Abstract
The NLRP3 inflammasome, a multiprotein cytosolic complex that activates the IL-1 family of cytokines, plays an important role in atherosclerosis (AS). High-sensitivity c-reactive protein (hs-CRP) is widely recognized as a major cardiovascular risk predictor and recent studies name NLRP3 as a predictor of CRP levels. Mounting evidence has indicated that subendothelial retention of apolipoprotein B100-containing lipoproteins, such as low-density lipoprotein (LDL), is the initial step of atherogenesis, and is usually termed the “response to retention hypothesis.” We previously reported that CRP promotes AS by directly increasing LDL transcytosis across endothelial cells (ECs). The present study aims to investigate the effects of CRP on NLRP3 inflammasome activation and the role of the NLRP3 inflammasome in CRP-induced LDL transcytosis. We found that CRP upregulated NF-κB activity, the NF-κB inhibitor (BAY-11-7082) and Fcγ receptors (FcγRs) inhibitor (CD32/64Ab) blocked CRP-induced NF-κB activation. CRP also induced expression of pro-IL-1β and NLRP3, while BAY and CD32/64 Ab suppressed CRP-mediated expression of NLRP3 and pro-IL-1β. Moreover, CRP activated the NLRP3 inflammasome in ECs. NADPH oxidase inhibitor, diphenylene iodonium (DPI) and dithiothreitol (DTT), a broad-spectrum P2 receptor inhibitor, oxidized ATP (oATP), and a broad inhibitor of cysteine proteases, E-64d, inhibited CRP-induced NLRP3 inflammasome activation. Furthermore, NLRP3 siRNA and caspase-1 inhibitor blocked CRP-mediated LDL transcytosis across ECs. In conclusion, NLRP3 inflammasome activation was shown to be involved in CRP-mediated LDL transcytosis across ECs. CRP not only increased the expression of pro-IL-1β and NLRP3 via the FcγRs/NF-κB pathway, but also promoted NLRP3 inflammasome activation and IL-1β maturation by upregulation of reactive oxygen species (ROS) levels, purinergic receptor signaling, and activation of cysteine proteases.
Highlights
Recent studies have suggested low-grade systemic inflammation is closely related with metabolic syndrome
We first studied the effect of C-reactive protein (CRP) on NLRP3 inflammasome activation in endothelial cells (ECs)
HUVECs were transfected with NLRP3 siRNA to knock down NLRP3 expression, which blunted the effects of CRP-induced NLRP3, pro-caspase-1/caspase-1, and proIL-1β/IL-1β expression (Figures 1A–E)
Summary
Recent studies have suggested low-grade systemic inflammation is closely related with metabolic syndrome. Low-grade chronic inflammation is characterized by a 2-3-fold increase in the systemic concentrations of cytokines including TNF-α, IL-6, and C-reactive protein (CRP). Excessive levels of proinflammatory cytokines may have facilitate the shift of the endothelial cell (EC) phenotype from a quiescent to an activated phenotype. Controversies regarding CRP as a causal agent for atherothrombosis remain, no study has denied that CRP serves as a biomarker for cardiovascular events. Phospholipase A2-dependent dissociation of circulating pentameric CRP (pCRP) to monomeric CRP (mCRP) localized and aggravated inflammation in AS and myocardial infarction (Braig et al, 2017). 1,6-bis(phosphocholine)-hexane inhibited the proinflammatory activity of mCRP by stabilizing pCRP (Braig et al, 2017). We found that CRP accelerates AS by directly increasing LDL transcytosis across ECs (Bian et al, 2014)
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