric pressures. The aim of this thesis was to explore the feasibility and potential clinical value of non-invasive measurements of respiratory mechanics and work of breathing. The work is presented in three sections. Firstly, conventional methods for measuring resistance, elastance and mechanical work of breathing were reviewed and the methods for the non-invasive approaches to be used were described in detail. The results from the non-invasive methods were then validated by comparison with conventional techniques in both ventilated patients and in subjects in the pulmonary function laboratory where oesophagal and gastric manometry were performed. Finally, the non-invasive methods were evaluated in three clinical scenarios: bronchodilator reversibility testing, assessment of progression in interstitial lung disease, and monitoring recovery from exacerbation of chronic obstructive pulmonary disease

Biomarkers of progressive lung fibrosis

Interstitial lung diseases (ILD) encompass a heterogeneous group of immuno-inflammatory and fibrotic diseases of the lung parenchyma. The most common and severe ILD is idiopathic pulmonary fibrosis (IPF), a chronic progressive fibrotic lung disease of unknown aetiology associated with poor prognosis. A substantial proportion of individuals with ILDs other than IPF also develop progressive fibrosis with clinical, radiological, and genetic similarities, suggesting a shared final common pathway across progressive fibrotic ILDs irrespective of aetiology. Study of shared mechanisms of progression has the potential to aid prognostication, enable a precise approach to therapeutic strategies and allow stratification into clinical trials. Biomarkers are objectively measured and reproducible characteristics that enable stratification of disease phenotypes. The aim of this thesis was to examine and characterise the role of clinical biomarkers in fibrotic lung diseases to enable early identification of progressive fibrotic phenotypes. An evidence synthesis of blood biomarkers as prognosticators in IPF highlighted several biomarkers with prognostic potential and identified priorities for future blood biomarker research. The first individual participant data (IPD) meta-analysis in IPF of matrix metalloproteinase-7 demonstrated baseline measurements were independently associated with disease outcomes. To evaluate the role of physiological variables as prognostic biomarkers and as surrogate trial endpoints, the largest analysis of interventional trial placebo arms in IPF was performed using robust IPD methodology. Baseline and three-month change in physiological variables, particularly FVC were independently associated with disease outcomes, supporting their role as prognostic biomarkers. The association between short-term change in FVC and disease outcomes were replicated in individuals receiving anti-fibrotics using pooled analysis of pirfenidone and nintedanib treatment arms. Moreover, a difference in FVC change over three-months between treatment and placebo arms was observed, supporting three-month FVC as a surrogate endpoint in future IPF trials. An ongoing prospective multi-centre observational cohort study (INJUSTIS) to assess longitudinal disease behaviour and the role of biomarkers in other fibrotic lung diseases was established. Interim analysis suggested a significant proportion of individuals with non-IPF fibrotic ILD had progressive phenotypes that were comparable with disease behaviour in IPF. Lung function, particularly FVC change over three-months was independently associated with poorer outcomes. The role of home spirometry in fibrotic ILD was assessed, and though measurements were accurate and reliable when compared with hospital spirometry, daily FVC measurements were unable to predict mortality at earlier timepoints. An exploratory blood biomarker analysis performed in individuals with extremes of IPF offered further support for the role of CA-125 as a prognostic biomarker and identified several biomarkers and biological pathways for more focussed assessment in the complete INJUSTIS cohort. Taken collectively, the data presented in this thesis strongly support an important role for biomarkers in fibrotic ILD to identify progressive fibrotic phenotypes and enable personalised approaches to patient management. Whilst the COVID-19 pandemic was severely disruptive, the work presented forms the basis for further study of biomarkers in progressive pulmonary fibrosis