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1.
Mucosal Immunol ; 11(2): 415-426, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28832026

RESUMO

Barrier dysfunction has been implicated in the pathophysiology of eosinophilic esophagitis (EoE). Transforming growth factor-ß1 (TGF-ß1), a potent pleiotropic molecule, is increased in EoE; however, no study has evaluated its influence on esophageal epithelial barrier. We hypothesized that TGF-ß1 regulates barrier dysfunction in EoE. We aimed to determine the role of TGF-ß1 in the epithelial barrier in models of EoE. To examine the impact of TGF-ß1 on esophageal barrier, immortalized human esophageal epithelial (EPC2-hTERT) cells were exposed to TGF-ß1 during the three-dimensional air-liquid interface (3D-ALI) model in vitro. TGF-ß1 exposure diminished EPC2-hTERT barrier function as measured by transepithelial electrical resistance (TEER) and 3 kDa Fluorescein isothiocyanate dextran paracellular flux (FITC Flux), and hematoxylin and eosin (H&E) assessment revealed prominent cellular separation. In analysis of epithelial barrier molecules, TGF-ß1 led to the specific reduction in expression of the tight-junction molecule, claudin-7 (CLDN7), and this was prevented by TGF-ß-receptor I inhibitor. Short hairpin ribonucleic acid (shRNA)-mediated CLDN7 knockdown diminished epithelial barrier function, whereas CLDN7 overexpression resulted in protection from TGF-ß1-mediated barrier dysfunction. In pediatric EoE biopsies CLDN7 expression was decreased and altered localization was observed with immunofluorescence analysis, and the TGF-ß1 downstream transcription factor, phosphorylated SMAD2/3 (pSMAD2/3), was increased. Our data suggest that TGF-ß1 participates in esophageal epithelial barrier dysfunction through CLDN7 dysregulation.


Assuntos
Claudinas/metabolismo , Esofagite Eosinofílica/imunologia , Eosinófilos/imunologia , Células Epiteliais/fisiologia , Esôfago/patologia , Junções Íntimas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Biópsia , Técnicas de Cultura de Células , Células Cultivadas , Criança , Claudinas/genética , Regulação para Baixo , Impedância Elétrica , Células Epiteliais/patologia , Humanos , RNA Interferente Pequeno/genética
2.
Allergy ; 72(8): 1232-1242, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27926989

RESUMO

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic antigen-mediated clinicopathologic disease of the esophagus characterized by an eosinophil-predominant inflammatory infiltrate. A clinical hallmark is extensive tissue remodeling including basal zone hyperplasia, fibrosis, and angiogenesis. However, the cellular mechanisms responsible for these processes are not fully defined. We hypothesized that targeting granulocyte-macrophage colony-stimulating factor (GM-CSF; an agonist cytokine linked with eosinophil survival and activation) would be protective in a preclinical model of EoE. METHODS: Eosinophilic esophagitis-like esophageal inflammation was induced in the L2-IL5OXA EoE mouse model, and GM-CSF production was assessed by mRNA and protein analyses. Granulocyte-macrophage colony-stimulating factor-receptor-alpha expression patterns were examined by flow cytometric and immunofluorescence analysis. L2-IL5OXA EoE mice were treated with anti-GM-CSF neutralizing antibody or isotype control and assessed for histopathological indices of eosinophilia, epithelial hyperplasia, and angiogenesis by immunohistochemistry and RT-PCR. RESULTS: Significantly increased levels of esophageal GM-CSF expression was detected in the L2-IL5OXA mouse EoE model during active inflammation. Granulocyte-macrophage colony-stimulating factor-receptor-alpha was predominantly expressed on esophageal eosinophils during EoE, in addition to select cells within the lamina propria. Anti-GM-CSF neutralization in L2-IL5OXA EoE mice resulted in a significant diminution of epithelial eosinophilia in addition to basal cell hyperplasia and vascular remodeling. This treatment response was independent of effects on esophageal eosinophil maturation or activation. CONCLUSION: Granulocyte-macrophage colony-stimulating factor is a potential therapeutic target to reduce esophageal eosinophilia and remodeling.


Assuntos
Esofagite Eosinofílica/metabolismo , Esofagite Eosinofílica/patologia , Mucosa Esofágica/metabolismo , Mucosa Esofágica/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Remodelação Vascular , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Transformada , Fatores Quimiotáticos de Eosinófilos/imunologia , Modelos Animais de Doenças , Esofagite Eosinofílica/genética , Esofagite Eosinofílica/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Eosinófilos/patologia , Mucosa Esofágica/imunologia , Feminino , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Masculino , Camundongos , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/imunologia
4.
Mucosal Immunol ; 8(6): 1324-38, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25850656

RESUMO

Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b(-/-) mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2b(fl/fl)VeCadCre(+)) or intestinal epithelia (Adora2b(fl/fl)VillinCre(+)) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses.


Assuntos
Colite/patologia , Células Epiteliais/metabolismo , Mucosa Intestinal/patologia , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais , Doença Aguda , Animais , Western Blotting , Colite/metabolismo , Modelos Animais de Doenças , Células Epiteliais/patologia , Citometria de Fluxo , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologia
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