3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

Preconceptional smoking alters spermatozoal miRNAs of murine fathers and affects offspring's body weight.

BACKGROUND: Active smoking has been reported among 7% of teenagers worldwide, with ages ranging from 13 to 15 years. An epidemiological study suggested that preconceptional paternal smoking is associated with adolescent obesity in boys. We developed a murine adolescent smoking model before conception to investigate the paternal molecular causes of changes in offspring's phenotype. METHOD: Male and female C57BL/6J mice were exposed to increasing doses of mainstream cigarette smoke (CS) from onset of puberty for 6 weeks and mated with room air (RA) controls. RESULTS: Thirteen miRNAs were upregulated and 32 downregulated in the spermatozoa of CS-exposed fathers, while there were no significant differences in the count and morphological integrity of spermatozoa, as well as the proliferation of spermatogonia between CS- and RA-exposed fathers. Offspring from preconceptional CS-exposed mothers had lower body weights (p = 0.007). Moreover, data from offspring from CS-exposed fathers suggested a potential increase in body weight (p = 0.062). CONCLUSION: We showed that preconceptional paternal CS exposure regulates spermatozoal miRNAs, and possibly influences the body weight of F1 progeny in early life. The regulated miRNAs may modulate transmittable epigenetic changes to offspring, thus influence the development of respiratory- and metabolic-related diseases such as obesity, a mechanism that warrants further studies for elaborate explanations.

Spotlight on microRNAs in allergy and asthma.

In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post-transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.

Host-microbe cross-talk in the lung microenvironment: implications for understanding and treating chronic lung disease.

Chronic respiratory diseases are highly prevalent worldwide and will continue to rise in the foreseeable future. Despite intensive efforts over recent decades, the development of novel and effective therapeutic approaches has been slow. However, there is new and increasing evidence that communities of micro-organisms in our body, the human microbiome, are crucially involved in the development and progression of chronic respiratory diseases. Understanding the detailed mechanisms underlying this cross-talk between host and microbiota is critical for development of microbiome- or host-targeted therapeutics and prevention strategies. Here we review and discuss the most recent knowledge on the continuous reciprocal interaction between the host and microbes in health and respiratory disease. Furthermore, we highlight promising developments in microbiome-based therapies and discuss the need to employ more holistic approaches of restoring both the pulmonary niche and the microbial community.

The need for clean air: The way air pollution and climate change affect allergic rhinitis and asthma.

Air pollution and climate change have a significant impact on human health and well-being and contribute to the onset and aggravation of allergic rhinitis and asthma among other chronic respiratory diseases. In Westernized countries, households have experienced a process of increasing insulation and individuals tend to spend most of their time indoors. These sequelae implicate a high exposure to indoor allergens (house dust mites, pets, molds, etc), tobacco smoke, and other pollutants, which have an impact on respiratory health. Outdoor air pollution derived from traffic and other human activities not only has a direct negative effect on human health but also enhances the allergenicity of some plants and contributes to global warming. Climate change modifies the availability and distribution of plant- and fungal-derived allergens and increases the frequency of extreme climate events. This review summarizes the effects of indoor air pollution, outdoor air pollution, and subsequent climate change on asthma and allergic rhinitis in children and adults and addresses the policy adjustments and lifestyle changes required to mitigate their deleterious effects.

Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development.

BACKGROUND: miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. METHODS: NHBE cells were cultured at air-liquid interface and treated with interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV-associated miRNAs were identified by a RT-qPCR-based profiling. Significant candidates were confirmed in EVs isolated by size-exclusion chromatography from nasal lavages of children with mild-to-moderate (n = 8) or severe asthma (n = 9), and healthy controls (n = 9). RESULTS: NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL-13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR-92b, miR-210, and miR-34a significantly correlated with lung function parameters in children (FEV1 FVC%pred and FEF25-75%pred ), thus lower sEV-miRNA levels in nasal lavages associated with airway obstruction. Subsequent ingenuity pathway analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation. CONCLUSION: Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR-34a, miR-92b, and miR-210, might be involved in the early development of a Th2 response in the airways and asthma.

miR-142-3p is associated with aberrant WNT signaling during airway remodeling in asthma.

Asthma is characterized by a chronic inflammation and remodeling of the airways. Although inflammation can be controlled, therapeutic options to revert remodeling do not exist. Thus, there is a large and unmet need to understand the underlying molecular mechanisms to develop novel therapies. We previously identified a pivotal role for miR-142-3p in regulating airway smooth muscle (ASM) precursor cell proliferation during lung development by fine-tuning the Wingless/Integrase I (WNT) signaling. Thus, we here aimed to investigate the relevance of this interaction in asthma. We performed quantitative RT-PCR and immune staining in a murine model for ovalbumin-induced allergic airway inflammation and in bronchial biopsies from patients with asthma and isolated primary fibroblasts thereof. miR-142-3p was increased in hyperproliferative regions of lung in murine and human asthma, whereas this microRNA (miRNA) was excluded from regions with differentiated ASM cells. Increases in miR-142-3p were associated with a decrease of its known target Adenomatous polyposis coli. Furthermore, we observed a differential expression of miR-142-3p in bronchial biopsies from patients with early or late onset severe asthma, which coincided with a differential WNT signature. Our data suggest that miR-142-3p is involved in regulating the balance between proliferation and differentiation of ASM cells in asthma, possibly via controlling WNT signaling. Thus, this miRNA might be an interesting target to prevent ASM hyperproliferation in asthma.

In utero exposure to cigarette smoke and effects across generations: A conference of animals on asthma.

BACKGROUND: The prevalence of asthma and chronic obstructive pulmonary disease (COPD) has risen markedly over the last decades and is reaching epidemic proportions. However, underlying molecular mechanisms are not fully understood, hampering the urgently needed development of approaches to prevent these diseases. It is well established from epidemiological studies that prenatal exposure to cigarette smoke is one of the main risk factors for aberrant lung function development or reduced fetal growth, but also for the development of asthma and possibly COPD later in life. Of note, recent evidence suggests that the disease risk can be transferred across generations, that is, from grandparents to their grandchildren. While initial studies in mouse models on in utero smoke exposure have provided important mechanistic insights, there are still knowledge gaps that need to be filled. OBJECTIVE: Thus, in this review, we summarize current knowledge on this topic derived from mouse models, while also introducing two other relevant animal models: the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. METHODS: This review is based on an intensive review of PubMed-listed transgenerational animal studies from 1902 to 2018 and focuses in detail on selected literature due to space limitations. RESULTS: This review gives a comprehensive overview of mechanistic insights obtained in studies with the three species, while highlighting the remaining knowledge gaps. We will further discuss potential (dis)advantages of all three animal models. CONCLUSION/CLINICAL RELEVANCE: Many studies have already addressed transgenerational inheritance of disease risk in mouse, zebrafish or fly models. We here propose a novel strategy for how these three model organisms can be synergistically combined to achieve a more detailed understanding of in utero cigarette smoke-induced transgenerational inheritance of disease risk.

D-tryptophan from probiotic bacteria influences the gut microbiome and allergic airway disease.

BACKGROUND: Chronic immune diseases, such as asthma, are highly prevalent. Currently available pharmaceuticals improve symptoms but cannot cure the disease. This prompted demands for alternatives to pharmaceuticals, such as probiotics, for the prevention of allergic disease. However, clinical trials have produced inconsistent results. This is at least partly explained by the highly complex crosstalk among probiotic bacteria, the host's microbiota, and immune cells. The identification of a bioactive substance from probiotic bacteria could circumvent this difficulty. OBJECTIVE: We sought to identify and characterize a bioactive probiotic metabolite for potential prevention of allergic airway disease. METHODS: Probiotic supernatants were screened for their ability to concordantly decrease the constitutive CCL17 secretion of a human Hodgkin lymphoma cell line and prevent upregulation of costimulatory molecules of LPS-stimulated human dendritic cells. RESULTS: Supernatants from 13 of 37 tested probiotic strains showed immunoactivity. Bioassay-guided chromatographic fractionation of 2 supernatants according to polarity, followed by total ion chromatography and mass spectrometry, yielded C11H12N2O2 as the molecular formula of a bioactive substance. Proton nuclear magnetic resonance and enantiomeric separation identified D-tryptophan. In contrast, L-tryptophan and 11 other D-amino acids were inactive. Feeding D-tryptophan to mice before experimental asthma induction increased numbers of lung and gut regulatory T cells, decreased lung TH2 responses, and ameliorated allergic airway inflammation and hyperresponsiveness. Allergic airway inflammation reduced gut microbial diversity, which was increased by D-tryptophan. CONCLUSIONS: D-tryptophan is a newly identified product from probiotic bacteria. Our findings support the concept that defined bacterial products can be exploited in novel preventative strategies for chronic immune diseases.

microRNA cluster 106a~363 is involved in T helper 17 cell differentiation.

T-helper cell type 17 (Th17) mediated inflammation is associated with various diseases including autoimmune encephalitis, inflammatory bowel disease and lung diseases such as chronic obstructive pulmonary disease and asthma. Differentiation into distinct T helper subtypes needs to be tightly regulated to ensure an immunological balance. As microRNAs (miRNAs) are critical regulators of signalling pathways, we aimed to identify specific miRNAs implicated in controlling Th17 differentiation. We were able to create a regulatory network model of murine T helper cell differentiation by combining Affymetrix mRNA and miRNA arrays and in silico analysis. In this model, the miR-212~132 and miR-182~183 clusters were significantly up-regulated upon Th17 differentiation, whereas the entire miR-106~363 cluster was down-regulated and predicted to target well-known Th17 cell differentiation pathways. In vitro transfection of miR-18b, miR-106a and miR-363-3p into primary murine Cd4+ lymphocytes decreased expression of retinoid-related orphan receptor c (Rorc), Rora, Il17a and Il17f, and abolished secretion of Th17-mediated interleukin-17a (Il17a). Moreover, we demonstrated target site-specific regulation of the Th17 transcription factors Rora and nuclear factor of activated T cells (Nfat) 5 by miR-18b, miR-106a and miR-363-3p through luciferase reporter assays. Here, we provide evidence that miRNAs are involved in controlling the differentiation and function of T helper cells, offering useful tools to study and modify Th17-mediated inflammation.

Altered Extracellular Vesicle-Derived Protein and microRNA Signatures in Bronchoalveolar Lavage Fluid from Patients with Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease for which there is no cure. Accumulating research results suggest a role for extracellular vesicles (EVs) in the pathogenesis of COPD. This study aimed to uncover the involvement of EVs and their molecular cargo in the progression of COPD by identification of EV-associated protein and microRNA (miRNA) profiles. We isolated EVs from the bronchial alveolar lavage fluid (BALF) of 18 patients with COPD and 11 healthy controls using size-exclusion chromatography. EV isolates were characterized using nanoparticle tracking analysis and protein content. Proteomic analysis revealed a higher abundance of 284 proteins (log2FC > 1) and a lower abundance of 3 proteins (log2FC < -1) in EVs derived from patients with COPD. Ingenuity pathway analysis showed that proteins enriched in COPD-associated EVs trigger inflammatory responses, including neutrophil degranulation. Variances in surface receptors and ligands associated with COPD EVs suggest a preferential interaction with alveolar cells. Small RNAseq analysis identified a higher abundance of ten miRNAs and a lower abundance of one miRNA in EVs from COPD versus controls (Basemean > 100, FDR < 0.05). Our data indicate that the molecular composition of EVs in the BALF of patients with COPD is altered compared to healthy control EVs. Several components in COPD EVs were identified that may perpetuate inflammation and alveolar tissue destruction.

Inspiring stories: the impact that being part of ERS activities can have on a professional career.

In this article, we provide an overview of the impact that being part of the @EuroRespSoc activities can have on a professional career, through the voices of Early Career Members (@EarlyCareerERS) https://bit.ly/32sHNW2.

Oral application of vancomycin alters murine lung microbiome and pulmonary immune responses.

Early life exposures to antibiotics negatively impact respiratory health and are associated with an increased risk of childhood asthma. It is explained that the lung is inclined to develop chronic inflammatory phenotypes due to early antibiotic alteration in the gut microbiome. We investigated whether a gut-targeted antibiotic has an impact on the lung microbiome and on pulmonary immunity. Fourteen-day old C57BL/6 mice were administered with vancomycin via oral gavage for 3 days (1 time/day). Control groups were treated with clarithromycin and phosphate-buffered saline (PBS), respectively. Five days after treatment, the cecum and lung microbiome, and pulmonary immune response were analyzed. Vancomycin treatment decreased the relative abundance of the genera Clostridium XIVa and Alistipes and the family Lachnospiraceae in the cecum. Furthermore, the relative abundance of the family Parabacteroidetes and the genus Lactobacillus were increased, whereas the abundance of the phylum Firmicutes was decreased. In the lung, vancomycin treatment reduced bacteria belonging to Clostridium XIVa and the family Lachnospiraceae as compared to those in the clarithromycin treated group. Lung cells from the vancomycin-treated mice released higher levels of interleukin (IL)-4 and IL-13 compared to those from the PBS group, and increased levels of IL-6, IFN-γ, and TNFα compared to lung cells from the clarithromycin and PBS treated mice. Our pilot study suggests that alteration in the gut microbiome could affect bacterial composition and immunity of the lung hence proposes a gut-lung microbiome axis in early life.

Isolation of extracellular vesicles with combined enrichment methods.

Extracellular vesicles (EVs) are currently of tremendous interest in many research disciplines and EVs have potential for development of EV diagnostics or therapeutics. Most well-known single EV isolation methods have their particular advantages and disadvantages in terms of EV purity and EV yield. Combining EV isolation methods provides additional potential to improve the efficacy of both purity and yield. This review assesses the contribution and efficacy of using combined EV isolation methods by performing a two-step systematic literature analysis from all papers applying EV isolation in the year 2019. This resulted in an overview of the various methods being applied for EV isolations. A second database was generated for all studies within the first database that fairly compared multiple EV isolation methods by determining both EV purity and EV yield after isolation. From these databases it is shown that the most used EV isolation methods are not per definition the best methods based on EV purity or EV yield, indicating that more factors play a role in the choice which EV isolation method to choose than only the efficacy of the method. From the included studies it is shown that ~60% of all the included EV isolations were performed with combined EV isolation methods. The majority of EV isolations were performed with differential ultracentrifugation alone or in combination with differential ultrafiltration. When efficacy of EV isolation methods was determined in terms of EV purity and EV yield, combined EV isolation methods clearly outperformed single EV isolation methods, regardless of the type of starting material used. A recommended starting point would be the use of size-exclusion chromatography since this method, especially when combined with low-speed centrifugation, resulted in the highest EV purity, while still providing a reasonable EV yield.

Extracellular Vesicles as Mediators of Cellular Cross Talk in the Lung Microenvironment.

The human lung is a complex tissue subdivided into several regions that differ in size, function, and resident cell types. Despite years of intensive research, we still do not fully understand the cross talk between these different regions and diverse cell populations in the lung and how this is altered in the development of chronic respiratory disease. The discovery of extracellular vesicles (EVs), small membrane vesicles released from cells for intercellular communication, has added another layer of complexity to cellular cross talk in the complex lung microenvironment. EVs from patients with chronic obstructive pulmonary disease, asthma, or sarcoidosis have been shown to carry microRNAs, proteins, and lipids that may contribute to inflammation or tissue degeneration. Here, we summarize the contribution of these small vesicles in the interplay of several different cell types in the lung microenvironment, with a focus on the development of chronic respiratory diseases. Although there are already many studies demonstrating the adverse effects of EVs in the diseased lung, we still have substantial knowledge gaps regarding the concrete role of EV involvement in lung disease, which should be addressed in future studies.

A sneak peek into the Early Career Members' session at the ERS International Congress and the experience of organising an ERS Research Seminar.

A sneak peek into the @EarlyCareerERS session at #ERSCongress 2020 and the experience of organising an @EuroRespSoc Research Seminar http://bit.ly/39yncgO.

Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1.

Smokers with apparently "healthy" lungs suffer from more severe and frequent viral respiratory infections, but the mechanisms underlying this observation are still unclear. Epithelial cells and dendritic cells (DC) form the first line of defense against inhaled noxes such as smoke or viruses. We therefore aimed to obtain insight into how cigarette smoke affects DCs and epithelial cells and how this influences the response to viral infection. Female C57BL/6J mice were exposed to cigarette smoke (CS) for 1 h daily for 24 days and then challenged i.n. with the viral mimic and Toll-like receptor 3 (TLR3) ligand poly (I:C) after the last exposure. DC subpopulations were analyzed 24 h later in whole lung homogenates by flow cytometry. Calu-3 cells or human precision-cut lung slices (PCLS) cultured at air-liquid interface were exposed to CS or air and subsequently inoculated with influenza H1N1. At 48 h post infection cytokines were analyzed by multiplex technology. Cytotoxic effects were measured by release of lactate dehydrogenase (LDH) and confocal imaging. In Calu-3 cells the trans-epithelial electrical resistance (TEER) was assessed. Smoke exposure of mice increased numbers of inflammatory and plasmacytoid DCs in lung tissue. Additional poly (I:C) challenge further increased the population of inflammatory DCs and conventional DCs, especially CD11b+ cDCs. Smoke exposure led to a loss of the barrier function in Calu-3 cells, which was further exaggerated by additional influenza H1N1 infection. Influenza H1N1-induced secretion of antiviral cytokines (IFN-α2a, IFN-λ, interferon-γ-induced protein 10 [IP-10]), pro-inflammatory cytokine IL-6, as well as T cell-associated cytokines (e.g., I-TAC) were completely suppressed in both Calu-3 cells and human PCLS after smoke exposure. In summary, cigarette smoke exposure increased the number of inflammatory DCs in the lung and disrupted epithelial barrier functions, both of which was further enhanced by viral stimulation. Additionally, the antiviral immune response to influenza H1N1 was strongly suppressed by smoke. These data suggest that smoke impairs protective innate mechanisms in the lung, which could be responsible for the increased susceptibility to viral infections in "healthy" smokers.

ERS International Congress, Madrid, 2019: highlights from the Basic and Translational Science Assembly.

In this review, the Basic and Translational Sciences Assembly of the European Respiratory Society (ERS) provides an overview of the 2019 ERS International Congress highlights. In particular, we discuss how the novel and very promising technology of single cell sequencing has led to the development of a comprehensive map of the human lung, the lung cell atlas, including the discovery of novel cell types and new insights into cellular trajectories in lung health and disease. Further, we summarise recent insights in the field of respiratory infections, which can aid in a better understanding of the molecular mechanisms underlying these infections in order to develop novel vaccines and improved treatment options. Novel concepts delineating the early origins of lung disease are focused on the effects of pre- and post-natal exposures on neonatal lung development and long-term lung health. Moreover, we discuss how these early life exposures can affect the lung microbiome and respiratory infections. In addition, the importance of metabolomics and mitochondrial function analysis to subphenotype chronic lung disease patients according to their metabolic program is described. Finally, basic and translational respiratory science is rapidly moving forward and this will be beneficial for an advanced molecular understanding of the mechanisms underlying a variety of lung diseases. In the long-term this will aid in the development of novel therapeutic targeting strategies in the field of respiratory medicine.