Novel Role for Tranilast in Regulating NLRP3 Ubiquitination, Vascular Inflammation, and Atherosclerosis

Abstract

Background Aberrant activation of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat-containing receptor family pyrin domain-containing 3) inflammasome is thought to play a causative role in atherosclerosis. NLRP3 is kept in an inactive ubiquitinated state to avoid unwanted NLRP3 inflammasome activation. This study aimed to test the hypothesis that pharmacologic manipulating of NLRP3 ubiquitination blunts the assembly and activation of the NLRP3 inflammasome and protects against vascular inflammation and atherosclerosis. Since genetic studies yielded mixed results about the role for this inflammasome in atherosclerosis in low-density lipoprotein receptor- or apolipoprotein E-deficient mice, this study attempted to clarify the discrepancy with the pharmacologic approach using both models. Methods and Results We provided the first evidence demonstrating that tranilast facilitates NLRP3 ubiquitination. We showed that tranilast restricted NLRP3 oligomerization and inhibited NLRP3 inflammasome assembly. Tranilast markedly suppressed NLRP3 inflammasome activation in low-density lipoprotein receptor- and apolipoprotein E-deficient macrophages. Through reconstitution of the NLRP3 inflammasome in human embryonic kidney 293T cells, we found that tranilast directly limited NLRP3 inflammasome activation. By adopting different regimens for tranilast treatment of low-density lipoprotein receptor- and apolipoprotein E-deficient mice, we demonstrated that tranilast blunted the initiation and progression of atherosclerosis. Mice receiving tranilast displayed a significant reduction in atherosclerotic lesion size, concomitant with a pronounced decline in macrophage content and expression of inflammatory molecules in the plaques compared with the control group. Moreover, tranilast treatment of mice substantially hindered the expression and activation of the NLRP3 inflammasome in the atherosclerotic lesions. Conclusions Tranilast potently enhances NLRP3 ubiquitination, blunts the assembly and activation of the NLRP3 inflammasome, and ameliorates vascular inflammation and atherosclerosis in both low-density lipoprotein receptor- and apolipoprotein E-deficient mice.

Keywords: NLRP3 ubiquitination; atherosclerosis; inflammasome; tranilast; vascular inflammation.

Figure 1. Tranilast (TR) enhances K63‐linked ubiquitination of NLRP3 (nucleotide‐binding oligomerization domain, leucine‐rich repeat–containing receptor family pyrin domain‐containing 3).

A and B, Human embryonic kidney 293T cells were transfected as indicated. Cell lysates were immunoprecipitated with anti‐Flag (F). The immunoprecipitates and lysates were immunoblotted with the indicated antibodies. C, J774A.1 cells were treated as indicated. Cell lysates were immunoprecipitated with anti‐NLRP3 and then immunoblotted with anti‐ubiquitin. Immunoblotting (IB) was conducted to examine the expression of indicated proteins. D through F, J774A.1 macrophages were treated as indicated. IB was conducted to examine the expression of proteins indicated. ASC indicates apoptosis‐associated speck‐like protein containing a caspase recruitment domain; DMSO, dimethyl sulfoxide; IgHC, heavy chain of immunoglobulin G; IP, immunoprecipitation; LPS, lipopolysaccharides; R, arginine; and Ub, ubiquitin.

Figure 2. Tranilast (TR) blunts NLRP3 (nucleotide‐binding oligomerization domain, leucine‐rich repeat–containing receptor family pyrin domain‐containing 3) inflammasome assembly.

A and B, J774A.1 cells were treated as indicated. Cell lysates were immunoprecipitated with anti–apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) (A) or anti–caspase‐1 (B). Immunoblotting was performed to assay for the expression of indicated proteins in the immunoprecipitates and whole cell lysates (WCLs). C, Human embryonic kidney (HEK) 293T cells were transfected as indicated. Cell lysates were immunoprecipitated with anti‐Flag. The immunoprecipitates and lysates were immunoblotted with the indicated antibodies. HA‐NEK7 indicates never in mitosis gene a‐related kinase 7; IP immunoprecipitation; and LPS, lipopolysaccharides.

Figure 3. Tranilast (TR) inhibits NLRP3 (nucleotide‐binding oligomerization domain, leucine‐rich repeat–containing receptor family pyrin domain‐containing 3) oligomerization and activation.

Low‐density lipoprotein receptor–deficient (Ldlr^−/−) bone marrow–derived macrophages (BMDMs) (A through C), apolipoprotein E–deficient (ApoE^−/−) BMDMs (D and E), wild‐type BMDMs (F and G), and J774A.1 macrophages (H through K) were treated as indicated. The supernatants (SNs), whole cell lysates (WCL) and the Triton‐insoluble fractions were analyzed by immunoblotting to determine the oligomerization of NLRP3 and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), caspase‐1 activation, and the release of interleukin (IL)‐1β and ASC. L, Human embryonic kidney 293T cells were transfected as indicated. At 8 hours before harvest, cells were treated without (–) or with TR (50 μmol/L). The SN and WCL were analyzed by immunoblotting as indicated. DSS indicates disuccinimidyl suberate; F, Flag; HA‐NEK7, never in mitosis gene a‐related kinase 7; LPS, lipopolysaccharides; SDD‐AGE, semi‐denaturing detergent agarose gel electrophoresis.

Figure 4. Tranilast (TR) inhibits the initiation of atherosclerosis in low‐density lipoprotein receptor–deficient (Ldlr^−/−) mice (group 1).

A, Shown is the regimen for treatment of Ldlr^−/− mice (6 to 8 weeks old) with TR (n=9) or vehicle (n=8). The atherosclerotic lesions in the whole aortas (B) and in the aortic sinuses (D) were visualized by Oil Red O staining. The lesional size of the aortas (C) and aortic sinuses (D) were measured. E, The collagen was visualized by Sirius Red staining and the collagen content in the plaque was measured. F, The fibrous caps were stained by Verhöeff elastic fiber dye, outlined with red dashed line and quantified; the necrotic core areas of the plaques were calculated. G through I, The expression of the indicated proteins in the aortic sinuses was detected by immunohistochemical staining. The positive area of each protein was measured. J, The profile of the plasma lipids and lipoproteins. DM indicates vehicle containing dimethyl sulfoxide; NS, no significant difference; TC, total cholesterol; TG, triacylglycerol; and α‐SMA, α‐smooth muscle actin. *P<0.05; **P<0.01.

Figure 5. Tranilast (TR) suppresses the progression of atherosclerosis in low‐density lipoprotein receptor–deficient (Ldlr^−/−) mice (group 2).

A, Diagramed is the regimen for treatment of Ldlr^−/− mice (6 to 8 weeks old) with TR (n=8) or vehicle (n=8). The atherosclerotic lesions (B through D), the collagen content in the plaque (E), the fibrous caps and the necrotic core (F), the expression of the indicated proteins (G through I), and the profile of the plasma lipids and lipoproteins (J) in mice were analyzed and quantified as described in Figure 3.

Figure 6. Tranilast (TR) represses the initiation of atherosclerosis in apolipoprotein E–deficient (ApoE^−/−) mice (group 3).

A, Illustrated is the regimen for treatment of ApoE^−/− mice (6 to 8 weeks old) with TR (n=8) or vehicle (n=9). The atherosclerotic lesions (B through D), the collagen content in the plaque (E), the fibrous caps and the necrotic core (F), the expression of the indicated proteins (G through I), and the profile of the plasma lipids and lipoproteins (J) in mice were evaluated and quantified as described in Figure 3. *P<0.05; **P<0.01; ***P<0.001.

Figure 7. Tranilast (TR) curbs the progression of atherosclerosis in apolipoprotein E–deficient (ApoE^−/−) mice (group 4).

A, Depicted is the regimen for treatment of ApoE^−/− mice (6 to 8 weeks old) with TR (n=12) or vehicle (n=10). The atherosclerotic lesions (B through D), the collagen content in the plaque (E), the fibrous caps and the necrotic core (F), the expression of the indicated proteins (G through I), as well as the profile of the plasma lipids and lipoproteins (J) in mice were examined and quantified as described in Figure 3.

Figure 8. Tranilast (TR) predominantly targeted the NLRP3 (nucleotide‐binding oligomerization domain, leucine‐rich repeat–containing receptor family pyrin domain‐containing 3) inflammasome in macrophages.

The frozen section of the aortic sinuses from mice treated with TR or vehicle containing dimethyl sulfoxide (DM) were analyzed by dual immunofluorescence staining. Double immunostaining was conducted with the antibodies against α–smooth muscle actin (α‐SMA) and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) (A), Mac3 and ASC (A), CD68 and interleukin (IL)‐1β (B).