Ibrutinib Regulates Ox-LDL-induced Macrophage Foam Cell Formation And Pyroptosis Via Inhibition Of NLRP3 Inflammasome

IntroductionMonocytes are recruited by chemokines released by endothelial cells in the early stages of atherosclerosis. Once monocytes adhere to the endothelial cells they penetrate into subendothelial space, where they differentiate into macrophages and ingest ox-LDL(oxidized low-density lipoprotein) which results in foam cell formation. Foam cells undergo death, and these dead cells contribute to the formation of necrotic core and weaken fibrous cap which disrupt the stability of atherosclerotic plaque. In general, macrophage foam cell formation and death play key roles in the development and progression of atherosclerosis.Inflammation is closely linked to atherosclerosis. Many studies which have been conducted recently demonstrated the important roles of NLRP3(nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome not only in atherogenesis but also in atherosclerotic plaque destabilization. Exact mechanism is still unclear but it has been postulated that it could be related with the effect of caspase-1(cysteine- requiring Aspartate Protease-1) or IL-1β(Interleukin-1β). Ox-LDL is able to induce NLRP3 inflammasome activation and this activated inflammasome can cause macrophage pyroptosis and may also be involved in macrophage lipid deposition.Ibrutinib is a covalent inhibitor of BTK(Bruton’s tyrosine kinase). A recent research has shown that ibrutinib was able to inhibit the activation of NLRP3 inflammasome by alum. It was still unknown if this inhibiting property of ibrutinib towards NLRP3 inflammasome was applicable to the other stimuli. Thus why we hypothesized that ibrutinib can regulate ox-LDL-induced macrophage foam cell formation and pyroptosis via NLRP3 inflammasome. ObjectiveTo investigate the effects of ibrutinib on ox-LDL-induced macrophage foam cell formation and pyroptosis, and to explore the roles that ibrutinib plays in them. MethodsPMA was used to induce THP-1 or THP-1- defNLRP3 monocytes to differentiate into macrophages. Before ox-LDL was added macrophages were pre-treated with ibrutinib, MCC950(specific inhibitor of NLRP3 inflammasome) or VX765(specific inhibitor of caspase-1). The effects of those treatments on NLRP3 were analyzed by using ELISA and Western Blot. The effect of ibrutinib on macrophage foam cell formation was evaluated by using oil red O staining, flow cytometry, immunofluorescence, and Western Blot. Macrophage pyroptosis was evaluated by using LDH release assay, Hoechst 33342/PI double staining and TUNEL staining. Migration and adhesion abilities of THP-1 cells were assayed by transwell chamber, wound healing assay and adhesion assay. Results(1) Ibrutinib inhibited ox-LDL-induced NLRP3 Inflammasome activation.(2) Ibrutinib, MCC950 and THP-1-defNLRP3 reduced macrophages ox-LDL uptake and increased macrophages cholesterol efflux via regulation of CD36, ABCA1 and SR-BI expression, thus inhibited foam cell formation.(3) Ibrutinib, MCC950, vx765 and THP-1-defNLRP3 suppressed LDH release, PI count and DNA strands break(TUNEL) induced by ox-LDL.(4) Ibrutinib and MCC950 inhibited THP-1 cell migration, but had no effect on adhesion. ConclusionIbrutinib may attenuate ox-LDL-induced macrophage foam cell formation and pyroptosis via inhibition of NLRP3 Inflammasome.