Background: Asthma is a complex chronic inflammatory disease characterised by airway inflammation, remodelling and hyperresponsiveness (AHR). Members of the AP-1 transcription factor family play important roles in the activation of the immune system and the control of cellular responses; however, their role in the development of asthma has not been well studied. We aimed to investigate the role of the lesser known AP-1 family member, Fra2 in experimental asthma. Methods: Phenotypic characterisation and gene expression profiling was performed on Fra2 (TG) overexpressing and wild-type mice. The efficacy of therapeutic interventions in regulating the Fra2 phenotype was determined. Results: Transcriptional profiling of TG mice revealed a high abundance of regulated genes associated with airway remodelling, inflammation and mucus production. A concomitant increase in peribronchial collagen deposition, smooth muscle thickening and mucus production was observed. TG mice possessed increased inflammatory infiltration in the lung, predominantly consisting of eosinophils and T-cells and elevated expression of Th2 cytokines and eotaxin. Furthermore, TG mice possessed severe AHR in response to increasing doses of methacholine. Glucocorticoid treatment led to a partial improvement of the asthma phenotype, whereas blockade of IL-13 via neutralising antibodies ameliorated AHR and mucus production, but had no effect on collagen deposition. Conclusion: We here describe a novel model for non-allergic asthma that does not require the application of exogenous allergens, which mimics several key features of the disease, such as airway inflammation, remodelling and hyperresponsiveness. Fra2 may represent a key molecule coordinating multiple aspects of asthma pathogenesis.

Natural killer (NK) cells play an important role in recognizing and killing pathogen-infected or malignant cells. Changes in their numbers or activation can contribute to several diseases and pathologies including systemic sclerosis (SSc), an autoimmune disease characterized by inflammation and tissue remodeling. In these patients, increased expression of the AP-1 transcription factor, Fra-2 was reported. In mice ectopic overexpression of Fra-2 (TG) leads to SSc with strong pulmonary fibrosis, pulmonary hypertension, and inflammation. Analysis of the underlying immune cell profile in the lungs of young TG mice, which do not yet show any signs of lung disease, revealed increased numbers of eosinophils and T cells but strongly reduced NK numbers. Therefore, we aimed to identify the cause of the absence of NK cells in the lungs of these mice and to determine the potential role of Fra-2 in NK development. Examination of inflammatory cell distribution in TG mice revealed similar NK deficiencies in the spleen, blood, and bone marrow. Deeper analysis of the WT and TG bone marrow revealed a potential NK cell developmental defect beginning at the preNKP stage. To determine whether this defect was cell-intrinsic or extrinsic, mixed bone marrow chimera and in vitro differentiation experiments were performed. Both experiments showed that the defect caused by Fra-2 was primarily cell-intrinsic and minimally dependent on the environment. Closer examination of surface markers and transcription factors required for NK development, revealed the expected receptor distribution but changes in transcription factor expression. We found a significant reduction in Nfil3, which is essential for the transition of common lymphoid cells to NK committed precursor cells and an AP-1 binding site in the promotor of this gene. In Summary, our data demonstrates that regulation of Fra-2 is essential for NK development and maturation, and suggests that the early NK dysfunction plays an important role in the pathogenesis of systemic sclerosis.

Pulmonary vascular remodeling is the main pathological hallmark of pulmonary hypertension disease. We undertook a comprehensive and multilevel approach to investigate the origin of smooth muscle actin-expressing cells in remodeled vessels. Transgenic mice that allow for specific, inducible, and permanent labeling of endothelial (Cdh5-tdTomato), smooth muscle (Acta2-, Myh11-tdTomato), pericyte (Cspg4-tdTomato), and fibroblast (Pdgfra-tdTomato) lineages were used to delineate the cellular origins of pulmonary vascular remodeling. Mapping the fate of major lung resident cell types revealed smooth muscle cells (SMCs) as the predominant source of cells that populate remodeled pulmonary vessels in chronic hypoxia and allergen-induced murine models. Combining in vivo cell type-specific, time-controlled labeling of proliferating cells with a pulmonary artery phenotypic explant assay, we identified proliferation of SMCs as an underlying remodeling pathomechanism. Multicolor immunofluorescence analysis showed a preserved pattern of cell type marker localization in murine and human pulmonary arteries, in both donors and idiopathic pulmonary arterial hypertension (IPAH) patients. Whilst neural glial antigen 2 (chondroitin sulfate proteoglycan 4) labeled mostly vascular supportive cells with partial overlap with SMC markers, PDGFRα-expressing cells were observed in the perivascular compartment. The luminal vessel side was lined by a single cell layer expressing endothelial markers followed by an adjacent and distinct layer defined by SMC marker expression and pronounced thickening in remodeled vessels. Quantitative flow cytometric analysis of single cell digests of diverse pulmonary artery layers showed the preserved separation into two discrete cell populations expressing either endothelial cell (EC) or SMC markers in human remodeled vessels. Additionally, we found no evidence of overlap between EC and SMC ultrastructural characteristics using electron microscopy in either donor or IPAH arteries. Lineage-specific marker expression profiles are retained during pulmonary vascular remodeling without any indication of cell type conversion. The expansion of resident SMCs is the major underlying and evolutionarily conserved paradigm of pulmonary vascular disease pathogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Pulmonary hypertension (PH) is a fatal disease characterized by remodeling processes such as increased migration and proliferation of pulmonary arterial smooth muscle cells (PASMC), enhanced matrix deposition, and dysregulation of cytoskeletal proteins. However, the contribution of cytoskeletal proteins in PH is still not fully understood. In this study, we have used a yeast two-hybrid screen to identify novel binding partners of the cytoskeletal adaptor protein four-and-a-half LIM domains 1 (Fhl-1). This identified paxillin as a new Fhl-1 interacting partner, and consequently we assessed its contribution to vascular remodeling processes. Native protein-protein binding was confirmed by co-immunoprecipitation studies in murine and human PASMC. Both proteins co-localized in PASMC in vitro and in vivo. In lung samples from idiopathic pulmonary arterial hypertension patients, paxillin expression was increased on mRNA and protein levels. Laser-microdissection of murine intrapulmonary arteries revealed elevated paxillin expression in hypoxia-induced PH. Furthermore, hypoxia-dependent upregulation of paxillin was HIF-1α dependent. Silencing of paxillin expression led to decreased PASMC adhesion, proliferation, and increased apoptosis. Regulation of these processes occurred via Akt and Erk1/2 kinases. In addition, adhesion of PASMC to the extracellular matrix protein fibronectin was critically dependent on paxillin expression. To summarize, we identified paxillin as a new regulator protein of PASMC growth.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to healthcare systems worldwide. Binding of the virus to angiotensin-converting enzyme 2 (ACE2) is an important step in the infection mechanism. However, it is unknown if ACE2 expression in patients with chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary arterial hypertension (IPAH), or pulmonary fibrosis (PF), is changed as compared to controls. We used lung samples from patients with COPD (n = 28), IPAH (n = 10), and PF (n = 10) as well as healthy control donor (n = 10) tissue samples to investigate the expression of ACE2 and related cofactors that might influence the course of SARS-CoV-2 infection. Expression levels of the ACE2 receptor, the putative receptor CD147/BSG, and the viral entry cofactors TMPRSS2 (transmembrane serine protease 2), EZR, and FURIN were determined by quantitative PCR and in open-access RNA sequencing datasets. Immunohistochemical and single-cell RNA sequencing (scRNAseq) analyses were used for localization and coexpression, respectively. Soluble ACE2 (sACE2) plasma levels were analyzed by enzyme-linked immunosorbent assay. In COPD as compared to donor, IPAH, and PF lung tissue, gene expression of ACE2, TMPRSS2, and EZR was significantly elevated, but circulating sACE2 levels were significantly reduced in COPD and PF plasma compared to healthy control and IPAH plasma samples. Lung tissue expressions of FURIN and CD147/BSG were downregulated in COPD. None of these changes were associated with changes in pulmonary hemodynamics. Histological analysis revealed coexpression of ACE2, TMPRSS2, and Ezrin in bronchial regions and epithelial cells. This was confirmed by scRNAseq analysis. There were no significant expression changes of the analyzed molecules in the lung tissue of IPAH and idiopathic PF as compared to control. In conclusion, we reveal increased ACE2 and TMPRSS2 expression in lung tissue with a concomitant decrease of protective sACE2 in COPD patients. These changes represent the possible risk factors for an increased susceptibility of COPD patients to SARS-CoV-2 infection.

Extracellular high-mobility group box-1 (HMGB1) acts as a signalling molecule during inflammation, cell differentiation and angiogenesis. Increased abundance of HMGB1 is associated with several pathological disorders such as cancer, asthma and chronic obstructive pulmonary disease (COPD). In this study, we investigated the relevance of HMGB1 in the pathological remodelling present in patients with idiopathic pulmonary arterial hypertension (IPAH) and pulmonary hypertension (PH) associated with COPD. Remodelled vessels present in COPD with PH and IPAH lung samples were often surrounded by HMGB1-positive cells. Increased HMGB1 serum levels were detected in both patient populations compared to control samples. The effects of physiological HMGB1 concentrations were then examined on cellular responses in vitro. HMGB1 enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC) and primary human arterial endothelial cells (PAEC). HMGB1 stimulated p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation. Furthermore, activation of the downstream AP-1 complex proteins c-Fos and c-Jun was observed. Silencing of c-Jun ablated the HMGB1-induced proliferation in PASMC. Thus, an inflammatory component such as HMGB1 can contribute to PASMC and PAEC proliferation and therefore potentially to vascular remodelling and PH pathogenesis.

Particulates from air pollution are implicated in causing or exacerbating respiratory and systemic cardiovascular diseases and are thought to be among the leading causes of morbidity and mortality. However, the contribution of ambient particulate matter to diseases affecting the pulmonary circulation, the right heart, and especially pulmonary hypertension is much less documented. Our own work and that of other groups has demonstrated that prolonged exposure to antigens via the airways can cause severe pulmonary arterial remodeling. In addition, vascular changes have been well documented in a typical disease of the airways, asthma. These experimental and clinical findings link responses in the airways with responses in the lung's vasculature. It follows that particulate air pollution could cause, or exacerbate, diseases in the pulmonary circulation and associated pulmonary hypertension. This perspective details the literature for support of this concept. Data regarding the health effects of particulate matter from air pollution on the lung's vasculature, with emphasis on the lung's inflammatory responses to particulate matter deposition and pulmonary hypertension, are discussed. A deeper understanding of the health implications of exposure to ambient particulate matter will improve our knowledge of how to improve the management of lung diseases, including diseases of the pulmonary circulation. As man-made ambient particulate air pollution is typically linked to economic growth, a better understanding of the health effects of exposure to particulate air pollution is expected to integrate the global goal of achieving healthy living for all.

Increasing evidence points towards an inflammatory component underlying pulmonary hypertension. However, the conclusive characterisation of multiple inflammatory cell populations in the lung is challenging due to the complexity of marker specificity and tissue inaccessibility. We used an unbiased computational flow cytometry approach to delineate the inflammatory landscape of idiopathic pulmonary arterial hypertension (IPAH) and healthy donor lungs.Donor and IPAH samples were discriminated clearly using principal component analysis to reduce the multidimensional data obtained from single-cell flow cytometry analysis. In IPAH lungs, the predominant CD45+ cell type switched from neutrophils to CD3+ T-cells, with increases in CD4+, CD8+ and γδT-cell subsets. Additionally, diversely activated classical myeloid-derived dendritic cells (CD14-HLA-DR+CD11c+CD1a+/-) and nonclassical plasmacytoid dendritic cells (pDCs; CD14-CD11c-CD123+HLA-DR+), together with mast cells and basophils, were more abundant in IPAH samples. We describe, for the first time, the presence and regulation of two cell types in IPAH, γδT-cells and pDCs, which link innate and adaptive immunity.With our high-throughput flow cytometry with multidimensional dataset analysis, we have revealed the interactive interplay between multiple inflammatory cells is a crucial part of their integrative network. The identification of γδT-cells and pDCs in this disease potentially provides a missing link between IPAH, autoimmunity and inflammation.

In idiopathic pulmonary arterial hypertension (IPAH), global transcriptional changes induce a smooth muscle cell phenotype characterised by excessive proliferation, migration, and apoptosis resistance. Long non-coding RNAs (lncRNAs) are key regulators of cellular function. Using a compartment-specific transcriptional profiling approach, we sought to investigate the link between transcriptional reprogramming by lncRNAs and the maladaptive smooth muscle cell phenotype in IPAH. Transcriptional profiling of small remodelled arteries from 18 IPAH patients and 17 controls revealed global perturbations in metabolic, neuronal, proliferative, and immunological processes. We demonstrated an IPAH-specific lncRNA expression profile and identified the lncRNA PAXIP1-AS1 as highly abundant. Comparative transcriptomic analysis and functional assays revealed an intrinsic role for PAXIP1-AS1 in orchestrating the hyperproliferative and migratory actions of IPAH smooth muscle cells. Further, we showed that PAXIP1-AS1 mechanistically interferes with the focal adhesion axis via regulation of expression and phosphorylation of its downstream target paxillin. Overall, we show that changes in the lncRNA transcriptome contribute to the disease-specific transcriptional landscape in IPAH. Our results suggest that lncRNAs, such as PAXIP1-AS1, can modulate smooth muscle cell function by affecting multiple IPAH-specific transcriptional programmes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In the era of personalized medicine, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as asthma phenotypes including obesity-associated asthma, are urgently needed. Peripheral blood was drawn from 10 obese, non-atopic asthmatic adults with a high body mass index (BMI; 36.67 ± 6.90); 10 non-obese, non-atopic asthmatic adults with normal BMI (23.88 ± 2.73); and 10 healthy controls with normal BMI (23.62 ± 3.74). All asthmatic patients were considered to represent a low type-2 asthma phenotype according to selective clinical parameters. RNA sequencing (RNA-Seq) was conducted on peripheral blood CD4+ T cells. Thousands of differentially expressed genes were identified in both asthma groups compared with heathy controls. The expression of interferon (IFN)-stimulated genes associated with IFN-related signaling pathways was specifically affected in obese asthmatics, while the gap junction and G protein-coupled receptor (GPCR) ligand binding pathways were enriched in both asthma groups. Furthermore, obesity gene markers were also upregulated in CD4+ T cells from obese asthmatics compared with the two other groups. Additionally, the enriched genes of the three abovementioned pathways showed a unique correlation pattern with various laboratory and clinical parameters. The specific activation of IFN-related signaling and viral infection pathways might provide a novel view of the molecular mechanisms associated with the development of the low type-2 obesity-associated asthma phenotype, which is a step ahead in the development of new stratified therapeutic approaches.

No abstract available

The coronavirus disease-2019 (COVID-19) pandemic has caused an enormous loss of lives. Various clinical trials of vaccines and drugs are being conducted worldwide; nevertheless, as of today, no effective drug exists for COVID-19. The identification of key genes and pathways in this disease may lead to finding potential drug targets and biomarkers. Here, we applied weighted gene co-expression network analysis and LIME as an explainable artificial intelligence algorithm to comprehensively characterize transcriptional changes in bronchial epithelium cells (primary human lung epithelium (NHBE) and transformed lung alveolar (A549) cells) during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study detected a network that significantly correlated to the pathogenicity of COVID-19 infection based on identified hub genes in each cell line separately. The novel hub gene signature that was detected in our study, including PGLYRP4 and HEPHL1, may shed light on the pathogenesis of COVID-19, holding promise for future prognostic and therapeutic approaches. The enrichment analysis of hub genes showed that the most relevant biological process and KEGG pathways were the type I interferon signaling pathway, IL-17 signaling pathway, cytokine-mediated signaling pathway, and defense response to virus categories, all of which play significant roles in restricting viral infection. Moreover, according to the drug-target network, we identified 17 novel FDA-approved candidate drugs, which could potentially be used to treat COVID-19 patients through the regulation of four hub genes of the co-expression network. In conclusion, the aforementioned hub genes might play potential roles in translational medicine and might become promising therapeutic targets. Further in vitro and in vivo experimental studies are needed to evaluate the role of these hub genes in COVID-19.

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

A central feature of progressive vascular remodeling is altered smooth muscle cell (SMC) homeostasis; however, the understanding of how different cell populations contribute to this process is limited. Here, we utilized single-cell RNA sequencing to provide insight into cellular composition changes within isolated pulmonary arteries (PAs) from pulmonary arterial hypertension and donor lungs. Our results revealed that remodeling skewed the balanced communication network between immune and structural cells, in particular SMCs. Comparative analysis with murine PAs showed that human PAs harbored heterogeneous SMC populations with an abundant intermediary cluster displaying a gradient transition between SMCs and adventitial fibroblasts. Transcriptionally distinct SMC populations were enriched in specific biological processes and could be differentiated into 4 major clusters: oxygen sensing (enriched in pericytes), contractile, synthetic, and fibroblast-like. End-stage remodeling was associated with phenotypic shift of preexisting SMC populations and accumulation of synthetic SMCs in neointima. Distinctly regulated genes in clusters built nonredundant regulatory hubs encompassing stress response and differentiation regulators. The current study provides a blueprint of cellular and molecular changes on a single-cell level that are defining the pathological vascular remodeling process.