Methods: Neutrophils isolated from peripheral blood of patients with severe asthma (SA, n=34) and non severe asthma (NSA, n=38) were left untreated or treated with IL-8 (100 ng/ml) for 16 hr. Autophagy level was evaluated by microtubule-associated light chain 3 (LC3) expression using Western blot and immunoflruorescent microscopy. NET production was measured by picogreen assay and immunoflruorescent microscopy. The effect of autophagy inhibitors (LY294002 and hydrochloroquine-HCQ) on neutrophil migration was assessed by neutrophil chemotaxis assay using ChemoTX® system.
Results: Both naïve and IL-8 treated neutrophils of the SA group produced significantly higher LC3 expressions than those of the NSA group (P<0.05 and P<0.01, respectively). IL-8 treated neutrophils generated significantly higher autophagy levels compared to naïve neutrophils in the SA group (P<0.001), which was not observed in the NSA group. NET production levels from naïve neutrophils were not different between the SA and NSA groups; however, NET levels produced by IL-8 treated neutrophils of the SA group were significantly higher than to those of the NSA group (P<0.01). A significantly positive correlation between autophagy and NET production was observed in both naïve and IL-8 treated neutrophils (P<0.001 for both). LY294002 (20 nM) inhibited LC3 expression while HCQ (10 μM) induced LC3 accumulation by blocking autophagosome degradation; both of which could significantly inhibit IL-8-induced migration of neutrophils (P<0.01 and P<0.05, respectively).
Conclusions: Autophagy and NET could contribute to the severity of asthma. Modulation of autophagy may help to improve asthma severity by reducing neutrophil migration into the asthmatic airways.