NLRP3 inflammasome is expressed and regulated in human islets

Abstract

NRLP3 inflammasome is a protein complex involved in the maturation of IL1β. In the onset of type 1 diabetes as well as in islet transplantation, IL-1β is one of the cytokines involved in the recruitment of immune cells in islets and eventually in islet destruction. Whether IL-1β is produced by islet cells is still under debate and NLRP3 inflammasome-dependent IL-1β production has not been yet determined in human islets. The aim of this study was to determine the expression and the regulation of the NRLP3 inflammasome in human islets. Human islets were stimulated with LPS and successively with ATP (LPS + ATP) in the presence or absence of the inflammasome inhibitor glyburide. Islets were also incubated in hypoxic or normoxic conditions for 24 h in the presence or absence of glyburide. Then, IL1B and NLRP3 expression was studied by real time PCR, protein expression by western blot, protein localization by immunofluorescence and protein secretion by ELISA. LPS + ATP increased gene expression of NRLP3 and IL1B. Glyburide partially prevented this effect. IL-1β protein was localized in β and non-β cells. Moreover, LPS + ATP increased IL-1β protein expression and production, which were prevented by glyburide. Hypoxia increased gene expression of NRLP3 and IL1B and induced IL-1β and caspase-1 production. Finally, hypoxia-induced cell death which was not prevented by inhibition of NLRP3 inflammasome. NRLP3 inflammasome is expressed and plays a role in IL-1β production by human islets. By contrast, NRLP3 inflammasome activation is not involved in islet cell death induced by hypoxia.

Fig. 1. NLRP3 and IL-1β gene expression in human islets in response to inflammasome activation.

Human islets were stimulated or not (control, CTR) with 1 μg/ml LPS for 4 h and successively with 5 mM ATP for 30 min (LPS + ATP) in the presence or absence of 200 μM glyburide (GLY). NLRP3 (a) and IL1B (b) in human islets were quantified by qRT-PCR and expressed relative to CTR. Data are means ± SEM from four experiments (a) and three experiments (b). *p<0.05, **p<0.01, ****p<0.0001

Fig. 2. Content and release of IL-1β in human islets in response to inflammasome activation.

Human islets were stimulated or not (control, CTR) with 1 μg/ml LPS for 4 h and successively with 5 mM ATP for 30 min (LPS + ATP) in the presence or absence of 200 μM glyburide (GLY). Protein levels of pro- and mature IL-1β forms were analyzed by Western blot (a) and density of IL-1β bands quantified (b). IL-1β cell localization was analyzed by immunofluorescence (c). The red staining (corresponding to IL-1β) was quantified by morphometry and expressed relative to CTR; a total of 79, 88, and 63 cells from three experiments were analyzed for CTR, LPS + ATP and LPS + ATP + GLY, respectively. IL-1β released in the supernatant was quantified by ELISA (e). Data are means ± SEM from three (b and d) and five experiments (e). *p<0.05, **p<0.01, ****p<0.0001

Fig. 3. Human islets respond to hypoxia.

Human islets were incubated either in control condition (Normoxia) or in 1% O₂ hypoxia chamber (Hypoxia) for 24 h, and VEGF expression (a), VEGF released (b) and HIF cell content (c) analyzed. a VEGF was evaluated by qRT-PCR. b Secreted VEGF was quantified by ELISA. c Protein level of HIF was performed by Western blot. Data are means ± SEM from three (a) and six experiments (b). **p<0.01

Fig. 4. Activation of NLRP3 inflammasome by hypoxia in human islets.

Human islets were incubated in control condition (Normoxia) or in 1% O₂ hypoxia chamber (Hypoxia) for 24 h. NLRP3 (a) and IL1B (b) were quantified by qRT-PCR and expressed relative to normoxia. Caspase-1 (c) and IL-1β (d) released were quantified by ELISA. Data are means ± SEM from 6 (a), 9 (b), 4 (c) and four experiments (d). *p<0.05, **p<0.01, ***p<0.001

Fig. 5. Glyburide prevents activation of NLRP3 inflammasome induced by hypoxia in human islets.

Human islets were incubated in 1% O₂ hypoxia chamber for 24 h in the absence (Hypoxia) or presence or of 200 μM glyburide (Hypoxia + GLY). NLRP3 (a) and IL1B (b) were quantified by qRT-PCR and expressed relative to Hypoxia. Caspase-1 (c) and IL-1β (d) released were quantified by ELISA and results expressed relative to Hypoxia. Data are means ± SEM from three experiments. *p<0.05, **p<0.01

Fig. 6. Role of NLRP3 inflammasome in hypoxia-induced cell death in human islets.

Human islets were incubated in control condition (Normoxia) or in 1% O₂ hypoxia chamber (Hypoxia) for 24 h in the presence or absence of 200 μM glyburide (GLY). Cell death was evaluated by Apoptosense^® ELISA kits. M65 values express both apoptosis and necrosis (a). M30 values express apoptosis (b). M65-M30 values (difference between the M65 and M30 values) express necrosis (c). NS no significant. Data are means ± SEM from seven experiments. *p<0.05