Background Cigarette smoking is proved to be an independent risk factor for the development and progression of rheumatoid arthritis (RA). Neutrophil extracellular traps (NETs) are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.
Objectives In the current study, we analyzed the formation and magnitude of NETs from circulationg and infiltrating neutrophils, which triggered by smoking in RA patient.
Methods Circulating neutrophils were separated from peripheral blood of healthy volunteers (n=7) and RA patients (n=15). Infiltrating neutrophils were separated from knee synovial tissue, which were obtained with auto biopsy gun under B ultrasound-guided from smoking and non-smoking RA patients. These neutrophils were incubated with 0, 5, 10% cigarette smoke extract (CSE). The formation of Nets were observed using immunofluorescence (IF), labbed by anti-MPO/anti-H3, then autophagy were inspected by anti-LC3/anti-beclin-1. The magnitude of Nets was detected by fluorescent quantitative assay with PICO green.
Results Smoking RA patients showed a significant augment of NETs in both neutrophils circulating in peripheral blood and infiltrating in synovial tissues, which is dramatically higher than no-smoking patients and healthy volunteers. Meanwhile, CSE treated neutrophils highly increased NETs in non-smoking RA, but showed much slightly effect in healthy volunteers. Furthermore, synovial tissues demonstrated the colocalization of neutrophils with NETs and autophagy. Additionally, autophagy was idencitified as the orchestrator of NETs formation, as shown by inhibition studies using wortmannin or bafilomycin A1.
Conclusions Our data show that cigarette smoke probably triggered NETs of both circulating and infiltrating neutrophils by autophagy dependent way in RA patients.
Disclosure of Interest None declared
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