Tracheal Epithelium Research Articles

Epidemic and evolutionary characteristics of peste des petits ruminants virus infecting Procapra przewalskii in Western China

Due to the migration or transboundary spread of domestic and wild animals, peste des petits ruminants virus posed a high potential threat to them. In this study, we initially detected that a class of animal named Procapra przewalskii was infected with peste des petits ruminants virus (PPRV ChinaGS2018) in Gansu province. According to phylogenetic relationships analysis, we found that ChinaGS2018 comprised of 15,954 nucleotides and was classified into IV genotypes. In addition, indirect immunofluorescence assay (IFA) showed that ChinaGS2018 could infect isolated primary goat tracheal epithelium cells (GTC). Comparing with full-length genome sequences revealed that ChinaGS2018 strain has high identity to the reference complete genomes (87.16–99.55%) at the nucleotide level. Multiple sequence alignment showed that F protein has the highest identity of 99.8%, and H protein has the highest nucleotide substitution ratio. Our study also suggested this strain may be transmitted from Xinjiang, China. Along with the migratory of Procapraprzewalskii, this wild ruminant infected with PPRV can pose a huge threat to other wild ruminants and domestic ones. This is the first report describing infected with PPRV which will provide insights into the epidemiology and pathogenesis of this important virus.

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Airway M Cells Arise in the Lower Airway Due to RANKL Signaling and Reside in the Bronchiolar Epithelium Associated With iBALT in Murine Models of Respiratory Disease.

Microfold (M) cells residing in the follicle-associated epithelium of mucosa-associated lymphoid tissues are specialized for sampling luminal antigens to initiate mucosal immune responses. In the past decade, glycoprotein 2 (GP2) and Tnfaip2 were identified as reliable markers for M cells in the Peyer's patches of the intestine. Furthermore, RANKL–RANK signaling, as well as the canonical and non-canonical NFκB pathways downstream, is essential for M-cell differentiation from the intestinal stem cells. However, the molecular characterization and differentiation mechanisms of M cells in the lower respiratory tract, where organized lymphoid tissues exist rarely, remain to be fully elucidated. Therefore, this study aimed to explore M cells in the lower respiratory tract in terms of their specific molecular markers, differentiation mechanism, and functions. Immunofluorescence analysis revealed a small number of M cells expressing GP2, Tnfaip2, and RANK is present in the lower respiratory tract of healthy mice. The intraperitoneal administration of RANKL in mice effectively induced M cells, which have a high capacity to take up luminal substrates, in the lower respiratory epithelium. The airway M cells associated with lymphoid follicles were frequently detected in the pathologically induced bronchus-associated lymphoid tissue (iBALT) in the murine models of autoimmune disease as well as pulmonary emphysema. These findings demonstrate that RANKL is a common inducer of M cells in the airway and digestive tracts and that M cells are associated with the respiratory disease. We also established a two-dimensional culture method for airway M cells from the tracheal epithelium in the presence of RANKL successfully. This model may be useful for functional studies of M cells in the sampling of antigens at airway mucosal surfaces.

Regulatory effect of dexamethasone on tracheal calcium processing proteins and mucosal secretion.

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.

A novel 3D-printed silk fibroin-based scaffold facilitates tracheal epithelium proliferation in vitro.

The functional epithelial regeneration is important for repairing tracheal defects. However, the potential of 3D-printed SF-based scaffolds for tracheal epithelial regeneration is still unknown. In this study, we developed a novel silk fibroin-based scaffold prepared by 3D printing of silk fibroin/hydroxypropyl methyl cellulose (SF/HPMC) thixotropic hydrogel and evaluated the tracheal epithelium proliferation on this scaffold in vitro. Combined with the freeze-dried technology, the 3D-printed SF/HPMC scaffolds had porous structures in the printed bars. After evaluation of their pore sizes, porosities, water contents and mechanical properties, the scaffolds were co-cultured with the normal human bronchial epithelial cell line (BEAS-2B) for seven days. We detected the BEAS-2B cells proliferation on the scaffolds using a CCK-8 assay, determined their mucin secretion and intercellular tight junction formation by immunofluorescence, as well as observed their cell viability and morphology by live/dead staining and scanning electronic microscopy. The results showed that the SF/HPMC scaffolds had good porosity, water content and mechanical properties. In addition, the BEAS-2B cells proliferated well on SF/HPMC scaffolds, during the seven-day co-culture, with high viability, mucin expression, and intercellular tight junction formation. In summary, these results demonstrated that the BEAS-2B cells could attach and proliferate on the 3D-printed SF/HPMC scaffolds, which were expected to have potential for facilitating tracheal epithelial regeneration.

Investigations of Pathological Immunohistochemical and Immunocytochemical Findings in Natural Infection with Mycoplasma gallisepticum in Laying Hens

Background: Mycoplasmosis is an infectious disease caused by Mycoplasma gallisepticum (MG), usually seen in the respiratory system of chickens, chick and turkeys, that causing great economic loss. The disease is characterized by respiratory system lesions such as sinusitis, tracheitis, airsacculitis, pneumonia and other symptoms such as loss of yield, arthritis, tenosynovitis. In this study, it was aimed to investigate diagnose of the disease by pathologic and molecular techniques in hens that naturally infected with MG as well as the usability of immunocytochemical (ICC) method in diagnose of the disease.Materials, Methods & Results: For this purpose, 98 hens were collected from 10 different coops that serologically positive. After necropsy, routine pathological procedures were performed to samples taken from nose, sinus, larynx, trachea, lung and air sacs. Scraping samples taken from lungs and tracheas were evaluated by ICC. Immunohistochemical (IHC) staining was performed to samples taken from nose, sinus, larynx, trachea, lung and air sacs. Indirect immunoperoxidase method was applied in the both IHC and ICC staining. Rabbit polyclonal anti MG antibody was used as primer antibody in the IHC and ICC staining. Additionally, culture and PCR techniques were applied to tracheas of all hens for MG. The GPO3 and MGSO genes were made for PCR analysis.In the tracheal examinations, 23 cases were positive for PCR, 17 cases ICC positive, 16 cases IHC positive and 10 culture samples found positive. All of culture positive cases were also positive for other three methods. When findings in all organs were evaluated, in 37 cases were detected positive by IHC (38%) and 23 cases were positive by ICC (23.5%). In the IHC positive cases, the first order was trachea in 16 cases followed by in 11 cases in sinus, in 8 cases in lung, in 6 cases air sac and 4 cases in nose, respectively. In 8 cases, IHC positivity was found in at least two organs. IHC positivity was detected in the nose, sinus and tracheal epithelia as well as in the macrophages within subepithelial lymphoid infiltration, vascular walls and endothelium. As the disease became chronic, it was found that the agents were seen more in the lymphoid tissue than the epithelium. In ICC staining positivity was found in 17 cases in the trachea and 11 cases in the lung. There were only 5 cases positive by ICC in both organs.Discussion: Clinical and pathological findings as well as serological, microbiological, molecular techniques and immunohistochemical methods are to be important methods in the diagnosis of the disease. While the culture results are shown as the gold standard in diagnosis of the disease, it is possible to obtain the results in the earliest 7-10 days in cultures and at least 20 days must be passed in order to say a cultural negative. In addition, in the field studies, it mentioned the use of vaccines, antibiotics and protective drugs affected the results of microbiology and serology; the importance of using techniques such as IHC and PCR for the diagnosis of the causative agents. The results of the present study indicate that the most important organ in the diagnosis of the disease is the trachea, and the most effective method is PCR followed by IHC and ICC methods. It was concluded that the results of ICC staining close to IHC staining, and ICC could be used for diagnostic purposes in positive reactions obtained from the tracheas or the other organs.

Collagen sponge scaffolds containing growth factors for the functional regeneration of tracheal epithelium

Tracheal epithelia have barrier and mucociliary clearance functions that prevent invasion of extraneous particles and infectious materials. Hence, following tracheal reconstructions, functional and morphological regeneration of epithelia is required to prevent respiratory declines and infectious diseases. Although growth factors (GFs) promote the regeneration of tracheal epithelial morphologies, it remains unclear whether tracheal grafts containing GFs are beneficial for regeneration of tracheal epithelial functions. Thus, we fabricated collagen sponge scaffolds containing insulin-like GF-1 (IGF-1) and the basic fibroblast, hepatocyte, and epidermal GFs (bFGFs, HGFs, and EGFs, respectively), and we evaluated the effects of the grafts on the functional regeneration of tracheal epithelia. Partial tracheal defects were imposed surgically, and collagen sponges containing IGF-1, bFGF, HGF, or EGF were then transplanted to defect sites. Subsequent immunofluorescence studies suggested that EGF and bFGF contribute to regular distributions of tight junction molecules, and tracer permeability assays suggested that EGF and bFGF promote regeneration of barrier function. Increased ciliogenesis was also observed using scanning electron microscopy in reconstructed regions treated with EGF- and bFGF-supplemented collagen sponges. However, bFGF-supplemented collagen sponges led to greater microsphere transport than did EGF-supplemented sponges. The present data suggested that collagen sponge scaffold containing bFGF promotes functional regeneration of tracheal epithelial tissues.

Evaluation of Cilia Function in Rat Trachea Reconstructed Using Collagen Sponge Scaffold Seeded with Adipose Tissue-Derived Stem Cells.

The tracheal lumen is essential for conducting air to the lung alveoli and for voice production. However, patients with severe tracheal stenosis and malignant tumors invading the trachea often require tracheal resection. Recently, various reported tissue engineering methods for tracheal reconstruction show that regeneration of ciliated epithelium in the reconstructed areas, as well as preservation of the luminal structure is possible. However, only few studies report on the mucociliary transport function in reconstructed tracheae. We investigated mucociliary transport function within rat tracheal epithelium, reorganized after autologous adipose tissue-derived stem cell (ASC) transplantation. Rat ASCs were expanded in culture, and then seeded in a collagen sponge, which was physically supported with a polypropylene framework. The ASC-seeded collagen sponge was transplanted into the rat tracheal defect. We then examined the motility and transport function of cilia generated in the transplanted area using ciliary beat frequency (CBF) and microsphere movement analyses. Our data suggested that autologous ASC transplantation promoted ciliogenesis, consistent with previous reports. The CBF analysis revealed that motility of the cilia generated in the ASC group was comparable to that observed in the normal rat tracheal epithelium. Transport function in the ASC group was higher than that in the control group. These data suggested that autologous ASC transplantation increased ciliated cells in the reconstructed area without significantly disrupting cilia motility, thereby promoting transport function regeneration. Autologous ASC transplantation is expected to be beneficial in morphological and functional regeneration of tracheal epithelium. Anat Rec, 303:471-477, 2020. © 2019 American Association for Anatomy.

Functional characterization of various channel-expressing central airway epithelial cells from mouse induced pluripotent stem cells.

Functional central airway epithelial cells (CAECs) from induced pluripotent stem cells (iPSCs) are an attractive potential cell source for central airway regeneration. The central airway epithelium, such as the tracheal epithelium, is composed of ciliated cells, goblet cells, and basal cells and has physiologically important functions such as the regulation of water volume on the airway surface by Cl- and water channels and the elimination of particles inhaled from the external environment by ciliary movement. Previous work from our group and from other research groups has reported the generation of airway epithelial cells from iPSCs. However, it remains unclear whether iPSC-derived CAECs express the various channels that are required for the regulation of water volume on the airway surface and whether these channels function properly. In this study, we generated CAECs from iPSCs supplemented with activin and bFGF using air-liquid interface culture. We then evaluated the physiological functioning of the iPSC-derived CAECs by examining the gene expression and transport functions of Cl - channels using a halide ion-sensitive yellow fluorescent protein and ciliary movement. Reverse-transcription polymerase chain reaction and immunohistochemistry indicated that various channel markers such as cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin (AQP) were present in iPSC-derived CAECs. Furthermore, the transport functions of Cl - channels and CFTR were successfully confirmed. Finally, ciliary movement was measured, and a ciliary beating frequency (CBF) of approximately 10 Hz was observed. These results demonstrate that CAECs generated by our method have physiological functions similar to those of native CAECs.

Wnt/Fgf crosstalk is required for the specification of basal cells in the mouse trachea.

Basal progenitor cells are crucial for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperone protein Gpr177 (also known as Wntless) in mouse tracheal epithelium causes a significant reduction in the number of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants, which maintain partial cell-cell adhesion but not the transcription regulation function of β-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage, concomitant with reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of Wnt/Fgf crosstalk in early tracheal development.

The ameliorating effect of some immune-stimulants on bursal and tracheal lesions of broiler chickens challenged with ND virus

BackgroundNowadays, immuno-stimulants have great importance in improving the immune system and minimising the effect of infectious diseases in poultry flocks. This study was designed to demonstrate the impact of using different immune-stimulants on the reduction of the harmful implications of virulent ND virus through evaluation of histopathological lesion scores on bursa and trachea of vaccinated and infected broiler chickens with Newcastle disease (ND).ResultsEighty Ross breed broiler chicks at the age of 1 day were randomly divided into four groups, each group formed of 20 birds. Group I was vaccinated against AI, ND and IBD and used as a control. Groups II, III and IV were immunised as group I and also had immuno-stimulant supplement, Imutrix® (oregano oils plus ß-glucan), Evit liquid® (Vitamin E plus selenium) and Immunair 17.5 (Propionibacterium acnes and E. coli lipopolysaccharides), respectively. All groups were experimentally infected with virulent ND virus at the age of 28 days. The morbidity rates for groups I, II, III and IV were 15%, 5%, 5% and 10% respectively. Histopathological examination of bursa revealed hyperplasia of lymphoid follicles in group I which became more evident in groups II, III and IV. Group III showed hyperplasia of tracheal epithelium with lymphocytes and heterophils infiltration in lamina propria. CD79A-positive cells were significantly higher in bursa of group III in comparison to other groups during an immunohistochemical examination.ConclusionIt was concluded that Evit liquid® (vitamin E plus selenium) supplementation might have potent immunomodulatory effect in chickens.

Hydrogen sulfide exposure triggers chicken trachea inflammatory injury through oxidative stress-mediated FOS/IL8 signaling.

Hydrogen sulfide (H2S) is well known to cause irritation and damage to airway following inhalation, but the mechanism by which H2S contributes to airway toxicity is unclear. In order to assess the respiratory toxicity of H2S inhalation in chicken trachea, we investigated the change of oxidative stress parameters, tracheal tissue structure and transcriptome profiles of chicken trachea exposed to H2S for 42 days. The results showed H2S exposure induced oxidative stress and inflammation in trachea. The ultrastructural analysis revealed loss of cilia and accumulation of mucus in tracheal epithelium. Differentially expressed genes (DEGs) analysis indicated 454 genes were significantly changed, including 136 genes upregulated and 318 genes downregulated. Gene ontology and KEGG analysis showed many genes involved in response to oxidative stress, inflammatory and immune response, which might contribute to H2S-induced tracheal inflammatory injury. Among those genes, N-acetyl-L-cysteine (NAC) treatment blocked the H2S-triggered expression of FOS and IL8. Silencing FOS by siRNA inhibited H2S-induced expression of IL8. Taken together, we concluded that H2S induced oxidative stress leads to tracheal inflammation through FOS/IL8 signaling, leading to excessive mucus secretion and absence of cilia. These results provide new insights for unveiling the biological effects of H2S in vivo and in vitro.

Replacement of Rat Tracheas by Layered, Trachea-Like, Scaffold-Free Structures of Human Cells Using a Bio-3D Printing System.

Current scaffold-based tissue engineering approaches are subject to several limitations, such as design inflexibility, poor cytocompatibility, toxicity, and post-transplant degradation. Thus, scaffold-free tissue-engineered structures can be a promising solution to overcome the issues associated with classical scaffold-based materials in clinical transplantation. The present study seeks to optimize the culture conditions and cell combinations used to generate scaffold-free structures using a Bio-3D printing system. Human cartilage cells, human fibroblasts, human umbilical vein endothelial cells, and human mesenchymal stem cells from bone marrow are aggregated into spheroids and placed into a Bio-3D printing system with dedicated needles positioned according to 3D configuration data, to develop scaffold-free trachea-like tubes. Culturing the Bio-3D-printed structures with proper flow of specific medium in a bioreactor facilitates the rearrangement and self-organization of cells, improving physical strength and tissue function. The Bio-3D-printed tissue forms small-diameter trachea-like tubes that are implanted into rats with the support of catheters. It is confirmed that the tubes are viable in vivo and that the tracheal epithelium and capillaries proliferate. This tissue-engineered, scaffold-free, tubular structure can represent a significant step toward clinical application of bioengineered organs.

Isl1 Regulation of Nkx2.1 in the Early Foregut Epithelium Is Required for Trachea-Esophageal Separation and Lung Lobation

The esophagus and trachea arise from the dorsal and ventral aspects of the anterior foregut, respectively. Abnormal separation of the esophagus and trachea leads to the formation of the common birth defect esophageal atresia with/without tracheoesophageal fistula (EA/TEF). Although loss of multiple signaling molecules and transcription factors has been implicated in EA/TEF formation, the underlying cellular mechanisms remain unknown. Here, we combine Xenopus and mouse genetic models to identify the transcription factor Isl1 as a new player regulating trachea-esophageal separation. Significantly, we find that Isl1 orchestrates the separation process through modulating a specific epithelial progenitor cell population (Isl1+ Nkx2.1+ Sox2+) located at the dorsal-ventral boundary of the foregut. Our lineage tracing experiments show that this population contributes to both tracheal and esophageal epithelium. Moreover, Isl1 is required for the transcription of Nkx2.1 in this unique population. Finally, deletion of the chromosomal region spanning the ISL1 gene has been found in patients with abnormal trachea-esophageal separation. Our studies thus provide definitive evidence that ISL1 is a new player in the process of foregut morphogenesis, acting in a small progenitor population of boundary cells.

Effect of E Cigarette Emissions on Tracheal Cells Monitored at the Air-Liquid Interface Using an Organic Electrochemical Transistor.

E-cigarettes have been suggested as a potentially healthier alternative to cigarettes based on studies using cell viability, DNA damage, and transcriptional response assays. However, little is known about the effect of e-cigarette aerosols on the integrity of the tracheal epithelium, specifically with respect to barrier resistance. This is partly due to the lack of methods for monitoring epithelia at the air-liquid interface (ALI), i.e., under physiological conditions. Here, it is shown that an organic electrochemical transistor can be adapted for the measurement of barrier resistance at the ALI. This technology enables accurate, continuous quantification of tracheal barrier integrity through the use of a conformable gate electrode placed on top of the cell-secreted mucus, obviating the need for addition of culture medium or buffer as a conductance medium for rigid electrodes. This platform allows for the detection of a dose-dependent, rapid decrease in barrier resistance of an in vitro model of human bronchial epithelium (MucilAir) after E-cigarette aerosols exposure. The system represents a powerful tool to study tissue responses of the human airway epithelium to inhaled smoke. The same technology will have broad applications for toxicology studies on other tissues with ALI, including other airway tissues and skin.

Experimental animal model for assessment of tracheal epithelium regeneration.

To develop an experimental model in rabbits for assessment of tracheal epithelium regeneration through application of either natural or artificial polymer scaffolds. First, we identified the size of full-thickness mucosal defect, which does not allow self-healing (a "critical defect"), thus representing an adequate experimental model for regenerative therapy of tracheal epithelium damage. Then, two methods of polymer scaffold fixation at the site of the epithelium defect were compared: suturing and fixation with a stent. This was done through: 1) formation of a full-thickness anterolateral mucosal defect by tracheal mucosa excision; and 2) fixation of the scaffold at the site of the tracheal epithelium defect using sutures (through a tracheal wall "window") or a vascular stent (through a small tracheal incision). The dimension of a critical anterolateral mucosal defect of the trachea for rabbits was found to be 1.5 cm in length and more than 50% of the tracheal circumference. Fixation of the scaffold with a stent proved to be more efficient due to a uniform distribution of the pressure over the entire surface of the scaffold, whereas the suturing of the scaffold provided unsatisfactory results. In addition, fixation of the scaffold by suturing required formation of a large "window" in the tracheal wall. Thus, using the stent appeared to be technically less complicated and much less traumatic as compared to suturing. We present an experimental in vivo animal model of tracheal epithelium injury and recovery. It can be effectively used with certain further modifications as a basis for routine testing of bioengineered constructs. NA Laryngoscope, 129:E213-E219, 2019.

Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid.

Acute airway acidification is a potent stimulus of sensory nerves and occurs commonly with gastroesophageal reflux disease, cystic fibrosis, and asthma. In infants and adults, airway acidification can acutely precipitate asthma-like symptoms, and treatment-resistant asthma can be associated with gastroesophageal reflux disease. Airway protective behaviors, such as mucus secretion and airway smooth muscle contraction, are often exaggerated in asthma. These behaviors are manifested through activation of neural circuits. In some populations, the neural response to acid might be particularly important. For example, the immune response in infants is relatively immature compared with adults. Infants also have a high frequency of gastroesophageal reflux. Thus, in the current study, we compared the transcriptomes of an airway-nervous system circuit (e.g., tracheal epithelia, nodose ganglia, and brain stem) in neonatal piglets challenged with intra-airway acid. We hypothesized that the identification of parallel changes in the transcriptomes of two neutrally connected tissues might reveal the circuit response, and, hence, molecules important for the manifestation of asthma-like features. Intra-airway acid induced airway hyperreactivity and airway obstruction in male piglets. In contrast, female piglets displayed airway obstruction without airway hyperreactivity. Pairwise comparisons revealed parallel changes in genes directly implicated in airway hyperreactivity ( scn10a) in male acid-challenged piglets, whereas acid-challenged females exhibited parallel changes in genes associated with mild asthma ( stat 1 and isg15). These findings reveal sex-specific responses to acute airway acidification and highlight distinct molecules within a neural circuit that might be critical for the manifestation of asthma-like symptoms in pediatric populations.

TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1

The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.

A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.

The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury-repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell typethat we call the 'pulmonary ionocyte', which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium.

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.

We combine single-cell RNA-seq and in vivo lineage tracing to study the cellular composition and hierarchy of the murine tracheal epithelium. We identify a new rare cell type, the FoxI1-positive pulmonary ionocyte; functional variations in club cells based on their proximodistal location; a distinct cell type that resides in high turnover squamous epithelial structures that we named “hillocks”; and disease-relevant subsets of tuft and goblet cells. With a new method, Pulse-Seq, we show that tuft, neuroendocrine, and ionocyte cells are continually and directly replenished by basal progenitor cells. Remarkably, the cystic fibrosis gene, CFTR, is predominantly expressed in the pulmonary ionocytes of both mouse and human. Foxi1 loss in murine ionocytes causes a loss of Cftr expression and disrupts airway fluid and mucus physiology, which are also altered in cystic fibrosis. By associating cell type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.