Jae Murphy1, Mahnaz Ramezanpour1, Natalia Stach2,3, Grzegorz Dubin2,3, Alkis James Psaltis1, Peter-John Wormald1, Sarah Vreugde1. 1. Department of Surgery-Otolaryngology, Head and Neck Surgery, The University of Adelaide, Woodville South, SA, Australia. 2. Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. 3. Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
Abstract
OBJECTIVE: Staphylococcus aureus (S. aureus) infection is known to contribute to the severity and recalcitrance of chronic rhinosinusitis (CRS), and its secreted products have been shown to alter the airway barrier. Extracellular proteases secreted by S. aureus are thought to be important in epithelial infection and immune evasion; however, their effect on airway mucosal barrier function is not known. METHODS: To investigate the impact of extracellular proteases on airway epithelial integrity, the purified S. aureus proteases V8 protease, Staphopain A, Staphopain B, Exfoliative toxin A, and serine protease-like A-F were applied to human nasal epithelial cell air-liquid interface (HNEC-ALI) cultures. Transepithelial electrical resistance (TEER), permeability (Papp) measurements, and immuno-localization of the tight junction proteins claudin-1 and ZO-1 were used to assess barrier integrity. Effects of the proteases on inflammation and cell viability were measured using interleukin-6 (IL-6) ELISA and a lactate dehydrogenase assay. RESULTS: Application of V8 protease to HNEC-ALI cultures caused a significant concentration and time-dependent decrease in TEER (22.67%, P < 0.0001), a reciprocal Papp increase (20.14-fold, P < 0.05), and a discontinuous ZO-1 immuno-localization compared to control. IL-6 production was significantly reduced in V8 protease-treated cells (153.5 pg/mL, P = 0.0069) compared to control (548.3 pg/mL), whereas no difference in cell viability was observed. CONCLUSION: S. aureus V8 protease causes dysfunction of mucosal barrier structure and function indicative of a leaky barrier. A reduction in IL-6 levels suggests that the mucosal immunity is impaired by this protease and thus has the potential to contribute to CRS recalcitrance. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E8-E15, 2018.
OBJECTIVE:Staphylococcus aureus (S. aureus) infection is known to contribute to the severity and recalcitrance of chronic rhinosinusitis (CRS), and its secreted products have been shown to alter the airway barrier. Extracellular proteases secreted by S. aureus are thought to be important in epithelial infection and immune evasion; however, their effect on airway mucosal barrier function is not known. METHODS: To investigate the impact of extracellular proteases on airway epithelial integrity, the purified S. aureus proteases V8 protease, Staphopain A, Staphopain B, Exfoliative toxin A, and serine protease-like A-F were applied to human nasal epithelial cell air-liquid interface (HNEC-ALI) cultures. Transepithelial electrical resistance (TEER), permeability (Papp) measurements, and immuno-localization of the tight junction proteins claudin-1 and ZO-1 were used to assess barrier integrity. Effects of the proteases on inflammation and cell viability were measured using interleukin-6 (IL-6) ELISA and a lactate dehydrogenase assay. RESULTS: Application of V8 protease to HNEC-ALI cultures caused a significant concentration and time-dependent decrease in TEER (22.67%, P < 0.0001), a reciprocal Papp increase (20.14-fold, P < 0.05), and a discontinuous ZO-1 immuno-localization compared to control. IL-6 production was significantly reduced in V8 protease-treated cells (153.5 pg/mL, P = 0.0069) compared to control (548.3 pg/mL), whereas no difference in cell viability was observed. CONCLUSION:S. aureus V8 protease causes dysfunction of mucosal barrier structure and function indicative of a leaky barrier. A reduction in IL-6 levels suggests that the mucosal immunity is impaired by this protease and thus has the potential to contribute to CRS recalcitrance. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E8-E15, 2018.
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