Submucosal Gland Cells Research Articles

Targeted inhibition of m6A demethylase FTO by FB23 attenuates allergic inflammation in the airway epithelium.

Epithelial cells play a crucial role in asthma, contributing to chronic inflammation and airway hyperresponsiveness. m6A modification, which involves key proteins such as the demethylase fat mass and obesity-associated protein (FTO), is crucial in the regulation of various diseases, including asthma. However, the role of FTO in epithelial cells and the development of asthma remains unclear. In this study, we investigated the demethylase activity of FTO using a small-molecule inhibitor FB23 in epithelial cells and allergic inflammation invivo and invitro. We examined the FTO-regulated transcriptome-wide m6A profiling by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq under FB23 treatment and allergic inflammation conditions. Immunofluorescence staining was performed to assess the tissue-specific expression of FTO in asthmatic bronchial mucosa. We demonstrated that FB23 alleviated allergic inflammation in IL-4/IL-13-treated epithelial cells and house dust mite (HDM)-induced allergic airway inflammation mouse model. The demethylase activity of FTO contributed to the regulation of TNF-α signaling via NF-κB and epithelial-mesenchymal transition-related pathways under allergic inflammation conditions in epithelial cells. FTO was expressed in epithelial, submucosal gland, and smooth muscle cells in human bronchial mucosa. In conclusion, FB23-induced inhibition of FTO alleviates allergic inflammation in epithelial cells and HDM-induced mice, potentially through diverse cellular processes and epithelial-mesenchymal transition signaling pathways, suggesting that FTO is a potential therapeutic target in asthma management.

TNFRSF1B Signaling Blockade Protects Airway Epithelial Cells from Oxidative Stress.

Progressive respiratory airway destruction due to unresolved inflammation induced by periodic infectious exacerbation episodes is a hallmark of cystic fibrosis (CF) lung pathology. To clear bacteria, neutrophils release high amounts of reactive oxygen species (ROS), which inflict collateral damage to the neighboring epithelial cells causing oxidative stress. A former genome-wide small interfering RNA (siRNA) screening in CF submucosal gland cells, instrumental for mucociliary clearance, proposed tumor necrosis factor receptor superfamily member 1B (TNFRSF1B; TNFR2) as a potential hit involved in oxidative stress susceptibility. Here, we demonstrate the relevance of TNFRSF1B transcript knock-down for epithelial cell protection under strong oxidative stress conditions. Moreover, a blockade of TNFR signaling through its ligand lymphotoxin-α (LTA), overexpressed in airway epithelial cells under oxidative stress conditions, using the anti-tumor necrosis factor (TNF) biologic etanercept significantly increased the viability of these cells from a toxic oxidizing agent. Furthermore, bioinformatic analyses considering our previous RNA interference (RNAi) screening output highlight the relevance of TNFRSF1B and of other genes within the TNF pathway leading to epithelial cell death. Thus, the inhibition of the LTα3-TNFR2 axis could represent a useful therapeutic strategy to protect the respiratory airway epithelial lining from the oxidative stress challenge because of recurrent infection/inflammation cycles faced by CF patients.

205 Airway submucosal gland cell biology, physiology, and pathophysiology in cystic fibrosis

205 Airway submucosal gland cell biology, physiology, and pathophysiology in cystic fibrosis

Role of mucociliary clearance system in respiratory diseases.

Mucociliary clearance system is the primary innate defense mechanism of the lung. It plays a vital role in protecting airways from microbes and irritants infection. Mucociliary clearance system, which is mediated by the actions of airway and submucosal gland epithelial cells, plays a critical role in a multilayered defense system via secreting fluids, electrolytes, antimicrobial and anti-inflammatory proteins, and mucus onto airway surfaces. Changes in environment, drugs or diseases can lead to mucus overproduction and cilia dysfunction, which in turn decrease the rate of mucociliary clearance and enhance mucus gathering. The dysfunction of mucociliary clearance system often occurs in several respiratory diseases, such as primary ciliary dysfunction, cystic fibrosis, asthma and chronic obstructive pulmonary disease, which are characterized by goblet cell metaplasia, submucosal gland cell hypertrophy, mucus hypersecretion, cilia adhesion, lodging and loss, and airway obstruction.

A spatially resolved atlas of the human lung characterizes a gland-associated immune niche

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new ‘gland-associated immune niche’ has implications for respiratory health.

From the Editor’s Desk…

From the Editor’s Desk…

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential

Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers. In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. Invitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. Invivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver. A cell population with clonal growth and organoid formation capability, which has liver differentiation potency invitro and invivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver. Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Age-Related Increase of Collagen/Fibrin Deposition and High PAI-1 Production in Human Nasal Polyps.

Objective: Our previous studies showed an age-related increased prevalence of nasal polyps (NP) and reduced production of S100A8/9 in elderly patients with chronic rhinosinusitis with NP (CRSwNP). In this study, we investigated an unbiased age-related gene expression profile in CRSwNP subjects and healthy controls, and further identified the differences in their tissue remodeling. Methods: Microarrays using NP and uncinate tissues from health controls (elderly, age ≥65 vs. non-elderly, age 18–49) were performed, and differentially regulated genes were analyzed. Quantitative real-time PCR (qPCR), Immunostaining, Periodic acid-Schiff (PAS), trichrome staining, Western blot, and ELISA were performed for further investigation. Results: Microarrays identified differentially expressed genes according to disease and age; 278 in NP vs. controls, 75 in non-elderly NP vs. non-elderly controls, and 32 in elderly NP vs. elderly controls. qPCR confirmed that the PLAT gene was downregulated and the SERPINB2 gene upregulated in NP vs. controls. The serous glandular cell-derived antimicrobial protein/peptide-related genes such as BPIFB3, BPIFB2, LPO, and MUC7 were remarkably reduced in NP, regardless of age. SERPINE1 gene (plasminogen activator inhibitor-1, PAI-1) expression was significantly increased in elderly NP versus elderly controls. IHC and western blot confirmed significantly decreased production of MUC7 and LPO in NP versus controls. There was a trend of age-related reduction of submucosal gland cells in normal controls. Trichrome and immunofluorescence staining demonstrated an age-related increase of collagen and fibrin deposition in NP, consistent with increased PAI-1 production. Conclusion: This study demonstrated age-related differential glandular remodeling patterns and fibrosis in NP and normal controls. PAI-1 expression was significantly increased in elderly NP versus elderly controls, suggesting PAI-1 as a potential treatment target in elderly NP.

IRBIT as a Regulator of Bicarbonate Transport in the Small Intestine

BackgroundSmall intestinal bicarbonate transport, is critical for epithelial protection, digestion, and absorption. Membrane trafficking and/or recycling of transporters is a critical means to modulate their function, however, the cellular mechanisms that drive these processes in the duodenum remain unclear. The inositol 1,4,5‐trisphosphate (IP3) receptor binding protein released with IP3 (IRBIT) is an intracellular protein that has been shown to regulate localization of bicarbonate transporters outside of the intestine, yet its role in regulating duodenal bicarbonate secretion is unknown.AimTo examine duodenal expression and function of IRBIT in regulating bicarbonate transport.MethodsDuodenal endoscopic biopsies from subjects without gross or histological evidence of disease were used directly or cultured using traditional apical‐in or flipped apical‐out enteroids. mRNA expression was evaluated using qPCR and re‐analysis of a duodenal sc‐RNAseq dataset. Protein expression and localization was determined by Western blot and confocal immunofluorescence, respectively. In vivo bicarbonate secretion was measured in anesthetized mice by duodenal perfusion and back‐titration.ResultsIRBIT is highly expressed in human duodenal biopsies, with protein densitometric analysis showing ≥3.5‐fold higher expression than Calu‐3 airway submucosal gland cells or HEK293 kidney‐derived cells (n=1 each). Analysis of duodenal sc‐RNAseq data (1,625 cells) showed ACHYL1 in crypts and villi. Immunofluorescence staining of human duodenal biopsies (n=3) showed IRBIT staining along the crypt‐villus axis, primarily localizing in E‐cadherin positive epithelial cells, but also present in the lamina propria. In patient‐derived duodenal enteroids, IRBIT was expressed in both crypt‐like (undifferentiated) and villus‐like (differentiated) enteroids (n=3 each), with greater IRBIT mRNA and protein expression in the latter, a pattern similar to SLC26A3 and SLC26A6 chloride/bicarbonate exchangers (n=3 each). In sc‐RNAseq analysis, AHCYL1 and SLC26A3 were co‐expressed in 38% of all villus cells, compared to only 5% co‐expressing AHCYL1 and SLC26A6. CFTR‐independent duodenal bicarbonate secretion stimulated by linaclotide (10‐7 M) was significantly inhibited by phospholipase C inhibition (U7312, 10‐6 M; 91±11%, n=3), which we showed disrupts IRBIT membrane localization, or SLC26A3 inhibition (DRAinh‐A250, 10‐5 M; 68±8%, n=10). Linaclotide stimulation of apical‐out enteroids increased both IRBIT (n=3) and SLC26A3 (n=12) membrane trafficking.Summary and ConclusionsIRBIT is highly expressed in the duodenum and appears to functionally interact with SLC26A3. IRBIT may be an attractive target to modulate small intestinal pH in diseases like cystic fibrosis, where impaired bicarbonate secretion contributes to intestinal disease and malnutrition.

Chloride channel accessory 1 gene deficiency causes selective loss of mucus production in a new pig model.

Morbidity and mortality of respiratory diseases are linked to airway obstruction by mucus but there are still no specific, safe, and effective drugs to correct this phenotype. The need for better treatment requires a new understanding of the basis for mucus production. In that regard, studies of human airway epithelial cells in primary culture show that a mucin granule constituent known as chloride channel accessory 1 (CLCA1) is required for inducible expression of the inflammatory mucin MUC5AC in response to potent type 2 cytokines. However, it remained uncertain whether CLCLA1 is necessary for mucus production in vivo. Conventional approaches to functional biology using targeted gene knockout were difficult due to the functional redundancy of additional Clca genes in mice not found in humans. We reasoned that CLCA1 function might be better addressed in pigs that maintain the same four-member CLCA gene locus and the corresponding mucosal and submucosal populations of mucous cells found in humans. Here we develop to our knowledge the first CLCA1-gene-deficient (CLCA1-/-) pig and show that these animals exhibit loss of MUC5AC+ mucous cells throughout the airway mucosa of the lung without affecting comparable cells in the tracheal mucosa or MUC5B+ mucous cells in submucosal glands. Similarly, CLCA1-/- pigs exhibit loss of MUC5AC+ mucous cells in the intestinal mucosa without affecting MUC2+ mucous cells. These data establish CLCA1 function for controlling MUC5AC expression as a marker of mucus production and provide a new animal model to study mucus production at respiratory and intestinal sites.

Distinct Secretion of MUC5AC and MUC5B in Upper and Lower Chronic Airway Diseases

The human airway is protected by a defensive mucus barrier. The most prominent components of mucus are the mucin glycoproteins MUC5AC and MUC5B. They are produced by goblet cells and submucosal gland cells in the upper and lower airways. Hyperplasia of these cells and hypersecretion of MUC5AC and MUC5B characterize chronic inflammatory diseases of the upper and lower airways. Recent studies have revealed that MUC5AC and MUC5B are expressed at specific sites in the respiratory tract through different molecular mechanisms and have distinct functions. Morphometric and histochemical studies have also examined the roles of goblet cells, submucosal gland cells, MUC5AC, and MUC5B in different chronic airway diseases individually. The individual study of goblet cells, submucosal gland cells, MUC5AC, and MUC5B in airway diseases would be helpful for precisely diagnosing chronic inflammatory diseases of the airway and establishing optimal treatments. This review focuses on the distinct secretion of MUC5AC and MUC5B and their producing cells in chronic inflammatory diseases of the upper and lower airway.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity.

Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing “protective” drugs. This approach could also prove useful to other respiratory pathologies.

Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis

BackgroundAllergic rhinitis (AR) is caused by allergic reaction to allergens such as pollen. Galanin (GAL), a neuropeptide that regulates inflammatory processes, is widely expressed in the central and peripheral nervous systems. Although neuropeptides are implicated in arthritis and chemically induced ileitis, their roles in AR remain unclear. MethodsWe developed a murine model of AR and generated control, systemic sensitization, mild AR, and severe AR groups. We examined GAL and GAL receptor (GALR) mRNA and protein levels and localization patterns in each group using reverse transcription PCR, western blotting, and immunohistochemical analyses. Additionally, we evaluated the effects of M871, a GALR2 antagonist, on mice with severe AR. ResultsGal and Galr2 are expressed in nasal mucosa and brain (control) samples from control and AR mice. GAL and GALR2 were expressed at similar levels and localized to ciliated epithelial and submucosal gland cells of the nasal mucosa in all four groups. Intranasal M871 administration significantly reduced the incidence of nose rubbing behaviors and sneezing (p < 0.001 in 30 min, respectively) in severe AR mice relative to that in controls. Mechanistically, we postulate that GALR2 is expressed in B cells, and M871 administration reduces IgE production, as well as the number of B cells in tissues. ConclusionsGAL signaling may not change progressively with increasing nasal sensitization, suggesting that this signaling process exacerbates, rather than directly trigger, AR. GAL–GALR2 signaling likely mediates AR development, suggesting that its inhibition represents a novel therapeutic strategy for AR.

17,18-Epoxyeicosatetraenoic Acid Inhibits TNF-α-Induced Inflammation in Cultured Human Airway Epithelium and LPS-Induced Murine Airway Inflammation.

17,18-Epoxyeicosatetraenoic acid (17,18-EpETE), an eicosapentaenoic acid metabolite, is generated from dietary oil in the gut, and antiinflammatory activity of 17,18-EpETE was recently reported. To evaluate the inhibitory effects of 17,18-EpETE in airway inflammation, we examined in vitro and in vivo effects on mucus production, neutrophil infiltration, and cytokine/chemokine production in airway epithelium. Nasal tissue localization of G protein-coupled receptor 40 (GPR40), a receptor of 17,18-EpETE, was determined by immunohistochemical staining. Expression of GPR40 mRNA in nasal mucosa of chronic rhinosinusitis (CRS) patients and control subjects was determined by reverse transcription-polymerase chain reaction (RT-PCR). The in vitro effects on airway epithelial cells were examined using normal human bronchial epithelial cells and NCI-H292 cells. To examine the in vivo effects of 17,18-EpETE on airway inflammation, we induced goblet cell metaplasia, mucus production, and neutrophil infiltration in mouse nasal epithelium by intranasal lipopolysaccharide (LPS) instillation. GPR40 is mainly expressed in human nasal epithelial cells and submucosal gland cells. RT-PCR analysis revealed that the expression of GPR40 mRNA was increased in nasal tissues from CRS patients compared with those from control subjects. 17,18-EpETE significantly inhibited tumor necrosis factor (TNF)-α-induced production of interleukin (IL)-6 , IL-8, and mucin from cultured human airway epithelial cells dose dependently, and these antiinflammatory effects on cytokine production were abolished by GW1100, a selective GPR40 antagonist. Intraperitoneal injection or intranasal instillation of 17,18-EpETE significantly attenuated LPS-induced mucus production and neutrophil infiltration in mouse nasal epithelium. Inflammatory cytokine/chemokine production in lung tissues and bronchoalveolar lavage fluids was also inhibited. These results indicate that 17,18-EpETE plays a regulatory role in mucus hypersecretion and neutrophil infiltration in nasal inflammation. Local or systemic administration may provide a new therapeutic approach for the treatment of intractable airway disease such as CRS.

Dissecting the cellular specificity of smoking effects and reconstructing lineages in the human airway epithelium

Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and non-smokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium.

Efficacy of Budesonide Nasal Spray on Neutrophilic Chronic Rhinosinusitis with Nasal Polyps: A Combined Clinical and Experimental Study

Background: Neutrophilic chronic rhinosinusitis with nasal polyps (CRSwNP) occur predominantly in Asian subjects. Appropriate treatments for this endotype have not been elucidated. This study aimed to evaluate the efficacy of budesonide nasal spray on neutrophilic CRSwNP. Materials and Methods: Fifteen neutrophilic CRSwNP patients were included, and then they received budesonide nasal spray treatment for 3 months. Biopsies of nasal polyps (NPs) were obtained from these subjects. Their clinical indexes were scored using Visual Analog Scale (VAS), Sino-Nasal Outcome Test (SNOT)-22, and Endoscopic Appearances (EAs). Histological analyses were used to assess numbers of neutrophils, goblet cells, and submucosal gland cells in NPs. Percentages of CD8⁺ T cells and CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) were evaluated using flow cytometry. Mucin 5AC (MUC5AC), MUC5B, myeloperoxidase (MPO), interferon (IFN)-γ, and interleukin (IL)-1β and their mRNAs were also examined. After that, we cultured NP tissues in vitro and evaluated the abovementioned inflammatory parameters before and after the administration of budesonide. Results: Budesonide nasal spray did not improve clinical evaluations including VAS, SNOT-22, and EA scores. Numbers of neutrophils and goblet cells, the score of submucosal gland cells, percentages of CD8⁺ T cells and Tregs, MUC5AC, MUC5B, MPO, IFN-γ, and IL-1β and their mRNAs were not decreased in NPs after the budesonide treatment. Furthermore, the administration of budesonide into NP cultures also did not reduce their levels in comparison with those before the treatment. Conclusion: These findings demonstrate that budesonide treatment may not alleviate the inflammatory condition in neutrophilic CRSwNP.

Pathogenesis and Cells of Origin of Barrett's Esophagus.

In patients with Barrett's esophagus (BE), metaplastic columnar mucosa containing epithelial cells with gastric and intestinal features replaces esophageal squamous mucosa damaged by gastroesophageal reflux disease. This condition is estimated to affect 5.6% of adults in the United States, and is a major risk factor for esophageal adenocarcinoma. Despite the prevalence and importance of BE, its pathogenesis is incompletely understood and there are disagreements over the cells of origin. We review mechanisms of BE pathogenesis, including transdifferentiation and transcommitment, and discuss potential cells of origin, including basal cells of the squamous epithelium, cells of esophageal submucosal glands and their ducts, cells of the proximal stomach, and specialized populations of cells at the esophagogastric junction (residual embryonic cells and transitional basal cells). We discuss the concept of metaplasia as a wound-healing response, and how cardiac mucosa might be the precursor of the intestinal metaplasia of BE. Finally, we discuss shortcomings in current diagnostic criteria for BE that have important clinical implications.

Su1040 – Early Activation of Sox9-Positive Cells in Esophageal Submucosal Glands After Injury

Su1040 – Early Activation of Sox9-Positive Cells in Esophageal Submucosal Glands After Injury

Prostaglandin E2‐Stimulated Cl− Secretion in Airway Epithelium Involves Protein Kinase A and Calcium Crosstalk

BackgroundMucociliary clearance is an integral defense mechanism against pulmonary infections and is impaired in Cystic Fibrosis (CF) airways. Prostaglandin E2 (PGE2) is abundant in infected airways and stimulates transepithelial Cl− secretion and mucociliary clearance, both of which are impaired in CF. Previously, we found that PGE2‐stimulated Cl− secretion was cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent in bronchial epithelial cells, but in submucosal gland cells utilized both CFTR and Ca2+‐activated Cl− channels (CaCC).AimTo determine the signaling mechanism(s) whereby PGE2 stimulates airway Cl− secretion in order to identify ways to circumvent the mucociliary defect in CF.MethodsHuman bronchial epithelial cell cultures (CFBE41o‐ with wildtype CFTR) were used for all experiments. Clsecretion was measured by short‐circuit current (Isc) with cell monolayers mounted in Ussing chambers and exposed to a serosal to mucosal Cl− gradient. These conditions minimize contribution from basolateral K+ channels and Isc reflects primarily Cl− transport. EP receptor expression was determined by qPCR.ResultsIn bronchial epithelial cell cultures, qPCR showed that EP4 receptor expression was 148% greater than EP3 receptor expression. CAY10598 (1 μM), a selective EP4 agonist, stimulated Cl− secretion to a similar magnitude as PGE2 (Cay: 85.5 +/− 8.6 μA/cm2 (n=4) vs. PGE2: 81.6 +/− 10.2 μA/cm2 (n=17); mean +/− SE). In contrast, sulprostone, an EP3 agonist, was less effective at 1 or 10 μM (5.6+/− 2.0 μA/cm2 (n=6) and 14.5+/− 7.3 μA/cm2 (n=2), respectively). Pre‐treatment with H‐89 (10 μM), a protein kinase A (PKA) inhibitor, decreased PGE2‐stimulated Cl− secretion by 74% (n=9). Similarly, pretreatment with BAPTA‐AM (100 μM), a Ca2+ chelator, inhibited PGE2‐stimulated Cl− secretion by 71% (n=7). Simultaneous pre‐treatment with H‐89 and BAPTA‐AM was additive and completely abolished PGE2‐stimulated Cl− secretion (94% inhibition, n=3). Additionally, wortmannin (1μM), a phosphoinositide 3‐kinase (PI3K) inhibitor, decreased PGE2‐stimulated Clsecretion by 56% (n=4).Summary and ConclusionsPGE2‐stimulated Cl− secretion in human bronchial epithelial cells predominantly occurs via EP4 receptors. While utilizing classic EP4 receptor signaling (PKA and PI3K), there is also substantial crosstalk with intracellular Ca2+. Interestingly, the latter does not activate CaCC, but rather CFTR. Ongoing experiments are aimed at further delineating the intracellular signaling mechanisms involved in PGE2‐stimulated Cl− secretion in bronchial epithelial cells, as well as comparing these mechanisms to PGE2‐stimulated Cl− secretion in submucosal gland cells. Through this we hope to identify potential ways to augment PGE2‐stimulated mucociliary clearance in CF, resulting in improved clearance of pulmonary infections.Support or Funding InformationCystic Fibrosis Foundation (Z.M.S), Elizabeth Nash Foundation (BI)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.