Annatto Tocotrienol Attenuates NLRP3 Inflammasome Activation in Macrophages

Abstract

Accumulating evidence suggests that aberrant innate immunity is closely linked to metabolic diseases, including type 2 diabetes. In particular, activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome and subsequent secretion of interleukin 1β (IL-1β) are critical determinants that precipitate disease progression. The seeds of annatto (Bixa orellana L.) contain tocotrienols (T3s), mostly (>90%) in the δ form (δT3). The aim of this study was to determine whether annatto T3 is effective in attenuating NLRP3 inflammasome activation in macrophages. Our results showed that annatto δT3 significantly attenuated NLRP3 inflammasome by decreasing IL-1β reporter activity, IL-1β secretion, and caspase-1 cleavage against lipopolysaccharide (LPS) followed by nigericin stimulation. With regard to mechanism, annatto δT3 1) reduced LPS-mediated priming of the inflammasome and 2) dampened reactive oxygen species production, the second signal required for assembly of the NLRP3 inflammasome in macrophages. Our work suggests that annatto δT3 may hold therapeutic potential for delaying the onset of NLRP3 inflammasome-associated chronic metabolic diseases.

Keywords: IL-1β; NLRP3 inflammasome; ROS production; annatto; delta-tocotrienol.

FIGURE 1

δT3 inhibited NLRP3 inflammasome activation. iJ774 macrophages were pretreated with δT3 (0, 1, 2.5, and 5 μM), and then stimulated with LPS/Ng. (A) Structure of the iGLuc (NLRP3 inflammasome and caspase activity reporter construct). (B) Relative Gaussia luciferase activity measured by luminometer. (C) IL-1β secretion in medium quantified by ELISA. (D) Released iGLuc fusion protein after caspase-1 activation. (E) RAW 264.7 macrophages were pretreated (1 or 2.5 μM) and stimulated with LPS (100 ng/mL) for 1 h. mRNA expression of Nlrp3, Il1b, and Tnfa were quantified by qPCR. Values in panels B, C, and E are means ± SEMs; n = 6–7. Means not sharing a common letter differ, P < 0.05 (1-factor ANOVA). Results shown in panel D are representative of triplicate samples. HPRT, hypoxanthine-guanine phosphoribosyltransferase; iGLuc, IL-1β–Gaussia luciferase fusion construct; iJ774, J774 macrophage stably expressing iGLuc reporter construct; LPS/Ng, LPS followed by nigericin; NLRP3, NOD-like receptor family pyrin domain–containing 3; RLU, relative luminescence unit; Tnfa, tumor necrosis factor α δT3, δ-tocotrienol.

FIGURE 2

δT3 inhibits NLRP3 inflammasome priming and ROS production in macrophages. RAW (A–D) or iJ774 (E–G) macrophages were pretreated with 1 μM of either δT3 or γT3, then stimulated with LPS alone (A, B) or LPS/Ng (C–G). (A) Proinflammatory gene expression of Nlrp3, Il1b, Tnfa, and Il18 by qPCR analysis (n = 6). (B) Western blot analysis of MAPKs of p-p38, p-ERK, p-JNK, IκBα degradation, and NLRP3. Relative intensity was quantified by Image J software (NIH; n = 3). (C) ROS production by MitoSOX Red (left panels) and quantification of relative fluorescence intensity (right panel; n = 5). (D) Cellular ROS production detected by DCFDA fluorescence (n = 8). (E) Relative Gaussia luciferase activity (n = 6). (F) IL-1β secretion in medium quantified by ELISA (n = 6). (G) Released iGLuc fusion protein and cleaved caspase-1 by Western blot analysis. Results are shown as means ± SEMs. Means not sharing a common letter differ, P < 0.05 (1-factor ANOVA). Panels C and D: ***P < 0.001 compared with control (Student's t test). Cont, control; DCFDA, 2,7-dichlorofluorescin diacetate; HPRT, hypoxanthine-guanine phosphoribosyltransferase; iGLuc, IL-1β–Gaussia luciferase fusion construct; iJ774, J774 macrophage stably expressing iGLuc reporter construct; IκBα, inhibitor of κB; LPS/Ng, LPS followed by nigericin; NLRP3, NOD-like receptor family pyrin domain–containing 3; p-ERK, phosphorylated ERK MAPKinase; p-JNK, phosphorylated JNK MAPKinase; p-p38, phosphorylated p38 MAPKinase; RLU, relative luminescence unit; ROS, reactive oxygen species; t-ERK, total levels of ERK MAPKinse; Tnfa, tumor necrosis factor α Trt, treatment; T3, tocotrienol; δT3, δ-tocotrienol; γT3, γ-tocotrienol.