The discovAIR project:a roadmap towards the Human Lung Cell Atlas

The Human Cell Atlas (HCA) consortium aims to establish an atlas of all organs in the healthy human body at single-cell resolution to increase our understanding of basic biological processes that govern development, physiology and anatomy, and to accelerate diagnosis and treatment of disease. The lung biological network of the HCA aims to generate the Human Lung Cell Atlas as a reference for the cellular repertoire, molecular cell states and phenotypes, and the cell-cell interactions that characterise normal lung homeostasis in healthy lung tissue. Such a reference atlas of the healthy human lung will facilitate mapping the changes in the cellular landscape in disease. The discovAIR project is one of six pilot actions for the HCA funded by the European Commission in the context of the H2020 framework program. DiscovAIR aims to establish the first draft of an integrated Human Lung Cell Atlas, combining single-cell transcriptional and epigenetic profiling with spatially resolving techniques on matched tissue samples, as well as including a number of chronic and infectious diseases of the lung. The integrated Lung Cell Atlas will be available as a resource for the wider respiratory community, including basic and translational scientists, clinical medicine, and the private sector, as well as for patients with lung disease and the interested lay public. We anticipate that the Lung Cell Atlas will be the founding stone for a more detailed understanding of the pathogenesis of lung diseases, guiding the design of novel diagnostics and preventive or curative interventions

Study of the role of bone morphogenetic proteins in asthma pathophysiology

Signaling by Bone Morphogenetic Proteins (BMP) has been implicated in the regulation of early lung development, adult lung homeostasis and tissue injury repair. However, the precise mechanism of action, the actualcellular targets and the spatio-temporal pattern of BMP signaling during these processes remainsinadequately described. In order to address this, we have utilized a transgenic mouse line harboring a BMP responsive eGFP reporter allele (BRE-eGFP) toconstruct the first detailed spatiotemporal map of canonical BMP pathway activation during lung development, homeostasis and adult lung injury/repair. Our studies demonstrate that during the pseudoglandular stage, when branching morphogenesis progresses in the developing lung, canonical BMP pathway is active mainly in the vascular network and the sub-epithelial smooth muscle layer of the proximal airways. Activation of the BMP pathway becomes evident in epithelial compartments of the lung only after E14.5 primarily in cells negative for epithelial lineage markers, located in the proximal portion of the airway tree and clusters adjacent to neuro-epithelial-bodies (NEBs), and in a substantial portion of immature alveolar epithelial cells. Epithelial cells with activated BMP pathway are highly enriched in progenitors capable of forming colonies in three-dimensional Matrigel cultures. As lung morphogenesis approaches completion, eGFP-expression declines and in adult lung its expression is barely detectable. However, upon tissue injury, either with naphthalene or bleomycin, the canonical BMP pathways is re-activated, in bronchial or alveolar epithelial cells respectively, ina manner reminiscent of early lung development and in tissue areas where reparatory progenitor cells reside. Our studies illustrate the dynamic activation of canonical BMP pathway during lung development and adult lung tissue repair and highlight involvement of this pathway in two important processes, namely, the early development of the pulmonary vasculature and the management of epithelial progenitor pools both during lung development and repair of adult lung tissue injury.Η υπερ-οικογένεια του Αυξητικού Παράγοντα Μεταμόρφωσης-β (TGF-β) περιλαμβάνει περισσότερα από 40 μέλη που εμπλέκονται σε πληθώρα βιολογικών διεργασιών τόσο κατά την ανάπτυξη όσο και την ομοιόσταση του οργανισμού. Προηγούμενες μελέτες έχουν συσχετίσει τις Μορφογενετικές Πρωτεΐνες των Οστών (BMPs) και την Ακτιβίνη-A με την ανάπτυξη και την παθολογία του πνεύμονα. Ωστόσο, αρκετά ερωτήματα παραμένουν αναπάντητα σχετικά με τον ακριβή ρόλο των παραπάνω των μορίων στις προαναφερθείσες διεργασίες. Τα πρώτο τμήμα της εργασίας πραγματεύεται το ρόλο της σηματοδότησης των BMPs κατά την ανάπτυξη και επιδιόρθωση ιστολογικών βλαβών στον ενήλικο ιστό. Η μελέτη στηρίζεται στη χρήση μίας διαγονιδιακής σειράς ποντικού που εκφράζει το Πράσινη Φθορίζουσα Πρωτεΐνη (eGFP) υπό τον έλεγχο ενός συνθετικού υποκινητή που αποκρίνεται στη σηματοδότηση των BMPs, ώστε αρχικά να χαρτογραφήσει το χωρο-χρονικό και κυτταρο-ειδικό πρότυπο ενεργοποίησης των BMPs κατά την ανάπτυξη και επιδιόρθωση του πνεύμονα αλλά και να απαντήσει ερωτήματα σχετικά με το βιολογικό ρόλο των BMPs σε αυτές τις διεργασίες. Συνοψίζοντας, από τα ευρήματα της παρούσας διατριβής συμπεραίνεται ότι η σηματοδότηση των BMPs διαδραματίζει πολύ πιο πολύπλοκο και δυναμικό ρόλο στη ανάπτυξη του πνεύμονα απ’ ότι πιστεύαμε μέχρι τώρα, καθώς παρατηρήθηκε ενεργή σηματοδότηση στο αγγειακό ενδοθήλιο, το στρώμα των Λείων Μυϊκών κυττάρων που επενδύουν του αεραγωγούς, το βρογχικό και κυψελιδικό επιθήλιο. Η ανάλυση του βιολογικού ρόλου των BMPs στην ανάπτυξη του πνεύμονα πραγματοποιήθηκε με in vitro πειράματα που επιβεβαίωσαν (i) τον αναπόσπαστο ρόλο των BMPs στην ανάπτυξη του αγγειακού δικτύου του πνεύμονα διότι η παρεμπόδιση της σηματοδότησης των BMPs με τη χρήση εκλεκτικών αναστολέων σε Ε12 εμβρυϊκούς πνεύμονες παρεμπόδισε τη ανάπτυξη του αγγειακού ενδοθηλίου και (ii) στη διατήρηση του προγονικού χαρακτήρα των επιθηλιακών κυττάρων του οργάνου περιορίζοντας τον πολλαπλασιασμό και τη διαφοροποίησή τους, καθώς η απομόνωση και καλλιέργεια των eGFP+ επιθηλιακών κυττάρων του πνεύμονα κατέδειξε την αυξημένη τους ικανότητα να σχηματίζουν αποικίες επιθηλιακών κυττάρων. Τέλος, προτείνεται κάποιος σημαντικός ρόλος των BMPs στην λειτουργία των επιδιορθωτικών μηχανισμών του ενήλικου οργάνου καθώς ενεργή σηματοδότηση παρατηρήθηκε μετά την πρόκληση βλάβης με Ναφθαλένιο και Μπλεομυκίνη στο ενήλικο όργανο όπου φυσιολογικά η σηματοδότηση έχει περιοριστεί σημαντικά. Το δεύτερο μέρος της διδακτορικής διατριβής αναφέρεται σε τμήμα των αποτελεσμάτων της εργασίας με τίτλο «Η υπερ-έκφραση της Ακτιβίνης-Α προκαλεί παθολογία που προσομοιάζει το Σύνδρομο Οξείας Πνευμονικής Δυσχέρειας (ARDS)» και παρουσιάζει τις επιπτώσεις της έκτοπης υπερ-έκφρασης της Ακτιβίνης-Α στην ομοιόσταση του πνεύμονα ενήλικων ποντικών. Συγκεκριμένα, η Ακτιβίνη-Α (i) προκαλεί αύξηση της έκφρασης του HMGB1, ενός παράγοντα κινδύνου που εμπλέκεται στη δημιουργία ενός προ-φλεγμονώδους περιβάλλοντος το οποίο συμβάλει στην παθολογία νόσων όπως η σήψη και το ARDS, και (ii) προκαλεί μείωση της έκφρασης των Σουρφακτανών C, B και Α στο πνευμονικό παρέγχυμα, το οποίο είναι ικανό να προκαλέσει παθολογία στον πνεύμονα

SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution.

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions

Spatial transcriptomic profiling of RespiratorySyncytial Virus (RSV) infection

Despite the fact that the human Respiratory Syncytial Virus (RSV) was first discoveredback in 1956, it remains one of the leading causes of morbidity and mortality inyoung children. Transcriptome-wide spatially resolved transcriptomics is a technologyunder rapid development that introduces a new modality for exploratory examinationof cellular behavior. With this modality, we examine how RSV infection changes thelocal cellular environment in the lung by infecting mice with RSV and comparing itto control samples four days after infection. We find viral presence in all compartmentsof the tissue, well-defined induced tertiary lymphoid tissue within some of thesamples, compartmentalized infiltration of innate immune cells, as well as functionalenrichment of airway epithelial repair pathways

Activin-A Overexpression in the Murine Lung Causes Pathology That Simulates Acute Respiratory Distress Syndrome

Rationale: Activin-A is up-regulated in various respiratory disorders. However, its precise role in pulmonary pathophysiology has not been adequately substantiated in vivo. Objectives: To investigate in vivo the consequences of dysregulated Activin-A expression in the lung and identify key Activin-A-induced processes that contribute to respiratory pathology. Methods: Activin-A was ectopically expressed in murine lung, and functional, structural, and molecular alterations were extensively analyzed. The validity of Activin-A as a therapeutic target was demonstrated in animals overexpressing Activin-A or treated with intratracheal instillation of LPS. Relevancy to human pathology was substantiated by demonstrating high Activin-A levels in bronchoalveolar lavage (BAL) samples from patients with acute respiratory distress syndrome (ARDS). Measurements and Main Results: Overexpression of Activin-A in mouse airways caused pulmonary pathology reminiscent of acute lung injury (ALI)/ARDS. Activin-A triggered a lasting inflammatory response characterized by acute alveolar cell death and hyaline membrane formation, sustained up-regulation of high-mobility group box 1, development of systemic hypercoagulant state, reduction of surfactant proteins SpC, SpB, and SpA, decline of lung compliance, transient fibrosis, and eventually emphysema. Therapeutic neutralization of Activin-A attenuated the ALI/ARDS-like pathology induced either by ectopic expression of Activin-A or by intratracheal instillation of LPS. In line with the similarity of the Activin-A-induced phenotype to human ARDS, selective up-regulation of Activin-A was found in BAL of patients with ARDS. Conclusions: Our studies demonstrate for the first time in vivo the pathogenic consequences of deregulated Activin-A expression in the lung, document novel aspects of Activin-A biology that provide mechanistic explanation for the observed phenotype, link Activin-A to ALI/ARDS pathophysiology, and provide the rationale for therapeutic targeting of Activin-A in these disorders

Activation of the Canonical Bone Morphogenetic Protein (BMP) Pathway during Lung Morphogenesis and Adult Lung Tissue Repair

<div><p>Signaling by Bone Morphogenetic Proteins (BMP) has been implicated in early lung development, adult lung homeostasis and tissue-injury repair. However, the precise mechanism of action and the spatio-temporal pattern of BMP-signaling during these processes remains inadequately described. To address this, we have utilized a transgenic line harboring a BMP-responsive eGFP-reporter allele (BRE-eGFP) to construct the first detailed spatiotemporal map of canonical BMP-pathway activation during lung development, homeostasis and adult-lung injury repair. We demonstrate that during the pseudoglandular stage, when branching morphogenesis progresses in the developing lung, canonical BMP-pathway is active mainly in the vascular network and the sub-epithelial smooth muscle layer of the proximal airways. Activation of the BMP-pathway becomes evident in epithelial compartments only after embryonic day (E) 14.5 primarily in cells negative for epithelial-lineage markers, located in the proximal portion of the airway-tree, clusters adjacent to neuro-epithelial-bodies (NEBs) and in a substantial portion of alveolar epithelial cells. The pathway becomes activated in isolated E12.5 mesenchyme-free distal epithelial buds cultured in Matrigel suggesting that absence of reporter activity in these regions stems from a dynamic cross-talk between endoderm and mesenchyme. Epithelial cells with activated BMP-pathway are enriched in progenitors capable of forming colonies in three-dimensional Matrigel cultures.</p> <p>As lung morphogenesis approaches completion, eGFP-expression declines and in adult lung its expression is barely detectable. However, upon tissue-injury, either with naphthalene or bleomycin, the canonical BMP-pathways is re-activated, in bronchial or alveolar epithelial cells respectively, in a manner reminiscent to early lung development and in tissue areas where reparatory progenitor cells reside. Our studies illustrate the dynamic activation of canonical BMP-pathway during lung development and adult lung tissue-repair and highlight its involvement in two important processes, namely, the early development of the pulmonary vasculature and the management of epithelial progenitor pools both during lung development and repair of adult lung tissue-injury.</p> </div

Spatial transcriptomic profiling of RespiratorySyncytial Virus (RSV) infection

Despite the fact that the human Respiratory Syncytial Virus (RSV) was first discoveredback in 1956, it remains one of the leading causes of morbidity and mortality inyoung children. Transcriptome-wide spatially resolved transcriptomics is a technologyunder rapid development that introduces a new modality for exploratory examinationof cellular behavior. With this modality, we examine how RSV infection changes thelocal cellular environment in the lung by infecting mice with RSV and comparing itto control samples four days after infection. We find viral presence in all compartmentsof the tissue, well-defined induced tertiary lymphoid tissue within some of thesamples, compartmentalized infiltration of innate immune cells, as well as functionalenrichment of airway epithelial repair pathways