Search

Epithelial cells in the larynx and trachea sense harmful cues and trigger protective reflexes.

Acetylcholine (ACh), which activates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), enhances airway ciliary beating by increasing the intracellular Ca 2+ concentration ([Ca 2+ ] i ). The mechanisms enhancing airway ciliary beating by nAChRs have remained largely unknown, although those by mAChRs are well understood. In this study, we focused on the effects of α7-nAChRs and voltage-gated Ca 2+ channels (Ca V s) on the airway ciliary beating. The activities of ciliary beating were assessed by frequency (CBF, ciliary beat frequency) and amplitude (CBD, ciliary bend distance) measured by high-speed video microscopy. ACh enhanced CBF and CBD by 25% mediated by an [Ca 2+ ] i increase stimulated by mAChRs and α7-nAChRs (a subunit of nAChR) in airway ciliary cells of mice. Experiments using PNU282987 (an agonist of α7-nAChR) and MLA (an inhibitor of α7-nAChR) revealed that CBF and CBD enhanced by α7-nAChR are approximately 50% of those enhanced by ACh. CBF, CBD, and [Ca 2+ ] i enhanced by α7-nAChRs were inhibited by nifedipine, suggesting activation of Ca V s by α7-nAChRs. Experiments using a high K + solution with/without nifedipine (155.5 mM K + ) showed that the activation of Ca V s enhances CBF and CBD via an [Ca 2+ ] i increase. Immunofluorescence and immunoblotting studies demonstrated that Cav1.2 and α7-nAChR are expressed in airway cilia. Moreover, IL-13 stimulated MLA-sensitive increases in CBF and CBD in airway ciliary cells, suggesting an autocrine regulation of ciliary beating by Ca V 1.2/α7-nAChR/ACh. In conclusion, a novel Ca 2+ signalling pathway in airway cilia, Ca V 1.2/α7-nAChR, enhances CBF and CBD and activates mucociliary clearance maintaining healthy airways., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) augments intracellular Ca 2+ signaling and contractile responses of airway smooth muscles, leading to airway hyperresponsiveness. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the cellular mechanism of the potentiated contraction of mouse tracheal smooth muscle induced by TNF-α. The results showed that TNF-α triggered facilitation of mouse tracheal smooth muscle contraction in an epithelium-independent manner. The TNF-α-induced hypercontractility could be suppressed by the protein kinase C inhibitor GF109203X, the tyrosine kinase inhibitor genistein, the Src inhibitor PP2, or the L-type voltage-dependent Ca 2+ channel blocker nifedipine. Following TNF-α incubation, the α 1C L-type Ca 2+ channel (Ca V 1.2) was up-regulated in cultured primary mouse tracheal smooth muscle cells. Pronounced phosphotyrosine levels were observed in mouse tracheas. In conclusion, this study shows that TNF-α enhanced airway smooth muscle contraction via protein kinase C-Src-Ca V 1.2 pathways, which provides novel insights into the pathologic role of proinflammatory cytokines in mediating airway hyperresponsiveness., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Human organ-on-a-chip models are powerful tools for preclinical research that can be used to study the mechanisms of disease and evaluate new targets for therapeutic intervention. Lung-on-a-chip models have been one of the most well-characterized designs in this field and can be altered to evaluate various types of respiratory disease and to assess treatment candidates prior to clinical testing. These systems are capable of overcoming the flaws of conventional two-dimensional (2-D) cell culture and in vivo animal testing due to their ability to accurately recapitulate the in vivo microenvironment of human tissue with tunable material properties, microfluidic integration, delivery of precise mechanical and biochemical cues, and designs with organ-specific architecture. In this review, we first describe an overview of currently available lung-on-a-chip designs. We then present how recent innovations in human stem cell biology, tissue engineering, and microfabrication can be used to create more predictive human lung-on-a-chip models for studying respiratory disease. Finally, we discuss the current challenges and future directions of lung-on-a-chip designs for in vitro disease modeling with a particular focus on immune and multiorgan interactions.

Pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. It is a growing clinical problem which can result in breathlessness or respiratory failure and has an average life expectancy of 3 years from diagnosis. Predominantly accumulation of M2 macrophages accelerates fibrosis progression by secreting multiple cytokines that promote fibroblast to myofibroblast transition and aberrant wound healing of epithelial cells. Targeting activated macrophages to inhibit the pro-fibrotic phenotype is considered as an approach for the potential treatment of PF. Clevudine is s a purine nucleoside analogue which in an oral formulation is approved for treatment of patients with hepatitis B virus (HBV). Here, we found that clevudine is capable of suppressing pro-fibrotic phenotype (i.e., CD206, Arg1 and YM1) of M2 macrophages while enhancing anti-fibrotic phenotype (i.e., CD86, IL-6 and IL-10) by inhibiting PI3K/Akt signaling pathway. This effect further alleviates M2-induced myofibroblast activation and epithelial-to-mesenchymal transition (EMT), thus resulting in a decline of collagen deposition, pro-fibrotic cytokines secretion, with a concomitant recover ofpulmonary functions in vivo. Less infiltration of M2 macrophages between α-SMA + cells was also found in clevudine treated mice. Our findings indicate a potential anti-fibrotic effect of clevudine by regulating macrophage polarization and might be meaningful in clinical settings., (Copyright © 2021. Published by Elsevier B.V.)

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.

Neutrophils are difficult to study, particularly in tissues, due to their short half-life and propensity for activation. We describe an organotypic airway model that uses patient airway fluid to enable the transmigration of blood neutrophils to acquire an airway-like phenotype in order to better understand their contribution to airway diseases. In particular, we showcase how conditioned neutrophils modulate their bacteria-killing abilities. For complete details on the use and execution of this protocol, please refer to Margaroli et al. (2021)., Competing Interests: Contents within this manuscript are included in patent USPTO 10670594., (© 2021 The Author(s).)

The airway epithelium protects us from environmental insults, which we encounter with every breath. Not only does it passively filter large particles, it also senses potential danger and alerts other cells, including immune and nervous cells. Together, these tissues orchestrate the most appropriate response, balancing the need to eliminate the danger with the risk of damage to the host. Each cell subset within the airway epithelium plays its part, and when impaired, may contribute to the development of respiratory disease. Here we highlight recent advances regarding the cellular and functional heterogeneity along the airway epithelium and discuss how we can use this knowledge to design more effective, targeted therapeutics., (© 2021. Crown.)

Non-targeted drug delivery systems have several limitations including the decreased bioavailability of the drug, poor stability and rapid clearance in addition to off-target distribution. Cell-specific targeted delivery approaches promise to overcome some of these limitations and enhance therapeutic selectivity. In this review, we aim to discuss cell-specific targeted approachesin the lung at the biochemical and molecular levels. These approaches include;a) directly administered small molecule drugs with intracellular action; b) targeted biologics and synthetic hybrids with extracellular action; c) site activateddrugs; and d) delivery systems.We discuss the pharmaceutical and biochemical parameters that govern the fate of drug molecules at delivery sites while presenting an overview of relevant literature surrounding this area of research and current advancements., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The National Heart, Lung, and Blood Institute of the National Institutes of Health, together with the Longfonds BREATH consortium, convened a working group to review the field of lung regeneration and suggest avenues for future research. The meeting took place on May 22, 2019, at the American Thoracic Society 2019 conference in Dallas, Texas, United States, and brought together investigators studying lung development, adult stem-cell biology, induced pluripotent stem cells, biomaterials, and respiratory disease. The purpose of the working group was 1 ) to examine the present status of basic science approaches to tackling lung disease and promoting lung regeneration in patients and 2 ) to determine priorities for future research in the field.

Background: Antioxidant and anti-inflammatory effects are two main pharmacological mechanisms of pirfenidone (PFD) besides the anti-fibrotic effect. This study aims to investigate whether PFD could mediate cigarette smoke extract (CSE) induced inflammation and oxidative stress in vitro and in vivo., Methods: BALB/C mice and alveolar epithelial (A549) cells treated with CSE were established as disease models in vivo and in vitro. Effects of PFD treatment on disease models were further measured. Hematoxylin and eosin (HE) staining was used to evaluate the pathological changes in lung tissues of mice. CCK-8 assay kit was applied to measure the viability of A549 cells treated by different concentrations of PFD. Inflammation cytokine expression in cell supernatants was measured with ELISA kits. The mRNA and protein levels of inflammation and oxidative stress-related factors were determined by real-time quantitative polymerase chain reaction analysis (RT-qPCR) and Western blotting. Furthermore, myeloperoxidase (MPO), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) were measured to detect the antioxidative activity of lung tissues. Moreover, an assay kit with fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) was used to evaluate the intracellular reactive oxygen species (ROS) generation., Results: In vitro and in vivo, PFD significantly reversed TNF-α, IL-6, CCL2, SOD1, and CAT mRNA level changes led by CSE; in addition, PFD significantly decreased the ratios of p-p65 to p65, p-ikBα to ikBα and increased Nrf-2 protein level compared with CSE group. In mice, high-dose (100 mg/kg/d) PFD significantly reversed MPO and MDA increases induced by CSE. However, PFD didn't significantly reverse T-AOC decrease induced by CSE. In A549 cell supernatant, PFD dramatically reversed the elevated levels of TNF-α and IL-1β induced by CSE. Furthermore, PFD could significantly reverse the increased level of ROS induced by CSE in A549 cells., Conclusion: Our study reveals the potential role of PFD in regulating inflammatory response and oxidative stress induced by CSE., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Bronchial thermoplasty regulates structural abnormalities involved in airway narrowing in asthma. In the present study we aimed to investigate the effect of bronchial thermoplasty on histopathological bronchial structures in distinct asthma endotypes/phenotypes., Methods: Endobronchial biopsies (n = 450) were collected from 30 patients with severe uncontrolled asthma before bronchial thermoplasty and after 3 sequential bronchial thermoplasties. Patients were classified based on blood eosinophils, atopy, allergy and smoke exposure. Tissue sections were assessed for histopathological parameters and expression of heat-shock proteins and glucocorticoid receptor. Proliferating cells were determined by Ki67-staining., Results: In all patients, bronchial thermoplasty improved asthma control (p < 0.001), reduced airway smooth muscle (p = 0.014) and increased proliferative (Ki67 +) epithelial cells (p = 0.014). After bronchial thermoplasty, airway smooth muscle decreased predominantly in patients with T2 high asthma endotype. Epithelial cell proliferation was increased after bronchial thermoplasty in patients with low blood eosinophils (p = 0.016), patients with no allergy (p = 0.028) and patients without smoke exposure (p = 0.034). In all patients, bronchial thermoplasty increased the expression of glucocorticoid receptor in epithelial cells (p = 0.018) and subepithelial mesenchymal cells (p = 0.033) and the translocation of glucocorticoid receptor in the nucleus (p = 0.036). Furthermore, bronchial thermoplasty increased the expression of heat shock protein-70 (p = 0.002) and heat shock protein-90 (p = 0.001) in epithelial cells and decreased the expression of heat shock protein-70 (p = 0.009) and heat shock protein-90 (p = 0.002) in subepithelial mesenchymal cells. The effect of bronchial thermoplasty on the expression of heat shock proteins -70 and -90 was distinctive across different asthma endotypes/phenotypes., Conclusions: Bronchial thermoplasty leads to a diminishment of airway smooth muscle, to epithelial cell regeneration, increased expression and activation of glucocorticoid receptor in the airways and increased expression of heat shock proteins in the epithelium. Histopathological effects appear to be distinct in different endotypes/phenotypes indicating that the beneficial effects of bronchial thermoplasty are achieved by diverse molecular targets associated with asthma endotypes/phenotypes.

The lungs are affected by illnesses including asthma, chronic obstructive pulmonary disease, and infections such as influenza and SARS-CoV-2. Physiologically relevant models for respiratory conditions will be essential for new drug development. The composition and structure of the lung extracellular matrix (ECM) plays a major role in the function of the lung tissue and cells. Lung-on-chip models have been developed to address some of the limitations of current two-dimensional in vitro models. In this review, we describe various ECM substitutes utilized for modeling the respiratory system. We explore the application of lung-on-chip models to the study of cigarette smoke and electronic cigarette vapor. We discuss the challenges and opportunities related to model characterization with an emphasis on in situ characterization methods, both established and emerging. We discuss how further advancements in the field, through the incorporation of interstitial cells and ECM, have the potential to provide an effective tool for interrogating lung biology and disease, especially the mechanisms that involve the interstitial elements.

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a disease that involves significant lung tissue damage. How SARS-CoV-2 infection leads to lung injury remains elusive. The open reading frame 8 (ORF8) protein of SARS-CoV-2 (ORF8 SARS-CoV-2 ) is a unique accessory protein, yet little is known about its cellular function. We examined the cellular distribution of ORF8 SARS-CoV-2 and its role in the regulation of human lung epithelial cell proliferation and antiviral immunity. Using live imaging and immunofluorescent staining analyses, we found that ectopically expressed ORF8 SARS-CoV-2 forms aggregates in the cytosol and nuclear compartments of lung epithelial cells. Using in silico bioinformatic analysis, we found that ORF8 SARS-CoV-2 possesses an intrinsic aggregation characteristic at its N-terminal residues 1-18. Cell culture did not reveal any effects of ORF8 SARS-CoV-2 expression on lung epithelial cell proliferation and cell cycle progression, suggesting that ORF8 SARS-CoV-2 aggregates do not affect these cellular processes. Interestingly, ectopic expression of ORF8 SARS-CoV-2 in lung epithelial cells suppressed basal expression of several antiviral molecules, including DHX58, ZBP1, MX1, and MX2. In addition, expression of ORF8 SARS-CoV-2 attenuated the induction of antiviral molecules by IFNγ but not by IFNβ in lung epithelial cells. Taken together, ORF8 SARS-CoV-2 is a unique viral accessory protein that forms aggregates when expressing in lung epithelial cells. It potently inhibits the expression of lung cellular anti-viral proteins at baseline and in response to IFNγ in lung epithelial cells, which may facilitate SARS-CoV-2 escape from the host antiviral innate immune response during early viral infection. In addition, it seems that formation of ORF8 SARS-CoV-2 aggregate is independent from the viral infection. Thus, it would be interesting to examine whether any COVID-19 patients exhibit persistent ORF8 SARS-CoV-2 expression after recovering from SARS-CoV-2 infection. If so, the pathogenic effect of prolonged ORF8 SARS-CoV-2 expression and its association with post-COVID symptoms warrant investigation in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Geng, Subramanian, Wu, Bu, Wang, Du, De Plaen and Tan.)

The airways receive a dense supply of sensory nerve fibers that are responsive to damaging or potentially injurious stimuli. These airway nociceptors are mainly derived from the jugular and nodose vagal ganglia, and when activated they induce a range of reflexes and sensations that play an essential role in airway protection. Jugular nociceptors differ from nodose nociceptors in their embryonic origins, molecular profile and termination patterns in the airways and the brain, and recent discoveries suggest that excessive activity in jugular nociceptors may be central to the development of chronic cough. For these reasons, targeting jugular airway nociceptor signaling processes at different levels of the neuraxis may be a promising target for therapeutic development. In this focused review, we present the current understanding of jugular ganglia nociceptors, how they may contribute to chronic cough and mechanisms that could be targeted to bring about cough suppression., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses , only eight years after the emergence of another zoonotic betacoronavirus , the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I ( HLA-A-24 -like) and II ( HLA-DPB1 -like), and innate immune response ( PTPN4, MAGOHB ), and in cilia coating the respiratory tract ( DNAH7 ). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses , although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.

Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows its role in immune surveillance against pathogens. Higher levels of serum SP-D have been reported in the patients with severe acute respiratory syndrome coronavirus (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro , in vivo and ex vivo . In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing human angiotensin converting enzyme 2 (hACE2). The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following treatment with rfhSP-D (10 µg/ml). These results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merit pre-clinical studies in animal models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hsieh, Beirag, Murugaiah, Chou, Kuo, Kao, Madan, Kishore and Wang.)

Here, we present a physiologically relevant model of the human pulmonary alveoli. This alveolar lung-on-a-chip platform is composed of a three-dimensional porous hydrogel made of gelatin methacryloyl with an inverse opal structure, bonded to a compartmentalized polydimethylsiloxane chip. The inverse opal hydrogel structure features well-defined, interconnected pores with high similarity to human alveolar sacs. By populating the sacs with primary human alveolar epithelial cells, functional epithelial monolayers are readily formed. Cyclic strain is integrated into the device to allow biomimetic breathing events of the alveolar lung, which, in addition, makes it possible to investigate pathological effects such as those incurred by cigarette smoking and severe acute respiratory syndrome coronavirus 2 pseudoviral infection. Our study demonstrates a unique method for reconstitution of the functional human pulmonary alveoli in vitro, which is anticipated to pave the way for investigating relevant physiological and pathological events in the human distal lung., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Epithelial cells of the conducting airways are a pivotal first line of defense against airborne pathogens and allergens that orchestrate inflammatory responses and mucociliary clearance. Nonetheless, the molecular mechanisms responsible for epithelial hyperreactivity associated with allergic asthma are not completely understood. Transcriptomic analysis of human airway epithelial cells (HAECs), differentiated in-vitro at air-liquid interface (ALI), showed 725 differentially expressed immediate-early transcripts, including putative long noncoding RNAs (lncRNAs). A novel lncRNA on the antisense strand of ICAM-1 or LASI was identified, which was induced in LPS-primed HAECs along with mucin MUC5AC and its transcriptional regulator SPDEF. LPS-primed expression of LASI, MUC5AC, and SPDEF transcripts were higher in ex-vivo cultured asthmatic HAECs that were further augmented by LPS treatment. Airway sections from asthmatics with increased mucus load showed higher LASI expression in MUC5AC + goblet cells following multi-fluorescent in-situ hybridization and immunostaining. LPS- or IL-13-induced LASI transcripts were mostly enriched in the nuclear/perinuclear region and were associated with increased ICAM-1, IL-6, and CXCL-8 expression. Blocking LASI expression reduced the LPS or IL-13-induced epithelial inflammatory factors and MUC5AC expression, suggesting that the novel lncRNA LASI could play a key role in LPS-primed trained airway epithelial responses that are dysregulated in allergic asthma.

Mucociliary transport in the respiratory epithelium depends on beating of cilia to move a mucus layer containing trapped inhaled particles toward the mouth. Little is known about the relationship between cilia beat frequency (CBF) and mucus transport velocity (MTV) in vivo under normal physiological conditions and when inspired air is dry or not fully humidified. This study was designed to use video-microscopy to simultaneously measure CBF and MTV in the tracheal epithelium through an implanted optical window in mechanically ventilated lambs. The inspired air in 6 animals was heated to body temperature and fully saturated with water for 4 hours as a baseline. In another series of experiments, 5 lambs were ventilated with air at different temperatures and humidities and the mucosal surface temperature was monitored with infrared macro-imaging. In the baseline experiments, during ventilation with fully humidified air at body temperature, CBF remained constant, mean 13.9 ± 1.6 Hz but MTV varied considerably between 0.1 and 26.1 mm/min with mean 11.0 ± 3.9 mm/min, resulting in a maximum mucus displacement of 34.2 µm/cilia beat. Fully humidified air at body temperature prevented fluctuations in the surface temperature during breathing indicating a thermodynamic balance in the airways. When lambs were ventilated with dryer air, the mucosal surface temperature and MTV dropped without a significant change in CBF. When inspired air was dry, mainly latent heat (92%) was transferred to air in the trachea, reducing the surface temperature by 5 °C. Reduced humidity of the inspired air lowered the surface temperature and reduced MTV in the epithelium during ventilation.

Non-typeable Haemophilus influenzae (NTHi) is the most common respiratory pathogen in patients with chronic obstructive disease. Limited data is available investigating the impact of NTHi infections on cellular re-differentiation processes in the bronchial mucosa. The aim of this study was to assess the effects of stimulation with NTHi on the bronchial epithelium regarding cellular re-differentiation processes using primary bronchial epithelial cells harvested from infection-free patients undergoing bronchoscopy. The cells were then cultivated using an air-liquid interface and stimulated with NTHi and TGF-β. Markers of epithelial and mesenchymal cells were analyzed using immunofluorescence, Western blot and qRT-PCR. Stimulation with both NTHi and TGF-ß led to a marked increase in the expression of the mesenchymal marker vimentin, while E-cadherin as an epithelial marker maintained a stable expression throughout the experiments. Furthermore, expression of collagen 4 and the matrix-metallopeptidases 2 and 9 were increased after stimulation, while the expression of tissue inhibitors of metallopeptidases was not affected by pathogen stimulation. In this study we show a direct pathogen-induced trans-differentiation of primary bronchial epithelial cells resulting in a co-localization of epithelial and mesenchymal markers and an up-regulation of extracellular matrix components.

Background: The Chr17q12-21.2 region is the strongest and most consistently associated region with asthma susceptibility. The functional genes or single nucleotide polymorphisms (SNPs) are not obvious due to linkage disequilibrium., Objectives: We sought to comprehensively investigate whole-genome sequence and RNA sequence from human bronchial epithelial cells to dissect functional genes/SNPs for asthma severity in the Severe Asthma Research Program., Methods: Expression quantitative trait loci analysis (n = 114), correlation analysis (n = 156) of gene expression and asthma phenotypes, and pathway analysis were performed in bronchial epithelial cells and replicated. Genetic association for asthma severity (426 severe vs 531 nonsevere asthma) and longitudinal asthma exacerbations (n = 273) was performed., Results: Multiple SNPs in gasdermin B (GSDMB) associated with asthma severity (odds ratio, >1.25) and longitudinal asthma exacerbations (P < .05). Expression quantitative trait loci analyses identified multiple SNPs associated with expression levels of post-GPI attachment to proteins 3, GSDMB, or gasdermin A (3.1 × 10 -9  -4 ). Higher expression levels of GSDMB correlated with asthma and greater number of exacerbations (P < .05). Expression levels of GSDMB correlated with genes involved in IFN signaling, MHC class I antigen presentation, and immune system pathways (false-discovery rate-adjusted P < .05). rs1031458 and rs3902920 in GSDMB colocalized with IFN regulatory factor binding sites and associated with GSDMB expression, asthma severity, and asthma exacerbations (P < .05)., Conclusions: By using a unique set of gene expression data from lung cells obtained using bronchoscopy from comprehensively characterized subjects with asthma, we show that SNPs in GSDMB associated with asthma severity, exacerbations, and GSDMB expression levels. Furthermore, its expression levels correlated with asthma exacerbations and antiviral pathways. Thus, GSDMB is a functional gene for both asthma susceptibility and severity., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The tracheobronchial tree is lined by a mucociliary epithelium containing millions of multiciliated cells. Their integrated oscillatory activity continuously propels an overlying pollution-protecting mucus layer in cranial direction, leading to mucociliary clearance - the primary defence mechanism of the airways. Mucociliary transport is commonly thought to co-emerge with the collective ciliary motion pattern under appropriate geometrical and rheological conditions. Proper ciliary alignment is therefore considered essential to establish mucociliary clearance in the respiratory system. Here, we used volume electron microscopy in combination with high-speed reflection contrast microscopy in order to examine ciliary orientation and its spatial organization, as well as to measure the propagation direction of metachronal waves and the direction of mucociliary transport on bovine tracheal epithelia with reference to the tracheal long axis (TLA). Ciliary orientation is measured in terms of the basal body orientation (BBO) and the axonemal orientation (AO), which are commonly considered to coincide, both equivalently indicating the effective stroke as well as the mucociliary transport direction. Our results, however, reveal that only the AO is in line with the mucociliary transport, which was found to run along a left-handed helical trajectory, whereas the BBO was found to be aligned with the TLA. Furthermore, we show that even if ciliary orientation remains consistent between adjacent cells, ciliary orientation exhibits a gradual shift within individual cells. Together with the symplectic beating geometry, this intracellular orientational pattern could provide for the propulsion of highly viscous mucus and likely constitutes a compromise between efficiency and robustness., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: To aid in the diagnosis of Primary Ciliary Dyskinesia (PCD) and to evaluate the respiratory epithelium in respiratory disease, normal age-related reference ranges are needed for ciliary beat frequency (CBF), beat pattern and ultrastructure. Our aim was to establish reference ranges for healthy Chinese children., Methods: Ciliated epithelial samples were obtained from 135 healthy Chinese children aged below 18 years by brushing the inferior nasal turbinate. CBF and beat pattern were analysed from high speed video recordings. Epithelial integrity and ciliary ultrastructure were assessed using transmission electronic microscopy., Results: The mean CBF from 135 children studied was 10.1 Hz (95% CI 9.8 to 10.4). Approximately 20% (ranged 18.0-24.2%) of ciliated epithelial edges were found to have areas of dyskinetically beating cilia. Normal beat pattern was observed in ciliated epithelium from all subjects. We did not find any effect of exposure to second hand smoke on CBF in our subjects. Microtubular defects were found in 9.3% of all of the cilia counted in these children, while other ciliary ultrastructural defects were found in less than 3%., Conclusions: We established the reference range for CBF, beat pattern and ultrastructure in healthy Chinese children. Using similar methodology, we found a lower overall mean CBF than previously obtained European values. This study highlights the need to establish normative data for ciliary function in different populations.

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.

Infections with respiratory syncytial virus (RSV) occurs repeatedly throughout life because sustained, protective memory responses fail to develop. Why this occurs is not known. During RSV infection the recognition of the virus via the cytosolic RIG-I like receptors and signaling via the adaptor protein MAVS is crucial for mounting an innate immune response. However, if this signaling pathway is important for T cell responses during primary infection and during re-infection is not fully elucidated. We describe a second peak of pro-inflammatory mediators during the primary immune response to RSV that coincides with the arrival of T cells into the lung. This second peak of cytokines/chemokines is regulated differently than the early peak and is largely independent of signaling via MAVS. This was concurrent with Mavs -/- mice mounting a strong T cell response to primary RSV infection, with robust IFN-γ; and Granzyme B production. However, after RSV re-infection, Mavs -/- mice showed fewer CD4 + and CD8 + short term memory T cells and their capacity to produce IFN-γ; and Granzyme B, was decreased. In sum, cytosolic recognition of RSV is important not only for initiating innate anti-viral responses but also for generating or maintaining efficient, short term T cell memory responses., (Copyright © 2020 Paulsen, Varese, Pinpathomrat, Kirsebom, Paulsen and Johansson.)

Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

Background: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases., Methods: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing., Results: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes., Conclusions: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

In the lung, the airway surface is protected by mucus, whose transport and evacuation is ensured through active ciliary beating. The mechanisms governing the long-range directional organization of ciliary beats, required for effective mucus transport, are much debated. Here, we experimentally show on human bronchial epithelium reconstituted in-vitro that the dynamics of ciliary-beat orientation is closely connected to hydrodynamic effects. To examine the fundamental mechanisms of this self-organization process, we build a two-dimensional model in which the hydrodynamic coupling between cilia is provided by a streamwise-alignment rule governing the local orientation of the ciliary forcing. The model reproduces the emergence of the mucus swirls observed in the experiments. The predicted swirl sizes, which scale with the ciliary density and mucus viscosity, are in agreement with in-vitro measurements. A transition from the swirly regime to a long-range unidirectional mucus flow allowing effective clearance occurs at high ciliary density and high mucus viscosity. In the latter case, the mucus flow tends to spontaneously align with the bronchus axis due to hydrodynamic effects.

Background: Targeted lung denervation (TLD), a novel bronchoscopic procedure which attenuates pulmonary nerve input to the lung to reduce the clinical consequences of neural hyperactivity, may be an important emerging treatment for COPD. While procedural safety and impact on clinical outcomes have recently been reported, the mechanism of action has not been reported. We explored the long-term pathologic and histopathologic effects in a sheep model of ablation of bronchial branches of the vagus nerve using a novel dual-cooled radiofrequency ablation catheter., Methods: Nineteen sheep underwent circumferential ablation of both main bronchi with simultaneous balloon surface cooling using a targeted lung denervation system (Nuvaira, Inc., USA). Animals were followed over an extended time course (30, 365, and 640 days post procedure). At each time point, lung denervation (axonal staining in bronchial nerves), and effect on peribronchial structures near the treatment site (histopathology of bronchial epithelium, bronchial cartilage, smooth muscle, alveolar parenchyma, and esophagus) were quantified. One way analysis of variance (ANOVA) was performed to reveal differences between group means on normal data. Non-parametric analysis using Kruskal-Wallis Test was employed on non-normal data sets., Results: No adverse clinical effects were observed in any sheep. Nerve axon staining distal to the ablation site was decreased by 60% at 30 days after TLD and efferent axon staining was decreased by >70% at 365 and 640 days. All treated airways exhibited 100% epithelial integrity. Effect on peribronchial structures was strictly limited to lung tissue immediately adjacent to the ablation site. Tissue structure 1 cm proximal and distal to the treatment area remained normal, and the pulmonary veins, pulmonary arteries, and esophagus were unaffected., Conclusions: The denervation of efferent axons induced by TLD therapy is durable and likely a contributing mechanism through which targeted lung denervation impacts clinical outcomes. Further, long term lung denervation did not alter the anatomy of the bronchioles or lung, as evaluated from both a gross and histologic perspective.

Although SARS-CoV-2 may primarily enter the cells of the lungs, the small bowel may also be an important entry or interaction site, as the enterocytes are rich in angiotensin converting enzyme (ACE)-2 receptors. The initial gastrointestinal symptoms that appear early during the course of Covid-19 support this hypothesis. Furthermore, SARS-CoV virions are preferentially released apically and not at the basement of the airway cells. Thus, in the setting of a productive infection of conducting airway epithelia, the apically released SARS-CoV may be removed by mucociliary clearance and gain access to the GI tract via a luminal exposure. In addition, post-mortem studies of mice infected by SARS-CoV have demonstrated diffuse damage to the GI tract, with the small bowel showing signs of enterocyte desquamation, edema, small vessel dilation and lymphocyte infiltration, as well as mesenteric nodes with severe hemorrhage and necrosis. Finally, the small bowel is rich in furin, a serine protease which can separate the S-spike of the coronavirus into two "pinchers" (S1 and 2). The separation of the S-spike into S1 and S2 is essential for the attachment of the virion to both the ACE receptor and the cell membrane. In this special review, we describe the interaction of SARS-CoV-2 with the cell and enterocyte and its potential clinical implications.

Asthma is characterized by airway hyperreactivity and inflammation. In the lungs, parasympathetic and sensory nerves control airway tone and induce bronchoconstriction. Dysregulation of these nerves results in airway hyperreactivity. Humans with eosinophilic asthma have significantly increased sensory nerve density in airway epithelium, suggesting that type 2 cytokines and inflammatory cells promote nerve growth. Similarly, mice with congenital airway eosinophilia also have airway hyperreactivity and increased airway sensory nerve density. Here, we tested whether this occurs during development. We show that transgenic mice that overexpress IL-5, a cytokine required for eosinophil hematopoiesis, give birth to wild-type offspring that have significantly increased airway epithelial nerve density and airway hyperreactivity that persists into adulthood. These effects are caused by in utero exposure to maternal IL-5 and resulting fetal eosinophilia. Allergen exposure of these adult wild-type offspring results in severe airway hyperreactivity, leading to fatal reflex bronchoconstriction. Our results demonstrate that fetal exposure to IL-5 is a developmental origin of airway hyperreactivity, mediated by hyperinnervation of airway epithelium.

The sinonasal mucosa has an essential role in defense mechanisms of the upper respiratory tract. The innate immune system presents the primary defense against noxious microorganisms followed by induction of the adaptive immune mechanisms as a consequence of the presence of pathogens. This well-known activation of adaptive immune system in response to presence of the antigen on mucosal surfaces is now broadly applicated in vaccinology research. Prevention of infectious diseases belongs to substantial challenges in maintaining the population health. Non-invasive, easily applicable mucosal vaccination purposes various research opportunities that could be usable in daily practice. However, the existence of multiple limitations such as rapid clearance of vaccine from nasal mucosa by means of mucociliary transport represents a great challenge in development of safe and efficient vaccines. Here we give an updated view on nasal functions with focus on nasal mucosal immunity and its potential application in vaccination in nearly future.

Viral infections of the airway can exacerbate respiratory diseases, such as asthma or chronic obstructive pulmonary disease (COPD), and accelerate disease progression. Phosphoinositide 3-kinase (PI3K)δ, a class 1A PI3K, has been studied as a potential target for achieving anti-oncogenic and anti-inflammatory effects. However, the role of PI3Kδ in antiviral responses is poorly understood. Using a synthetic double-stranded RNA poly I:C and a selective PI3Kδ inhibitor IC87114, we investigated the role of PI3Kδ signaling in poly I:C-induced expression of the T lymphocyte-inhibitory molecule programmed death 1 ligand 1 (PD-L1), inflammatory responses and antiviral interferon (IFN) responses. C57BL/6N mice were treated with IC87114 or vehicle by intratracheal (i.t.) instillation followed by i.t. administration of poly I:C. Poly I:C increased PD-L1 expression on epithelial cells, lymphocytes, macrophages, and neutrophils in the lungs and IC87114 suppressed poly I:C-induced PD-L1 expression on epithelial cells and neutrophils possibly via inhibition of the Akt/mTOR signaling pathway. IC87114 also attenuated poly I:C-induced increases in numbers of total cells, macrophages, neutrophils and lymphocytes, as well as levels of KC, IL-6 and MIP-1β in bronchoalveolar lavage fluid. Gene expression of IFNβ, IFNλ 2 and IFN-stimulated genes (ISGs) were upregulated in response to poly I:C and a further increase in gene expression was observed following IC87114 treatment. In addition, IC87114 enhanced poly I:C-induced phosphorylation of IRF3. We assessed the effects of IC87114 on human primary bronchial epithelial cells (PBECs). IC87114 decreased poly I:C-induced PD-L1 expression on PBECs and secretion of IL-6 and IL-8 into culture supernatants. IC87114 further enhanced poly I:C- induced increases in the concentrations of IFNβ and IFNλ 1/3 in culture supernatants as well as upregulated gene expression of ISGs in PBECs. Similar results were obtained in PBECs transfected with siRNA targeting the PIK3CD gene encoding PI3K p110δ, and stimulated with poly I:C. In human metapneumovirus (hMPV) infection of PBECs, IC87114 suppressed hMPV-induced PD-L1 expression and reduced viral replication without changing the production levels of IFNβ and IFNλ 1/3 in culture supernatants. These data suggest that IC87114 may promote virus elimination and clearance through PD-L1 downregulation and enhanced antiviral IFN responses, preventing prolonged lung inflammation, which exacerbates asthma and COPD., (Copyright © 2020 Fujita, Kan-o, Tonai, Yamamoto, Ogawa, Fukuyama, Nakanishi and Matsumoto.)

Previous pneumococcal experience establishes lung-resident IL-17A-producing CD4 + memory T RM cells that accelerate neutrophil recruitment against heterotypic pneumococci. Herein, we unravel a novel crosstalk between CD4 + T RM cells and lung epithelial cells underlying this protective immunity. Depletion of CD4 + cells in pneumococcus-experienced mice diminished CXCL5 (but not CXCL1 or CXCL2) and downstream neutrophil accumulation in the lungs. Epithelial cells from experienced lungs exhibited elevated mRNA for CXCL5 but not other epithelial products such as GM-CSF or CCL20, suggesting a skewing by CD4 + T RM cells. Genome-wide expression analyses revealed a significant remodeling of the epithelial transcriptome of infected lungs due to infection history, ~80% of which was CD4 + cell-dependent. The CD4 + T RM cell product IL-17A stabilized CXCL5 but not GM-CSF or CCL20 mRNA in cultured lung epithelial cells, implicating posttranscriptional regulation as a mechanism for altered epithelial responses. These results suggest that epithelial cells in experienced lungs are effectively different, owing to their communication with T RM cells. Our study highlights the role of tissue-resident adaptive immune cells in fine-tuning epithelial functions to hasten innate immune responses and optimize defense in experienced lungs, a concept that may apply broadly to mucosal immunology.

This study tested whether electrical stimulation of the pharyngeal mucosa is able to induce reliably the swallowing reflex in awake and asleep obstructive sleep apnea (OSA) patients, and whether the induced reflexes affect the sleep variables. In addition, the latency, occurrence, and morphology of swallows were evaluated. Eight patients received an esophageal catheter that was used on three consecutive nights for electrical stimulation and manometric recordings. The electrical stimulation proved itself safe, but its efficiency in inducing swallows sank from 80.0 % in awake to 37.4 % in sleeping subjects and was lowest in the sleep stage N3. The swallowing reflex was triggered with a mean latency of 3.69 ± 0.70 s, was predominantly induced in the hyperventilation phase, and had no significant effect on the subject's sleep variables. These findings indicate that electrical stimulation can more effectively trigger the swallowing reflex while the subjects are awake than during sleep without showing remarkable clinical benefits in terms of apnea-hypopnea index (AHI) improvement., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction : Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory airway disease characterized as tight junction loosening, inflammation, and mucosal remodeling. Epithelial cells form a barrier against allergens, bacteria, and proteases, and can also trigger or enhance the immune response by releasing various inflammatory mediators including cytokines, chemokines, and secreted proteins to promote the pathogenesis of CRSwNP. Areas covered: We review the epithelium-derived cytokine and secreted protein networks driving CRSwNP, and discuss these mediators in a cellular context. We illustrate their roles as potential mediators-biomarkers in clinical practice, which may help to understand the mechanisms underlying the pathologies of different endotypes of CRSwNP and to improve treatment outcomes in patients with CRSwNP based on the development of novel predictors for CRSwNP management. Expert opinion : The understanding of the role of epithelium-derived inflammatory mediators helps to investigate the pathophysiological mechanisms of CRSwNP endotypes. An increasing number of studies show that these mediators target immune cells and promote the recruitment, activation or regulation of the proliferation or apoptosis of these cells. Based on this achievement, further investigations are necessary to explore the multi-dimensional role of epithelium-derived inflammatory mediators in CRSwNP.

Pathological remodeling of the airway epithelium is commonly observed in cystic fibrosis (CF). Thus, tissue repair is critical to restore integrity and maintenance of the epithelial barrier function. Epithelial repair is a multi-step process initiated by progenitor cell migration into the injured area, proliferation, and re-differentiation into all of the cell types that contribute to the function of a normal airway epithelium. Recent technological advances applied to relevant animal and cell injury models have helped in understanding the complexity of progenitor cell differentiation. This short review will introduce the current knowledge of the mechanisms regulating airway epithelial cell (AEC) regeneration and repair, with a focus on the specification of two rare cell types/states: ionocytes and deuterosomal cells., Competing Interests: Declaration of Competing Interest PB, AC, and MC do not have any conflicts of interest relevant to this publication. ABJ and LWP are employees of Novartis Institutes of BioMedical Research. ABJ is a stockholder in Novartis., (Copyright © 2019 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Motile cilia are a striking example of the functional cellular organelle, conserved across all the eukaryotic species. Motile cilia allow the swimming of cells and small organisms and transport of liquids across epithelial tissues. Whilst the molecular structure is now very well understood, the dynamics of cilia is not well established either at the single cilium level nor at the level of collective beating. Indeed, a full understanding of this requires connecting together behaviour across various lengthscales, from the molecular to the organelle, then at the cellular level and up to the tissue scale. Aside from the fundamental interest in this system, understanding beating is important to elucidate aspects of embryonic development and a variety of health conditions from fertility to genetic and infectious diseases of the airways., (© 2020 The Author(s).)

Acute respiratory distress syndrome (ARDS) is a multifactorial, inflammatory lung injury disease with high morbidity and mortality. However, the underlying pathogenic mechanism remains unknown. In this study, lipopolysaccharide (LPS)-stimulated alveolar epithelial cells were used to mimic the inflammatory pathogenesis of ARDS in vitro. We here investigated the role of miR-424 in LPS-stimulated alveolar epithelial cells and found it to be substantially downregulated. Overexpression of miR-424 inhibited apoptosis and inflammation in LPS-stimulated alveolar epithelial cells, and the miR-424 inhibitor exhibited the opposite effect. A bioinformatic analysis revealed a potential binding site of miR-424 in the 3'-UTR of fibroblast growth factor 2 (FGF2). A luciferase reporter assay suggested that miR-424 targeted FGF2 in alveolar epithelial cells. The level of FGF2 protein was inhibited by miR-424 mimic, whereas was significantly upregulated after miR-424 suppression in LPS-stimulated alveolar epithelial cells. MiR-424 also exhibited the protective role in LPS-induced apoptosis and inflammation by directly targeting FGF2 via the NF-κB pathway. In conclusion, our results demonstrate that miR-424 had a protective role in LPS-induced apoptosis and inflammation of alveolar epithelial cells by targeting FGF2 via regulating NF-κB pathway. This might contribute novel evidence to help identify a therapeutic target for treating ARDS., (Copyright © 2019. Published by Elsevier Inc.)

Background:  Trachea reconstruction requires creation of a functional lining, semirigid support, and vascularity. We aimed to design composite flaps with these three components in a rabbit model., Methods:  Circumferential ( n  = 9) and partial anterior ( n  = 8) tracheal defects were created in rabbits. A circumferential defect was reconstructed with a tubed ear flap incorporating cartilage for support and skin for lining. This was pedicled on the posterior auricular vessels and tunneled into the neck to bridge the defect. In the second experiment, a longitudinal anterior trachea defect was patched with a pedicled rib cartilage and intercostal muscle flap based on the internal mammary vessels. The vascularized fascia over the intercostal muscles replaced the lining while the cartilage provided support. Postoperatively, the rabbits were monitored clinically and endoscopically. The tracheal constructs were examined histologically after the animals were sacrificed., Results:  Rabbits with circumferential defects reconstructed with the ear flap survived up to 6 months. Histology demonstrated vascularized cartilage with good integration of the flap with native trachea. However, hair growth and skin desquamation resulted in airway obstruction in the long term. In the second experiment, all the rabbits survived without respiratory distress, and the intercostal muscle fascia was completely covered by native respiratory epithelium., Conclusion:  We described two experimental techniques using autologous composite flaps for single-stage trachea reconstruction in a rabbit model. Skin was a poor lining replacement, whereas vascularized muscle fascia became covered with respiratory epithelium. A rib cartilage and muscle flap could potentially be used for reconstruction of partial defects in humans., Competing Interests: None declared., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Purpose of Review: Epithelial barrier defects are being appreciated in various inflammatory disorders; however, causal underlying mechanisms are lacking. In this review, we describe the disruption of the airway epithelium with regard to upper and lower airway diseases, the role of epigenetic alterations underlying this process, and potential novel ways of interfering with dysfunctional epithelial barriers as a novel therapeutic approach., Recent Findings: A defective epithelial barrier, impaired innate defence mechanisms or hampered epithelial cell renewal are found in upper and lower airway diseases. Barrier dysfunction might facilitate the entrance of foreign substances, initiating and facilitating the onset of disease. Latest data provided novel insights for possible involvement of epigenetic alterations induced by inflammation or other unknown mechanisms as a potential mechanism responsible for epithelial defects. Additionally, these mechanisms might precede disease development, and represent a novel therapeutic approach for restoring epithelial defects., Summary: A better understanding of the role of epigenetics in driving and maintaining epithelial defects in various inflammatory diseases, using state-of-the-art biology tools will be crucial in designing novel therapies to protect or reconstitute a defective airway epithelial barrier.

Background: Hypothermia with xenon gas has been used to reduce brain injury and disability rate after perinatal hypoxia-ischemia. We evaluated xenon gas therapy effects in an in vitro model with or without hypothermia on cultured human airway epithelial cells (Calu-3)., Methods: Calu-3 monolayers were grown at an air-liquid interface and exposed to one of the following conditions: 1) 21% FiO2 at 37°C (control); 2) 45% FiO2 and 50% xenon at 37°C; 3) 21% FiO2 and 50% xenon at 32°C; 4) 45% FiO2 and 50% xenon at 32°C for 24 hours. Transepithelial resistance (TER) measurements were performed and apical surface fluids were collected and assayed for total protein, IL-6, and IL-8. Three monolayers were used for immunofluorescence localization of zonula occludens-1 (ZO-1). The data were analyzed by one-way ANOVA., Results: TER decreased at 24 hours in all treatment groups. Xenon with hyperoxia and hypothermia resulted in greatest decrease in TER compared with other groups. Immunofluorescence localization of ZO-1 (XY) showed reduced density of ZO-1 rings and incomplete ring-like staining in the 45% FiO2- 50% xenon group at 32°C compared with other groups. Secretion of total protein was not different among groups. Secretion of IL-6 in 21% FiO2 with xenon group at 32°C was less than that of the control group. The secretion of IL-8 in 45% FiO2 with xenon at 32°C was greater than that of other groups., Conclusion: Hyperoxia and hypothermia result in detrimental epithelial cell function and inflammation over 24-hour exposure. Xenon gas did not affect cell function or reduce inflammation.

The pulmonary epithelium separates the gaseous intraluminal space of the airways and the aqueous interstitium. This compartimentalization is required for appropriate lung function, it is established during perinatal periods and can be disturbed in lung edema. Herein we elaborated the impact of the air-liquid interface (ALI) on the function of the pulmonary epithelium. We used NCI-H441 epithelia as a well-established and characterized model of distal airway epithelia, which were cultivated either at ALI or (at submerged conditions) at liquid-liquid interface conditions (LLI). Our study revealed that paracellular permeability was increased and claudin 1 (CLDN1) expression levels were reduced under LLI conditions. This was accompanied by elevated c-FOS, c-JUN and retinoic acid receptor α (RARA) expression, as well as cellular retinoic acid (RA) content. Exposure of epithelia to RA derivatives of ALI cultivated epithelia mimicked effects of LLI. The increase in RA content was in line with the identified upregulation of retinoic acid anabolizing enzymes ALDH1A3 and DHRS3. CLDN1 promoter analysis revealed c-FOS and c-JUN as activating transcription factors, whereas activation of RARA reduced CLDN1 promoter activity. We then concluded that ALI/LLI dependent modulation of CLDN1 expression and TJ permeability is under the control of RA synthesis. Activation of RARA results in an inhibition of c-FOS/c-JUN dependent CLDN1 promoter activation and increased TJ permeability. Our results underscore RA signalling as a pivotal mechanism in adjusting TJ properties, which could play a role during birth when the lung changes from LLI to ALI conditions., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Airway remodeling is accepted to be a determining component within the natural history of asthma. It is a phenomenon characterized by changes in the airways structures that marches in parallel with and can be influenced by airway inflammation, floating at the interface between both natural and adaptive immunity and physical and mechanical cells behavior. In this review we aimed to highlight the comprehensive, yet not exhaustive, evidences of how immune cells induce, regulate and adapt to the recognized markers of airway remodeling. Mucous cell hyperplasia, epithelial dysfunction and mesenchymal transition, extracellular matrix protein synthesis and restructuration, fibroblast to myofibroblast transition, airway smooth muscle proliferation, bioactive and contractile properties, and vascular remodeling encompass complex physiopathological mechanisms that can be induced, suppressed or regulated by different cellular and molecular pathways. Growth factors, cytokines, chemokines and adhesion molecules expressed or derived either from the immune network of cells infiltrating the asthmatic airways and involving T helper lymphocytes, immune lymphoid cells, dendritic cells, eosinophils, neutrophils, mast cells or by the structural components such as epithelial cells, fibroblasts, myocytes, airway smooth muscle cells concur with protein cellular matrix component and metalloproteases in modifying the airway structure in a detrimental way. The consequences in lung function decline, fixed airway obstruction and clinical severity of the disease suggest the possibility of identify among the immune molecular pathway of remodeling some biological parameters or signal pathway to be either a good tracer for monitoring the disease evolution or a target for hypothetical phenotypes and endotypes. In the era of personalized medicine, a biomarker of remodeling might predict a response to small-molecule inhibitors or biologicals potentially targeting a fundamental aspect of asthma pathogenesis that impacts on the low responsiveness to airway inflammation directed treatments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Key Points: Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca 2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca 2+ elevation by TRPV4 stimulation leads to CFTR channel activation., Abstract: TMEM16A is a Ca 2+ -activated Cl - channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca 2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca 2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl - channel. Our study reveals the existence of separate Ca 2+ signalling pathways linked to different Cl - secretory processes., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)