Hypoxia Disrupts The Nasal Epithelial Barrier By Inhibiting PTPN2 In Chronic Rhinosinusitis With Nasal Polyps

ObjectiveHypoxia is involved in inflammation and immune response;however,the role of hypoxia in the development of chronic rhinosinusitis with nasal polyps（CRSwNP）is not fully understood.We aimed to investigate the mechanisms by which hypoxia disrupts the nasal epithelial barrier in CRSwNP.Methods1.The expression of hypoxia-inducible factor-1α（HIF-1α）,protein tyrosine phosphatase non-receptor type 2（PTPN2）,and tight junction（TJ）components（claudin-4,occludin,and ZO-1）was detected in the middle turbinate or uncinate process tissues of the control group,and in the nasal polyps of the Eosinophilic CRSwNP（Eos CRSwNP）and non-Eos CRSwNP groups using immunohistochemistry,western blotting,and q RT-PCR.2.Primary human nasal epithelial cells（HNECs）were treated with Cobalt chloride（Co Cl₂）,a HIF-1αstabilizer and a hypoxia mimetic agent,and treated with HIF-1αinhibitor PX-478.The expressions of HIF-1α,PTPN2 and TJs were detected by western blotting and q RT-PCR,and TJs were detected by immunofluorescence staining.Paracellular flux was assessed using Fluorescein isothiocyanate dextran 4k Da（FD4）.PTPN2 was knocked down by si RNA or PTPN2 was overexpressed by lentivirus infection in BEAS-2B cells,and the expression of PTPN2 and TJs was detected by q RT-PCR and western blotting after Co Cl₂ treatment.3.Ovalbumin and Staphylococcus aureus enterotoxin B were used to establish a mouse model of Eos CRSwNP,and PX-478 and PTPN2 overexpression lentivirus were used to treat the mouse model.FD4 was used to evaluate the permeability of the nasal mucosa.The polypoid lesions of the nasal mucosa were assessed by hematoxylin and eosin staining,and the expression of HIF-1α,PTPN2 and TJs was detected in the nasal mucosa using immunohistochemical staining.Results1.HIF-1αprotein levels were higher in the non-Eos CRSwNP and Eos CRSwNP groups than in the control group.Additionally,HIF-1αprotein levels were higher in the Eos CRSwNP group than in the non-Eos CRSwNP group.The protein levels of PTPN2,claudin-4,occludin,and ZO-1 in the non-Eos and Eos CRSwNP groups were lower than those in the control group.In addition,the protein levels of PTPN2 and TJs in the Eos CRSwNP group were lower than those in the non-Eos CRSwNP group.Moreover,the m RNA levels of PTPN2,claudin-4,occludin,and ZO-1 were lower in the non-Eos CRSwNP and Eos CRSwNP groups than in the control group.2.The hypoxia mimetic agent Co Cl₂ decreased the expression of PTPN2,claudin-4,occludin,and ZO-1,and increased epithelial cell permeability in HNECs,which was blocked by PX-478.Immunofluorescence results showed that the intercellular TJs of Co Cl₂-treated HNECs were unconnected and cells had a loose morphology,whereas PX-478 partially restored TJ and cell morphology.Overexpression of PTPN2 inhibited hypoxia-induced downregulation of TJ expression in BEAS-2B cells,while knockdown of PTPN2 aggravated the effects of hypoxia.3.In the Eos CRSwNP mouse model,both PX-478 and PTPN2 overexpression reduced the formation of nasal polypoid lesions,permeability of the nasal epithelium,and restored TJ expression.ConclusionHypoxia-induced HIF-1αdownregulates TJ expression by inhibiting PTPN2,thereby disrupting the nasal epithelial barrier and promoting CRSwNP development.HIF-1αand PTPN2 may be potential targets for the treatment of CRSwNP.