Airway basal cells show a dedifferentiated KRT17highPhenotype and promote fibrosis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study, we focus on the properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing (scRNAseq) of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNAseq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrate that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

Computation and visualization of cell-cell signaling topologies in single-cell systems data using Connectome.

Single-cell RNA-sequencing data has revolutionized our ability to understand of the patterns of cell-cell and ligand-receptor connectivity that influence the function of tissues and organs. However, the quantification and visualization of these patterns in a way that informs tissue biology are major computational and epistemological challenges. Here, we present Connectome, a software package for R which facilitates rapid calculation and interactive exploration of cell-cell signaling network topologies contained in single-cell RNA-sequencing data. Connectome can be used with any reference set of known ligand-receptor mechanisms. It has built-in functionality to facilitate differential and comparative connectomics, in which signaling networks are compared between tissue systems. Connectome focuses on computational and graphical tools designed to analyze and explore cell-cell connectivity patterns across disparate single-cell datasets and reveal biologic insight. We present approaches to quantify focused network topologies and discuss some of the biologic theory leading to their design.

FeV1 and BMI influence King's Sarcoidosis Questionnaire score in sarcoidosis patients.

BACKGROUND: Sarcoidosis is granulomatous disease of unknown origin affecting organ function and quality of life. The King's Sarcoidosis Questionnaire (KSQ) serves as a tool to assess quality of life in sarcoidosis patients with general health and organ specific domains. A German translation has been validated in a German cohort. In this study we assessed, whether clinical parameters influence KSQ scores. METHODS: Clinical data (e.g. lung function, organ impairment, serological parameters) for the German validation cohort were extracted from clinical charts and investigated by correlation and linear regression analyses. RESULTS: KSQ subdomain scores were generally lower in patients with respective organ manifestation or on current therapy. LUNG subdomain was significantly predicted by lung functional parameters, however for general health status, only FeV1 exerted significant influence. GHS was not influenced by serological parameters, but was significantly negatively correlated with body mass index (BMI). KSQ provides additional information beyond lung function, clinical or serological parameters in sarcoidosis patients. Notably, high BMI is significantly negatively associated with patients' well-being as measured by KSQ-GHS. CONCLUSION: This observation may direct further studies investigating the effect of obesity on sarcoidosis-related quality of life and strategies to intervene with steroid-sparing therapies and measures of life style modifications. Trial registration This study was registered in the German Clinical Trials Register (reference number DRKS00010072). Registered January 2016.

Abnormal FeV1 and body mass index are associated with impaired cough-related quality of life in sarcoidosis patients.

Sarcoidosis is a granulomatous disease that mainly manifests within the lungs and may thereby impair lung function. Beyond and independently from organ impairment, sarcoidosis may affect quality of life which can be quantified by questionnaires. The Leicester Cough Questionnaire (LCQ) has been developed to assess cough-related quality of life. We analysed data from a prospectively collected cohort of sarcoidosis patients for validation of the German LCQ version. Our analyses demonstrated that LCQ values add additional information beyond routinely monitored parameters (e.g. lung function). Only FeV1 and BMI slightly influence LCQ scores, where all other parameters tested did not correlate with LCQ scores. In summary, LCQ is a valuable tool providing information on the patient' quality of life beyond routine follow-up parameters. FeV1 and BMI may represent treatable traits to reduce cough-related disease burden.

S2K Guideline for Diagnosis of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe and often fatal disease. Diagnosis of IPF requires considerable expertise and experience. Since the publication of the international IPF guideline in the year 2011 and the update 2018 several studies and technical advances have occurred, which made a new assessment of the diagnostic process mandatory. The goal of this guideline is to foster early, confident, and effective diagnosis of IPF. The guideline focusses on the typical clinical context of an IPF patient and provides tools to exclude known causes of interstitial lung disease including standardized questionnaires, serologic testing, and cellular analysis of bronchoalveolar lavage. High-resolution computed tomography remains crucial in the diagnostic workup. If it is necessary to obtain specimens for histology, transbronchial lung cryobiopsy is the primary approach, while surgical lung biopsy is reserved for patients who are fit for it and in whom a bronchoscopic diagnosis did not provide the information needed. After all, IPF is a diagnosis of exclusion and multidisciplinary discussion remains the golden standard of diagnosis.

Cathepsin B promotes collagen biosynthesis, which drives bronchiolitis obliterans syndrome.

Bronchiolitis obliterans syndrome (BOS) is a major complication after lung transplantation (LTx). BOS is characterised by massive peribronchial fibrosis, leading to air trapping-induced pulmonary dysfunction. Cathepsin B, a lysosomal cysteine protease, has been shown to enforce fibrotic pathways in several diseases. However, the relevance of cathepsin B in BOS progression has not yet been addressed. The aim of the study was to elucidate the function of cathepsin B in BOS pathogenesis.We determined cathepsin B levels in bronchoalveolar lavage fluid (BALF) and lung tissue from healthy donors (HD) and BOS LTx patients. Cathepsin B activity was assessed via a fluorescence resonance energy transfer-based assay and protein expression was determined using Western blotting, ELISA and immunostaining. To investigate the impact of cathepsin B in the pathophysiology of BOS, we used an in vivo orthotopic left LTx mouse model. Mechanistic studies were performed in vitro using macrophage and fibroblast cell lines.We found a significant increase of cathepsin B activity in BALF and lung tissue from BOS patients, as well as in our murine model of lymphocytic bronchiolitis. Moreover, cathepsin B activity was associated with increased biosynthesis of collagen and had a negative effect on lung function. We observed that cathepsin B was mainly expressed in macrophages that infiltrated areas characterised by a massive accumulation of collagen deposition. Mechanistically, macrophage-derived cathepsin B contributed to transforming growth factor-ß1-dependent activation of fibroblasts, and its inhibition reversed the phenotype.Infiltrating macrophages release active cathepsin B, thereby promoting fibroblast activation and subsequent collagen deposition, which drive BOS. Cathepsin B represents a promising therapeutic target to prevent the progression of BOS.

Single-cell connectomic analysis of adult mammalian lungs.

Efforts to decipher chronic lung disease and to reconstitute functional lung tissue through regenerative medicine have been hampered by an incomplete understanding of cell-cell interactions governing tissue homeostasis. Because the structure of mammalian lungs is highly conserved at the histologic level, we hypothesized that there are evolutionarily conserved homeostatic mechanisms that keep the fine architecture of the lung in balance. We have leveraged single-cell RNA sequencing techniques to identify conserved patterns of cell-cell cross-talk in adult mammalian lungs, analyzing mouse, rat, pig, and human pulmonary tissues. Specific stereotyped functional roles for each cell type in the distal lung are observed, with alveolar type I cells having a major role in the regulation of tissue homeostasis. This paper provides a systems-level portrait of signaling between alveolar cell populations. These methods may be applicable to other organs, providing a roadmap for identifying key pathways governing pathophysiology and informing regenerative efforts.

Translation and psychometric properties of the King's Sarcoidosis Questionnaire (KSQ) in German language.

BACKGROUND: King's Sarcoidosis Questionnaire (KSQ) is a novel, validated, health-related quality of life questionnaire on sarcoidosis with 5 scales and 29 items. For future multinational observational and interventional studies on sarcoidosis, a validated German version of the KSQ is needed. The objective of our study is to translate the original KSQ and develop a German version possessing good psychometric properties and with as few modifications as possible. METHODS: We translated the KSQ into German, tested it in structured interviews in sarcoidosis patients, and asked consecutive patients in an outpatient clinic to complete it. We relied on the KSQ's original version to achieve its psychometric properties in the German version. Structural validity, internal consistency, construct validity, and fit to Rasch model were assessed. Our procedure's logic meant that in the first step we optimized the item selection in the German version to maximize its psychometric quality. In step two, we assessed the unmodified version's properties in comparison to the modified version's. RESULTS: One hundred ninety-four patients with sarcoidosis were included and completed the questionnaires. Due to ambiguous factor loadings, four items of the scale "General Health Status" had to be eliminated. Another item was excluded to ensure the Rasch model fit. This modified, 24-item version of the KSQ shows acceptable Rasch model fit and good model fit in confirmatory factor analyses (TLI = 0.90, CFI = 0.91, RMSEA = 0.08). Cronbach's Alpha ranges from 0.82 to 0.91. Several hypotheses concerning construct validity (e.g., correlations with SF-36) are confirmed or partly confirmed. The measurement properties of the original unmodified version are similar in their construct validity and internal consistency; however, we were unable to confirm structural validity and fit to the Rasch model in the original version. CONCLUSIONS: We translated and validated the German KSQ and report good psychometric properties. The reduced 24-item version has the advantage that all scales are unidimensional and fulfil the requirements of the Rasch model, ensuring its benefits. The original 29-item version, on the other hand, allows us to compare German data to international data however, at the price, of less structural validity and the lack of fit to the Rasch model. TRIAL REGISTRATION: This study was registered in the German Clinical Trials Register (reference number DRKS00010072 ). Registered January 2016.

Psychometric properties of the German version of the Leicester Cough Questionnaire in sarcoidosis.

BACKGROUND: Cough is one of the most common symptoms in general and pulmonary medicine with profound negative impact on health-related quality of life (HRQL). The Leicester Cough Questionnaire (LCQ) is a validated HRQL questionnaire, yet a validated German version of the LCQ is not available and it has never been tested in a cohort with sarcoidosis. OBJECTIVES: To translate the LCQ into German and determine its psychometric properties. METHODS: The LCQ was translated in a forward-backward approach. Structured interviews in sarcoidosis patients were performed. Subsequently, sarcoidosis patients were asked to answer the German LCQ and comparative questionnaires. Distribution properties, item difficulty, concurrent validity, Rasch model fit and internal consistency of the German LCQ were determined. RESULTS: 200 patients with sarcoidosis were included. We provide evidence for reliability, unidimensionality and internal consistency. However, only a moderate correlation with general and respiratory-specific HRQL questionnaires, no Rasch model fit could be shown. Skewed responses caused by floor effects were detected. CONCLUSION: We demonstrate that the German LCQ is valid and reliable and its psychometric properties fulfil the standards required for its use in clinical settings as well as in interventional trials.

Phenotypes of organ involvement in sarcoidosis.

Sarcoidosis is a highly variable, systemic granulomatous disease of hitherto unknown aetiology. The GenPhenReSa (Genotype-Phenotype Relationship in Sarcoidosis) project represents a European multicentre study to investigate the influence of genotype on disease phenotypes in sarcoidosis.The baseline phenotype module of GenPhenReSa comprised 2163 Caucasian patients with sarcoidosis who were phenotyped at 31 study centres according to a standardised protocol.From this module, we found that patients with acute onset were mainly female, young and of Scadding type I or II. Female patients showed a significantly higher frequency of eye and skin involvement, and complained more of fatigue. Based on multidimensional correspondence analysis and subsequent cluster analysis, patients could be clearly stratified into five distinct, yet undescribed, subgroups according to predominant organ involvement: 1) abdominal organ involvement, 2) ocular-cardiac-cutaneous-central nervous system disease involvement, 3) musculoskeletal-cutaneous involvement, 4) pulmonary and intrathoracic lymph node involvement, and 5) extrapulmonary involvement.These five new clinical phenotypes will be useful to recruit homogenous cohorts in future biomedical studies.

Usefulness of Cyclophosphamide Pulse Therapy in Interstitial Lung Diseases.

BACKGROUND: Interstitial lung diseases (ILDs) are a group of disorders characterised by progressive lung function decline. Stabilisation of lung function under intermittent i.v. cyclophosphamide was shown in patients suffering from systemic sclerosis, yet data in ILD patients are scarce. OBJECTIVES: To retrospectively evaluate the usefulness of cyclophosphamide pulse therapy in ILD. METHODS: We retrospectively analysed all patients who received i.v. cyclophosphamide in our centre from 2002 to 2012. Lung function, survival status, and bronchoalveolar lavage cytology were recorded during a follow-up period of 18 months. RESULTS: Twenty-six patients with idiopathic pulmonary fibrosis, 6 with lymphocytic interstitial pneumonia (LIP), 8 with idiopathic non-specific interstitial pneumonia (NSIP), 7 with rheumatoid arthritis-associated ILD, and 7 with perinuclear anti-neutrophil cytoplasmic antibody-positive ILD (pANCA+ ILD) were included. Patients with LIP and NSIP had the best survival outcome, those with pANCA+ ILD the worst. In the total cohort, we found a significantly higher total lung capacity decline in the year before treatment compared to the year after treatment. CONCLUSIONS: This retrospective analysis of cyclophosphamide treatment shows a stabilisation of lung function in most patients with fibrotic ILDs, yet prospective studies in clearly defined diagnoses are urgently needed.

Immune response to Propionibacterium acnes in patients with sarcoidosis--in vivo and in vitro.

BACKGROUND: Propionibacterium acnes was found in lungs and lymph nodes of patients with sarcoidosis and may induce hypersensitivity type granuloma formation. Data regarding the immune response to P. acnes of European sarcoid patients are scarce. METHODS: We assessed the total IgG and IgA amount and specific antibodies to P. acnes and to Staphylococcus aureus, serving as a control, in BAL fluid of 64 patients with sarcoidosis and of 21 healthy volunteers. In a subcohort of sarcoid patients and controls, TNF-α and GM-CSF production of BAL cells stimulated with heat-killed P. acnes were measured. RESULTS: In sarcoid patients, the total IgG and IgA levels in BAL fluid were significantly elevated compared to healthy volunteers. IgG and IgA titres against P. acnes and S. aureus were increased in sarcoid patients, yet based on the total amount of antibodies, only antibodies directed against P. acnes were relatively and significantly increased. Furthermore, BAL cells of sarcoid patients produced significantly more TNF-α and GM-CSF upon stimulation with heat-killed P. acnes compared to controls. CONCLUSIONS: Patients with sarcoidosis had elevated levels of specific antibodies against P. acnes which suggest contact with this bacterium in the past. Furthermore, BAL cells of sarcoid patients produced inflammatory cytokines (TNF-α and GM-CSF) upon stimulation with P. acnes indicating potential involvement of this pathogen in the pathogenesis of sarcoidosis in some patients.

Macrophage activation in acute exacerbation of idiopathic pulmonary fibrosis.

BACKGROUND: Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) is a common cause of disease acceleration in IPF and has a major impact on mortality. The role of macrophage activation in AE of IPF has never been addressed before. METHODS: We evaluated BAL cell cytokine profiles and BAL differential cell counts in 71 IPF patients w/wo AE and in 20 healthy volunteers. Twelve patients suffered from AE at initial diagnosis while sixteen patients developed AE in the 24 months of follow-up. The levels of IL-1ra, CCL2, CCL17, CCL18, CCL22, TNF-α, IL-1ß, CXCL1 and IL-8 spontaneously produced by BAL-cells were analysed by ELISA. RESULTS: In patients with AE, the percentage of BAL neutrophils was significantly increased compared to stable patients. We found an increase in the production rate of the pro-inflammatory cytokines CXCL1 and IL-8 combined with an increase in all tested M2 cytokines by BAL-cells. An increase in CCL18 levels and neutrophil counts during AE was observed in BAL cells from patients from whom serial lavages were obtained. Furthermore, high baseline levels of CCL18 production by BAL cells were significantly predictive for the development of future AE. CONCLUSIONS: BAL cell cytokine production levels at acute exacerbation show up-regulation of pro-inflammatory as well as anti-inflammatory/ M2 cytokines. Our data suggest that AE in IPF is not an incidental event but rather driven by cellular mechanisms including M2 macrophage activation.