Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1β in type 2 diabetes

Abstract

Interleukin 1β (IL-1β) is an important inflammatory mediator of type 2 diabetes. Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, triggered the NLRP3 inflammasome and generated mature IL-1β. One therapy for type 2 diabetes, glyburide, suppressed IAPP-mediated IL-1β production in vitro. Processing of IL-1β initiated by IAPP first required priming, a process that involved glucose metabolism and was facilitated by minimally oxidized low-density lipoprotein. Finally, mice transgenic for human IAPP had more IL-1β in pancreatic islets, which localized together with amyloid and macrophages. Our findings identify previously unknown mechanisms in the pathogenesis of type 2 diabetes and treatment of pathology caused by IAPP.

Figure 1. Inflammasome activation and IL-1β production induced by human IAPP

(a) IAPP from different species or polystyrene beads (adjuvant) were incubated overnight with BMDC with and without 3 hours priming using LPS. Supernatants were then analyzed for IL-1β, IL-1α, IL-6 and TNF by ELISA. (b) Immunoblotting for IL-1β was performed using supernatants from BMDC primed with LPS, then activated with IAPP, MSU or Alum for 6 hours. (c) IL-1β secretion from BMDM and BMDC compared to purified mouse pancreatic islets or Rin-5F cells (a pancreatic beta cell line), stimulated with LPS and IAPP. Rin-5F cells were also cultured for different times with elevated glucose (25 mM D-glucose). (d) Flow cytometric analysis of BMDC stimulated with IAPP for 1 hour then treated with FAM-YVAD-fmk which covalently binds to active caspase-1 and fluoresces. Control is shaded under the curve, IAPP stimulated is unshaded. (e) Immortalized BMDM expressing YFP-ASC were primed with LPS, then activated with IAPP overnight and imaged with a fluorescent microscope (40x objective). Arrows indicates speck formation. Means ± SD, * p<0.01, ** p<0.001. All data representative of three independent experiments.

Figure 2. IAPP oligomers activate the Nlrp3 inflammasome which is prevented by glyburide, and inhibitors targeting phagocytosis, ROS and Cathepsin B

(a) WT or Nlrp3 deficient (Nlrp3^−/−) BMDM were primed with LPS and then activated with uric acid crystals (MSU), Alum or IAPP overnight after which IL-1β, TNF and IL-6 production was measured. (b) Thioflavin T fluorescence was used to indicate IAPP fibrillar content after reconstitution in H₂0 or PBS for different times at room temperature. These different preparations of IAPP were then used to activate BMDC as in (a), and IL-1β was measured. (c) IAPP reconstituted in H₂0 for different times was separated based on size into fractions >100 kDa and <100 kDa, then used to activate BMDC as in (a), and IL-1β was measured. (d) BMDC were treated as in (a) for IAPP, with the addition of various inhibitors 1 hour before inflammasome activation; 1 µg/ml caspase-1 inhibitor, 5 µM cytochalasin D, 250 µM bafilomycin A or 40 µM IAPP receptor inhibitor peptide (AC187). Starting at the highest concentration indicated, two 10 fold dilutions of the inhibitor were also tested, or in the case of AC187, two 4 fold dilutions. (e) BMDC were treated as in (a) for IAPP, with the addition of various inhibitors 1 hour before inflammasome activation; 1 µg/ml ROS inhibitor (APDC), 10 µM cathepsin B inhibitor (CA-074 Me) and 50 µM glyburide. Starting at the highest concentration indicated, two 10 fold dilutions of the inhibitor were also tested, or in the case of glyburide, two 4 fold dilutions. Means ± SD, * p<0.05, ** p<0.01, *** p<0.001. All data representative of three independent experiments.

Figure 3. Priming the inflammasome requires glucose metabolism

(a) BMDC were treated with 25 mM D- or L-glucose for different periods of time prior to LPS priming and inflammasome activation with IAPP, after which IL-1β production was measured. (b) BMDC were treated with increased amounts of 2-deoxy-D-glucose (2DG) either 3 hours before or 3 hours after priming with LPS. Cells were then activated with IAPP and the cytokines IL-1β, IL-6 and TNF were measured. (c) IL-1β mRNA levels were determined after 3 hours LPS stimulation of BMDC in the presence of increased amounts of D-glucose and its analogues for a total of 6 hours. Means ± SD, * p<0.001. All data representative of three independent experiments.

Figure 4. Minimally oxidized LDL can prime for inflammasome activation by IAPP

(a) IAPP activation of the inflammasome after 3 hrs priming with LPS, human plasma LDL, or LDL oxidized to different extents. LDL oxidation is quantified by relative electrophoretic mobility (REM). BMDM from C3H/HeN mice were compared to those from C3H/HeJ mice that do not have functional TLR4. (b) Dose response for minimally oxidized LDL (mmLDL) priming of IAPP inflammasome activation and IL-1β production. IL-6 and TNF are also produced in a dose-dependent fashion. (c) IL-1β and Nlrp3 mRNA levels were measured after priming with mmLDL for different times. Means ± SD, * p<0.05, ** p<0.01, *** p<0.001. All data representative of three independent experiments.

Figure 5. Increased IL-1β expression in islets of mice transgenic for human IAPP

(a) Immunofluorescent analysis (20x objective) of sections from the pancreas of wild-type (WT) and IAPP transgenic (IAPP-TG) mice, both fed a high fat diet for one year. Sections were stained for insulin (red) and IL-1β (green). (b) Pancreatic sections from IAPP-TG mice stained for amyloid (red) and IL-1β (green). (c) Pancreatic sections from WT and IAPP-TG mice stained for macrophages (red) and IL-1β (green). All images representative of three individual mice. (d) The area within the islet expressing insulin was quantified for multiple islets of three individual mice. (e) The area of amyloid deposition in the islet, measured as for insulin. (f) The islet area expressing IL-1β, measured as for insulin. (g) The islet area stained for macrophages, measured as for insulin. Means ± SD, * p=0.0427, ** p=0.0261, n=3.