Background

Activation of the NLRP3 inflammasome contributes to the pathogenesis of inflammatory lung diseases, modulating the release of inflammatory and remodeling factors. The impact of cigarette smoke (CS) on the activation of NLRP3 inflammasome and related caspases in lung fibroblasts is unknown. Recently, it has been discovered that activation of caspase-1 and -8 downstream targets gasdermins (both D and E) promote fibrosis in several preclinical models.

Aim

To investigate the impact of CS extract (CSE) on inflammasome-dependent responses in human lung fibroblasts MRC5.

Methods

MRC5 were treated with 5% and 10% CSE in the presence or not of the antioxidant N-acetyl cysteine (N-AC) and of inhibitors of the NLRP3 inflammasome (MCC950) and caspase-8 (z-IETDfmk). RNA was collected for qRT-PCR. Supernatants were collected to evaluate caspase activity and cell lysis (LDH assay). Mitochondrial Reactive Oxygen species (mROS) were evaluated using Mitosox Red. Cell lysates were collected for Western Blot (WB).  

Results

CSE activated caspase-1 -8 and -3/7 and increased mROS in a dose-dependent manner at 24h and 72h. Cell lysis and GSDME cleavage were observed at 72h. N-AC treatment inhibited CSE-induced mROS generation and caspase activation. Z-IETD inhibited CSE-induced Caspase-3/7 activation and consequent GSDME cleavage. NLRP3 was not expressed (qRT-PCR and WB). Caspase-1, GSDMD, GSDME, and the inflammasome adapter ASC were constitutively expressed at gene/protein level.

Conclusion

This study unveiled that CSE activates the inflammatory axis caspase-8/caspase-3/7/GSDME in MRC5 cells. Whether this has a role in the pathogenesis of CS-associated lung diseases needs further investigation.