Hydroxychloroquine inhibits IL-1β production from amyloid-stimulated human neutrophils

Hydroxychloroquine (HCQ) exerts various anti-inflammatory and immunomodulatory effects and is widely used for the treatment of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [1]. However, its mode of action is not completely understood [2]. According to the previous studies, the actions of HCQ on the immune system appear to involve its ability to interfere with lysosomal acidification and thereby affect antigen processing and Toll-like receptor (TLR) signaling [2‐4]. The NLRP3 inflammasome is a cytoplasmic macromolecular complex that orchestrates inflammatory responses in innate immunity by inducing caspase-1 activation and IL-1β processing [5]. Various danger signals, including reactive oxygen species (ROS), crystals, potassium efflux, and amyloid proteins, have been identified as possible activators of the NLRP3 inflammasome [6]. Recent studies demonstrated the pivotal roles of SAA in the regulation of immunity and inflammation. Cytokine-like activities of SAA for its induction of IL-1β, TNFα, and IL-8 had been demonstrated. The engagement of formyl peptide receptor-like 1 (FPRL1) by its ligand, SAA, initiates intracellular signaling such as nuclear factor-κB, leading to an activation of the innate immunity that is crucial to the development of inflammation [7]. Also, SAA has been identified as an endogenous activator of the NLRP3 inflammasome, which is critical for pro-IL-1β processing and activation [8]. Collectively, these findings illustrate proinflammatory functions of SAA including inflammasome activation.

Recent evidence suggests that the inflammasome is dysregulated in SLE and plays an important role in lupus-associated organ damage [9]. For example, NLRP3 inflammasome contributes to glomerular injuries and proteinuria in lupus nephritis [10]. Therefore, inflammasome-targeted therapies appear to be a new approach for lupus treatment. Because of the diverse effects of HCQ on pattern-recognition receptors [11], it is tempting to speculate that the inflammasome could be a target for HCQ during innate immune cell activation. On the basis of its high affinity for the lysosomal/endosomal compartment [3], we hypothesized that HCQ may affect the process for inflammasome activation. In the present study, we directly addressed the above hypothesis by determining the effects of HCQ on the NLRP3 inflammasome activation process using human neutrophils as representative innate immune cells. Our findings demonstrated that HCQ inhibited amyloid-mediated NLRP3 inflammasome activation and IL-1β secretion from human neutrophils. Taken together, the present findings provide novel insights toward understanding the anti-inflammatory effects of HCQ on the innate immune system.

We previously showed that serum amyloid A (SSA) is capable of inducing IL-1β secretion from human neutrophils without a priming signal [12]. In this study, we investigated the effect of HCQ on SAA-induced NLRP3 inflammasome activation and subsequent IL-1β secretion in human neutrophils. As shown in Fig. 1, SSA stimulation alone induced IL-1β secretion from human neutrophils and reached a plateau at 10 μg/ml. Neutrophils were pretreated with various HCQ concentrations for 1 h and exposed to SAA (10 μg/ml). The supernatants were analyzed for their IL-1β contents by ELISA. HCQ pretreatment suppressed IL-1β secretion from SAA-stimulated neutrophils in a dose-dependent manner (Fig. 2). It was reported that ATP-induced K⁺ efflux plays a key role in activating the NLRP3 inflammasome and HCQ inhibits this inflammasome activation process by inhibiting Ca⁺⁺-activated K⁺ channels (KCa) [13]. To address the involvement of Ca⁺⁺-activated K⁺ channels, we examined the effects of iberiotoxin (IBTX), a specific Ca⁺⁺-activated K⁺ channel inhibitor, against SAA-induced inflammasome activation. ATP-induced IL-1β secretion from LPS-primed neutrophils was blocked by IBTX as described previously [13] (data not shown), whereas IBRX pretreatment did not affect SAA-induced IL-1β secretion in neutrophils (Fig. 3), suggesting that KCa may not be involved in SAA-mediated inflammasome activation.

Next, we examined the effect of SAA on pro-IL-1β mRNA expression in human neutrophils. As shown in Fig. 4, SAA induced pro-IL-1β mRNA expression in these cells. HCQ pretreatment did not affect pro-IL-1β mRNA expression in SAA-stimulated neutrophils and SAA remained a potent inducer of pro-IL-1β mRNA expression.

As a key mediator of immunity, NF-κB plays a critical role in priming the NLRP3 inflammasome [14]. The NF-κB pathway is also involved in the transcription of pro-IL-1β [15]. Thus, we examined whether HCQ pretreatment (2 h) modulated the NF-κB pathway in SAA-stimulated neutrophils. Phosphorylation of NF-κB was induced in neutrophils by SAA stimulation. However, HCQ pretreatment had no influence on this SAA-induced NF-κB phosphorylation (Fig. 5). We then checked the protein expression of NLRP3, a major component of the inflammasome complex, in human neutrophils. Unstimulated neutrophils exhibited marginal expression of NLRP3, but the expression was enhanced in response to SAA stimulation. HCQ pretreatment did not affect the NLRP3 protein expression in SAA-stimulated neutrophils (Fig. 6).

It is generally accepted that the cleaved form of caspase-1, p20, is released along with processed IL-1β during NLRP3 inflammasome activation [16]. Therefore, culture supernatants were analyzed for secretion of cleaved caspase-1 by an ELISA specific for caspase-1 (p20). Consistent with the impaired IL-1β production, caspase-1 (p20) production was suppressed in neutrophils stimulated with SAA after pretreatment with HCQ in a dose-dependent manner (Fig. 7). Finally, to further confirm the biologic significance of the inhibitory effects of HCQ, we assessed SAA-induced IL-1β or cleaved caspase-1 (p20) secretion from neutrophils at the early time point. To minimize the apoptosis or pyrotosis induction compared to the inflammasome activation in neutrophils, we performed the same assay at an early time point (12 h). SAA stimulation induced IL-1β secretion from neutrophils in a dose-dependent manner even in the short duration of culture periods (Fig. 8a). HCQ pretreatment inhibited SAA-induced IL-1β (Fig. 8b) or caspase-1 (Fig. 8c) secretion from neutrophils in this short duration of culture periods.

Chloroquine and its analog HCQ, originally antimalarial drugs, are widely used for the treatment of rheumatic diseases because of their anti-inflammatory and immunosuppressive effects [1]. However, their effects on immunity and potential mechanisms remain unclear. Previous studies demonstrated that HCQ interferes with the TLR4 signaling pathway and reduces the production of cytokines in LPS-stimulated macrophages [17]. The diverse effects of HCQ on pattern-recognition receptor signaling suggest that the inflammasome could be a target for HCQ [18, 19]. Emerging evidence has suggested an important role for the NLRP3 inflammasome in the pathogenesis of rheumatic diseases [20]. In addition to classical immune cells, activation of NLRP3 was demonstrated in neutrophil-mediated inflammatory processes [21]. In this study, we found that HCQ inhibited SAA-induced IL-1β production in human neutrophils. An exciting clinical implication of the present findings is the identification of HCQ as a potent modulator of autoinflammation by affecting the NLRP3 inflammasome.

Activation of the NLRP3 inflammasome requires two steps that are controlled by different mechanisms [22]. In the first step, NLRP3 expression is induced, and in the second step, NLRP3 activation leads to caspase-1 activation and subsequent pro-IL-1β processing [23]. As a key mediator of immunity, NF-κB plays a critical role for priming of the NLRP3 inflammasome [24]. The NF-κB pathway is also involved in the transcription of pro-IL-1β and NLRP3 expression, which are the limiting steps for NLRP3 inflammasome activation [25].

We found that SAA stimulation led to expression of both pro-IL-1β mRNA and NLRP3 protein. In addition, NF-κB activation was required for NLRP3 protein induction. HCQ pretreatment did not affect SAA-induced NF-κB signaling, a known activator of NLRP3, suggesting that HCQ did not inhibit the priming of the NLRP3 inflammasome. Attenuated NF-κB activation may result in reduced SAA-induced NLRP3 protein expression or pro-IL-1β mRNA expression. However, our data clearly indicated that HCQ pretreatment did not affect SAA-induced pro-IL-1β mRNA or NLRP3 protein expressions. Therefore, HCQ appears to inhibit NLRP3 inflammasome activation by affecting the SAA-mediated NLRP3 activation steps without affecting priming steps.

We showed that HCQ inhibits SAA-induced IL-1β secretion and cleaved caspase-1 (p20) secretion from human neutrophils. These findings suggest that HCQ inhibits SAA-mediated proinflammatory properties by inhibiting the NLRP3 inflammasome activation process. Previous studies demonstrated that stress-induced proteins, including amyloid protein, activate the NLRP3 inflammasome [8]. Consistent with previous reports, our data in human neutrophils indicated that NLRP3 inflammasome activation was induced in amyloid-stimulated neutrophils. We also demonstrated that HCQ limits danger signal-induced IL-1β release from human neutrophils. However, the mechanism by which HCQ exerts its inhibitory effects on inflammasome activation is still incompletely understood.

Because of the diverse effects of HCQ on pattern-recognition receptors and lysosomal function, it is tempting to speculate that the inflammatome could be a target for HCQ during innate immune cell activation. Our data indicated that HCQ may have a protective effect on amyloid-mediated inflammatory processes at the level of neutrophils, as representative innate immune cells. However, it remains unclear how HCQ mediates this inhibition. Recent studies demonstrated that ROS can induce lysosomal damage, leading to enhanced NLRP3 inflammasome activation [26]. HCQ was shown to inhibit ROS production in activated macrophages [27]. Thus, it is possible that HCQ modulates the NLRP3 inflammasome activation process by affecting mitochondrial ROS accumulation.

There is a limitation to this study. Although the HCQ concentrations used in this study were higher than those measured in serum from patients on regimens used to treat SLE or RA, the maximum serum levels we could achieve were 5–10 μM. However, it was reported that the use of higher concentrations of chloroquine for treatment of leukocytes in vitro resulted in intracellular concentrations comparable to those obtained in vivo during chloroquine therapy [28].

We have demonstrated that HCQ can inhibit SAA-induced IL-1β secretion from human neutrophils. Our results suggest that HCQ may inhibit NLRP3 inflammasome activation by affecting the activation steps, rather than the priming steps. These findings provide insights into the novel anti-inflammatory mechanisms of HCQ and suggest a new strategy for targeting autoinflammatory disorders.