The MFGE8/integrin β3 axis mitigates experimental neutrophilic asthma by suppressing NLRP3-Caspase-1 pathway-mediated NETosis

Neutrophilic asthma, characterized by the relative accumulation of neutrophils in the airways, constitutes a distinct endotype of asthma resistant to corticosteroid and associated with severe and uncontrolled cases. Milk fat globule-EGF factor 8 (MFGE8) is a soluble glycoprotein functioning in phagocytosis, tissue repair, angiogenesis, and the regulation of neutrophil activity. However, the role of this glycoprotein in neutrophilic asthma has not been thoroughly investigated. MFGE8 concentrations were assessed in asthmatic patients with various endotypes. Utilizing an ovalbumin (OVA)/complete Freund's adjuvant (CFA)-induced mouse model of neutrophilic asthma, we investigated the critical roles of MFGE8 in neutrophilic asthma in MFGE8-knockout (Mfge8-/-) mice by assessment of H&E/PAS staining, bronchoalveolar lavage (BAL) cell counting, and lung function tests. Bioinformatic analyses were conducted to determine downstream functions, and mechanistic experiments were performed on primary cultures of neutrophils isolated from the mouse's lungs. Recombinant MFGE8 was administered to the mice to evaluate its therapeutic effects. Our results demonstrated that MFGE8 is expressed in neutrophils, and its protein levels are significantly reduced in the sputum supernatant of patients suffering from neutrophilic and paucigranulocytic asthma. Mfge8-/- mice exacerbated airway neutrophil infiltration, mucus secretion, and hyperresponsiveness. Further mechanistic studies, involving gene sequencing of sputum cells and lung tissue biopsies of asthmatic patients from GEO databases, identified neutrophil extracellular traps (NETs) as the critical factor in the progression of neutrophilic asthma. MFGE8 was shown to inhibit the formation of NETs (NETosis) through interaction with integrin β3. The absence of MFGE8 or the application of integrin β3 inhibitor was found to enhance NETosis and promote the release of pro-inflammatory cytokines via NLRP3-Caspase-1 pathway. Upstream mechanism indicated that USP14 mediated the ubiquitination of MFGE8 and participated in the development of NETosis. Moreover, recombinant MFGE8 was observed to effectively mitigate neutrophilic airway inflammation. This study underscored the significance of the MFGE8/integrin β3 axis in ameliorating NETosis and neutrophilic airway inflammation, suggesting MFGE8 as a potential therapeutic target for neutrophilic asthma. Not applicable.