NLRP3 Inflammasome Expression and Activation in Human Atherosclerosis

Abstract

Background: The NLR family, pyrin domain containing 3 (NLRP3) inflammasome is an interleukin (IL)-1β and IL-18 cytokine processing complex that is activated in inflammatory conditions. The role of the NLRP3 inflammasome in the pathogenesis of atherosclerosis and myocardial infarction is not fully understood.

Methods and results: Atherosclerotic plaques were analyzed for transcripts of the NLRP3 inflammasome, and for IL-1β release. The Swedish First-ever myocardial Infarction study in Ac-county (FIA) cohort consisting of DNA from 555 myocardial infarction patients and 1016 healthy individuals was used to determine the frequency of 4 single nucleotide polymorphisms (SNPs) from the downstream regulatory region of NLRP3. Expression of NLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 (CASP1), IL1B, and IL18 mRNA was significantly increased in atherosclerotic plaques compared to normal arteries. The expression of NLRP3 mRNA was significantly higher in plaques of symptomatic patients when compared to asymptomatic ones. CD68-positive macrophages were observed in the same areas of atherosclerotic lesions as NLRP3 and ASC expression. Occasionally, expression of NLRP3 and ASC was also present in smooth muscle cells. Cholesterol crystals and ATP induced IL-1β release from lipopolysaccharide-primed human atherosclerotic lesion plaques. The minor alleles of the variants rs4266924, rs6672995, and rs10733113 were associated with NLRP3 mRNA levels in peripheral blood mononuclear cells but not with the risk of myocardial infarction.

Conclusions: Our results indicate a possible role of the NLRP3 inflammasome and its genetic variants in the pathogenesis of atherosclerosis.

Keywords: NLRP3; inflammasome; interleukin‐1β; myocardial infarction; polymorphism.

Figure 1

Expression of (A) NLR family, pyrin domain containing 3 (NLRP3) (P<0.00001); (B) Apoptosis‐associated speck‐like protein containing a CARD (ASC) (P<0.00000001); (C) Caspase‐1 (P<0.000001); (D) Interleukin (IL)‐1β (P<0.0000000000001), and (E) IL‐18 (P<0.00000000001) mRNA in human atherosclerotic carotid lesions compared to transplant donor vessels from the Biobank of Karolinska Endarterectomies (BiKE) cohort. F, NLRP3 mRNA expression in asymptomatic plaques compared to symptomatic plaques from the BiKE cohort (P<0.035).

Figure 2

Immunohistochemical staining of NLR family, pyrin domain containing 3 (NLRP3), Apoptosis‐associated speck‐like protein containing a CARD (ASC), CD68 and smooth muscle actin (SMA) in carotid plaque. Lower panels show the magnified image. Left panels show the immunostaining of NLRP3/ASC (red) and CD68 (green). Right panels show immunostaining of NLRP3/ASC (red) and SMA (green). Arrows indicate co‐localization of NLRP3 or ASC in CD68‐ (left) or SMA‐positive cells (right). The scale bars for ×1.25 and ×40 objectives are 1 mm and 100 μm, respectively.

Figure 3

A, IL‐1β release from human carotid plaque (n=9) subjected to lipopolysaccharide (LPS) (113.8±138.6 pg/mL), ATP (78±129.0 pg/mL), or LPS+ATP (413.8±412.7 pg/mL) treatment and in control (9±12.7 pg/mL). B, IL‐1β release from human carotid plaque (n=7) subjected to LPS (145.8±112.7 pg/mL), cholesterol crystal (38.3±8.3 pg/mL), or LPS+ cholesterol crystal (351.1±206.4 pg/mL) treatment and in control (15.2 pg/mL). ^# P<0.05 vs LPS. **P<0.005 vs unstim. ***P<0.001 vs unstim.

Figure 4

Association between genotype of variants and expression level of the NLR family, pyrin domain containing 3 (NLRP3) gene in plaque and peripheral blood mononuclear cells (PBMCs). The Y axis represents −log10 (P), calculated using additive model for the association between genotype and expression level. The X axis represents different genotypes of the variants. The minimum and maximum values are represented by whiskers, the first and third quartiles (box), the median values (medlines), outliers are displayed as circles. The Y axis represents the mRNA expression level of NLRP3.