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Background & aimsEosinophilic esophagitis (EoE) is an antigen-mediated eosinophilic disease of the esophagus that involves fibroblast activation and progression to fibrostenosis. Cytokines produced by T-helper type 2 cells and transforming growth factor beta 1 (TGFβ1) contribute to the development of EoE, but other cytokines involved in pathogenesis are unknown. We investigate the effects of tumor necrosis factor superfamily member 14 (TNFSF14, also called LIGHT) on fibroblasts in EoE.MethodsWe analyzed publicly available esophageal CD3+ T-cell single-cell sequencing data for expression of LIGHT. Esophageal tissues were obtained from pediatric patients with EoE or control individuals and analyzed by immunostaining. Human primary esophageal fibroblasts were isolated from esophageal biopsy samples of healthy donors or patients with active EoE. Fibroblasts were cultured; incubated with TGFβ1 and/or LIGHT; and analyzed by RNA sequencing, flow cytometry, immunoblots, immunofluorescence, or reverse transcription polymerase chain reaction. Eosinophils were purified from peripheral blood of healthy donors, incubated with interleukin 5, cocultured with fibroblasts, and analyzed by immunohistochemistry.ResultsLIGHT was up-regulated in the esophageal tissues from patients with EoE, compared with control individuals, and expressed by several T-cell populations, including T-helper type 2 cells. TNF receptor superfamily member 14 (TNFRSF14, also called HVEM) and lymphotoxin beta receptor are receptors for LIGHT that were expressed by fibroblasts from healthy donors or patients with active EoE. Stimulation of esophageal fibroblasts with LIGHT induced inflammatory gene transcription, whereas stimulation with TGFβ1 induced transcription of genes associated with a myofibroblast phenotype. Stimulation of fibroblasts with TGFβ1 increased expression of HVEM; subsequent stimulation with LIGHT resulted in their differentiation into cells that express markers of myofibroblasts and inflammatory chemokines and cytokines. Eosinophils tethered to esophageal fibroblasts after LIGHT stimulation via intercellular adhesion molecule-1.ConclusionsT cells in esophageal tissues from patients with EoE express increased levels of LIGHT compared with control individuals, which induces differentiation of fibroblasts into cells with inflammatory characteristics. TGFβ1 increases fibroblast expression of HVEM, a receptor for LIGHT. LIGHT mediates interactions between esophageal fibroblasts and eosinophils via ICAM1. This pathway might be targeted for the treatment of EoE.

There is an increasing prevalence of esophageal diseases but intact human tissue platforms to study esophageal function, disease mechanisms, and the interactions between cell types in situ are lacking. To address this, we utilized full thickness human donor esophagi to create and validate the ex vivo function of mucosa and smooth muscle (n = 25). Explanted tissue was tested for contractile responses to carbachol and histamine. We then treated ex vivo human esophageal mucosa with a cytokine cocktail to closely mimic the Th2 and inflammatory milieu of eosinophilic esophagitis (EoE) and assessed alterations in smooth muscle and extracellular matrix function and stiffening. We found that full thickness human esophagus as well as the individual layers of circular and longitudinal muscularis propria developed tension in response to carbachol ex vivo and that mucosa demonstrated squamous cell differentiation. Treatment of mucosa with Th2 and fibrotic cytokines recapitulated the majority of the clinical Eosinophilic Esophagitis Diagnostic Profile (EDP) on fluidic transcriptional microarray. Transforming growth factor-beta-1 (TGFβ1) increased gene expression of fibronectin, smooth muscle actin, and phospholamban (p

BackgroundEosinophilic esophagitis (EoE) is a chronic TH2-assocated inflammatory condition accompanied by substantial impairments in epithelial barrier function and increased numbers of interleukin 9 (IL-9) expressing inflammatory cells. While IL-9 is known to affect barrier function in the intestine, the functional effects of IL-9 on the esophagus are unclear. Herein we aimed to understand the expression of the IL-9 receptor and effects of IL-9 on the epithelium in EoE.MethodsWe used esophageal biopsies from pediatric EoE patients with active and inactive disease to analyze the expression of the IL-9 receptor, the adherens junction protein E-cadherin and the tight junction protein claudin-1. We treated primary human esophageal epithelial cells with IL-9 to understand its effects on E-cadherin expression and function.ResultsActive EoE subjects had increased epithelial expression of IL-9 receptor mRNA and protein (P

BackgroundGroup 2 innate lymphoid cells (ILC2s) expand in the lungs of mice during type 2 inflammation induced by the fungal allergen Alternaria alternata. The increase in ILC2 numbers in the lung has been largely attributed to local proliferation and whether ILC2s migrate from the circulation to the lung after Alternaria exposure is unknown.ObjectiveWe examined whether human (lung, lymph node, and blood) and mouse lung ILC2s express β1 and β2 integrin adhesion molecules and whether these integrins are required for trafficking of ILC2s into the lungs of mice.MethodsHuman and mouse ILC2s were assessed for surface expression of β1 and β2 integrin adhesion molecules by using flow cytometry. The role of β1 and β2 integrins in ILC2 trafficking to the lungs was assessed by in vivo blocking of these integrins before airway exposure to Alternaria in mice.ResultsBoth human and mouse lung ILC2s express high levels of β1 and β2 integrin adhesion receptors. Intranasal administration of Alternaria challenge reduced ILC2 numbers in the bone marrow and concurrently increased blood and lung ILC2 numbers. In vivo blocking of β2 integrins (CD18) significantly reduced ILC2 numbers in the lungs but did not alter ILC2 proliferation, apoptosis, and function. In contrast, in vivo blocking of β1 integrins or α4 integrins did not affect lung ILC2 numbers.ConclusionILC2 numbers increase in the mouse lung not only through local proliferation but also through trafficking from the circulation into the lung using β2 rather than β1 or α4 integrins.

Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway-hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMBZp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMB-overexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma but whose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium.

Background and purposeAsthma manifests as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyperresponsiveness (AHR). Although the molecular mechanisms remain unclear, activation of specific PI3K isoforms mediate inflammation and AHR. We aimed to determine whether inhibition of PI3Kδ evokes dilation of airways and to elucidate potential mechanisms.Experimental approachHuman precision cut lung slices from non-asthma donors and primary human airway smooth muscle (HASM) cells from both non-asthma and asthma donors were utilized. Phosphorylation of Akt, myosin phosphatase target subunit 1 (MYPT1) and myosin light chain (MLC) were assessed in HASM cells following either PI3K inhibitor or siRNA treatment. HASM relaxation was assessed by micro-pattern deformation. Reversal of constriction of airways was assessed following stimulation with PI3K or ROCK inhibitors.Key resultsSoluble inhibitors or PI3Kδ knockdown reversed carbachol-induced constriction of human airways, relaxed agonist-contracted HASM and inhibited pAkt, pMYPT1 and pMLC in HASM. Similarly, inhibition of Rho kinase also dilated human PCLS airways and suppressed pMYPT1 and pMLC. Baseline pMYPT1 was significantly elevated in HASM cells derived from asthma donors in comparison with non-asthma donors. After desensitization of the β2 -adrenoceptors, a PI3Kδ inhibitor remained an effective dilator. In the presence of IL-13, dilation by a β agonist, but not PI3K inhibitor, was attenuated.Conclusion and implicationsPI3Kδ inhibitors act as dilators of human small airways. Taken together, these findings provide alternative approaches to the clinical management of airway obstruction in asthma.

Segmental allergen challenge induced increased levels of bronchoalveolar (BAL) follistatin like 1 (FSTL1). BAL macrophages both expressed FSTL1, and could be induced by FSTL1 to express MMP9 suggesting a potential role of FSTL1 in remodeling.

Group 2 innate lymphoid cells (ILC2s) produce high levels of IL-5 and IL-13, both of which are important pathogenic mediators in eosinophilic esophagitis (EoE). ILC2s have not been previously described in EoE. Our study demonstrates the novel finding that ILC2s are increased in esophageal biopsies from EoE patients with active disease compared with inactive EoE and non-diseased controls, implicating these cells in EoE pathogenesis.

Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although [beta]-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that [beta]-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the [beta]-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing [[beta].sub.2]-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or tO an alteration of the ERK/ MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways. [[beta].sub.2]-adrenergic receptor; epidermal growth factor receptor; asthma; bronchospasm

Wound healing is a response to injury that is initiated to reconstruct damaged tissue. In skin, reepithelialization involves both epithelial cells and fibroblasts and contributes to the reformation of a barrier between the external environment and internal milieu. Growth factors including epidermal growth factor (EGF) play important roles in promoting this process. In the present studies we employed CV-1 fibroblasts in a tissue culture model of reepithelialization to develop strategies for optimizing wound closure stimulated by EGF. We found that EGF enhanced cell motility within 6-8 h of EGF treatment in serum-free medium but wounds failed to close within 24 h. However, if medium on these cultures was exchanged for medium containing serum, cells pretreated with EGF closed new scrape wounds more rapidly than did cells that were not pretreated. These results indicate that serum factors work in concert with EGF to coordinate cell motility for efficient wound closure. Indeed. EGF enhanced the rate of wound closure in the presence of serum, and this effect also persisted for at least 24 h after EGF was removed. This coordination of EGF-induced cell motility was accompanied by an increase in the transient phosphorylation of ERK1 and ERK2. The persistent effects of EGF were blocked by transient exposure to reversible inhibitors of transcription and translation, indicating that the expression of new proteins mediated this response. We propose that EGF-stimulated CV-I fibroblast motility is coordinated by a serum component that induces cell-cell adhesive properties consistent with an epithelial phenotype, thereby enhancing the reepithelialization process. wound healing; cell motility

On binding to its receptor, epidermal growth factor (EGF) initiates a cascade of events leading to cell proliferation or differentiation. In addition, the EGF receptor itself is downregulated to attenuate mitogenic signaling. Downregulation occurs through trafficking of receptors to lysosomes, culminating in proteolytic destruction of both the receptor and ligand; however, endocytic sorting mechanisms that underlie lysosomal targeting remain obscure. The goal of this study was to explore one aspect of the molecular basis for ligand-induced lysosomal targeting and degradation of EGF receptors. In this study, we identify a tyrosine-leucine motif ([sup.954]YLVI) that is essential for transit of ligand-receptor complexes to lysosomes. When this motif is mutated, HEK 293 cells expressing the mutant receptors demonstrate impaired lysosomal targeting and downregulation compared with wild-type receptors. [sup.954]YLVI is highly conserved among EGF receptors from various mammalian and invertebrate species and is critical for receptor downregulation. We propose that [sup.954]YLVI works in concert with at least two additional regions within the EGF receptor cytoplasmic domain that are essential for efficiently targeting ligand-receptor complexes to the lysosome. degradation; lysosomes; ubiquitination; targeting

Enzyme assays following cotransfection of membrane lipid desaturase (MLD) gene with epidermal growth factor receptor were utilized to identify the gene encoding a desaturase and to determine the effect on biosynthesis of the transmembrane receptor for epidermal growth factor (EGF). Results revealed that MLD has properties of a membrane protein of endoplasmic reticulum. It was also shown that MLD specifically inhibits the biosynthesis of the EGF receptor following overexpression in transfected cells.

Abstract Background The β2-adrenergic receptor (β2AR) is a primary target for medications used to treat asthma. Due to the low abundance of β2AR, very few studies have reported its localization in tissues. However, the intracellular location of β2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β2AR in primary cell cultures. Methods A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β2AR were identified and used to localize native β2AR in primary cultures of rat airway smooth muscle and epithelial cells. β2AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA). Results Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β2AR (Ab-Bethyl) specifically recognized human but not rat β2AR. An antibody developed against the C-terminal domain of the mouse β2AR (Ab-sc570) specifically recognized rat but not human β2AR. An antibody developed against 78 amino acids of the C-terminus of the human β2AR (Ab-13989) was capable of recognizing both rat and human β2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. Conclusion Antibodies have been identified that recognize human β2AR, rat β2AR or both rat and human β2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β2AR in tissues.

The recent discovery of sensory (tastant and odorant) G proteincoupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases. [ABSTRACT FROM AUTHOR]