Expression And Regulation Of Nasal Epithelial Barrier Molecules In Chronic Rhinosinusitis

Part Ⅰ: p120 regulates E-cadherin expression in nasal epithelial cells in chronic rhinosinusitisObjective: The physical barrier formed by the epithelial cells in nasal cavity and paranasal sinus plays an important role in the pathogenesis of chronic rhinosinusitis(CRS).Tight junctions and adherens junctions play an essential role in maintaining the physiological conditions of the epithelial physical barrier.E-cadherin was considered to be an important player in maintaining normal adherens junctions structure and regulating immune response.However,the regulatory mechanism of E-cadherin in CRS remains unclear.Methods: The expression of E-cadherin and p120 was detected by real-time quantitative polymerase chain reaction(RT-q PCR)and western blotting.Human nasal epithelial cells(HNECs)cultured with submerged method were transfected with p120 specific small interfering RNA to explore the mechanism of E-cadherin by p120.HNECs differentiated with the air-liquid interface(ALI)method were stimulated with various cytokines and Tolllike receptor(TLR)agonists.The barrier properties of differentiated HNECs were assessed by measuring fluorescent dextran permeability.Results: E-cadherin and p120 expression was decreased in HNECs from patients with CRS,and p120 protein expression level was positively correlated with that of E-cadherin.Knocking down p120 in HNECs cultured with submerged method significantly reduced the E-cadherin protein expression.The Rac1 inhibitor NSC23766 reversed the protein expression of E-cadherin in p120 knockdown experiments.Inflammatory mediators,including IL-4,TNF-α,TGF-β1,LPS and IFN-γ,reduced E-cadherin and p120 protein expression and increased paracellular permeability.Conclusion: p120 is involved in regulating the expression of E-cadherin protein in CRS.Inflammatory mediators reduce the protein expression of E-cadherin and p120 and disrupt the integrity of epithelial barrier.Part Ⅱ: Expression of IL-22 BP in eosinophilic chronic rhinosinusitis with nasal polyps and its correlation with epithelial barrier moleculesObjective: Interleukin-22 binding protein(IL-22BP),as a naturally soluble neutralizing antibody against IL-22,plays an important role in maintaining the cell function maintained by IL-22.However,the function of IL-22 BP in CRSwNP is still unclear.Methods: Total eosinophil and inflammatory cell count in nasal tissue was determined by hematoxylin-eosin staining.The IL-22 BP m RNA and protein expression in both nasal mucosa from control subjects and nasal polyps from the patients with CRSwNP was detected by real-time quantitative polymerase chain reaction(RT-q PCR)and enzymelinked immunosorbent assay(ELISA).Immunofluorescence was used to determine the distribution of IL-22 BP.RT-q PCR and immunohistochemistry were used to analyze the expression of epithelial barrier molecules.The protein levels of tissue inflammatory mediators were measured using the Bio-Plex suspension chip method.Eosinophils isolated from peripheral blood cultured in medium and stimulated with different cytokines,IL-22 BP protein expression level in supernatant was detected by ELISA.Nasal polyp size,Visual analogue score(VAS),computed tomography(CT)and clinical symptoms in CRSwNP patients were scored for clinical relevance.Results: CRSwNP was divided into eosinophilic chronic rhinosinusitis with nasal polyps(EosCRSwNP)and noneosinophilic chronic rhinosinusitis with nasal polyps(Non-EosCRSwNP)according to the infiltration degree of tissue eosinophils.IL-22 BP m RNA and protein expression was significantly increased in nasal polyps from the patients with eosinophilic chronic rhinosinusitis with nasal polyps(EosCRSwNP)compared with control subjects and those from patients with noneosinophilic chronic rhinosinusitis with nasal polyp(Non-EosCRSwNP).In nasal polyps from the patients with EosCRSwNP,immunofluorescence revealed that eosinophils were the main IL-22 BP immunoreactive cells.IL-22 BP was positively correlated with type 2 inflammatory mediators(IL-5,IL-13,IL-25 and Ig E)in nasal polyps from the patients with EosCRSwNP.IL-22 BP and ECP protein levels were increased in cultured supernatants with IL-5 stimulation compared with those without stimulant,IL-4,or IL-13 stimulation.The results of RT-q PCR showed that the m RNA levels of tight junction molecule(ZO-2,occludin and claudin-4)and adherens junctions molecule(E-cadherin)decreased significantly in nasal epithelial cells(HNECs)from both the patients with Non-EosCRSwNP and the patients with EosCRSwNP compared with those from control subjects.Compared with HNECs from control subjects,ZO-1 m RNA expression was significantly down-regulated in HNECs from patients with EosCRSwNP.However,there was no significant difference in the m RNA expression of ZO-1,ZO-2,occludin,claudin-4 and E-cadherin in HNECs from both patients with EosCRSwNP and patients with Non-EosCRSwNP.Claudin-4 and occludin were negatively correlated with IL-22 BP at m RNA expression level in patients with EosCRSwNP.IL-22 BP protein levels was positively correlated with disease severity and degree of tissue inflammation in the patients with EosCRSwNP.Conclusion: The expression of IL-22 BP was increased in nasal polyps of EosCRSwNP patients.The type 2 inflammatory factors promote the production of IL-22 BP in eosinophils.IL-22 BP in nasal polyps from the patients with EosCRSwNP was negatively correlated with epithelial barrier junction molecules.IL-22 BP was positively correlated with disease severity and degree of tissue inflammation.