Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes

Abstract

Despite the recent attention focused on the roles of the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in the pathogenesis of type 2 diabetes, little is known about the ex vivo profile of inflammasome activation in type 2 diabetic patients. In this study, we investigated patterns of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) from drug-naïve patients with newly diagnosed type 2 diabetes. Type 2 diabetic subjects had significantly increased mRNA and protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and proinflammatory cytokines in MDMs cultured with autologous sera compared with healthy controls. Upregulated interleukin (IL)-1β maturation, IL-18 secretion, and caspase-1 cleavage were observed in MDMs from type 2 diabetic patients after stimulation with various danger molecules (ATP, high-mobility group protein B1, free fatty acids, islet amyloid polypeptide, and monosodium uric acid crystals). Mitochondrial reactive oxygen species and NLRP3 were required for IL-1β synthesis in MDMs. Finally, 2 months of therapy with the antidiabetic drug metformin significantly inhibited the maturation of IL-1β in MDMs from patients with type 2 diabetes through AMP-activated protein kinase (AMPK) activation. Taken together, these data suggest that NLRP3 inflammasome activation is elevated in myeloid cells from type 2 diabetic patients and that antidiabetic treatment with metformin contributes to modulation of inflammasome activation in type 2 diabetes.

FIG. 1.

Expression profiles of NLRP3, ASC, and inflammatory cytokines in MDMs and sera from patients with type 2 diabetes (T2D) and healthy controls (HCs). A, C–E: MDMs were obtained from 47 patients with T2D and 57 HCs. The MDMs were incubated with media containing autologous sera and stimulated with ultrapure LPS (A, C, and E; 10 ng/mL) and Pam3CSK4 (Pam, E; 10 ng/mL) for 4 h. B: MDMs were cultured from T2D patients (n = 10) and HCs (n = 10). The protein levels of NLRP3 and ASC were measured by Western blot analysis. The intensity of each band for each protein was quantified and normalized relative to the housekeeping gene β-actin (B, right). D: Sera collected from the peripheral blood of T2D patients (n = 47) and HCs (n = 57). The mRNA expression of Il1β, Nlrp3, and Asc (A) and Il6, Il8, Tnfα, and Ccl2 (E) was analyzed by quantitative real-time RT-PCR. IL-1β and IL-18 production in culture supernatants (C) and sera (D) were measured by ELISA. Serum cytokine levels were determined in samples pretreated with protease inhibitors (4% volume/volume). A and E: Results are expressed as the mean concentration of triplicate samples. Data are representative of two independent experiments. B–D: Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. HCs. U, untreated.

FIG. 2.

Upregulated activation of casapse-1, IL-1β maturation, and production of IL-1β and IL-18 in MDMs from patients with type 2 diabetes (T2D) compared with healthy controls (HCs). MDMs were isolated from T2D patients (n = 47) and HCs (n = 57) were primed with LPS (10 ng/mL) for 4 h and stimulated with various ligands: ATP (1 mmol/L for 1 h), MSU (100 μg/mL for 6 h), FFAs (200 μmol/L for 16 h), and IAPP (10 μmol/L for 16 h). A: Western blotting analysis to determine caspase-1 (Casp1) and IL-1β protein levels (cell lysates [Cell], Casp1 p45, and pro-IL-1β [31 kDa]; supernatants [SN], cleaved Casp1 p10, and mature IL-1β [17 kDa]). The intensity of each band for each protein was quantified and normalized to the housekeeping gene β-actin (A, bottom). Data are expressed as means ± SEM. B: ELISA of IL-1β (top) and IL-18 (bottom) levels in culture supernatants. Cells were left untreated (U; left) or treated with the indicated ligands. Results are expressed as the mean of triplicate samples. Data are representative of two independent experiments. ***P < 0.001 vs. HCs. A, ATP; M, MSU; F, FFAs; I, IAPP.

FIG. 3.

Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes (T2D) is mediated by mitochondrial ROS. A: PBMCs isolated from T2D patients (n = 9) and healthy controls (HCs; n = 9) were primed with LPS (10 ng/mL) for 4 h and then stimulated with ATP (1 mmol/L) or HMGB1 (10 ng/mL) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 μmol/L). Then, the cells were stained with MitoSOX, gated for the CD14⁺ population, and analyzed by flow cytometry. Representative images (left) and quantitative analysis of mean fluorescence intensities (right) are shown (A, right). Data are expressed as means ± SEM. B: MDMs from T2D patients (n = 5) and HCs (n = 5) were primed with LPS (10 ng/mL, for 4 h), and then stimulated with ATP (1 mmol/L) or HMGB1 (10 ng/mL) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 μmol/L). C: MDMs from T2D patients (n = 5) and HCs (n = 5) were transduced with nonspecific control shRNA lentiviral particles (shNS) or lentiviral shRNA specific for NLRP3 (shNLRP3) or ASC (shASC), primed with LPS, and stimulated with ATP (1 mmol/L for 1 h) or MSU (100 μg/mL for 6 h). ELISA analysis of IL-1β (B and C) and IL-18 (B). Data are expressed as means ± SEM. C: Representative images of gels run to assess transduction efficiency by semiquantitative RT-PCR analysis (top). ***P < 0.001 vs. HCs. U, untreated; A, ATP; M, MSU; SC, solvent control.

FIG. 4.

Metformin treatment inhibits the secretion of mature IL-1β and caspase-1 activation in MDMs from patients with type 2 diabetes (T2D). MDMs were isolated from T2D patients (n = 11) before (Before) and after (After) treatment with metformin for 2 months. MDMs were primed with LPS (10 ng/mL) for 4 h, and then stimulated with various ligands: ATP (1 mmol/L for 1 h), MSU (100 μg/mL for 6 h), and FFAs (200 μmol/L for 16 h). A: Western blotting analysis to determine caspase-1 (Casp1) and IL-1β protein levels (cell lysates [Cell], Casp1 p45, and pro-IL-1β [31 kDa]; supernatants [SN], cleaved Casp1 p10, and mature IL-1β [17 kDa]) (A, right). The intensity of each band for each protein was quantified and normalized to the housekeeping gene β-actin. Data are expressed as means ± SEM. ***P < 0.001 vs. control cultures. ELISA analysis of IL-1β (B) and IL-18 (C) levels in supernatants from cultured MDMs. Results are expressed as the mean of triplicate samples. Data are representative of two independent experiments. U, untreated; A, ATP; M, MSU; F, FFAs.

FIG. 5.

The antidiabetic drug metformin inhibits IL-1β and IL-18 production induced by various inflammasome stimuli in LPS-primed MDMs. Primary MDMs from healthy controls (n = 5) were primed with LPS (10 ng/mL for 4 h) in the presence of a high glucose concentration (15 mmol/L). They were then treated with metformin (Met) at the indicated doses (A, B, and D; 200 and 500 μmol/L for 60 min) or for the indicated periods of time (C; 200 μmol/L for 30, 60, or 120 min), and stimulated with ATP (A–C; 1 mmol/L for 1 h), MSU (C and D; 100 μg/mL for 6 h), or FFAs (D; 200 μmol/L for 16 h). A: Quantitative real-time RT-PCR analysis of Il1β and IL8 mRNA levels. B and D: ELISA analysis of IL-1β and IL-18. C: Western blotting analysis of IL-1β protein levels in cell lysates (Cell, pro-IL-1β [31 kDa]) and supernatants (SN; mature IL-1β [17 kDa]). A, B, and D: Data are expressed as means ± SEM of five independent experiments. ***P < 0.001 vs. solvent control (SC). U, untreated.

FIG. 6.

AMPK pathway activation inhibits the induction of IL-1β and IL-18 production by various inflammasome stimuli in LPS-primed MDMs. Primary MDMs were isolated from healthy controls (A and B; n = 5) or type 2 diabetic patients (C; n = 11) before and after treatment with metformin for 2 months. A: MDMs were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and then treated with compound C (Comp C; 5, 10, or 25 μmol/L) and stimulated with ATP (1 mmol/L for 1 h). Data are expressed as means ± SEM of five independent experiments. B: MDMs were transduced with nonspecific control shRNA lentiviral particles (shNS) or lentiviral shRNA specific for AMPK (shAMPK). Then, the cells were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and treated with ATP or MSU (100 μg/mL for 6 h). Data are expressed as means ± SEM of three independent experiments. Representative images of semiquantitative RT-PCR gels run to assess transduction efficiency (top). C: The cells were primed with LPS (10 ng/mL) for 4 h in the presence of autologous sera and then treated with ATP or MSU. A and B: ELISA of IL-1β and IL-18 levels. C: Western blotting analysis of p-AMPKα protein levels. The intensity of each band for each protein was quantified and normalized to the housekeeping protein β-actin (C, right). Data are expressed as means ± SEM. ***P < 0.001 vs. control cultures. U, untreated; SC, solvent control; A, ATP; M, MSU; Before, before treatment with metformin; After, after treatment with metformin.