Cold-inducible RNA-binding protein (CIRP) potentiates uric acid-induced IL-1β production

Abstract

Background: Gout is an autoinflammatory disease driven by interleukin-1 (IL-1) induction in response to uric acid crystals. IL-1β production is dependent on inflammasome activation, which requires a priming signal, followed by an activating signal. The cold-inducible RNA-binding protein (CIRP) has been recently identified as a damage-associated molecular pattern (DAMP). In this study, we evaluated the roles of CIRP in monosodium urate (MSU)-mediated IL-1β secretion using human neutrophils.

Methods: Human neutrophils were stimulated by MSU in the presence or absence of CIRP priming to determine NLRP3 inflammasome activation and subsequent caspase-1 activation and IL-1β production. Cellular supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine the presence of IL-1β or caspase-1 (p20). The cellular supernatants and lysates were also analyzed by immunoblotting using anti-cleaved IL-1β or anti-cleaved caspase-1 antibodies.

Results: Neither CIRP nor MSU stimulation alone induced sufficient IL-1β secretion from neutrophils. However, MSU stimulation induced IL-1β secretion from CIRP-primed neutrophils in a dose-dependent manner. This MSU-induced IL-1β secretion from CIRP-primed neutrophils was accompanied by the induction of cleaved IL-1β (p17), which was inhibited by the pretreatment of MCC950, a specific inhibitor for NLRP3. Furthermore, cleaved caspase-1 was induced in the cellular lysates of CIRP/MSU-treated neutrophils. Additionally, CIRP stimulation induced the protein expression of pro-IL-1β in neutrophils.

Conclusions: Our data indicate that CIRP, an endogenous stress molecule, triggers uric acid-induced mature IL-1β induction as a priming stimulus for NLRP3 inflammasome in human neutrophils. We propose that CIRP acts as an important proinflammatory stimulant that primes and activates inflammasome and pro-IL-1β processing in response to uric acid in innate immune cells.

Fig. 1

CIRP pretreatment induces MSU-mediated IL-1β synthesis from neutrophils in a dose-dependent manner. Neutrophils (2 × 10⁶/ml) were pretreated with various concentrations of CIRP for 6 h. After pretreatment, the cells were stimulated with or without MSU (200 μg/ml) for 18 h and supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of two independent experiments. Single asterisk indicates significant difference was observed at a threshold of p < 0.01compared to unstimulated control neutrophils. Double asterisk indicates significant difference was observed at a threshold of p < 0.001compared to unstimulated control neutrophils

Fig. 2

MSU induces IL-1β synthesis from CIRP-primed neutrophils. Neutrophils (2 × 106/ml) were pretreated with various concentrations of CIRP (300 ng/ml) for 6 h. After pretreatment, the cells were stimulated with various concentrations of MSU for 18 h and supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of two independent experiments

Fig. 3

MSU induces cleaved IL-1β secretion from CIRP- primed neutrophils. a Neutrophils were pretreated or untreated with the indicated concentrations of CIRP for 6 h. After pretreatment, the cells were stimulated with MSU (200 mg/ml) for 18 h and supernatants were analyzed by immunoblot for the presence of cleaved IL-1β (p17). Three experiments were performed using different neutrophils and a representative result is shown. b Neutrophils were pretreated with 300 ng/ml of CIRP or heat denatured CIRP (boiled for 30 min) for 6 h. After pretreatment, the cells were stimulated with MSU (200 mg/ml) for 18 h and supernatants were analyzed by immunoblot for the presence of cleaved IL-1β (p17). Two experiments were performed using different neutrophils, and a representative result is shown

Fig. 4

MSU induces caspase-1 (p20) synthesis from CIRP-primed neutrophils. Neutrophils (2 × 10⁶/ml) were pretreated with various concentrations of CIRP for 6 h. After pretreatment, the cells were stimulated MSU (200 μg/ml) for 18 h and supernatants were analyzed for the presence of caspase-1 (p20) by ELISA. Single asterisk indicates significant difference was observed at a threshold of p < 0.01compared to unstimulated control neutrophils

Fig. 5

Caspase-1 immunoblot analysis using the cellular lysates of MUS-stimulated neutrophils. Neutrophils were pretreated or untreated with the indicated concentrations of CIRP for 6 h. After pretreatment, the cells were stimulated with of MSU (200 μg/ml) for 18 h. Cellular lysates were analyzed by immunoblotting with antibody that recognize cleaved caspase-1 (p20). β-actin was the loading control. Three experiments were performed using different neutrophils, and a representative result is shown

Fig. 6

a Effects of MCC950 on MSU-induced IL-1β synthesis from CIRP-primed neutrophils. CIRP (300 ng/ml, for 6 h)-primed neutrophils were pretreated with or without MCC950 for 30 min, and then stimulated with MSU (200 μg/mL) for 18 h. Supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of two independent experiments. Significant difference was observed at a threshold of p < 0.01 between CIRP/MSU-stimulated neutrophils with and without pretreatment of MCC950 (10^− 6 M). b Effects of MCC950 on MSU-induced cleaved-IL-1β (p17) expression of CIRP-primed neutrophils. CIRP (300 ng/ml, for 6 h)-primed neutrophils were pretreated with or without MCC950 for 30 min, and then stimulated with MSU (200 μg/mL) for 18 h. Cellular lysates were analyzed by western blotting using anti-cleaved-IL-1β antibodies. Two experiments were performed using different neutrophils, and a representative result is shown

Fig. 7

CIRP-induced NLRP3 expression in neutrophils. CIRP (300 ng/ml, for 30 min)-primed neutrophils were pretreated with or without MCC950) for 30 min and stimulated with MSU (200 μg/mL) for 18 h. Cellular lysates were analyzed by western blotting using anti-NLRP3 antibodies. β-actin was the loading control. Two experiments were performed using different neutrophils, and a representative result is shown

Fig. 8

Pro-IL-1β immunoblot analysis using the cellular lysates of CIRP or MSU-stimulated neutrophils. Neutrophils were pretreated or untreated with CIRP (300 ng/ml) or MSU (200 μg/ml) for 18 h. Cellular lysates were analyzed by immunoblotting using anti-pro-IL-1β antibody. β-actin was the loading control. Three experiments were performed using different neutrophils, and a representative result is shown