Molecular insights of cigarette smoke condensate-activated NLRP3 inflammasome in THP-1 cells in a stage-specific atherogenesis

Abstract

Background: Smoking induces excessive inflammation which is associated with all the stages of atherosclerosis. Earlier, we reported Nod-like receptor protein 3 (NLRP3) inflammasome activation as a pro-atherosclerotic property of cigarette smoking. In the present study, we aimed to explore the underlying detailed upstream mechanism and the cellular status of putative downstream molecules of cigarette smoke condensate (CSC)-activated NLRP3 inflammasome in atherosclerotic disease.

Methods and results: THP-1 monocytes, macrophages and foam cells represent crucial stages of atherogenesis as initiation, progression and development. To determine the upstream molecular regulators of smoking-induced NLRP3 inflammasome in atherogenesis, Myeloid differentiation primary response 88/Nuclear Factor kappa-light-chain-enhancer of activated B cells (MyD88/NF-κB) and Suppressor of cytokine signaling 3/Signal transduction and activator of transcription 3 (SOCS3/STAT3) pathways were elucidated. Stage-specific THP-1 cells were treated with MyD88 and SOCS3/STAT3 inhibitors. The results showed that MyD88 inhibition markedly attenuated the expression of NLRP3 markers (NLRP3, caspase-1, Interleukin (IL)-1β and IL-18), IL-6, SOCS3 and NF-κB. Moreover, the secretory levels of pro-cytokines were also significantly reduced in culture media. In contrast, no changes were observed with SOCS3/STAT3 inhibitor. Further, ac-vyad-cmk, an inflammasome inhibitor was used to explore the downstream targets of CSC-activated NLRP3 inflammasome in atherosclerotic process. The transcriptional profiling of 25 atherosclerotic markers was carried out using ExProfile™ Custom Gene qPCR Arrays. CSC exposure upregulated the expression of 17 genes and downregulated 4 genes in a stage-specific manner. Inhibitory experiments showed aberrant changes in CSC-regulated genes. Altogether, 15 molecules were common in all three stages.

Conclusion: The findings may suggest that MyD88/NF-κB pathway is an upstream regulator of NLRP3 inflammasome underlying smoking-induced atherosclerosis. Notably, 15 atherosclerotic molecules associated with endothelial dysfunction, scavenger receptors, cholesterol esterification and matrix-metalloproteins were found downstream to CSC-activated NLRP3 inflammasome.

Keywords: Cigarette smoke condensate; Downstream targets; MyD88; NLRP3 inflammasome; SOCS3; THP-1 cells.