Mouse Tracheal Epithelial Cells Research Articles

CC16 drives VLA-2-dependent SPLUNC1 expression.

CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated. We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity. We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge. We identified 8 antimicrobial proteins significantly decreased by CC16-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden. Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.

The Impact of CC16 on Pulmonary Epithelial-Driven Host Responses during Mycoplasma pneumoniae Infection in Mouse Tracheal Epithelial Cells.

Club Cell Secretory Protein (CC16) plays many protective roles within the lung; however, the complete biological functions, especially regarding the pulmonary epithelium during infection, remain undefined. We have previously shown that CC16-deficient (CC16-/-) mouse tracheal epithelial cells (MTECs) have enhanced Mp burden compared to CC16-sufficient (WT) MTECs; therefore, in this study, we wanted to further define how the pulmonary epithelium responds to infection in the context of CC16 deficiency. Using mass spectrometry and quantitative proteomics to analyze proteins secreted apically from MTECs grown at an air-liquid interface, we investigated the protective effects that CC16 elicits within the pulmonary epithelium during Mycoplasma pneumoniae (Mp) infection. When challenged with Mp, WT MTECs have an overall reduction in apical protein secretion, whereas CC16-/- MTECs have increased apical protein secretion compared to their unchallenged controls. Following Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) assessment, many of the proteins upregulated from CC16-/- MTECS (unchallenged and during Mp infection) were related to airway remodeling, which were not observed by WT MTECs. These findings suggest that CC16 may be important in providing protection within the pulmonary epithelium during respiratory infection with Mp, which is the major causative agent of community-acquired pneumoniae.

Identification of a novel interleukin-9 receptor complex in non-hematopoietic cells

Abstract Beyond cell-cell contact, communication at a distance requires the secretion of cytokines that bind to specific receptors on responsive cells. Interleukin (IL)-9 is a cytokine with pleiotropic effects not only on immune cells that contribute to the progression of tumor and autoimmunity, but also on structural cells including airway and intestinal epithelial cells involved in the development of inflammatory and allergic diseases. Typically, IL-9 receptor (IL-9R) functions as a complex of two subunits – the α-chain (IL-9Rα) and a common γ (γc) chain, that is shared with other cytokine receptors. Although structural cells express IL-9Rα, they lack expression of the γc chain. Thus, it is still unclear how IL-9 transmits a signal through a single IL-9Rα chain in non-hematopoietic cells. This study aimed to explore the possibility that like the type II IL-4Rα, the IL-9Rα chain can pair with the IL-13Rα1 chain, forming a type II IL-9R and can mediate IL-9 responses in epithelial cells. We demonstrated that the IL-9Rα/IL-13Rα1 complex was detected in situin mouse tracheal epithelial cells and human small airway epithelial cells (SAEC) using proximity ligation assay. STAT3 was activated after exposure to IL-9 in vitroin SAEC when co-cultured with neutralizing antibody targeting γc chain but STAT3 activity returned to its basal level in the presence of anti-IL-13Rα1 antibody, providing further evidence of a type II IL-9R on epithelial cells. Strikingly, asthma-associated genes are upregulated in SAEC upon IL-9 stimulation in RNA-seq analysis. Collectively, our results identify a putative receptor complex - a type II IL-9R composed of IL-9Rα and IL-13Rα1, providing a mechanism for responses of structural cells to IL-9 and a potential role in asthma.

Low molecular weight hyaluronan inhibits lung epithelial ion channels by activating the calcium-sensing receptor

Herein, we tested the hypothesis that low molecular weight hyaluronan (LMW-HA) inhibits lung epithelial ions transport in-vivo, ex-vivo, and in-vitro by activating the calcium-sensing receptor (CaSR). Twenty-four hours post intranasal instillation of 50–150 µg/ml LMW-HA to C57BL/6 mice, there was a 75% inhibition of alveolar fluid clearance (AFC), a threefold increase in the epithelial lining fluid (ELF) depth, and a 20% increase in lung wet/dry (W/D) ratio. Incubation of human and mouse precision cut lung slices with 150 µg/ml LMW-HA reduced the activity and the open probability (Po) of epithelial sodium channel (ENaC) in alveolar epithelial type 2 (ATII) cells, and in mouse tracheal epithelial cells (MTEC) monolayers as early as 4 h. The Cl− current through cystic fibrosis transmembrane conductance regulator (CFTR) and the activity of Na,K-ATPase were both inhibited by more than 66% at 24 h. The inhibitory effects of LMW-HA on ion channels were reversed by 1 µM NPS-2143, or 150 µg/ml high molecular weight hyaluronan (HMW-HA). In HEK-293 cells expressing the calcium-sensitive Cl− channel TMEM16-A, CaSR was required for the activation of the Cl− current by LMW-HA. This is the first demonstration of lung ions and water transport inhibition by LMW-HA, and its mediation through the activation of CaSR.

Pre-existing atopy prevents mortality from respiratory viral infection via induction of neuregulin-1

Abstract Respiratory viral infections are associated with significant morbidity and mortality. RNA viruses, such as Respiratory Syncytial Virus (RSV) have been implicated in the development of asthma. We have a high-fidelity model of respiratory viral infection. Our mouse model utilizes a natural rodent pathogen, Sendai virus (SeV) similar to RSV, which faithfully replicates in mouse airway epithelial cells. We have shown that making these mice atopic (i.e, sensitized and challenged with house dust mite extracts) before viral infection prevents development of disease and more importantly prevents mice from dying of a normally lethal dose of SeV. Further, lungs and bronchoalveolar lavage fluid of atopic mice have significant increased levels of Neuregulin-1 (NRG1), potentially due to high expression of Nrg1 in dendritic cells as determined by RNAseq of CD11c+ cells. To determine a temporal relationship between NRG1 expression and respiratory viral infection, we utilized an in vitro system of well-differentiated human bronchial epithelial cells and mouse tracheal epithelial cells. Cells were treated on the basolateral side with NRG1 (10 to 100ng) for six-days before 4000 pfu of RSV-GFP or SeV-GFP inoculation, respectively. There was marked reduction in viral spreading (quantified by reduced GFP expression) with NRG1 treatment. Interleukin-6 (IL6) expression was reduced in NRG1 treated cells at 48h PI, as well. IL6 has been implicated with cellular stress and is involved in disease pathology. Therefore, reducing IL6 might affect the clinical presentation of serious infections. In conclusion our studies show that atopy induced NRG1 plays a novel role with a therapeutic potential in severe respiratory viral infections. Supported by Abigail Wexner Research Institute at Nationwide Children’s Hospital, Start-up fund to MHG

Cytotoxicity of 2D engineered nanomaterials in pulmonary and corneal epithelium

Two-dimensional (2D) engineered nanomaterials are widely used in consumer and industrial goods due to their unique chemical and physical characteristics. Engineered nanomaterials are incredibly small and capable of being aerosolized during manufacturing, with the potential for biological interaction at first-contact sites such as the eye and lung. The unique properties of 2D nanomaterials that make them of interest to many industries may also cause toxicity towards epithelial cells. Using murine and human respiratory epithelial cell culture models, we tested the cytotoxicity of eight 2D engineered nanomaterials: graphene (110 nm), graphene oxide (2 um), graphene oxide (400 nm), reduced graphene oxide (2 um), reduced graphene oxide (400 nm), partially reduced graphene oxide (400 nm), molybdenum disulfide (400 nm), and hexagonal boron nitride (150 nm). Non-graphene nanomaterials were also tested in human corneal epithelial cells for ocular epithelial cytotoxicity. Hexagonal boron nitride was found to be cytotoxic in mouse tracheal, human alveolar, and human corneal epithelial cells. Hexagonal boron nitride was also tested for inhibition of wound healing in alveolar epithelial cells; no inhibition was seen at sub-cytotoxic doses. Nanomaterials should be considered with care before use, due to specific regional cytotoxicity that also varies by cell type. Supported by U01ES027288 and T32HL007013 and T32ES007059.

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung.

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3−/− in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

Airway Basal Cells Mediate Hypoxia-Induced EMT by Increasing Ribosome Biogenesis.

Excessive secretion of airway mucus and fluid accumulation are the common features of many respiratory diseases, which, in turn, induce cell hypoxia in the airway epithelium, resulting in epithelial–mesenchymal transition (EMT) and ultimately fibrosis. However, the mechanisms of EMT induced by hypoxia in the airway are currently unclear. To mimic the status of edematous fluid retention in the airway, we cultured primary mouse tracheal epithelial cells (MTECs) in a liquid–liquid interface (LLI) mode after full differentiation in a classic air–liquid interface (ALI) culture system. The cell hypoxia was verified by the physical characteristics and lactate production in cultured medium as well as HIF expression in MTECs cultured by LLI mode. EMT was evidenced and mainly mediated by basal cells, supported by flow cytometry and immunofluorescence assay. The differently expressed genes of basal and other airway epithelial cells were found to be enriched in the ribosome by our analysis of an MTEC single-cell RNA sequencing data set and Myc, the global regulator of ribosome biogenesis was identified to be highly expressed in basal cells. We next separated basal cells from bulk MTECs by flow cytometry, and the real-time PCR results showed that ribosome biogenesis was significantly upregulated in basal cells, whereas the inhibition of ribosome biogenesis alleviated the phosphorylation of the mammalian target of rapamycin/AKT and abrogated hypoxia-induced EMT in MTECs. Collectively, these observations strongly suggest that basal cells in the airway epithelium may mediate the process of hypoxia-induced EMT, partly through enhancing ribosome biogenesis.

Ferulic acid ameliorates lipopolysaccharide-induced tracheal injury via cGMP/PKGII signaling pathway

BackgroundTracheal injury is a common clinical condition that still lacks an effective therapy at present. Stimulation of epithelial sodium channel (ENaC) increases Na+ transport, which is a driving force to keep tracheal mucosa free edema fluid during tracheal injury. Ferulic acid (FA) has been proved to be effective in many respiratory diseases through exerting anti-oxidant, anti-inflammatory, and anti-thrombotic effects. However, these studies rarely involve the level of ion transport, especially ENaC.MethodsC57BL/J male mice were treated intraperitoneally with normal saline or FA (100 mg/kg) 12 h before, and 12 h after intratracheal administration of lipopolysaccharide (LPS, 5 mg/kg), respectively. The effects of FA on tracheal injury were not only assessed through HE staining, immunofluorescence assay, and protein/mRNA expressions of ENaC located on tracheas, but also evaluated by the function of ENaC in mouse tracheal epithelial cells (MTECs). Besides, to explore the detailed mechanism about FA involved in LPS-induced tracheal injury, the content of cyclic guanosine monophosphate (cGMP) was measured, and Rp-cGMP (cGMP inhibitor) or cGMP-dependent protein kinase II (PKGII)-siRNA (siPKGII) were applied in primary MTECs, respectively.ResultsHistological examination results demonstrated that tracheal injury was obviously attenuated by pretreatment of FA. Meanwhile, FA could reverse LPS-induced reduction of both protein/mRNA expressions and ENaC activity. ELISA assay verified cGMP content was increased by FA, and administration of Rp-cGMP or transfection of siPKGII could reverse the FA up-regulated ENaC protein expression in MTECs.ConclusionsFerulic acid can attenuate LPS-induced tracheal injury through up-regulation of ENaC at least partially via the cGMP/PKGII pathway, which may provide a promising new direction for preventive and therapeutic strategy in tracheal injury.

CC16 Deficiency in the Context of Early-Life Mycoplasma pneumoniae Infection Results in Augmented Airway Responses in Adult Mice.

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16-/-) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults (early life infection model; ELIM) and compared to adult mice infected for only 3 days (adult infection model; AIM). CC16-/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16-/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16-/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16-/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16-/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Effective-component compatibility of Bufei Yishen formula protects COPD rats against PM2.5-induced oxidative stress via miR-155/FOXO3a pathway

Ambient particulate matter <2.5 µm (PM2.5) has been identified as a critical risk factor in chronic obstructive pulmonary disease (COPD) exacerbation, but therapies for this condition are limited. Effective-component compatibility of Bufei Yishen formula (ECC-BYF) exhibits beneficial efficacy on COPD rats. However, its effect on PM2.5-aggravated COPD rats are considered to be uncertain. In this study, we used an established PM2.5-aggravated COPD rat model in vivo to evaluate the protective effect of ECC-BYF, and focused on its antioxidative role in PM2.5-stimulated bronchial epithelial cells via regulating microRNA (miR)−155/ forkhead box class O3a (FOXO3a) pathway. As expected, PM2.5-aggravated COPD rats showed a reduction of lung function, persistent lung inflammation, and remodeling of lung tissue. In comparison, ECC-BYF administration significantly enhanced lung function, alleviated alveolar destruction, inflammatory cell infiltration, mucus hypersecretion, and collagen deposition, along with diminishing inflammatory cytokine production and oxidative stress. Furthermore, ECC-BYF pretreatment markedly decreased the fluorescence intensity of reactive oxygen species (ROS) in PM2.5-induced human bronchial epithelial (Beas-2B) cells and primary mouse tracheal epithelial cells (MTECs), as well as reversing the imbalance between oxidants and antioxidants in Beas-2B. Meanwhile, ECC-BYF elevated FOXO3a while inhibiting miR-155 expression dose -dependently. In vitro transfection of miR-155 mimic into Beas-2B significantly decreased FOXO3a protein expression, accompanied by the reduced superoxide dismutase 2 (SOD2) and catalase (CAT) expressions, thus eliminating the protective effect of ECC-BYF on PM2.5-evoked oxidative stress. Nonethless, FOXO3a overexpression could partially restore the antioxidative effect of ECC-BYF. In conclusion, ECC-BYF can protect pre-existing COPD against PM2.5 contamination by exerting a profound antioxidative influence via regulating miR-155/FOXO3a signaling.

DRP1-Mediated Mitochondrial Fission Regulates Lung Epithelial Response to Allergen.

Mitochondria regulate a myriad of cellular functions. Dysregulation of mitochondrial control within airway epithelial cells has been implicated in the pro-inflammatory response to allergens in asthma patients. Because of their multifaceted nature, mitochondrial structure must be tightly regulated through fission and fusion. Dynamin Related Protein 1 (DRP1) is a key driver of mitochondrial fission. During allergic asthma, airway epithelial mitochondria appear smaller and structurally altered. The role of DRP1-mediated mitochondrial fission, however, has not been fully elucidated in epithelial response to allergens. We used a Human Bronchial Epithelial Cell line (HBECs), primary Mouse Tracheal Epithelial Cells (MTECs), and conditional DRP1 ablation in lung epithelial cells to investigate the impact of mitochondrial fission on the pro-inflammatory response to house dust mite (HDM) in vitro and in vivo. Our data suggest that, following HDM challenge, mitochondrial fission is rapidly upregulated in airway epithelial cells and precedes production of pro-inflammatory cytokines and chemokines. Further, deletion of Drp1 in lung epithelial cells leads to decreased fission and enhanced pro-inflammatory signaling in response to HDM in vitro, as well as enhanced airway hyper-responsiveness (AHR), inflammation, differential mucin transcription, and epithelial cell death in vivo. Mitochondrial fission, therefore, regulates the lung epithelial pro-inflammatory response to HDM.

Malondialdehyde-Acetaldehyde Adduct Formation Decreases Immunoglobulin A Transport across Airway Epithelium in Smokers Who Abuse Alcohol

Alcohol misuse and smoking are risk factors for pneumonia, yet the impact of combined cigarette smoke and alcohol on pneumonia remains understudied. Smokers who misuse alcohol form lung malondialdehyde-acetaldehyde (MAA) protein adducts and have decreased levels of anti-MAA secretory IgA (sIgA). Transforming growth factor-β (TGF-β) down-regulates polymeric Ig receptor (pIgR) on mucosal epithelium, resulting in decreased sIgA transcytosis to the mucosa. It is hypothesized that MAA-adducted lung protein increases TGF-β, preventing expression of epithelial cell pIgR and decreasing sIgA. Cigarette smoke and alcohol co-exposure on sIgA and TGF-β in human bronchoalveolar lavage fluid and in mice instilled with MAA-adducted surfactant protein D (SPD-MAA) were studied herein. Human bronchial epithelial cells (HBECs) and mouse tracheal epithelial cells were treated with SPD-MAA and sIgA and TGF-β was measured. Decreased sIgA and increased TGF-β were observed in bronchoalveolar lavage from combined alcohol and smoking groups in humans and mice. CD204 (MAA receptor) knockout mice showed no changes in sIgA. SPD-MAA decreased pIgR in HBECs. Conversely, SPD-MAA stimulated TGF-β release in both HBECs and mouse tracheal epithelial cells, but not in CD204knockout mice. SPD-MAA stimulated TGF-β in alveolar macrophage cells. These data show thatMAA-adducted surfactant protein stimulates lung epithelial cell TGF-β, down-regulates pIgR, anddecreases sIgA transcytosis. These data provide a mechanism for the decreased levels of sIgA observed in smokers who misuse alcohol.

Obese adipose tissue modulates proinflammatory responses of mouse airway epithelial cells.

Although recognized as an important endocrine organ, little is known about the mechanisms through which adipose tissue can regulate inflammatory responses in distant tissues, such as lung that are affected by obesity. To explore potential mechanisms, male C57BL/6J mice were provided either high-fat diet, low-fat diet, or were provided a high-fat diet then switched to the low-fat diet to promote weight loss. Visceral adipocytes were then cultured in vitro to generate conditioned media (CM) that was used to treat both primary (mouse tracheal epithelial cells; MTECs) and immortalized (mouse-transformed club cells; MTCCs) airway epithelial cells. Adiponectin levels were greatly depressed in the CM from both obese and diet-switched adipocytes relative to mice continually fed the low-fat diet. MTECs from mice with obesity secreted higher baseline levels of inflammatory cytokines than MTECs from lean or diet-switched mice. MTECs treated with obese adipocyte CM increased their secretion of these cytokines compared with MTECs treated with lean CM. Diet-switched CM modestly decreased the production of cytokines compared with obese CM, and these effects were recapitulated when the CM was used to treat MTCCs. Adipose stromal vascular cells from mice with obesity expressed genes consistent with an M1 macrophage phenotype and decreased eosinophil abundance compared with lean stromal vascular fraction, a profile that persisted in the lean diet-switched mice despite substantial weight loss. Soluble factors secreted from obese adipocytes exert a proinflammatory effect on airway epithelial cells, and these alterations are attenuated by diet-induced weight loss, which could have implications for the airway dysfunction related to obese asthma and its mitigation by weight loss.

Genetic variation in surfactant protein-A2 alters responses to ozone.

BackgroundIncreased exposure to Ozone (O3) is associated with adverse health effects in individuals afflicted with respiratory diseases. Surfactant protein-A (SP-A), encoded by SP-A1 and SP-A2, is the largest protein component in pulmonary surfactant and is functionally impaired by O3-oxidation.ObjectiveWe used humanized SP-A2 transgenic mice with allelic variation corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 in the lectin domain to determine the impact of this genetic variation in regards to O3 exposure.MethodsMice were exposed to 2ppm O3 or Filtered Air (FA) for 3 hours and 24 hrs post-challenge pulmonary function tests and other parameters associated with inflammation were assessed in the bronchoalveolar lavage (BAL) fluid and lung tissue. Additionally, mouse tracheal epithelial cells were cultured and TEER measurements recorded for each genotype to determine baseline epithelial integrity.ResultsCompared to FA, O3 exposure led to significantly increased sensitivity to methacholine challenge in all groups of mice. SP-A2 223Q variant mice were significantly protected from O3-induced AHR compared to SP-A-/- and SP-A2 223K mice. Neutrophilia was observed in all genotypes of mice post O3-exposure, however, SP-A2 223Q mice had a significantly lower percentage of neutrophils compared to SP-A-/- mice. Albumin levels in BAL were unchanged in O3-exposed SP-A2 223Q mice compared to their FA controls, while levels were significantly increased in all other genotypes of O3-exposed mice. SP-A 223Q MTECS has significant higher TEER values than all other genotypes, and WT MTECS has significantly higher TEER than the SP-A KO and SP-A 223K MTECS.SignificanceTaken together, our study suggests that expression of a glutamine (Q) as position 223 in SP-A2, as opposed to expression of lysine (K), is more protective in acute exposures to ozone and results in attenuated O3-induced AHR, neutrophilia, and vascular permeability.

Respiratory Epithelial Cells Respond to Lactobacillus plantarum but Provide No Cross-Protection against Virus-Induced Inflammation.

Virus-induced inflammation plays a critical role in determining the clinical outcome of an acute respiratory virus infection. We have shown previously that the administration of immunobiotic Lactobacillus plantarum (Lp) directly to the respiratory tract prevents lethal inflammatory responses to subsequent infection with a mouse respiratory virus pathogen. While Lp-mediated protective responses involve non-redundant contributions of both Toll-like receptor 2 (TLR2) and NOD2, the cellular basis of these findings remains unclear. Here, we address the impact of Lp and its capacity to suppress inflammation in virus-infected respiratory epithelial cells in two cell culture models. We found that both MLE-12 cells and polarized mouse tracheal epithelial cells (mTECs) were susceptible to infection with Influenza A and released proinflammatory cytokines, including CCL2, CCL5, CXCL1, and CXCL10, in response to replicating virus. MLE-12 cells express NOD2 (81 ± 6.3%) and TLR2 (19 ± 4%), respond to Lp, and are TLR2-specific, but not NOD2-specific, biochemical agonists. By contrast, we found that mTECs express NOD2 (81 ± 17%) but minimal TLR2 (0.93 ± 0.58%); nonetheless, mTECs respond to Lp and the TLR2 agonist, Pam2CSK4, but not NOD2 agonists or the bifunctional TLR2-NOD2 agonist, CL-429. Although MLE-12 cells and mTECS were both activated by Lp, little to no cytokine suppression was observed in response to Lp followed by virus infection via a protocol that replicated experimental conditions that were effective in vivo. Further study and a more complex approach may be required to reveal critical factors that suppress virus-induced inflammatory responses.

Nicotine stimulates ion transport via metabotropic β4 subunit containing nicotinic ACh receptors.

Background and PurposeMucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown.Experimental ApproachTransepithelial short circuit currents (ISC) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+]i were studied on freshly dissociated mouse tracheal epithelial cells.Key ResultsApical application of the nAChR agonist nicotine transiently increased ISC. The nicotine effect was abolished by the nAChR antagonist mecamylamine. α‐Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3β2, α4β2, α4β4 and α3β4) and A‐85380 (α4β2 and α3β4) increased ISC. The antagonists dihydro‐β‐erythroidine (α4β2, α3β2, α4β4 and α3β4), α‐conotoxin MII (α3β2) and α‐conotoxin PnIA (α3β2) reduced the nicotine effect. Nicotine‐ and epibatidine‐induced currents were unaltered in β2−/−mice, but in β4−/− mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+‐ATPase inhibitor) or the ryanodine receptor antagonists JTV‐519 and dantrolene there was a reduction in the nicotine‐effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H‐89 and the TMEM16A (Ca2+‐activated chloride channel) inhibitor T16Ainh‐A01 significantly reduced the nicotine‐effect.Conclusion and Implicationsα3β4 nAChRs are responsible for the nicotine‐induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.

Mesenchymal Stem Cell-Conditioned Medium Rescues LPS-Impaired ENaC Activity in Mouse Trachea via WNK4 Pathway.

Airway epithelium plays an essential role in maintaining the homeostasis and function of respiratory system as the first line of host defense. Of note, epithelial sodium channel (ENaC) is one of the victims of LPS-induced airway injury. Regarding the great promise held by mesenchymal stem cells (MSCs) for regenerative medicine in the field of airway injury and the limitations of cell-based MSCs therapy, we focused on the therapeutic effect of MSCs conditioned medium (MSCs-CM) on the ENaC activity in mouse tracheal epithelial cells. Ussing chamber apparatus was applied to record the short-circuit currents in primary cultured mouse tracheal epithelial cells, which reflects the ENaC activity. Expressions of α and γ ENaC were measured at the protein and mRNA levels by western blot and real-time PCR, respectively. The expression of with-no-lysinekinase- 4 (WNK4) and ERK1/2 were measured at protein levels, and the relationship between WNK4 and ERK1/2 was determined by WNK4 knockdown. MSCs-CM restored the LPS-impaired ENaC activity, as well as enhanced the mRNA and protein expressions of ENaC in primary cultured mouse tracheal epithelial cells. Meanwhile, WNK4 and ERK1/2, both negative-regulators of ENaC, were suppressed accordingly after the administration of MSCs-CM in LPS-induced airway injury. After WNK4 gene was knocked down by siRNA, the level of ERK1/2 phosphorylation decreased. In light of the key role of ENaC in fluid reabsorption and the beneficial effects of MSCs-CM in the injury of airway epithelium, our results suggest that MSCs-CM is effective in alleviating LPS-induced ENaC dysfunction through WNK4-ERK1/2 pathway, which will provide a potent direction for the therapy of airway injury.

The Transient Receptor Potential Channel Vanilloid 1 Is Critical in Innate Airway Epithelial Responses to Protease Allergens.

The airway epithelium plays a critical role in innate responses to airborne allergens by secreting IL-1 family cytokines such as IL-1α and IL-33 as alarmins that subsequently orchestrate appropriate immune responses. Previous studies revealed that epithelial IL-33 secretion by allergens such as Alternaria alternata or house dust mite involves Ca2+-dependent signaling, via initial activation of ATP-stimulated P2YR2 (type 2 purinoceptor) and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase DUOX1. We sought to identify proximal mechanisms by which epithelial cells sense these allergens and here highlight the importance of PAR2 (protease-activated receptor 2) and TRP (transient receptor potential) Ca2+ channels such as TRPV1 (TRP vanilloid 1) in these responses. Combined studies of primary human nasal and mouse tracheal epithelial cells, as well as immortalized human bronchial epithelial cells, indicated the importance of both PAR2 and TRPV1 in IL-33 secretion by both Alternaria alternata and house dust mite, based on both pharmacological and genetic approaches. TRPV1 was also critically involved in allergen-induced ATP release, activation of DUOX1, and redox-dependent activation of EGFR (epidermal growth factor receptor). Moreover, genetic deletion of TRPV1 dramatically attenuated allergen-induced IL-33 secretion and subsequent type 2 responses in mice in vivo. TRPV1 not only contributed to ATP release and P2YR2 signaling but also was critical in downstream innate responses to ATP, indicating potentiating effects of P2YR2 on TRPV1 activation. In aggregate, our studies illustrate a complex relationship between various receptor types, including PAR2 and P2YR2, in epithelial responses to asthma-relevant airborne allergens and highlight the central importance of TRPV1 in such responses.

Parental centrioles are dispensable for deuterosome formation and function during basal body amplification.

Mammalian epithelial cells use a pair of parental centrioles and numerous deuterosomes as platforms for efficient basal body production during multiciliogenesis. How deuterosomes form and function, however, remain controversial. They are proposed to arise either spontaneously for massive de novo centriole biogenesis or in a daughter centriole-dependent manner as shuttles to carry away procentrioles assembled at the centriole. Here, we show that both parental centrioles are dispensable for deuterosome formation. In both mouse tracheal epithelial and ependymal cells (mTECs and mEPCs), discrete deuterosomes in the cytoplasm are initially procentriole-free. They emerge at widely dispersed positions in the cytoplasm and then enlarge, concomitant with their increased ability to form procentrioles. More importantly, deuterosomes still form efficiently in mEPCs whose daughter centriole or even both parental centrioles are eliminated through shRNA-mediated depletion or drug inhibition of Plk4, a kinase essential to centriole biogenesis in both cycling cells and multiciliated cells. Therefore, deuterosomes can be assembled autonomously to mediate de novo centriole amplification in multiciliated cells.