Mechanotransduction and the extracellular matrix: Key drivers of lung pathologies and drug responsiveness.

The lung is a biomechanically active organ, with multiscale mechanical forces impacting the organ, tissue and cellular responses within this microenvironment. In chronic lung diseases, such as chronic obstructive pulmonary disease, pulmonary fibrosis and others, the structure of the lung is drastically altered impeding gas exchange. These changes are, in part, reflected in alterations in the composition, amount and organization of the extracellular matrix within the different lung compartments. The transmission of mechanical forces within lung tissue are broadcast by this complex mix of extracellular matrix components, in particular the collagens, elastin and proteoglycans and the crosslinking of these components. At both a macro and a micro level, the mechanical properties of the microenvironment have a key regulatory role in ascertaining cellular responses and the function of the lung. Cells adhere to, and receive signals from, the extracellular matrix through a number of different surface receptors and complexes which are important for mechanotransduction. This review summarizes the multiscale mechanics in the lung and how the mechanical environment changes in lung disease and aging. We then examine the role of mechanotransduction in driving cell signaling events in lung diseases and finish with a future perspective of the need to consider how such forces may impact pharmacological responsiveness in lung diseases.

Lung fibroblast-derived extracellular vesicles and soluble factors alleviate elastase-induced lung injury.

One of the main pathological features of chronic obstructive pulmonary disease (COPD) is the loss of functional alveolar tissue as a consequence of impaired regenerative capacities (emphysema). Recent research suggests that the secretome from mesenchymal cells, particularly extracellular vesicles (EVs), may possess regenerative properties beneficial for lung repair. However, the regenerative potential of the soluble factors (SFs) within the secretome remains largely unexplored in COPD. To this extent, we purified EVs and SFs secreted by lung fibroblasts to generate EV-enriched and SF-enriched fractions, and evaluated their effects on elastase-induced lung injury in both precision-cut lung slices (PCLS) and a mouse model. EV- and SF-enriched fractions were concentrated and purified from the conditioned medium of cultured MRC-5 lung fibroblasts using a combination of ultrafiltration and size exclusion chromatography, and were subsequently characterized according to the MISEV guidelines. Treatment with EV- or SF-enriched concentrates prevented and improved elastase-induced emphysema in PCLS, leading to reduced lung injury and upregulated markers of alveolar epithelial cells (aquaporin 5 and surfactant protein C), indicating potential parenchymal regeneration. Accordingly, prophylactic intratracheal treatment with lung fibroblast-derived EV- and SF-enriched concentrates in vivo attenuated elastase-induced lung tissue destruction, improved lung function, and enhanced gene expression of alveolar epithelial cell markers. Here, alveolar repair not only serves the purpose of facilitating gas exchange, but also by reinstating the essential parenchymal tethering required for optimal airway mechanics. In conclusion, this study highlights the therapeutic potential of both lung fibroblast-derived EV- and SF-enriched concentrates for the treatment of lung injury and emphysema.

Novel high-yield potato protease inhibitor panels block a wide array of proteases involved in viral infection and crucial tissue damage.

Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis. KEY MESSAGES: Protease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation. Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro. Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases. Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.

Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

An epithelial gene signature of trans-IL-6 signaling defines a subgroup of type 2-low asthma.

BACKGROUND: Asthma is stratified into type 2-high and type 2-low inflammatory phenotypes. Limited success has been achieved in developing drugs that target type 2-low inflammation. Previous studies have linked IL-6 signaling to severe asthma. IL-6 cooperates with soluble-IL-6Rα to activate cell signaling in airway epithelium. OBJECTIVE: We sought to study the role of sIL-6Rα amplified IL-6 signaling in airway epithelium and to develop an IL-6+ sIL-6Rα gene signature that may be used to select asthma patients who potentially respond to anti-IL-6 therapy. METHODS: Human airway epithelial cells were stimulated with combinations of IL-6, sIL-6Rα, and inhibitors, sgp130 (Olamkicept), and anti-IL-6R (Tocilizumab), to assess effects on pathway activation, epithelial barrier integrity, and gene expression. A gene signature was generated to identify IL-6 high patients using bronchial biopsies and nasal brushes. RESULTS: Soluble-IL-6Rα amplified the activation of the IL-6 pathway, shown by the increase of STAT3 phosphorylation and stronger gene induction in airway epithelial cells compared to IL-6 alone. Olamkicept and Tocilizumab inhibited the effect of IL-6 + sIL-6Rα on gene expression. We developed an IL-6 + sIL-6Rα gene signature and observed enrichment of this signature in bronchial biopsies but not nasal brushes from asthma patients compared to healthy controls. An IL-6 + sIL-6Rα gene signature score was associated with lower levels of sputum eosinophils in asthma. CONCLUSION: sIL-6Rα amplifies IL-6 signaling in bronchial epithelial cells. Higher local airway IL-6 + sIL-6Rα signaling is observed in asthma patients with low sputum eosinophils.

Mitochondrial transplantation rescues neuronal cells from ferroptosis.

Ferroptosis is a type of oxidative cell death that can occur in neurodegenerative diseases and involves damage to mitochondria. Previous studies demonstrated that preventing mitochondrial dysfunction can rescue cells from ferroptotic cell death. However, the complexity of mitochondrial dysfunction and the timing of therapeutic interventions make it difficult to develop an effective treatment strategy against ferroptosis in neurodegeneration conditions. In this study, we explored the use of mitochondrial transplantation as a novel therapeutic approach for preventing ferroptotic neuronal cell death. Our data showed that isolated exogenous mitochondria were incorporated into both healthy and ferroptotic immortalized hippocampal HT-22 cells and primary cortical neurons (PCN). The mitochondrial incorporation was accompanied by increased metabolic activity and cell survival through attenuating lipid peroxidation and mitochondrial superoxide production. Further, the function of mitochondrial complexes I, III and V activities contributed to the neuroprotective activity of exogenous mitochondria. Similarly, we have also showed the internalization of exogenous mitochondria in mouse PCN; these internalized mitochondria were found to effectively preserve the neuronal networks when challenged with ferroptotic stimuli. The administration of exogenous mitochondria into the axonal compartment of a two-compartment microfluidic device induced mitochondrial transportation to the cell body, which prevented fragmentation of the neuronal network in ferroptotic PCN. These findings suggest that mitochondria transplantation may be a promising therapeutic approach for protecting neuronal cells from ferroptotic cell death.

Current and future developments in the pharmacology of asthma and COPD: ERS seminar, Naples 2022.

Pharmacological management of airway obstructive diseases is a fast-evolving field. Several advances in unravelling disease mechanisms as well as intracellular and molecular pathways of drug action have been accomplished. While the clinical translation and implementation of in vitro results to the bedside remains challenging, advances in comprehending the mechanisms of respiratory medication are expected to assist clinicians and scientists in identifying meaningful read-outs and designing clinical studies. This European Respiratory Society Research Seminar, held in Naples, Italy, 5-6 May 2022, focused on current and future developments of the drugs used to treat asthma and COPD; on mechanisms of drug action, steroid resistance, comorbidities and drug interactions; on prognostic and therapeutic biomarkers; on developing novel drug targets based on tissue remodelling and regeneration; and on pharmacogenomics and emerging biosimilars. Related European Medicines Agency regulations are also discussed, as well as the seminar's position on the above aspects.

Inhaled pan-phosphodiesterase inhibitors ameliorate ovalbumin-induced airway inflammation and remodeling in murine model of allergic asthma.

Asthma is a heterogeneous, chronic respiratory disease characterized by airway inflammation and remodeling. Phosphodiesterase (PDE) inhibitors represent one of the intensively studied groups of potential anti-asthmatic agents due to their affecting both airway inflammation and remodeling. However, the effect of inhaled pan-PDE inhibitors on allergen induced asthma has not been reported to date. In this study we investigated the impact of two, representative strong pan-PDE inhibitors from the group of 7,8-disubstituted derivatives of 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione: compound 38 and 145, on airway inflammation and remodeling in murine model of ovalbumin (OVA)-challenged allergic asthma. Female Balb/c mice were sensitized and challenged with OVA, 38 and 145 were administrated by inhalation, before each OVA challenge. The inhaled pan-PDE inhibitors markedly reduced the OVA-induced airway inflammatory cell infiltration, eosinophil recruitment, Th2 cytokine level in bronchoalveolar lavage fluid, as well as both, total and OVA-specific IgE levels in plasma. In addition, inhaled 38 and 145 decreased many typical features of airway remodeling, including goblet cell metaplasia, mucus hypersecretion, collagen overproduction and deposition, as well as Tgfb1, VEGF, and α-SMA expression in airways of allergen challenged mice. We also demonstrated that both 38 and 145 alleviate airway inflammation and remodelling by inhibition of the TGF-ß/Smad signaling pathway activated in OVA-challenged mice. Taken together, these results suggest that the investigated pan-PDE inhibitors administered by inhalation are dual acting agents targeting both airway inflammation and remodeling in OVA-challenged allergic asthma and may represent promising, anti-asthmatic drug candidates.

Interleukin-11 disrupts alveolar epithelial progenitor function.

Background: Interleukin-11 (IL-11) is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF), since IL-11 induces myofibroblast differentiation and stimulates their excessive collagen deposition in the lung. In IPF there is disrupted alveolar structural architecture, yet the effect of IL-11 on the dysregulated alveolar repair remains to be elucidated. Methods: We hypothesised that epithelial-fibroblast communication associated with lung repair is disrupted by IL-11. Thus, we studied whether IL-11 affects the repair responses of alveolar lung epithelium using mouse lung organoids and precision-cut lung slices (PCLS). Additionally, we assessed the anatomical distribution of IL-11 and IL-11 receptor (IL-11R) in human control and IPF lungs using immunohistochemistry. Results: IL-11 protein was observed in airway epithelium, macrophages and in IPF lungs, also in areas of alveolar type 2 (AT2) cell hyperplasia. IL-11R staining was predominantly present in smooth muscle and macrophages. In mouse organoid co-cultures of epithelial cells with lung fibroblasts, IL-11 decreased organoid number and reduced the fraction of Prosurfactant Protein C-expressing organoids, indicating dysfunctional regeneration initiated by epithelial progenitors. In mouse PCLS exposed to IL-11, ciliated cell markers were increased. The response of primary human fibroblasts to IL-11 on gene expression level was minimal, though bulk RNA-sequencing revealed IL-11 modulated various processes which are associated with IPF, including unfolded protein response, glycolysis and Notch signalling. Conclusions: IL-11 disrupts alveolar epithelial regeneration by inhibiting progenitor activation and suppressing the formation of mature alveolar epithelial cells. Evidence for a contribution of dysregulated fibroblast-epithelial communication to this process is limited.

Association of asthma genetic variants with asthma-associated traits reveals molecular pathways of eosinophilic asthma.

INTRODUCTION: Asthma is a complex, polygenic, heterogenous inflammatory disease. Recently, we generated a list of 128 independent single nucleotide polymorphisms (SNPs) associated with asthma in genome-wide association studies. However, it is unknown if asthma SNPs are associated with specific asthma-associated traits such as high eosinophil counts, atopy, and airway obstruction, revealing molecular endotypes of this disease. Here, we aim to identify the association between asthma SNPs and asthma-associated traits and assess expression quantitative trait locus (e-QTLs) to reveal their downstream functional effects and find drug targets. METHODS: Association analyses between 128 asthma SNPs and associated traits (blood eosinophil numbers, atopy, airway obstruction, airway hyperresponsiveness) were conducted using regression modelling in population-based studies (Lifelines N = 32,817/Vlagtwedde-Vlaardingen N = 1554) and an asthma cohort (Dutch Asthma genome-wide association study N = 917). Functional enrichment and pathway analysis were performed with genes linked to the significant SNPs by e-QTL analysis. Genes were investigated to generate novel drug targets. RESULTS: We identified 69 asthma SNPs that were associated with at least one trait, with 20 SNPs being associated with multiple traits. The SNP annotated to SMAD3 was the most pleiotropic. In total, 42 SNPs were associated with eosinophil counts, 18 SNPs with airway obstruction, and 21 SNPs with atopy. We identified genetically driven pathways regulating eosinophilia. The largest network of eosinophilia contained two genes (IL4R, TSLP) targeted by drugs currently available for eosinophilic asthma. Several novel targets were identified such as IL-18, CCR4, and calcineurin. CONCLUSION: Many asthma SNPs are associated with blood eosinophil counts and genetically driven molecular pathways of asthma-associated traits were identified.

Age-associated differences in the human lung extracellular matrix.

Extracellular matrix (ECM) remodeling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study, we aimed to identify and localize age-associated ECM differences in human lungs using comprehensive transcriptomic, proteomic, and immunohistochemical analyses. Our previously identified age-associated gene expression signature of the lung was re-analyzed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (false discovery rate, FDR < 0.05) with a profile of age-associated proteins identified from a lung tissue proteomics dataset from nine control patients (49-76 years) (FDR < 0.05). Extensive immunohistochemical analysis was used to localize and semi-quantify the age-associated ECM differences in lung tissues from 62 control patients (18-82 years). Comparative analysis of transcriptomic and proteomic data identified seven ECM proteins with higher expression with age at both gene and protein levels: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4, and LUM. With immunohistochemistry, we demonstrated higher protein levels with age for COL6A2 in whole tissue, parenchyma, airway wall, and blood vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in lung parenchyma. Our study revealed that higher age is associated with lung ECM remodeling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility to developing chronic lung diseases.NEW & NOTEWORTHY We identified seven age-associated extracellular matrix (ECM) proteins, i.e., COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4, and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for COL6A2 in whole tissue, parenchyma, airway wall, and vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in parenchyma. Our findings lay a new foundation for the investigation of ECM differences in age-associated chronic lung diseases.

IL-1ß Induces a Proinflammatory Fibroblast Microenvironment that Impairs Lung Progenitors' Function.

Chronic obstructive pulmonary disease (COPD) is characterized by a persistent inflammatory state in the lungs and defective tissue repair. Although the inflammatory response in patients with COPD is well characterized and known to be exaggerated during exacerbations, its contribution to lung injury and abnormal repair is still unclear. In this study, we aimed to investigate how the inflammatory microenvironment affects the epithelial progenitors and their supporting mesenchymal niche cells involved in tissue repair of the distal lung. We focused on IL-1ß, a key inflammatory mediator that is increased during exacerbations of COPD, and used an organoid model of lung epithelial cells and fibroblasts to assess the effect of IL-1ß treatment on these cells' transcriptome and secreted factors. Whereas direct treatment of the lung organoids with IL-1ß promoted organoid growth, this switched toward inhibition when it was added as fibroblast pretreatment followed by organoid treatment. We then investigated the IL-1ß-driven mechanisms in the fibroblasts and found an inflammatory response related to (C-X-C motif) ligand (CXCL) chemokines; we confirmed that these chemokines were responsible for the impaired organoid growth and found that targeting their C-X-C chemokine receptors 1/2 (CXCR1/2) receptors or the IL-1ß intracellular signaling reduced the proinflammatory response and restored organoid growth. These data demonstrate that IL-1ß alters the fibroblasts' state by promoting a distinct inflammatory response, switching their supportive function on epithelial progenitors toward an inhibitory one in an organoid assay. These results imply that chronic inflammation functions as a shift toward inhibition of repair, thereby contributing to chronic inflammatory diseases like COPD.

Targeted lung denervation modulates the mucosal epithelial transcriptome in COPD.

This study shows that TLD reduces airway epithelial expression of genes related to acetylcholine processing and airway inflammation, which may help to elucidate the mechanism for its effect of reducing severe exacerbations in COPD https://bit.ly/3dWcqZk.

Function-specific IL-17A and dexamethasone interactions in primary human airway epithelial cells.

Asthmatics have elevated levels of IL-17A compared to healthy controls. IL-17A is likely to contribute to reduced corticosteroid sensitivity of human airway epithelium. Here, we aimed to investigate the mechanistic underpinnings of this reduced sensitivity in more detail. Differentiated primary human airway epithelial cells (hAECs) were exposed to IL-17A in the absence or presence of dexamethasone. Cells were then collected for RNA sequencing analysis or used for barrier function experiments. Mucus was collected for volume measurement and basal medium for cytokine analysis. 2861 genes were differentially expressed by IL-17A (Padj < 0.05), of which the majority was not sensitive to dexamethasone (< 50% inhibition). IL-17A did inhibit canonical corticosteroid genes, such as HSD11B2 and FKBP5 (p < 0.05). Inflammatory and goblet cell metaplasia markers, cytokine secretion and mucus production were all induced by IL-17A, and these effects were not prevented by dexamethasone. Dexamethasone did reverse IL-17A-stimulated epithelial barrier disruption, and this was associated with gene expression changes related to cilia function and development. We conclude that IL-17A induces function-specific corticosteroid-insensitivity. Whereas inflammatory response genes and mucus production in primary hAECs in response to IL-17A were corticosteroid-insensitive, corticosteroids were able to reverse IL-17A-induced epithelial barrier disruption.

Differential roles for lysyl oxidase (like), family members in chronic obstructive pulmonary disease; from gene and protein expression to function.

Chronic obstructive pulmonary disease (COPD) is characterized by long-term airflow obstruction with cigarette smoke as a key risk factor. Extracellular matrix (ECM) alterations in COPD may lead to small airway wall fibrosis. Altered collagen cross-linking, potentially mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4), orchestrates disturbed ECM homeostasis. In this study, we investigated the effects of smoking status and presence and severity of COPD on LOs gene and protein expression in the airways and the impact of LOs inhibition on airway contraction in an ex vivo mouse model. We used gene expression data from bronchial brushings, airway smooth muscle (ASM) cells in vitro and immunohistochemistry in lung tissue to assess smoke- and COPD-associated differences in LOs gene and protein expression in the small airways. We found higher LOX expression in current- compared to ex-smokers and higher LOXL1 expression in COPD compared to non-COPD patients. LOX and LOXL2 expression were upregulated in COPD ASM cells treated with cigarette smoke extract. LOXL1 and LOXL2 protein levels were higher in small airways from current- compared to non-smokers. In COPD patients, higher LOXL1 and lower LOX protein levels were observed, but no differences for LOXL2, LOXL3, and LOXL4 protein were detected in small airways. Inhibiting LOs activity increased airway contraction in murine lung slices. COPD-associated changes in LOs, in particular LOX and LOXL1, may be related to smoking and contribute to impaired airway function, providing potential novel targets for preventing or treating small airways changes in COPD.

Pan-Phosphodiesterase Inhibitors Attenuate TGF-ß-Induced Pro-Fibrotic Phenotype in Alveolar Epithelial Type II Cells by Downregulating Smad-2 Phosphorylation.

Airway remodeling is a pathological process that accompanies many chronic lung diseases. One of the important players in this process are epithelial cells, which under the influence of pro-inflammatory and pro-fibrotic factors present in the airway niche, actively participate in the remodeling process by increasing extracellular matrix secretion, acquiring migration properties, and overproducing pro-fibrotic transducers. Here, we investigated the effect of three new 8-arylalkylamino- and 8-alkoxy-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl-N-(5-(tert-butyl)-2-hydroxyphenyl)butanamides (1, 2, and 3), representing prominent pan-phosphodiesterase (pan-PDE) inhibitors on transforming growth factor type ß (TGF-ß)-induced alveolar epithelial type II cells (A549 cell line) of a pro-fibrotic phenotype. Our results demonstrate for the first time the strong activity of pan-PDE inhibitors in the prevention of TGF-ß-induced mesenchymal markers' expression and A549 cells' migration. We also showed an increased p-CREB and decreased p-Smad-2 phosphorylation in TGF-ß-induced A549 cells treated with 1, 2, and 3 derivatives, thereby confirming a pan-PDE inhibitor mesenchymal phenotype reducing effect in alveolar epithelial type II cells via suppression of the canonical Smad signaling pathway. Our observations confirmed that PDE inhibitors, and especially those active against various isoforms involved in the airway remodeling, constitute an interesting group of compounds modulating the pro-fibrotic response of epithelial cells.

Diesel exhaust particles distort lung epithelial progenitors and their fibroblast niche.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by inflammation and impaired tissue regeneration, and is reported as the fourth leading cause of death worldwide by the Centers for Disease Control and Prevention (CDC). Environmental pollution and specifically motor vehicle emissions are known to play a role in the pathogenesis of COPD, but little is still known about the molecular mechanisms that are altered following diesel exhaust particles (DEP) exposure. Here we used lung organoids derived from co-culture of alveolar epithelial progenitors and fibroblasts to investigate the effect of DEP on the epithelial-mesenchymal signaling niche in the distal lung, which is essential for tissue repair. We found that DEP treatment impaired the number as well as the average diameter of both airway and alveolar type of lung organoids. Bulk RNA-sequencing of re-sorted epithelial cells and fibroblasts following organoid co-culture shows that the Nrf2 pathway, which regulates antioxidants' activity, was upregulated in both cell populations in response to DEP; and WNT/ß-catenin signaling, which is essential to promote epithelial repair, was downregulated in DEP-exposed epithelial cells. We show that pharmacological treatment with anti-oxidant agents such as N-acetyl cysteine (NAC) or Mitoquinone mesylate (MitoQ) reversed the effect of DEP on organoids growth. Additionally, a WNT/ß-catenin activator (CHIR99021) successfully restored WNT signaling and promoted organoid growth upon DEP exposure. We propose that targeting oxidative stress and specific signaling pathways affected by DEP in the distal lung may represent a strategy to restore tissue repair in COPD.

A transcriptomics-guided drug target discovery strategy identifies receptor ligands for lung regeneration.

Currently, there is no pharmacological treatment targeting defective tissue repair in chronic disease. Here, we used a transcriptomics-guided drug target discovery strategy using gene signatures of smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke, identifying druggable targets expressed in alveolar epithelial progenitors, of which we screened the function in lung organoids. We found several drug targets with regenerative potential, of which EP and IP prostanoid receptor ligands had the most profound therapeutic potential in restoring cigarette smoke-induced defects in alveolar epithelial progenitors in vitro and in vivo. Mechanistically, we found, using single-cell RNA sequencing analysis, that circadian clock and cell cycle/apoptosis signaling pathways were differentially expressed in alveolar epithelial progenitor cells in patients with COPD and in a relevant model of COPD, which was prevented by prostaglandin E2 or prostacyclin mimetics. We conclude that specific targeting of EP and IP receptors offers therapeutic potential for injury to repair in COPD.