PARK2 attenuates house dust mite-induced inflammatory reaction, pyroptosis and barrier dysfunction in BEAS-2B cells by ubiquitinating NLRP3

Objective: PARK2, a Parkinson's disease-associated gene, functions as an E3 ubiquitin ligase regulating the degradation of proteins via ubiquitination. Our study was designed to explore its role in allergic asthma and the underlying mechanisms. Methods: Airway epithelial cell line BEAS-2B was treated with house dust mite (HDM) to mimic allergic asthma in vitro. Lentivirus oePARK2 and siPARK2 were constructed to overexpress and knock down PARK2 expression, respectively. RT-qPCR, western blot, co-immunoprecipitation, and ubiquitination assay were performed to investigate the interaction between PARK2 and NLRP3. NLRP3 inflammasome activity, IL-1 beta and IL-18 secretion, pyroptosis, and epithelial barrier integrity were detected to explore the role of PARK2 in allergic asthma. Results: PARK2 expression was remarkably down-regulated in HDM-treated BEAS-2B cells. In BEAS-2B cells, NLRP3 protein was reduced by PARK2 overexpression and increased by PARK2 knockdown. Interestingly, PARK2 overexpression and knockdown didn't affect NLRP3 mRNA. Co-immunoprecipitation assay showed that PARK2 interacted with NLRP3. Proteasome inhibitor MG132 abolished PARK2 overexpression-induced down-regulation of NLRP3 protein. Ubiquitination assays showed that PARK2 overexpression enhanced the ubiquitination of NLRP3. Collectively, PARK2 negatively regulates NLRP3 protein via ubiquitination. In HDM-treated BEAS-2B cells, PARK2 overexpression repressed HDM-induced NLRP3 inflammasome activation, IL-1 beta and IL-18 secretion, pyroptosis, and epithelial barrier dysfunction. In BEAS-2B cells, PARK2 knockdown promoted NLRP3 inflammasome activation, IL-1 beta and IL-18 secretion, pyroptosis, and barrier impairment, while its effects were abrogated by NLRP3 inhibitor INF39. Conclusion: Our study demonstrates that PARK2 attenuates HDM-induced NLRP3 inflammasome activation, the release of inflammatory cytokines, pyroptosis, and barrier impairment in airway epithelial cells by ubiquitinating NLRP3.