SARS-CoV-2 Infection of Airway Cells

In a laboratory setting, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was inoculated into human bronchial epithelial cells

Mucus clearance in the respiratory tract: A new concept? [Un nouveau concept de mécanisme de clairance respiratoire ?]

Dans toutes les muqueuses, respiratoire, gastro-intestinale ou encore celle des voies génitales, la présence d’un film viscoélastique de mucus est nécessaire pour protéger l’organisme contre l’invasion d’agents pathogènes, qu’il s’agisse de virus, bactéries ou autres polluants. Ce mucus est constitué principalement de larges glycoprotéines, appelées mucines, qui doivent être parfaitement hydratées pour maintenir les propriétés viscoélastiques du mucus. Dans les voies respiratoires, la couche de mucus séquestre les agents inhalés et progresse, grâce aux battements ciliaires des cellules sous-jacentes, vers la glotte. Le mucus est continuellement avalé ou expectoré, ce qui définit le mécanisme de clairance pulmonaire. Depuis des décennies, le dogme veut que les cellules ciliées battent librement dans un milieu aqueux et propulsent le mucus qui flotte sur l’épithélium respiratoire, mais nous avons récemment réfuté ce concept dans une étude publiée dans Science [1]. En effet, l’ancienne notion ne permet pas d’expliquer l’incidence des plaques de mucus observées dans les maladies caractérisées par l’obstruction pulmonaire (par exemple : bronchite chronique, mucoviscidose ou asthme). Notre étude montre que l’espace périciliaire est en fait occupé par de larges glycoprotéines organisées de manière spécifique en un réseau ayant une densité supérieure à celle de la couche mobile de mucus sus-jacente. Ce réseau dense de macromolécules est attaché aux cellules ciliées et possède les mêmes propriétés qu’un gel

Accumulation de mucus - Le point de départ de la pathogenèse pulmonaire chez les patients atteints de mucoviscidose

La mucoviscidose (en anglais, cystic fibrosis ou CF) est une maladie génétique grave affectant principalement la population caucasienne dans une proportion d’une naissance sur 4 000. Des mutations du gène codant la protéine CFTR (cystic fibrosis transmembrane conductance regulator) entraînent des changements de flux ioniques à travers la membrane plasmique de cellules épithéliales, qui altèrent le réseau de mucines dans plusieurs organes comme les poumons et l’intestin [1-3]. Les mucines polymériques sont de grandes glycoprotéines qui organisent la couche de mucus et assurent la lubrification et la protection des muqueuses contre l’invasion d’agents infectieux. Chez les patients atteints de mucoviscidose, le dysfonctionnement du canal ionique CFTR modifie le mucus, qui devient visqueux et adhère aux parois des bronches et du tube digestif. Dans les poumons, l’obstruction des voies aériennes est propice aux infections bactériennes [4]. De plus, les propriétés antibactériennes de la couche de mucus sont diminuées chez des animaux modèles développant certains symptômes de la maladie [5]. Une infection précède généralement le développement d’une inflammation, il est tentant de penser qu’une infection précoce puisse déclencher la pathogénèse pulmonaire et provoquer une réaction inflammatoire chronique chez les patients atteints de mucoviscidose. A l’appui de cette hypothèse, des lésions pulmonaires causées par l’inflammation peuvent être détectées très tôt par imagerie médicale fondée sur la tomodensitométrie (en anglais, computed tomography ou CT). En effet, 22 % des patients atteints de mucoviscidose présentent des lésions dès l’âge de 1 an [6]. Les descriptions de la progression de la maladie font donc souvent référence au cercle vicieux « obstruction-infection-inflammation » (dans cet ordre), car l’inflammation est fréquemment associée à la présence de microorganismes pathogènes. Cependant, notre étude récemment publiée dans le journal Science Translational Medicine démontre que l’inflammation précède l’infection bactérienne et que le mucus pourrait être à l’origine de cette réponse inflammatoire [7]. L’accumulation de mucus est, de plus, détectée avant les premiers signes de lésions pulmonaires, suggérant, là encore, que les propriétés anormales du mucus chez ces patients déclenchent une réponse immunitaire et constituent ainsi le point de départ de la maladie de la mucoviscidose

Mucins and CFTR: Their Close Relationship

Mucociliary clearance is a critical defense mechanism for the lungs governed by regionally coordinated epithelial cellular activities, including mucin secretion, cilia beating, and transepithelial ion transport. Cystic fibrosis (CF), an autosomal genetic disorder caused by the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, is characterized by failed mucociliary clearance due to abnormal mucus biophysical properties. In recent years, with the development of highly effective modulator therapies, the quality of life of a significant number of people living with CF has greatly improved; however, further understanding the cellular biology relevant to CFTR and airway mucus biochemical interactions are necessary to develop novel therapies aimed at restoring CFTR gene expression in the lungs. In this article, we discuss recent advances of transcriptome analysis at single-cell levels that revealed a heretofore unanticipated close relationship between secretory MUC5AC and MUC5B mucins and CFTR in the lungs. In addition, we review recent findings on airway mucus biochemical and biophysical properties, focusing on how mucin secretion and CFTR-mediated ion transport are integrated to maintain airway mucus homeostasis in health and how CFTR dysfunction and restoration of function affect mucus properties

Mucus, mucins, and cystic fibrosis

Cystic fibrosis (CF) is both the most common and most lethal genetic disease in the Caucasian population. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is characterized by the accumulation of thick, adherent mucus plaques in multiple organs, of which the lungs, gastrointestinal tract and pancreatic ducts are the most commonly affected. A similar pathogenesis cascade is observed in all of these organs: loss of CFTR function leads to altered ion transport, consisting of decreased chloride and bicarbonate secretion via the CFTR channel and increased sodium absorption via epithelial sodium channel upregulation. Mucosa exposed to changes in ionic concentrations sustain severe pathophysiological consequences. Altered mucus biophysical properties and weakened innate defense mechanisms ensue, furthering the progression of the disease. Mucins, the high-molecular-weight glycoproteins responsible for the viscoelastic properties of the mucus, play a key role in the disease but the actual mechanism of mucus accumulation is still undetermined. Multiple hypotheses regarding the impact of CFTR malfunction on mucus have been proposed and are reviewed here. (a) Dehydration increases mucin monomer entanglement, (b) defective Ca2+ chelation compromises mucin expansion, (c) ionic changes alter mucin interactions, and (d) reactive oxygen species increase mucin crosslinking. Although one biochemical change may dominate, it is likely that all of these mechanisms play some role in the progression of CF disease. This article discusses recent findings on the initial cause(s) of aberrant mucus properties in CF and examines therapeutic approaches aimed at correcting mucus properties

Munc13-2 −/− baseline secretion defect reveals source of oligomeric mucins in mouse airways: Muc5b secretion defect in Munc13-2−/−mouse airways

Since the airways of control mouse lungs contain few alcian blue/periodic acid–Schiff's (AB/PAS)+ staining ‘goblet’ cells in the absence of an inflammatory stimulus such as allergen sensitization, it was surprising to find that the lungs of mice deficient for the exocytic priming protein Munc13-2 stain prominently with AB/PAS under control conditions. Purinergic agonists (ATP/UTP) stimulated release of accumulated mucins in the Munc13-2-deficient airways, suggesting that the other airway isoform, Munc13-4, supports agonist-regulated secretion. Notably, however, not all of the mucins in Munc13-2-deficient airways were secreted, suggesting a strict Munc13-2 priming requirement for a population of secretory granules. AB/PAS+ staining of Munc13-2-deficient airways was not caused by an inflammatory, metaplastic-like response: bronchial–alveolar lavage leucocyte numbers, Muc5ac and Muc5b mRNA levels, and Clara cell ultrastructure (except for increased secretory granule numbers) were all normal. A Muc5b-specific antibody indicated the presence of this mucin in Clara cells of wildtype (WT) control mice, and increased amounts in Munc13-2-deficient mice. Munc13-2 therefore appears to prime a regulated, baseline secretory pathway, such that Clara cell Muc5b, normally secreted soon after synthesis, accumulates in the gene-deficient animals, making them stain AB/PAS+. The defective priming phenotype is widespread, as goblet cells of several mucosal tissues appear engorged and Clara cells accumulated Clara cell secretory protein (CCSP) in Munc13-2-deficient mice. Additionally, because in the human airways, MUC5AC localizes to the surface epithelium and MUC5B to submucosal glands, the finding that Muc5b is secreted by Clara cells under control conditions may indicate that it is also secreted tonically from human bronchiolar Clara cells

Molecular organization of the mucins and glycocalyx underlying mucus transport over mucosal surfaces of the airways

Mucus, with its burden of inspired particulates, and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be ‘watery’ and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate, a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed keratan sulfate-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190–1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, while the smaller AAV penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies

Ablation of IL-33 suppresses Th2 responses but is accompanied by sustained mucus obstruction in the Scnn1b transgenic mouse model

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33

A Periciliary Brush Promotes the Lung Health by Separating the Mucus Layer from Airway Epithelia

Mucus clearance is the primary defense mechanism that protects airways from inhaled infectious and toxic agents. In the current Gel-on-Liquid mucus clearance model mucus gel is propelled on top of a “watery” periciliary layer surrounding the cilia. However, this model fails to explain the formation of distinct mucus layer in health or why mucus clearance fails in disease. We propose a Gel-on-Brush model in which the periciliary layer is occupied by membrane spanning mucins and mucopolysaccharides densely tethered to the airway surface. This brush prevents mucus penetration into the periciliary space and causes mucus to form a distinct layer. The relative osmotic moduli of the mucus and periciliary brush layers explain both the stability of mucus clearance in health and its failure in airway disease