NLRP3-mediated epithelial pyroptosis involved in airway hyperresponsiveness in combined eosinophilic and neutrophilic...

BACKGROUND: Airway hyperresponsiveness (AHR) contributes to asthma pathogenesis, yet targeted therapies remain limited, especially for combined eosinophilic-neutrophilic (EOS+NEU+) inflammation. OBJECTIVE: To investigate the role of the NOD-like receptor protein 3 (NLRP3) signalling in EOS+NEU+ asthma-associated AHR. METHODS: We prospectively enrolled 48 patients with asthma and paired with 8 healthy controls. Patients with asthma were classified as non-eosinophilic (non-EOS), eosinophilic without neutrophilic (EOS+NEU−), or EOS+NEU+ asthma mainly based on induced sputum type 2 inflammation markers. Large and small airway function, inflammatory markers, and NLRP3 expression were analysed. In vivo and in vitro models mimicking each phenotype were used to investigate the effects of inhibiting NLRP3 (MCC950 or knockout), Gasdermin D (GSDMD) (disulfiram or gene silencing), and interleukin (IL)-1β signalling (IL-1R knockout) on AHR. RESULTS: Compared to patients with EOS+NEU− inflammation, those with EOS+NEU+ inflammation had higher sputum eosinophils and neutrophils. Large and small airway function declined across all asthma subgroups versus controls, worsening from non-EOS to EOS+NEU− and further in EOS+NEU+. Sputum NLRP3 and IL-1β increased progressively across asthma groups and correlated positively with FeNO and sputum eosinophils, while NLRP3 positively correated with neutrophils but negatively with lung function.Meanwhile, EOS+NEU+ mice showed higher NLRP3 and IL-1β and worse small airway function than those in EOS+NEU− mice. Mimicking EOS+NEU+ inflammation in vitro, IL-13 combined with IL-17 A stimulation induced NLRP3 activation, GSDMD-N-terminal (NT) upregulation, pore formation in human bronchial epithelial cells, with IL-1β/lactate dehydrogenase (LDH) release—indicative of epithelial cell pyroptosis, more obvious than single IL-13 or IL-17 A stimulation. In vivo, NLRP3 knockout suppressed GSDMD-NT and IL-1β, alleviating AHR. In vivo and in vitro, GSDMD inhibition/silencing reduced IL-1β and LDH release, mitigating AHR, while IL-1R knockout reduced airway smooth muscle tension, also improving AHR. CONCLUSIONS: EOS+NEU+ asthma showed more severe AHR compaired with non-EOS and EOS+NEU− asthma. Enhanced NLRP3/GSDMD/IL-1β signalling in EOS+NEU+ asthma, drives more severe AHR via epithelial pyroptosis and granulocytic inflammation. Targeting NLRP3/GSDMD/IL-1β offers therapeutic potential, with sputum NLRP3 and IL-1β as promising biomarkers for risk factor of progression. CLINICAL TRIAL REGISTRATION: ChiCTR2500104677.