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Hydroxychloroquine Inhibits IL-1β Production From Amyloid-Stimulated Human Neutrophils

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Hydroxychloroquine Inhibits IL-1β Production From Amyloid-Stimulated Human Neutrophils

Yuya Fujita et al. Arthritis Res Ther.

Abstract

Background: Hydroxychloroquine (HCQ) is used for the treatment of patients with rheumatic diseases. We tested the hypothesis that HCQ affects the NLRP3 inflammasome, which is involved in autoinflammation.

Methods: Human neutrophils were stimulated with serum amyloid A (SAA) in vitro and measured for IL-1β and caspase-1 (p20) secretion by ELISA. Pro-IL-1β mRNA expression in human neutrophils was quantified by real-time RT-PCR.

Results: SAA stimulation induced significant production of IL-1β in human neutrophils. SAA stimulation also induced NF-κB activation, pro-IL-1β mRNA expression, and NLRP3 protein expression in human neutrophils. HCQ pretreatment significantly inhibited the SAA-induced IL-1β production in human neutrophils, but did not affect the SAA-induced NF-κB activation, pro-IL-1β mRNA expression, and NLRP3 protein expression. Furthermore, SAA stimulation induced cleaved caspase-1 (p20) secretion from human neutrophils, and this release was suppressed by HCQ pretreatment.

Conclusions: Treatment with HCQ was associated with impaired production of IL-1β in SAA-stimulated human neutrophils without affecting the priming process of the NLRP3 inflammasome such as pro-IL-1β or NLRP3 induction. These findings suggest that HCQ affects the NLRP3 activation process, resulting in the impaired IL-1β production in human neutrophils, as representative innate immune cells.

Keywords: Amyloid; Hydroxychloroquine; Inflammasome; Interleukin-1 beta; NLR family pyrin domain containing 3; Neutrophils; Serum amyloid A.

Conflict of interest statement

KM has received research grants from Chugai, Pfizer, and AbbVie. The rest of the authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
SAA induces IL-1β synthesis from neutrophils in a dose-dependent manner. Neutrophils (2 × 106/ml) were incubated with the indicated concentrations of SAA for 24 h, and supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of two independent experiments
Fig. 2
Fig. 2
Hydroxychloroquine inhibits the IL-1β synthesis from SAA-stimulated neutrophils. Neutrophils were pretreated with the indicated concentrations of hydroxychloroquine for 1 h and stimulated with SAA (10 μg/ml) for 24 h, and supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of two independent experiments. *p < 0.01 compared to SAA-stimulated neutrophils. **p < 0.001 compared to SAA-stimulated neutrophils
Fig. 3
Fig. 3
Hydroxychloroquine inhibits the IL-1β synthesis from SAA-stimulated neutrophils. Neutrophils were pretreated with the indicated concentrations of hydroxychloroquine or iberiotoxin (IBTX) for 1 h and stimulated with SAA (10 μg/ml) for 24 h, and supernatants were analyzed for IL-1β production by ELISA. Values represent the mean ± SD of two independent experiments
Fig. 4
Fig. 4
SAA induces the transcription of pro-IL-1β in human neutrophils. Neutrophils were pretreated with the indicated concentrations of hydroxychloroquine for 1 h and stimulated with SAA (10 μg/ml) for 8 h. The cells were harvested and analyzed for pro-IL-1β and GAPDH mRNA levels by real-time PCR. Values represent the mean ± SD of two independent experiments. *NS (not significant) compared to SAA-stimulated neutrophils
Fig. 5
Fig. 5
Phosphorylation of NF-κB p65 in SAA-treated neutrophils. Neutrophils were pretreated with the indicated concentrations of hydroxychloroquine for 2 h and stimulated with SAA (10 μg/ml) for 20 min. Cells were lysed and cellular lysates were subjected to Western blot using anti-phosphor-NF-κB and β-actin antibodies. Data are representative of two independent experiments
Fig. 6
Fig. 6
NLRP3 expression in neutrophils. Neutrophils were pretreated with the indicated concentrations of hydroxychloroquine for 1 h and stimulated with SAA (10 μg/ml) for 24 h. Cellular lysates were analyzed by Western using anti-NLRP3 or anti-β-actin antibodies. Three experiments were performed using different neutrophils, and a representative result is shown
Fig. 7
Fig. 7
Hydroxychloroquine inhibits the caspase-1 (p20) release from SAA-stimulated neutrophils. Neutrophils were pretreated with the indicated concentrations of hydroxychloroquine for 1 h and stimulated with SAA (10 μg/ml) for 24 h, and supernatants were analyzed for caspase-1 (p20) by ELISA. Values represent the mean ± SD of two independent experiments. *p < 0.05 compared to SAA-stimulated neutrophils
Fig. 8
Fig. 8
Hydroxychloroquine inhibits IL-1β and caspase-1 (p20) release from SAA-stimulated neutrophils. Neutrophils (2 × 106/ml) were incubated with the indicated concentrations of SAA for 12 h, and supernatants were analyzed for IL-1β production by ELISA (a). Neutrophils were pretreated with the indicated concentrations of hydroxychloroquine for 1 h and stimulated with SAA (10 μg/ml) for 12 h, and supernatants were analyzed for IL-1β (b) or caspase-1 (c; p20) by ELISA. Two experiments were performed using different neutrophils, and a representative result is shown

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