Respiratory Syncytial Virus Infection Does Not Induce Epithelial-Mesenchymal Transition.

Respiratory syncytial virus (RSV) infection does not cause severe disease in most of us despite suffering from multiple RSV infections during our lives. However, infants, young children, older adults, and immunocompromised patients are unfortunately vulnerable to RSV-associated severe diseases. A recent study suggested that RSV infection causes cell expansion, resulting in bronchial wall thickening in vitro. Whether the virus-induced changes in the lung airway resemble epithelial-mesenchymal transition (EMT) is still unknown. Here, we report that RSV does not induce EMT in three different in vitro lung models: the epithelial A549 cell line, primary normal human bronchial epithelial cells, and pseudostratified airway epithelium. We found that RSV increases the cell surface area and perimeter in the infected airway epithelium, which is distinct from the effects of a potent EMT inducer, transforming growth factor ß1 (TGF-ß1), driving cell elongation-indicative of cell motility. A genome-wide transcriptome analysis revealed that both RSV and TGF-ß1 have distinct modulation patterns of the transcriptome, which suggests that RSV-induced changes are distinct from EMT. IMPORTANCE We have previously shown that RSV infects ciliated cells on the apical side of the lung airway. RSV-induced cytoskeletal inflammation contributes to an uneven increase in the height of the airway epithelium, resembling noncanonical bronchial wall thickening. RSV infection changes epithelial cell morphology by modulating actin-protein 2/3 complex-driven actin polymerization. Therefore, it is prudent to investigate whether RSV-induced cell morphological changes contribute to EMT. Our data indicate that RSV does not induce EMT in at least three different epithelial in vitro models: an epithelial cell line, primary epithelial cells, and pseudostratified bronchial airway epithelium.

Respiratory epithelial cell types, states and fates in the era of single-cell RNA-sequencing.

Standalone and consortia-led single-cell atlases of healthy and diseased human airways generated with single-cell RNA-sequencing (scRNA-seq) have ushered in a new era in respiratory research. Numerous discoveries, including the pulmonary ionocyte, potentially novel cell fates, and a diversity of cell states among common and rare epithelial cell types have highlighted the extent of cellular heterogeneity and plasticity in the respiratory tract. scRNA-seq has also played a pivotal role in our understanding of host-virus interactions in coronavirus disease 2019 (COVID-19). However, as our ability to generate large quantities of scRNA-seq data increases, along with a growing number of scRNA-seq protocols and data analysis methods, new challenges related to the contextualisation and downstream applications of insights are arising. Here, we review the fundamental concept of cellular identity from the perspective of single-cell transcriptomics in the respiratory context, drawing attention to the need to generate reference annotations and to standardise the terminology used in literature. Findings about airway epithelial cell types, states and fates obtained from scRNA-seq experiments are compared and contrasted with information accumulated through the use of conventional methods. This review attempts to discuss major opportunities and to outline some of the key limitations of the modern-day scRNA-seq that need to be addressed to enable efficient and meaningful integration of scRNA-seq data from different platforms and studies, with each other as well as with data from other high-throughput sequencing-based genomic, transcriptomic and epigenetic analyses.

Identification of a Novel Subset of Human Airway Epithelial Basal Stem Cells.

The basal cell maintains the airway's respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell. To address functional heterogeneity amongst the basal cell population, we developed a novel monoclonal antibody, HLO1-6H5, that identifies a subset of KRT5+ (cytokeratin 5) basal cells. We used HLO1-6H5 and other known basal cell-reactive reagents to isolate viable airway subsets from primary human airway epithelium by Fluorescence Activated Cell Sorting. Isolated primary cell subsets were assessed for the stem cell capabilities of self-renewal and differentiation in the bronchosphere assay, which revealed that bipotent stem cells were, at minimum 3-fold enriched in the HLO1-6H5+ cell subset. Crosslinking-mass spectrometry identified the HLO1-6H5 target as a glycosylated TFRC/CD71 (transferrin receptor) proteoform. The HLO1-6H5 antibody provides a valuable new tool for identifying and isolating a subset of primary human airway basal cells that are substantially enriched for bipotent stem/progenitor cells and reveals TFRC as a defining surface marker for this novel cell subset.

PM2.5 Induces Pyroptosis via Activation of the ROS/NF-κB Signaling Pathway in Bronchial Epithelial Cells.

Background and Objectives: Fine particulate matter, PM2.5, is becoming a major threat to human health, particularly in terms of respiratory diseases. Pyroptosis is a recently discovered and distinct form of cell death, characterized by pore formation in the cell membrane and secretions of proinflammatory cytokines. There has been little research on the effect of PM2.5 on pyroptosis, especially in airway epithelium. We investigated whether PM2.5-related oxidative stress induces pyroptosis in bronchial epithelial cells and defined the underlying mechanisms. Materials and Methods: After exposure of a BEAS-2B cell line to PM2.5 concentration of 20 µg/mL, reactive oxygen species (ROS) levels, parameters related to pyroptosis, and NF-κB signaling were measured by Western blotting, immunofluorescence, and ELISA (Enzyme-linked immunosorbent assay). Results: PM2.5 induced pyroptotic cell death, accompanied by LDH (Lactate dehydrogenase) release and increased uptake of propidium iodide in a dose-dependent manner. PM2.5 activated the NLRP3-casp1-gasdermin D pathway, with resulting secretions of the proinflammatory cytokines IL-1ß and IL-18. The pyroptosis activated by PM2.5 was alleviated significantly by NLRP3 inhibitor. In PM2.5-exposed BEAS-2B cells, levels of intracellular ROS and NF-κB p65 increased. ROS scavenger inhibited the expression of the NLRP3 inflammasome, and the NF-κB inhibitor attenuated pyroptotic cell death triggered by PM2.5 exposure, indicating that the ROS/NF-κB pathway is involved in PM2.5-induced pyroptosis. Conclusions: These findings show that PM2.5 exposure can cause cell injury by NLRP3-inflammasome-mediated pyroptosis by upregulating the ROS/NF-κB pathway in airway epithelium.

Preterm ovine respiratory epithelial cell responses to mechanical ventilation, lipopolysaccharide, and interleukin-13.

Mechanical ventilation causes airway injury, respiratory epithelial cell proliferation, and lung inflammation in preterm sheep. Whether preterm epithelial cells respond similarly to adult epithelial cells or are altered by mechanical ventilation is unknown. We test the hypothesis that mechanical ventilation alters the responses of preterm airway epithelium to stimulation in culture. Respiratory epithelial cells from the trachea, left mainstem bronchi (LMSB), and distal bronchioles were harvested from unventilated preterm lambs, ventilated preterm lambs, and adult ewes. Epithelial cells were grown in culture or on air-liquid interface (ALI) and challenged with combinations of either media only, lipopolysaccharide (LPS; 10 ng/mL), bronchoalveolar fluid (BALF), or interleukin-13 (IL-13). Cell lysates were evaluated for mRNA changes in cytokine, cell type markers, Notch pathway, and acute phase markers. Mechanical ventilation altered preterm respiratory epithelium cell types. Preterm respiratory epithelial cells responded to LPS in culture with larger IL-8 induction than adults, and mechanical ventilation further increased cytokines IL-1ß and IL-8 mRNA induction at 2 h. IL-8 protein is detected in cell media after LPS stimulation. The addition of BALF from ventilated preterm animals increased IL-1ß mRNA to LPS (fivefold) in both preterm and adult cells and suppressed IL-8 mRNA (twofold) in adults. Preterm respiratory epithelial cells, when grown on ALI, responded to IL-13 with an increase in goblet cell mRNA. Preterm respiratory epithelial cells responded to LPS and IL-13 with responses similar to adults. Mechanical ventilation or exposure to BALF from mechanically ventilated animals alters the responses to LPS.NEW & NOTEWORTHY Preterm lamb respiratory epithelial cells can be extracted from the trachea and bronchi and frozen, and the preterm cells can respond in culture to stimulation with LPS or IL-13. Brief mechanical ventilation changes the distribution and cell type of preterm respiratory cells toward an adult phenotype, and mechanical ventilation alters the response to LPS in culture. Bronchoalveolar lavage fluid from preterm lambs receiving mechanical ventilation also alters unventilated preterm and adult responses to LPS.

Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection.

Allergy and rhinovirus (RV) infections are major triggers for rhinitis and asthma, causing a socioeconomic burden. As RVs and allergens may act synergistically to promote airway inflammation, simultaneous treatment strategies for both causative agents would be innovative. We have previously identified the transmembrane glycoprotein intercellular adhesion molecule 1 (ICAM-1) as an anchor for antibody conjugates bispecific for ICAM-1 and Phleum pratense (Phl p) 2, a major grass pollen allergen, to block allergen transmigration through the epithelial barrier. Since ICAM-1 is a receptor for the major group RVs, we speculated that our bispecific antibody conjugates may protect against RV infection. Therefore, we created antibody conjugates bispecific for ICAM-1 and the major grass pollen allergen Phl p 5 and analyzed their capacity to affect allergen penetration and RV infection. Bispecific antibody conjugates significantly reduced the trans-epithelial migration of Phl p 5 and thus the basolateral Phl p 5 concentration and allergenic activity as determined by humanized rat basophilic leukemia cells and inhibited RV infection of cultured epithelial cells. A reduction in allergenic activity was obtained only through the prevention of allergen transmigration because the Phl p 5-specific IgG antibody did not block the allergen-IgE interaction. Our results indicate the potential of allergen/ICAM-1-specific antibody conjugates as a topical treatment strategy for allergy and RV infections.

Bordetella pertussis Induces Interferon Gamma Production by Natural Killer Cells, Resulting in Chemoattraction by Respiratory Epithelial Cells.

BACKGROUND: Whooping cough is caused by infection of the airways with Bordetella pertussis (Bp). As interferon gamma (IFN-γ) is essential for protective immunity against Bp, we investigated how IFN-γ is induced by Bp or the virulence antigens filamentous hemagglutinin adhesin, pertactin, or pertussis toxin, and how IFN-γ contributes to local immune responses in humans. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors and/or respiratory epithelial cells were stimulated with soluble antigens or inactivated intact Bp and the presence or absence of blocking antibodies or chemokines. Supernatants and cells were analyzed for IFN-γ and chemokine production, and lymphocyte migration was tested using epithelial supernatants. RESULTS: The soluble antigens failed to induce IFN-γ production, whereas inactivated Bp induced IFN-γ production. Natural killer (NK) cells were the main source of IFN-γ production, which was enhanced by interleukin 15. Epithelial-PBMC co-cultures showed robust IFN-γ-dependent CXCL9 and CXCL10 production by the epithelial cells following stimulation with IFN-γ and Bp. The epithelial-derived chemokines resulted in CXCR3-dependent recruitment of NK and T cells. CONCLUSIONS: Inactivated Bp, but not antigens, induced potent IFN-γ production by NK cells, resulting in chemoattraction of lymphocytes toward the respiratory epithelium. These data provide insight into the requirements for IFN-γ production and how IFN-γ enhances local immune responses to prevent Bp-mediated disease.

Metformin-induced alterations in gills of the freshwater fish Astyanax lacustris (Lütken, 1875) detected by histological and scanning electron microscopy.

The antidiabetic drug metformin is widely prescribed and found in different concentrations in the environment around the world, raising concern about potential impacts on aquatic life. Analyses of the effects of exposure of biological models to aquatic contaminants are important for assessing pollution effects on fish health. The gills of fishes represent primary targets of disturbance by pollutants, mainly because of the large surface of the respiratory epithelium and the high perfusion rate, which both help the entry of pollutants into this tissue. In this context, the aim of this work was to use gill histological analyses biomarkers to evaluate the toxicity of metformin on aquatic environmental systems, by means of chronic exposure for 90 days of Astyanax lacustris (lambari), an ecologically important neotropical species that can be used as an environmental bioindicator. Histopathological analyses were performed using Light and Scanning Electron Microscopy. The main changes were lamellar fusion, telangiectasia hyperplasia and disappearance of microridges. The morphological changes observed possibly interfere with the gill physiology, indicating an unfavorable situation to the presence of metformin in the water, pointing to a concern that metformin may pose a risk to Astyanax lacustris and likely to other fish species, compromising the dynamics of the aquatic ecosystem as a whole. Graphical abstract.

SARS-CoV-2 Infection Dysregulates Cilia and Basal Cell Homeostasis in the Respiratory Epithelium of Hamsters.

Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.

Air sac and gill vasotocin receptor gene expression in the air-breathing catfish Heteropneustes fossilis exposed to water and air deprivation conditions.

Heteropneustes fossilis is a facultative air-breathing freshwater catfish and inhabits ponds, ditches, swamps, marshes and rivers that dry up in summers. It possesses a pair of unique tubular accessory respiratory organ (air sac), which is a modification of the gill chamber and enables it to live in water-air transition zones. In the catfish, three vasotocin (Vt) receptor gene paralogs viz., v1a1, v1a2 and v2a were identified for Vt actions. In the present study, the receptor gene transcripts were localized in the gill and air sac by in situ hybridization, and their expression levels in relation to water and air deprivation conditions were investigated by quantitative RT-PCR. The catfish were exposed to 1 h and 2 h in gonad inactive (resting) and gonad active (prespawning) phases. The gene paralogs showed overlapping distribution in the respiratory epithelium of primary and secondary lamellae of gills and reduced lamellae of the air sacs. In water deprivation (forced aerial mode of respiration) experiment, v2a expression showed a high fold increase in the air sac, which was unchanged or inhibited in the gill. Both v1a1 and v1a2 expression was significantly upregulated in the air sac but showed varied responses in the gill. The gill v1a1 expression was unchanged in the resting phase and modestly upregulated in the prespawning phase. The gill v1a2 expression was modestly upregulated at 1 h in both phases but unchanged at 2 h. In the air deprivation experiment (forced aquatic respiration), the v2a expression in the air sac was inhibited except for a mild stimulation at 1 h in the prespawning phase. In the gill, the v2a expression was stimulated with a steep upregulation at 2 h in the prespawning phase. Both v1a1 and v1a2 expression was significantly high in the gill but only modestly increased or unchanged in the air sac. The expression patterns point to a functional distinction; the V2 type receptor expression was higher in the air sac during forced aerial respiration, and the V1 type receptor expression was highly prominent in the gill during forced aquatic respiration. Water and air deprivation treatments caused a significant increase in plasma cortisol level, and the stimulation was higher in the water deprivation fish in the resting phase but equally prominent in the water and air deprivation groups in the prespawning phase. The results indicate that the changes in the expression patterns of Vt receptor genes may be a sequel to stress (hypoxic, metabolic and osmotic), and both Vt and cortisol may interact to counter the stress responses. This study shows that Vt has a new role in the control of air sac functions.

Evaluation of the usefulness of vitamin D as a predictor of mortality in patients with COVID-19.

INTRODUCTION: One of the functions of vitamin D is to regulate respiratory epithelium inflammatory response; therefore, deficiency of this vitamin in the context of COVID-19 could constitute a predictive biomarker of the disease outcome. OBJECTIVE: To evaluate the usefulness of vitamin D for predicting mortality in patients with COVID-19. METHODS: Observational, retrospective study in which 154 patients diagnosed with COVID-19 were included, out of whom 111 survived and 43 died. Vitamin D concentration was determined in all of them. RESULTS: A log-rank p-value < 0.032 was obtained for survival when vitamin D concentration was used as a categorical variable (≤ 20 ng/mL and > 20 ng/mL). On Cox proportional analysis, age and vitamin D concentration were shown to be risk factors associated with mortality in patients with COVID-19 (age: HR = 1.036, 95% CI = 1.016-1.058, p < 0.001; vitamin D: HR (≤ 20 ng/mL and > 20 ng/mL) = 0.478, 95% CI = 0.237-0.966, p < 0.040). CONCLUSION: Age and vitamin D concentration were predictive factors for mortality in COVID-19-infected patients.

INTRODUCCIÓN: Una de las funciones de la vitamina D es regular la respuesta inflamatoria del epitelio respiratorio; por ello, la deficiencia de esa vitamina en el contexto de COVID-19 podría constituir un biomarcador preditivo del desenlace de COVID-19. OBJETIVO: Evaluar la utilidad de la vitamina D para predecir la mortalidad en pacientes con COVID-19. MÉTODOS: Estudio observacional y retrospectivo en el que se incluyeron 154 pacientes con diagnóstico de COVID-19, de los cuales 111 sobrevivieron y 43 fallecieron. En todos se determinó la concentración de vitamina D. RESULTADOS: Se obtuvo un valor log-rank de p < 0.032 para la supervivencia al utilizar la concentración de vitamina D como variable categórica (≤ 20 ng/mL y > 20 ng/mL). Mediante análisis proporcional de Cox se encontró que la edad y concentración de vitamina D mostraron ser factores de riesgo asociados a la mortalidad en pacientes con COVID-19 (edad: HR = 1.036, IC 95 % = 1.016-1.058, p < 0.001; vitamina D: HR ≤ 20 ng/mL y > 20 ng/mL = 0.478, IC 95 % = 0.237-0.966, p < 0.040). CONCLUSIÓN: La edad y la concentración de vitamina D constituyeron factores predictivos de mortalidad en pacientes infectados por COVID-19.

Stem Cell-derived Respiratory Epithelial Cell Cultures as Human Disease Models.

Advances in stem cell biology and the understanding of factors that determine lung stem cell self-renewal have enabled long-term in vitro culture of human lung cells derived from airway basal and alveolar type II cells. Improved capability to expand and study primary cells long term, including in clonal cultures that are recently derived from a single cell, will allow experiments that address fundamental questions about lung homeostasis and repair, as well as translational questions in asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and lung cancer research. Here, we provide a brief history of postnatal lung epithelial cell culture and describe recent methodological advances. We further discuss the applications of primary cultures in defining "normal" epithelium, in modeling lung disease, and in future cell therapies.

The interplay between airway epithelium and the immune system - A primer for the respiratory clinician.

The respiratory epithelium is one of the primary interfaces between the body's immune system and the external environment. This review discusses the innate and adaptive immunomodulatory effects of the respiratory epithelium, highlighting the physiologic immune responses associated with health and the disease-causing sequelae when these physiologic responses go awry. Airway macrophages, dendritic cells, and innate lymphoid cells are discussed as orchestrators of physiological and pathological innate immune responses and T cells, B cells, mast cells, and granulocytes (eosinophils and neutrophils) as orchestrators of physiologic and pathologic adaptive immune responses. The interplay between the airway epithelium and the varied immune cells as well as the interplay between these immune cells is discussed, highlighting the importance of the dose of noxious stimuli and pathogens in immune programming and the timing of their interaction with the immune cells that determine the pattern of immune responses. Although each cell type has been researched individually, this review highlights the need for simultaneous temporal investigation of immune responses from these varied cells to noxious stimuli and pathogens.

Evaluating Feasibility of Human Tissue Engineered Respiratory Epithelium Construct as a Potential Model for Tracheal Mucosal Reconstruction.

The normal function of the airway epithelium is vital for the host's well-being. Conditions that might compromise the structure and functionality of the airway epithelium include congenital tracheal anomalies, infection, trauma and post-intubation injuries. Recently, the onset of COVID-19 and its complications in managing respiratory failure further intensified the need for tracheal tissue replacement. Thus far, plenty of naturally derived, synthetic or allogeneic materials have been studied for their applicability in tracheal tissue replacement. However, a reliable tracheal replacement material is missing. Therefore, this study used a tissue engineering approach for constructing tracheal tissue. Human respiratory epithelial cells (RECs) were isolated from nasal turbinate, and the cells were incorporated into a calcium chloride-polymerized human blood plasma to form a human tissue respiratory epithelial construct (HTREC). The quality of HTREC in vitro, focusing on the cellular proliferation, differentiation and distribution of the RECs, was examined using histological, gene expression and immunocytochemical analysis. Histological analysis showed a homogenous distribution of RECs within the HTREC, with increased proliferation of the residing RECs within 4 days of investigation. Gene expression analysis revealed a significant increase (p < 0.05) in gene expression level of proliferative and respiratory epithelial-specific markers Ki67 and MUC5B, respectively, within 4 days of investigation. Immunohistochemical analysis also confirmed the expression of Ki67 and MUC5AC markers in residing RECs within the HTREC. The findings show that calcium chloride-polymerized human blood plasma is a suitable material, which supports viability, proliferation and mucin secreting phenotype of RECs, and this suggests that HTREC can be a potential candidate for respiratory epithelial tissue reconstruction.

Respiratory microbiome and epithelial interactions shape immunity in the lungs.

The airway epithelium represents a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli including microbes and allergens. However, lung epithelial cells are increasingly recognized as active effectors of microbial defence, contributing to both innate and adaptive immune function in the lower respiratory tract. These cells express an ample repertoire of pattern recognition receptors with specificity for conserved microbial and host motifs. Modern molecular techniques have uncovered the complexity of the lower respiratory tract microbiome. The interaction between the microbiota and the airway epithelium is key to understanding how stable immune homeostasis is maintained. Loss of epithelial integrity following exposure to infection can result in the onset of inflammation in susceptible individuals and may culminate in lung disease. Here we discuss the current knowledge regarding the molecular and cellular mechanisms by which the pulmonary epithelium interacts with the lung microbiome in shaping immunity in the lung. Specifically, we focus on the interactions between the lung microbiome and the cells of the conducting airways in modulating immune cell regulation, and how defects in barrier structure and function may culminate in lung disease. Understanding these interactions is fundamental in the search for more effective therapies for respiratory diseases.

Key role of the epithelium in chronic upper airways diseases.

The respiratory epithelium of the upper airways is a first-line defence against inhaled irritants, pathogens and allergens. It ensures a physical barrier provided by apical junctions and mucociliary clearance to avoid excessive activation of the immune system. The epithelium also forms a chemical and immunological barrier, extensively equipped to protect the airways against external aggressions before the adaptive immune system is required. Under normal circumstances, the epithelium is capable of recovering rapidly after damage. This manuscript reviews these main properties of the upper airway epithelium as well as its reported impairments in chronic inflammatory diseases. The knowledge on normal epithelial functions and their dysregulation in upper airway diseases should help to design new epithelial-targeted treatments.

T2 and T17 cytokines alter the cargo and function of airway epithelium-derived extracellular vesicles.

BACKGROUND: Asthma is a common and heterogeneous disease that includes subgroups characterized by type 2 (T2) or type 17 (T17) immune responses for which there is a need to identify the underlying mechanisms and biomarkers in order to develop specific therapies. These subgroups can be defined by airway epithelium gene signatures and the airway epithelium has also been implicated to play a significant role in asthma pathology. Extracellular vesicles (EVs) carry functional biomolecules and participate in cell-to-cell communication in both health and disease, properties that are likely to be involved in airway diseases such as asthma. The aim of this study was to identify stimulus-specific proteins and functionality of bronchial epithelium-derived EVs following stimulation with T2 or T17 cytokines. METHODS: EVs from cytokine-stimulated (T2: IL-4 + IL-13 or T17: IL-17A + TNFα) human bronchial epithelial cells cultured at air-liquid interface (HBEC-ALI) were isolated by density cushion centrifugation and size exclusion chromatography and characterized with Western blotting and electron microscopy. Transcriptomic (cells) and proteomic (EVs) profiling was also performed. RESULTS: Our data shows that EVs are secreted and can be isolated from the apical side of HBEC-ALI and that cytokine stimulation increases EV release. Genes upregulated in cells stimulated with T2 or T17 cytokines were increased also on protein level in the EVs. Proteins found in T17-derived EVs were suggested to be involved in pathways related to neutrophil movement which was supported by assessing neutrophil chemotaxis ex vivo. CONCLUSIONS: Together, the results suggest that epithelial EVs are involved in airway inflammation and that the EV proteome may be used for discovery of disease-specific mechanisms and signatures which may enable a precision medicine approach to the treatment of asthma.

Expression of SARS-CoV-2 entry receptors in the respiratory tract of healthy individuals, smokers and asthmatics.

SARS-CoV-2 is causing a pandemic with currently > 29 million confirmed cases and > 900,000 deaths worldwide. The locations and mechanisms of virus entry into the human respiratory tract are incompletely characterized. We analyzed publicly available RNA microarray datasets for SARS-CoV-2 entry receptors and cofactors ACE2, TMPRSS2, BSG (CD147) and FURIN. We found that ACE2 and TMPRSS2 are upregulated in the airways of smokers. In asthmatics, ACE2 tended to be downregulated in nasal epithelium, and TMPRSS2 was upregulated in the bronchi. Furthermore, respiratory epithelia were negative for ACE-2 and TMPRSS2 protein expression while positive for BSG and furin, suggesting a possible alternative entry route for SARS-CoV-2.

How the Respiratory Epithelium Senses and Reacts to Influenza Virus.

The human lung is constantly exposed to the environment and potential pathogens. As the interface between host and environment, the respiratory epithelium has evolved sophisticated sensing mechanisms as part of its defense against pathogens. In this review, we examine how the respiratory epithelium senses and responds to influenza A virus, the biggest cause of respiratory viral deaths worldwide.

Interleukin-33 in the developing lung-Roles in asthma and infection.

It has become increasingly clear that interleukin-33 (IL-33) plays a crucial role in initiation of type 2 immunity. The last decade of intense research has uncovered multiple mechanisms through which IL-33 targets key effector cells of the allergic immune response. Recently, IL-33 has been implicated in shaping the immune system of the lungs early in life, at a time which is crucial in the subsequent development of allergic asthma. In this review, we will address the current literature describing the role of IL-33 in the healthy and diseased lung. In particular, we will focus on the evidence for IL-33 in the development of immune responses in the lung, including the role of IL-33-responsive immune cells that may explain susceptibility to allergic sensitization at a young age and the association between genetic variants of IL-33 and asthma in humans. Finally, we will indicate areas for potential therapeutic modulation of the IL-33 pathway.