The Antimalarial Chloroquine Suppresses LPS-Induced NLRP3 Inflammasome Activation and Confers Protection against Murine Endotoxic Shock

Abstract

Activation of the NLRP3 inflammasome, which catalyzes maturation of proinflammatory cytokines like IL-1β and IL-18, is implicated and essentially involved in many kinds of inflammatory disorders. Chloroquine (CQ) is a traditional antimalarial drug and also possesses an anti-inflammatory property. In this study, we investigated whether CQ suppresses NLRP3 inflammasome activation and thereby confers protection against murine endotoxic shock. CQ attenuated NF-κB and MAPK activation and prohibited expression of IL-1β, IL-18, and Nlrp3 in LPS treated murine bone marrow-derived macrophages (BMDMs), demonstrating its inhibitory effect on the priming signal of NLRP3 activation. Then, CQ was shown to inhibit caspase-1 activation and ASC specks formation in BMDMs, which indicates that CQ also suppresses inflammasome assembly, the second signal for NLRP3 inflammasome activation. In a murine endotoxic shock model, CQ effectively improved survival and markedly reduced IL-1β and IL-18 production in serum, peritoneal fluid, and lung tissues. Moreover, CQ reduced protein levels of NLRP3 and caspases-1 p10 in lung homogenates of mice with endotoxic shock, which may possibly explain its anti-inflammatory activity and life protection efficacy in vivo. Overall, our results demonstrate a new role of CQ that facilitates negative regulation on NLRP3 inflammasome, which thereby confers protection against lethal endotoxic shock.

Figure 1

CQ suppress IL-1β and IL-18 secretion in LPS- and ATP-primed BMDMs. BMDMs were pretreated with or without 0.4, 4, and 40 μM CQ for 1 h and then stimulated with LPS (100 ng/ml) for 4 h and ATP (2 mM) for an additional 1 h. The IL-1β and IL-18 levels in culture supernatants were determined by ELISA (n = 3). ^∗∗P < 0.01 versus LPS plus ATP group (a-b). LDH release from BMDMs was measured by LDH assay (c). ^∗P < 0.05, ^∗∗P < 0.01 versus LPS plus ATP group. All data were expressed as mean ± SEM in triplicate and are representative of three independent experiments.

Figure 2

CQ inhibits transcription and maturation of IL-1β and IL-18. (a) BMDMs were pretreated with CQ (4, 40 μM) for 1 h, primed with LPS (100 ng/ml) for 4 h, and stimulated with ATP (2 mM) for an additional 1 h. Cell lysates and supernatants were prepared and IL-1β level was detected by ELISA. ^∗∗P < 0.01 versus LPS plus ATP group. (b) IL-1β and IL-18 mRNA expressions were measured by qRT-PCR. BMDMs were pretreated with CQ (40 μM) for 1 h and then primed with LPS (100 ng/ml) for 2 h. Data are shown as the means ± SEM (n = 3). ^∗∗P < 0.01 versus LPS group; ^##P < 0.01 versus LPS plus ATP group.

Figure 3

CQ restrains NF-κB p65 phosphorylation and nuclear localization. (a) Western blot of phospho-NF-κB p65 (Ser536). Cells were pretreated with 40 μM CQ for 1 h and then 100 ng/ml LPS treated for 2 h and 4 h, respectively. Total cell protein was collected for western blot of NF-κB p65 phosphorylation and GAPDH. ^#P < 0.05 versus LPS group (2 h). ^∗∗P < 0.01 versus LPS group (4 h). (b) Cells were pretreated with 40 μM CQ for 1 h and then stimulated by LPS for 30 min. IκB-α and phospho-IκB-α levels were detected by western blot. (c) Confocal images of nuclear translocation of NF-κB p65. Cells were pretreated with different concentrations of CQ for 1 h, followed by 100 ng/ml LPS treatment for 2 h. Cells were stained by anti-NF-κB p65 (red) antibodies and DAPI (blue) for nuclear staining. (d) Cells were treated as in (b). ERK, p38, and JNK and their phosphorylated forms (p-IκB-α, p-ERK, p-p38, and p-JNK) in cell lysates were detected by western blot.

Figure 4

CQ dampens NLRP3 inflammasome activation. BMDMs were pretreated with CQ (4, 40 μM) for 1 h and then stimulated with LPS (100 ng/ml) for 4 h and ATP (2 mM) for an additional 1 h. (a) Nlrp3 mRNA expression was measured by qRT-PCR. (b) NLRP3 and GAPDH were measured in cell lysates by western blot. (c) Western blot analysis of cleaved caspase-1 (p10) in culture supernatants (Sup) or of procaspase-1, ASC, and tubulin in cell lysates (Lys). (d) BMDMs were pretreated with CQ at the indicated concentration for 1 h and then stimulated with LPS (100 ng/ml) for 4 h and nigericin (10 μM) for 30 min. Production of mature IL-1β in supernatants was measured by ELISA. Data are shown as means ± SEM; ^∗P < 0.05, ^∗∗P < 0.01 versus LPS plus ATP group.

Figure 5

CQ restrains ASC speck formation and potassium efflux. (a) Representative immunofluorescence images of ASC speck formation in LPS-primed BMDMs stimulated with ATP in the presence or absence of CQ (20 μM) were acquired by confocal microscopy. (b) A percentage of cells containing specks were quantified in at least five distinct fields. (c, d) LPS-primed BMDMs were treated for 1 h with 2 mM ATP in a medium containing the specified [K⁺], and the secreted IL-1β was measured. (e) CQ pretreatment BMDMs were treated with LPS plus ATP, and the intracellular content of K⁺ was detected. Data are shown as means ± SEM; ^##P < 0.01 versus CQ pretreatment (4 μM) group; ^∗P < 0.05, ^∗∗P < 0.01 versus LPS plus ATP group.

Figure 6

CQ protects mice against lethal endotoxic shock by suppressing NLRP3 inflammasome activation. CQ improved survival in endotoxic shock murine model. BALB/c murine mice (n = 16 per group) were injected with a lethal dose of LPS (15 mg/kg) alone or together with CQ (10, 30 mg/kg). (a) Survival in each group was observed for 168 h and the log-rank test was used for intergroup comparison. ^∗P < 0.05 versus LPS group. (b) IL-18 and IL-1β levels in serum at 6 h were measured by ELISA. (c) IL-18 and IL-1β levels in lung homogenates at 6 h were measured by ELISA (n = 7 per group). (d) IL-18 and IL-1β levels in peritoneal lavage fluid at 6 h were measured by ELISA (n = 6 per group). Data are shown as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01 versus LPS group. (e) Caspase-1 activation and NLRP3 expression in the lung were detected by western blot (n = 3).

Figure 7

The proposed mechanism of CQ for the inhibition of NLRP3 inflammasome activation.