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Anti-inflammatory effect of artemisinin on uric acid-induced NLRP3 inflammasome activation through blocking interaction between NLRP3 and NEK7
Biochemical and Biophysical Research Communications 517 (2019) 338e345
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Anti-inflammatory effect of artemisinin on uric acid-induced NLRP3 inflammasome activation through blocking interaction between NLRP3 and NEK7 Seong-Kyu Kim a, *, Jung-Yoon Choe a, Ki-Yeun Park b a b
Division of Rheumatology, Department of Internal Medicine, Catholic University of Daegu School of Medicine, Daegu, Republic of Korea Arthritis and Autoimmunity Research Center, Catholic University of Daegu, Daegu, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 July 2019 Accepted 23 July 2019 Available online 26 July 2019
Objective: Artemisinin is a potent anti-malarial agent that plays a potent role in regulating inflammatory disorders. NEK7 is a major interacting partner with NLRP3 in NLRP3 inflammasome. The aim of this study was to clarify the anti-inflammatory effect of artemisinin on activation of uric acid-induced NLRP3 inflammasome through regulation of NEK7. Methods: Human macrophage U937 cells treated with lipopolysaccharide (LPS), monosodium urate (MSU) crystals, or artemisinin were used in in vitro study. Intracellular potassium (Kþ) level was measured in U937 cells treated with and without artemisinin. Expression of target genes or proteins NEK7, NLRP3, ASC, caspase-1, interleukin-1b (IL-1b), and NF-kB signaling molecules was measured. MSU crystal-induced arthritis model was used for in vivo study. Results: Gout patients showed higher NLRP3 and NEK7 mRNA expression, compared to controls. Enhanced expression of NLRP3, caspase-1, and IL-1b was noted in macrophages treated with LPS (10 ng/ ml) and MSU crystals (0.1 mg/ml), which was markedly suppressed by treatment with artemisinin (1, 10, and 100 mM). Artemisinin significantly inhibited interaction between NLRP3 and NEK7 in NLRP3 inflammasome activation. Artemisinin (10 and 100 mM) attenuated intracellular Kþ efflux in macrophages stimulated with LPS and MSU crystals. Artemisinin suppressed foot and ankle swelling in MSU crystal-induced arthritis mice. Conclusion: This study revealed that artemisinin inhibited activation of NLRP3 inflammasome by suppressing interaction between NEK7 and NLRP3 in uric acid-induced inflammation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Artemisinin NLRP3 Inflammasome Interleukin-1b NEK7 Uric acid
1. Introduction Artemisinin is an important therapeutic agent with prominent anti-malarial efficacy and was originally derived from Artemisia annua [1,2]. Recently, it has been well established that the antiinflammatory and immunoregulatory effects of artemisinin have clinical applications for diverse inflammatory and autoimmune rheumatic diseases such as alcohol-induced liver damage, tubulointerstitial nephritis, and rheumatoid arthritis (RA) [2,3]. Despite this clinical relevance, studies on the action mechanism of
* Corresponding author. Division of Rheumatology, Department of Internal Medicine, Arthritis and Autoimmunity Research Center, Daegu Catholic University School of Medicine, 33, Duryugongwon-ro 17-gil, Nam-gu, Daegu, 42472, Republic of Korea. E-mail address: [email protected] (S.-K. Kim). https://doi.org/10.1016/j.bbrc.2019.07.087 0006-291X/© 2019 Elsevier Inc. All rights reserved.
artemisinin have not been conducted. Artemisinin exerts a therapeutic effect by blocking the expression of proinflammatory cytokines such as tumor necrosis factor-a, interleukin-1b (IL-1b), and IL-17 by regulating reactive oxygen species generation, mitogen activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) signaling pathways, and pathogenic Th17 responses [4,5]. The NLRP3 inflammasome is a cytosolic protein complex formed by assembly of NLRP3, the adapter apoptosis-associated speck-like protein (ASC), and procaspase-1 [6,7]. Activation of NLRP3 inflammasome leads to maturation and secretion of IL-1b and IL-18 and then induced cellular damage and pyroptosis. Never in mitosis gene A (NIMA)-related kinase 7 (NEK7) has been recognized as a crucial molecule in regulation of NLRP3 inflammasome activation through interaction with NLRP3 [8,9]. Caspase-1 activation and NLRP3 oligomerization were not found in NEK7/ macrophages treated with monosodium urate (MSU) crystals and production of IL-1b in
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the intraperitoneal lavage fluid in mice stimulated with MSU crystals was significantly inhibited [8,9]. Some recent studies have explored the effect of artemisinin on NLRP3 inflammasome activation and demonstrated that artemisinin could potentially be used as a treatment for diverse NLRP3-mediated inflammatory diseases [10e13]. It has been well recognized that the NLRP3 inflammasome is a critical component for development of uric acid-induced inflammation, such as gout [14e16]. There is currently a lack of research on the molecular mechanism of artemisinin for NEK7 in activation of NLRP3 inflammasome stimulated by uric acid. Therefore, the purpose of this study was to investigate whether artemisinin could regulate NEK7-mediated NLRP3 inflammasome activation in uric acid-induced inflammation. 2. Subjects and methods 2.1. Subjects A total of 16 subjects (8 with gout and 8 healthy controls) were consecutively enrolled in this study to obtain peripheral blood mononuclear cells (PBMCs). Patients with gout met the classification criteria proposed by the American College of Rheumatology [17]. Subjects who did not show any clinical signs and symptoms that suggested inflammatory arthritis, such as RA, spondyloarthritis, or crystal-induced arthritis, were defined as healthy controls. Both patients with gout and healthy controls provided written informed consent at study enrollment. The protocol of this study was approved by the Institutional Review Board of Daegu Catholic University Medical Center (CR-16-077). 2.2. Cell cultures and reagents Human monocytic leukemia U937 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and PenStrep (100 U/mL Penicillin and 100 mg/mL Streptomycin) in a 5% CO2 atmosphere at 37 C. To differentiate monocytes into macrophages, cells were treated with 150 nM of phorbol 12-myristate 13-acetate [PMA (St. Louis, MO, USA)] for 24 h. After exposure to PMA, cells were pretreated with artemisinin (1, 10, and 100 mM) for 24 h and then treated with LPS (St. Louis, MO, USA) and MSU crystals for 6 h. Artemisinin (St. Louis, MO, USA) was dissolved in 10% dimethyl sulfoxide (DMSO) and then diluted with 90% saline. 2.3. Intracellular potassium (Kþ) concentration The level of intracellular Kþ was measured using a FluxOR Potassium Ion Channel Assay kit (Molecular Probes, Eugene, OR, USA) following the manufacturer's protocol. Cells (1 104) were seeded in 96-well plates and after 24 h cells were pretreated with artemisinin followed by treatment with MSU and LPS for 6 h. The medium was removed, and the cells were added to 80 ml of loading buffer and 100 ml of assay buffer. The fluorescence intensity of intracellular Kþ concentration was measured using a microplate reader with excitation at 488 nm and emission at 540 nm. 2.4. MSU crystal-induced arthritis model Male C57BL/6 mice (6e8 weeks old) were used to construct MSU crystal-induced arthritis animal model (Hyochang Science, Daegu, Korea) and divided into three groups composed 5 mice each as follows: (1) control group (treated with saline), (2) MSU alone group (intraarticular injection of 0.5 mg/ml of MSU crystals for 24 h), and (3) artemisinin and MSU group (intraperitoneal pretreatment
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with 50 mg/kg and 100 mg/kg of artemisinin for 2 h and intraarticular injection of 0.5 mg/ml of MSU crystals for 24 h). MSU crystal-induced arthritis mice were sacrificed in a CO2 chamber, and ankle tissue was collected. Mice were monitored by measuring ankle thickness with calipers and by assessing swelling. All animal experiments were approved by the Animal Care and Use Committee of Catholic University of Daegu School of Medicine (DCIAFCR1907-01-Y). 2.5. Real-time quantitative polymerase chain reaction (RT-qPCR) Cells were pretreated with artemisinin for 24 h, followed by treatment with MSU crystals and LPS for 6 h. Total RNA from cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer's protocol. cDNA was synthesized from 1 mg of RNA using a ReverTra Ace-a-kit (Toyobo Osaka, Japan) according to the manufacturer's protocols. RT-qPCR was performed using SYBR Green PCR Master Mix (ToYoBo, Tokyo, Japan) on a Mini Option TM Real-time PCR system (Bio-Rad Hercules, CA, USA). The expression value of each target gene was calculated after normalizing to glyceraldehyde 3-phosphate dehydrogenase. Comparison of gene expression was analyzed using the 2eDDCT method. 2.6. Western blot analysis Cell extracts were separated by SDSePAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, California, USA). The membranes were probed with primary antibodies, followed by incubation with species-specific horseradish peroxidaseconjugated secondary antibody. For immunoprecipitation, cells lysates were precleared with protein A/G agarose beads (Santa Cruz, Dallas, TX, USA) for 1 h at 4 C. The beads were extensively washed and eluted with 5x Laemmli Sample buffer by boiling for 5 min at 95 C. Immunoreactive proteins were tested with the western blot detection system kit (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the Chemidoc XRS imaging system (Bio-Rad, Hercules, CA, USA). Ankle tissues were homogenized using the Tissue Lysis Buffer (TLA, Promega, Madison, WI, USA) for 20 min on ice. The homogenates were centrifuged at 12,000 rpm for 15 min to obtain the supernatants for use in Western blot assay. 2.7. Immunofluorescent staining Cells (4 103) were seeded in 4-well chamber slides and pretreated with artemisinin for 24 h, followed by treatment with LPS and MSU crystals for 12 h. Cells were fixed in 10% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.1% TritonX-100 for 10 min at room temperature. The cells were immunostained with primary NEK7 antibody (Abcam, Cambridge, UK) at 4 C overnight. After washing, cells were incubated with the secondary antibody conjugated with goat anti-rabbit IgGFITC (Santa Cruz, CA, USA) for 1 h at room temperature. Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAKO, Glostrup, Denmark) for 10 min at room temperature in the dark and images were examined using a confocal microscope (Nikon, Tokyo, Japan). 2.8. Statistical analysis The statistical analysis for comparison between two groups was assessed by Mann-Whitney U test. Correlation between NEK7 and NLRP3 expression was evaluated by Spearman's correlation analysis. Statistical significance was determined as less than 0.05.
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Statistical analysis was conducted with GraphPad Software (San Diego, CA, USA).
10 mM and 100 mM of artemisinin treatment (p < 0.05 and p < 0.001, respectively), but not by 1 mM of artemisinin.
3. Results
3.4. Effect of artemisinin in NEK7-mediated NLRP3 inflammasome in arthritis mice model
3.1. NLRP3 and NEK7 expression stimulated by uric acid We first compared the mRNA expression of NLRP3 and NEK7 between patients with gout (n ¼ 8) and controls (n ¼ 8) to determine the difference in expression of NLRP3 and NEK7 genes (Fig. 1A). The PBMCs from gout patients showed higher expression of NLRP3 and NEK7 genes compared to controls (p < 0.05 for both). There was a significant correlation between NEK7 and NLRP3 mRNA expression for all subjects (r ¼ 0.798, p < 0.001) (Fig. 1B). However, a close correlation between the two mRNA expressions was maintained in patients with gout (r ¼ 0.822, p ¼ 0.012). Human U937 macrophages stimulated by either LPS (10 ng/ml) or MSU crystals (0.1 mg/ml) did not induce significantly higher expression in NLRP3 and NEK7 genes than controls (p > 0.01 of both) (Fig. 1C). Macrophages stimulated with both LPS and MSU crystals enhanced mRNA expression of NLRP3 and NEK7 genes compared to controls (p < 0.001 for both). 3.2. Effect of artemisinin on uric acid-induced NF-kB pathway and NLRP3 inflammasome We investigated whether uric acid inflammation is mediated through NF-kB signal pathway and NLRP3 inflammasome and evaluated the effect of artemisinin on these signaling pathways. First, we noted that treatment with LPS and MSU crystals activated NF-kB signal pathway, showing phosphorylation of IkBa and p65 in U937 cells (Fig. 2A). Artemisinin treatment (1, 10, and 100 mM) markedly suppressed phosphorylation of IkBa and p65 in dose dependent manner. Considering the effect of artemisinin on NLRP3 inflammasome, enhanced production of NLRP3, caspase-1, and IL1b proteins under stimulation of LPS and MSU crystals was significantly attenuated by treatment with artemisinin in a dose dependent manner (Fig. 2B). Enhanced NLRP3, caspase-1, and IL-1b mRNA expression in human macrophages stimulated with LPS (10 ng/ml) and MSU crystals (0.1 mg/ml) was significantly inhibited by treatment with 100 mM of artemisinin but not with 1 mM and 10 mM of artemisinin (Fig. 2C). 3.3. Effect of artemisinin on interaction between NLRP3 and NEK7 We assessed whether interaction between NLRP3 and NEK7 is an essential component in NLRP3 inflammasome activation under stimulation with uric acid. In in vitro study using U937 cells, stimulation with LPS and MSU crystals induced endogenous interaction between NLRP3 and NEK7, which was suppressed by treatment with artemisinin (1, 10, and 100 mM) (Fig. 3A and B). Artemisinin also sequentially inhibited assembly of NLRP3 with ASC and caspase-1 in a dose-dependent manner (Fig. 3B). As shown in Fig. 3C, artemisinin (100 mM but not 1 mM and 10 mM) inhibited NEK7 m RNA expression in U937 cells treated with LPS (10 ng/ml) and MSU crystals (0.1 mg/ml). In immunofluorescence staining to assess the effect of artemisinin on NEK7 in macrophages, LPS and MSU crystals showed that intracellular NEK7 expression, compared to control cells, was significantly blocked by artemisinin (Fig. 3D). NEK7 is dependent on intracellular Kþ efflux under stimulation by cell danger signals. We evaluated whether artemisinin affected intracellular Kþ level in uric acid-induced inflammation (Fig. 3E). Stimulation with LPS and MSU crystals caused a decrease in Kþ level, which was blocked by
Thickness of foot and ankle swelling in mice induced by intraarticular injection with MSU crystals (0.5 mg/ml) was markedly increased compared to control mice and was significantly inhibited by artemisinin (50 mg/kg and/or 100 mg/kg) (Fig. 4A). Pretreatment with artemisinin (50 mg/kg and 100 mg/kg) inhibited phosphorylation of IkBa and p65 in synovial tissue extracted from MSU crystal-induced mice (Fig. 4B). In addition, activation of IL-1b and caspase-1 was significantly suppressed in synovial tissue of mice treated with 50 mg/kg and 100 mg/kg of artemisinin (Fig. 4C). Similarly, mRNA expression of IL-1b and caspase-1 in spleen tissue extracted from MSU crystal-induced arthritis mice was significantly inhibited by pretreatment with artemisinin (Fig. 4D). IP analysis showed that endogenous interaction of NLRP3 with NEK7 was markedly inhibited in synovial tissue of mice pretreated with artemisinin (Fig. 4E). The interaction of NLRP3 with ASC and caspase-1 was also significantly blocked by artemisinin treatment. The mRNA expression of NEK7 and NLRP3 in spleen tissue from MSU crystal-induced arthritis mice was significantly attenuated by pretreatment with artemisinin (Fig. 4F). 4. Discussion The NLRP3 inflammasome is a multiple protein complex that induces activation of pro-inflammatory cytokines, IL-1b and IL-18, and ultimately releases these cytokines and plays a crucial role in innate immunity in response to endogenous or exogenous pathogens and pathogenic microbes [6,7]. The NLRP3 inflammasome is found to be associated with the pathogenesis of various inflammatory and autoimmune diseases [18]. Therefore, possible treatment for these diseases have been suggested by identifying therapeutic candidates such as miR-223, parthenolide, MCC950, and tranilast that block activation of NLRP3 inflammasome through regulation of targets including inhibition of caspase-1, ASC oligomerization, and NLRP3 oligomerization [19e22]. In this study, we found that artemisinin inhibits NLRP3 inflammasome activation through down-regulating interaction between NLRP3 and NEK7. NEK7 is involved in NLRP3 oligomerization and activates the NLRP3 inflammasome using danger signals such as nigericin, ATP, and gramicidin [8]. NLRP3 inflammasome activators can induce the NLRP3-NKE7 interaction that is dependent on Kþ efflux. NEK7 binding to the leucine-rich repeat (LRR) domain of NLRP3 is required for NLRP3 inflammasome [9]. IL-1b production under stimulation with NLRP3 inducers such as nigericin, ATP, and gramicidin was significantly reduced in NEK7/ BMDMs compared to NEK7þ/þ BMDMs [8]. LPS-primed Nek7Cu/Cu macrophages treated with inducers of NLRP3 inflammasome such as ATP and alum significantly attenuated IL-1b production but not that of flagellin or poly (dA:dT), stimulators of the NLRC4 and AIM2 inflammasomes [9], which is consistent with the results of other studies [8]. In this study, increased NEK7 gene expression and protein production and subsequent activation of NLRP3 inflammasome were noted in LPS-primed macrophages stimulated with MSU crystals. These results suggest that NEK7 is an important mediator of the mechanism of NLRP3 inflammasome activation, and that it could also provide a potent therapeutic target for NLRP3 inflammasome-mediated inflammation. The NLRP3 inflammasome is an important pathogenic mechanism in diverse inflammatory disorders such as cryopyrinassociated periodic syndromes, gout, atherosclerosis, and type II
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Fig. 1. NLRP3 and NEK7 mRNA expression is increased in uric acid-induced condition. A) Comparison of NLRP3 and NEK7 mRNA expression from PBMCs between gout (n ¼ 8) and healthy controls (n ¼ 8). *p < 0.05 versus controls. B) Correlation between NLRP3 and NEK7 mRNA expression was analyzed in total subjects (n ¼ 16) and only gout (n ¼ 8). C) RTqPCR analysis of NLRP3 and NEK mRNA expression was measured in macrophages treated with LPS alone, MSU crystal alone, and LPS-primed MSU crystal. *p < 0.05 versus nonstimulated macrophages. Data are expressed as mean ± SEM. The images are representative of three independent experiments.
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Fig. 2. Artemisinin attenuates activation of NF-kB pathway and NLRP3 inflammasome in uric acid-induced inflammation. A) Effect of artemisinin on phosphorylation of IkBa and p65 (component of NF-kB) in LPS-primed macrophages treated with MSU crystals. B) Effect of artemisinin on NLRP3 expression and activation of caspase-1 and IL-1b in LPS-primed macrophages treated with MSU crystals. C) RT-qPCR analysis of NLRP3, caspase-1, and IL-1b mRNA expression in macrophages costimulated with LPS and MSU crystals. *p < 0.01, ** p < 0.05, and ***p < 0.001 versus LPS and MSU crystal-stimulated macrophages without artemisinin treatment. Data are expressed as mean ± SEM. The images are representative of three independent experiments.
diabetes mellitus [7,23]. Uric acid is also a potent stimulator in development of gouty arthritis pathogenically associated with NLRP3 inflammasome activation [14,15]. Based on the crucial role of NEK7 in NLRP3 inflammasome activation, He et al. demonstrated that uric acid reduced activation of caspase-1 and IL-1b release in NEK7/ BMDMs compared to NEK7þ/þ BMDMs [8]. The in vivo study showed that intraperitoneal stimulation with monosodium urate crystals promoted an NEK7-dependent inflammatory response and IL-1b production, which were associated with higher peritoneal exudate cells, neutrophils, or monocyte/macrophage recruitment in NEK7þ/þ mice rather than NEK7þ/Cu or NEK7Cu/Cu Cuties mutant mice [9]. In our study, NEK7 gene expression in PBMC of patients with gout was significantly increased compared with the PBMC of healthy controls, together with NLRP3 gene expression. In addition, increased NEK7 protein production was consistently observed in foot and ankle joint tissue from MSU crystal-induced arthritis model. These results indicate that NEK7 is involved in the pathogenesis of gouty arthritis. Several studies recently demonstrated that anti-inflammatory effect of artemisinin was associated with tight regulation of the NLRP3 inflammasome [10e13]. Activation of the NLRP3 inflammasome is a two-step signal model that involves priming (signal 1) and activation (signal 2). At the priming step, microbial or
endogenous danger stimuli lead to upregulation of NLRP3 and proIL-1b expression by activating the NF-kB transcription factor. Shi et al. showed the inhibitory effect of artemisinin on NF-kB activity and NLRP3 inflammasome activation in a neuroinflammation model using APPswe/PS1dE9 transgenic mice [12]. In another study, administration of artemisinin (100 mg/kg) for 16 weeks reduced tubulointerstitial inflammation and fibrosis in 5/6 subtotal nephrectomy rats [13]. The renal protective effect of artemisinin was found to be dependent on downregulation of NF-kB activity and NLRP3 inflammasome activation. We also consistently observed that artemisinin blocked the NF-kB signal pathway, showing reduced phosphorylation of IkBa and p65 in LPSpretreated macrophages stimulated by MSU crystals. In the same conditions, both IL-1b and NLRP3 gene expression in macrophages treated with MSU crystals was also markedly attenuated by artemisinin treatment. Other inhibitory mechanisms of artemisinin have been proposed at the step of assembly and activation in the NLRP3 inflammasome. Artemisinin suppressed NLRP3 inflammasome activation stimulated by danger signals such as ATP, nigericin, and silica through inhibition of ASC oligomerization and speck formation in LPS-primed BMDMs [10]. Here, artemisinin inhibited the effects on the NF-kB pathway and NLRP3 expression and reduced the assembly of NLRP3 inflammasome components during
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Fig. 3. Artemisinin suppresses the role of NEK7 in activation of MSU crystal-induced NLRP3 inflammasome. A) Effect of artemisinin on interaction of NLRP3 and NEK7 in LPS-primed U937 macrophages stimulated with MSU crystals. B) Effect of artemisinin on interaction of ASC and caspase-1 with NLRP3 in the uric acid-induced condition. C) RT-qPCR analysis for effect of artemisinin on NEK7 m RNA expression in LPS-primed U937 cells stimulated with MSU crystals. *p < 0.05 versus LPS and MSU crystal-stimulated macrophages without artemisinin treatment. D) Immunofluorescent staining for intracellular NEK7 expression with and without pretreatment with artemisinin for 24 h. E) Analysis of intracellular Kþ level in MSU crystal-stimulated U937 macrophages with and without artemisinin. *p < 0.05 and **p < 0.01 versus LPS and MSU crystal-stimulated macrophages without artemisinin treatment.
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Fig. 4. Artemisinin improves MSU crystal-induced arthritis. A) C57BL/6 male mice were treated with a) saline, b) MSU crystals (0.5 mg/ml for 24 h), c) artemisinin (50 mg/kg for 2 h), and d) artemisinin (100 mg/kg for 2 h). Thickness of foot and paw swelling was measured in experimental mice. *p < 0.05 and **p < 0.01 versus mice without artemisinin treatment. B) Effect of artemisinin on phosphorylation of IkBa and p65 (component of NF-kB) in synovial tissue of MSU crystal-induced arthritis mice. C) Effect of artemisinin on activation of caspase-1 and IL-1b in synovial tissue of MSU crystal-induced arthritis mice. D) RT-qPCR analysis for effect of artemisinin on caspase-1 and IL-1b mRNA expression in spleen tissue of MSU crystal-induced arthritis mice. *p < 0.05 and **p < 0.01 versus mice without artemisinin treatment. E) Interaction of NLRP3 with NEK7, ASC, and caspase-1 in synovial tissue of arthritis mice was determined by immunoprecipitation assay. F) RT-qPCR analysis for effect of artemisinin on NEK7 and NLRP3 mRNA expression in spleen tissue of MSU crystalinduced arthritis mice. *p < 0.05 and **p < 0.01 versus mice without artemisinin treatment.
NLRP3 inflammasome activation. The intracellular Kþ efflux (or lower intracellular Kþ concentration) in response to most NLRP3 inducers is a crucial mechanism for NLRP3 inflammasome activation [10,24,25]. High extracellular Kþ concentration attenuated inflammasome activation, which was supported by evidence that ASC expression was significantly blocked by extracellular KCl treatment to LPS-primed BMDMs under ATP, nigerisin, and silica stimulation [10]. Because NEK7 is required for NLRP3 inflammasome activation downstream of the Kþ efflux [8], the role of NEK7 is also affected by intracellular Kþ level via the Kþ efflux. Lower intracellular Kþ concentration enhanced NEK7 and NLRP3 interactions in NLRP3 inflammasome activation. In this study, MSU crystal stimulation in LPS-primed macrophages led to decreased intracellular Kþ concentration, which was reversed by artemisinin treatment. There is currently little research on the mechanism of intracellular Kþ increase induced artemisinin. However, inhibition of the Kþ efflux of artemisinin may be attributed to indirect anti-inflammatory effects rather than direct effects of the drug. This suggests that the inhibitory effect of artemisinin on intracellular Kþ efflux can be deduced to block NLRP3 inflammasome activation. Although NEK7 is a potent therapeutic target in activation of the NLRP3 inflammasome, there are limited potent pharmaceutical
agents that specifically control NEK7. Recently, Moraes et al. demonstrated that GSK-3 Inhibitor XIII suppressed the activity of hNEK7 through its non-ATP-competitive inhibition by approximately 46.3% [26]. Recently, He et al. demonstrated that oridonin, the main extract from Rabdosia Rubescens, specifically blocked NLRP3 inflammasome activation through cysteine 279 binding of the NACHT domain of NLRP3 and inhibition of the NLRP3-NEK7 interaction [27]. Although there are diverse anti-inflammatory effects associated with artemisinin, the action mechanism on NEK7 has not been determined. We found that NEK7-NLRP3 interaction was prominently noted in MSU crystal-stimulated macrophages and the uric acid-induced arthritis mice model and was significantly blocked by artemisinin treatment. Based on these observations, artemisinin also shows anti-inflammatory effects through regulation of NEK7 expression or activity. In conclusion, artemisinin downregulates NEK7-NLRP3 interaction in LPS-primed macrophages treated with MSU crystals and MSU crystal-induced arthritis mice and attenuates the intracellular Kþ efflux in the NLRP3 inflammasome process. The results of this study provide evidence that artemisinin may be a potent therapeutic agent for blocking NLRP3 inflammasome activation in uric acid-induced inflammation.
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Funding source This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2019R1F1A1061098). This work was supported by the grant of Research Institute of Medical Science, Catholic University of Daegu (2019). Disclosure statement The authors do not have an actual or potential conflict of interest or not get interest of any kind to declare with regards to this work. Acknowledgement None. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.087. References [1] S. Krishna, L. Bustamante, R.K. Haynes, H.M. Staines, Artemisinins: their growing importance in medicine, Trends Pharmacol. Sci. 29 (2008) 520e527. [2] W.E. Ho, H.Y. Peh, T.K. Chan, W.S. Wong, Artemisinins: pharmacological actions beyond anti-malarial, Pharmacol. Ther. 142 (2014) 126e139. [3] Y. Wu, W. Tang, J. Zuo, Development of artemisinin drugs in the treatment of autoimmune Diseases, Sci. Bull. 61 (2016) 37e41. [4] J.X. Wang, W. Tang, R. Zhou, J. Wan, L.P. Shi, Y. Zhang, Y.F. Yang, Y. Li, J.P. Zuo, The new water-soluble artemisinin derivative SM905 ameliorates collageninduced arthritis by suppression of inflammatory and Th17 responses, Br. J. Pharmacol. 153 (2008) 1303e1310. [5] W.D. Li, Y.J. Dong, Y.Y. Tu, Z.B. Lin, Dihydroarteannuin ameliorates lupus symptom of BXSB mice by inhibiting production of TNF-a and blocking the signaling pathway NF-kB translocation, Int. Immunopharmacol. 12 (2006) 1243e1250. ~ ez, Mechanism and regulation of NLRP3 inflammasome [6] Y. He, H. Hara, G. Nún activation, Trends Biochem. Sci. 41 (2016) 1012e1021. [7] V.A. Rathinam, K.A. Fitzgerald, Inflammasome complexes: emerging mechanisms and effector functions, Cell 165 (2016) 792e800. ~ ez, NEK7 is an essential mediator of [8] Y. He, M.Y. Zeng, D. Yang, B. Motro, G. Nún NLRP3 activation downstream of potassium efflux, Nature 530 (2016) 354e357. [9] H. Shi, Y. Wang, X. Li, X. Zhan, M. Tang, M. Fina, L. Su, D. Pratt, C.H. Bu, S. Hildebrand, S. Lyon, L. Scott, J. Quan, Q. Sun, J. Russell, S. Arnett, P. Jurek, D. Chen, V.V. Kravchenko, J.C. Mathison, E.M. Moresco, N.L. Monson, R.J. Ulevitch, B. Beutler, NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component, Nat. Immunol.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Anti-inflammatory effect of artemisinin on uric acid-induced NLRP3 inflammasome activation through blocking interaction between NLRP3 and NEK7 Seong-Kyu Kim a, *, Jung-Yoon Choe a, Ki-Yeun Park b a b
Division of Rheumatology, Department of Internal Medicine, Catholic University of Daegu School of Medicine, Daegu, Republic of Korea Arthritis and Autoimmunity Research Center, Catholic University of Daegu, Daegu, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 July 2019 Accepted 23 July 2019 Available online 26 July 2019
Objective: Artemisinin is a potent anti-malarial agent that plays a potent role in regulating inflammatory disorders. NEK7 is a major interacting partner with NLRP3 in NLRP3 inflammasome. The aim of this study was to clarify the anti-inflammatory effect of artemisinin on activation of uric acid-induced NLRP3 inflammasome through regulation of NEK7. Methods: Human macrophage U937 cells treated with lipopolysaccharide (LPS), monosodium urate (MSU) crystals, or artemisinin were used in in vitro study. Intracellular potassium (Kþ) level was measured in U937 cells treated with and without artemisinin. Expression of target genes or proteins NEK7, NLRP3, ASC, caspase-1, interleukin-1b (IL-1b), and NF-kB signaling molecules was measured. MSU crystal-induced arthritis model was used for in vivo study. Results: Gout patients showed higher NLRP3 and NEK7 mRNA expression, compared to controls. Enhanced expression of NLRP3, caspase-1, and IL-1b was noted in macrophages treated with LPS (10 ng/ ml) and MSU crystals (0.1 mg/ml), which was markedly suppressed by treatment with artemisinin (1, 10, and 100 mM). Artemisinin significantly inhibited interaction between NLRP3 and NEK7 in NLRP3 inflammasome activation. Artemisinin (10 and 100 mM) attenuated intracellular Kþ efflux in macrophages stimulated with LPS and MSU crystals. Artemisinin suppressed foot and ankle swelling in MSU crystal-induced arthritis mice. Conclusion: This study revealed that artemisinin inhibited activation of NLRP3 inflammasome by suppressing interaction between NEK7 and NLRP3 in uric acid-induced inflammation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Artemisinin NLRP3 Inflammasome Interleukin-1b NEK7 Uric acid
1. Introduction Artemisinin is an important therapeutic agent with prominent anti-malarial efficacy and was originally derived from Artemisia annua [1,2]. Recently, it has been well established that the antiinflammatory and immunoregulatory effects of artemisinin have clinical applications for diverse inflammatory and autoimmune rheumatic diseases such as alcohol-induced liver damage, tubulointerstitial nephritis, and rheumatoid arthritis (RA) [2,3]. Despite this clinical relevance, studies on the action mechanism of
* Corresponding author. Division of Rheumatology, Department of Internal Medicine, Arthritis and Autoimmunity Research Center, Daegu Catholic University School of Medicine, 33, Duryugongwon-ro 17-gil, Nam-gu, Daegu, 42472, Republic of Korea. E-mail address: [email protected] (S.-K. Kim). https://doi.org/10.1016/j.bbrc.2019.07.087 0006-291X/© 2019 Elsevier Inc. All rights reserved.
artemisinin have not been conducted. Artemisinin exerts a therapeutic effect by blocking the expression of proinflammatory cytokines such as tumor necrosis factor-a, interleukin-1b (IL-1b), and IL-17 by regulating reactive oxygen species generation, mitogen activated protein kinase (MAPK) and nuclear factor-kB (NF-kB) signaling pathways, and pathogenic Th17 responses [4,5]. The NLRP3 inflammasome is a cytosolic protein complex formed by assembly of NLRP3, the adapter apoptosis-associated speck-like protein (ASC), and procaspase-1 [6,7]. Activation of NLRP3 inflammasome leads to maturation and secretion of IL-1b and IL-18 and then induced cellular damage and pyroptosis. Never in mitosis gene A (NIMA)-related kinase 7 (NEK7) has been recognized as a crucial molecule in regulation of NLRP3 inflammasome activation through interaction with NLRP3 [8,9]. Caspase-1 activation and NLRP3 oligomerization were not found in NEK7/ macrophages treated with monosodium urate (MSU) crystals and production of IL-1b in
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the intraperitoneal lavage fluid in mice stimulated with MSU crystals was significantly inhibited [8,9]. Some recent studies have explored the effect of artemisinin on NLRP3 inflammasome activation and demonstrated that artemisinin could potentially be used as a treatment for diverse NLRP3-mediated inflammatory diseases [10e13]. It has been well recognized that the NLRP3 inflammasome is a critical component for development of uric acid-induced inflammation, such as gout [14e16]. There is currently a lack of research on the molecular mechanism of artemisinin for NEK7 in activation of NLRP3 inflammasome stimulated by uric acid. Therefore, the purpose of this study was to investigate whether artemisinin could regulate NEK7-mediated NLRP3 inflammasome activation in uric acid-induced inflammation. 2. Subjects and methods 2.1. Subjects A total of 16 subjects (8 with gout and 8 healthy controls) were consecutively enrolled in this study to obtain peripheral blood mononuclear cells (PBMCs). Patients with gout met the classification criteria proposed by the American College of Rheumatology [17]. Subjects who did not show any clinical signs and symptoms that suggested inflammatory arthritis, such as RA, spondyloarthritis, or crystal-induced arthritis, were defined as healthy controls. Both patients with gout and healthy controls provided written informed consent at study enrollment. The protocol of this study was approved by the Institutional Review Board of Daegu Catholic University Medical Center (CR-16-077). 2.2. Cell cultures and reagents Human monocytic leukemia U937 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and PenStrep (100 U/mL Penicillin and 100 mg/mL Streptomycin) in a 5% CO2 atmosphere at 37 C. To differentiate monocytes into macrophages, cells were treated with 150 nM of phorbol 12-myristate 13-acetate [PMA (St. Louis, MO, USA)] for 24 h. After exposure to PMA, cells were pretreated with artemisinin (1, 10, and 100 mM) for 24 h and then treated with LPS (St. Louis, MO, USA) and MSU crystals for 6 h. Artemisinin (St. Louis, MO, USA) was dissolved in 10% dimethyl sulfoxide (DMSO) and then diluted with 90% saline. 2.3. Intracellular potassium (Kþ) concentration The level of intracellular Kþ was measured using a FluxOR Potassium Ion Channel Assay kit (Molecular Probes, Eugene, OR, USA) following the manufacturer's protocol. Cells (1 104) were seeded in 96-well plates and after 24 h cells were pretreated with artemisinin followed by treatment with MSU and LPS for 6 h. The medium was removed, and the cells were added to 80 ml of loading buffer and 100 ml of assay buffer. The fluorescence intensity of intracellular Kþ concentration was measured using a microplate reader with excitation at 488 nm and emission at 540 nm. 2.4. MSU crystal-induced arthritis model Male C57BL/6 mice (6e8 weeks old) were used to construct MSU crystal-induced arthritis animal model (Hyochang Science, Daegu, Korea) and divided into three groups composed 5 mice each as follows: (1) control group (treated with saline), (2) MSU alone group (intraarticular injection of 0.5 mg/ml of MSU crystals for 24 h), and (3) artemisinin and MSU group (intraperitoneal pretreatment
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with 50 mg/kg and 100 mg/kg of artemisinin for 2 h and intraarticular injection of 0.5 mg/ml of MSU crystals for 24 h). MSU crystal-induced arthritis mice were sacrificed in a CO2 chamber, and ankle tissue was collected. Mice were monitored by measuring ankle thickness with calipers and by assessing swelling. All animal experiments were approved by the Animal Care and Use Committee of Catholic University of Daegu School of Medicine (DCIAFCR1907-01-Y). 2.5. Real-time quantitative polymerase chain reaction (RT-qPCR) Cells were pretreated with artemisinin for 24 h, followed by treatment with MSU crystals and LPS for 6 h. Total RNA from cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer's protocol. cDNA was synthesized from 1 mg of RNA using a ReverTra Ace-a-kit (Toyobo Osaka, Japan) according to the manufacturer's protocols. RT-qPCR was performed using SYBR Green PCR Master Mix (ToYoBo, Tokyo, Japan) on a Mini Option TM Real-time PCR system (Bio-Rad Hercules, CA, USA). The expression value of each target gene was calculated after normalizing to glyceraldehyde 3-phosphate dehydrogenase. Comparison of gene expression was analyzed using the 2eDDCT method. 2.6. Western blot analysis Cell extracts were separated by SDSePAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, California, USA). The membranes were probed with primary antibodies, followed by incubation with species-specific horseradish peroxidaseconjugated secondary antibody. For immunoprecipitation, cells lysates were precleared with protein A/G agarose beads (Santa Cruz, Dallas, TX, USA) for 1 h at 4 C. The beads were extensively washed and eluted with 5x Laemmli Sample buffer by boiling for 5 min at 95 C. Immunoreactive proteins were tested with the western blot detection system kit (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the Chemidoc XRS imaging system (Bio-Rad, Hercules, CA, USA). Ankle tissues were homogenized using the Tissue Lysis Buffer (TLA, Promega, Madison, WI, USA) for 20 min on ice. The homogenates were centrifuged at 12,000 rpm for 15 min to obtain the supernatants for use in Western blot assay. 2.7. Immunofluorescent staining Cells (4 103) were seeded in 4-well chamber slides and pretreated with artemisinin for 24 h, followed by treatment with LPS and MSU crystals for 12 h. Cells were fixed in 10% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.1% TritonX-100 for 10 min at room temperature. The cells were immunostained with primary NEK7 antibody (Abcam, Cambridge, UK) at 4 C overnight. After washing, cells were incubated with the secondary antibody conjugated with goat anti-rabbit IgGFITC (Santa Cruz, CA, USA) for 1 h at room temperature. Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAKO, Glostrup, Denmark) for 10 min at room temperature in the dark and images were examined using a confocal microscope (Nikon, Tokyo, Japan). 2.8. Statistical analysis The statistical analysis for comparison between two groups was assessed by Mann-Whitney U test. Correlation between NEK7 and NLRP3 expression was evaluated by Spearman's correlation analysis. Statistical significance was determined as less than 0.05.
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Statistical analysis was conducted with GraphPad Software (San Diego, CA, USA).
10 mM and 100 mM of artemisinin treatment (p < 0.05 and p < 0.001, respectively), but not by 1 mM of artemisinin.
3. Results
3.4. Effect of artemisinin in NEK7-mediated NLRP3 inflammasome in arthritis mice model
3.1. NLRP3 and NEK7 expression stimulated by uric acid We first compared the mRNA expression of NLRP3 and NEK7 between patients with gout (n ¼ 8) and controls (n ¼ 8) to determine the difference in expression of NLRP3 and NEK7 genes (Fig. 1A). The PBMCs from gout patients showed higher expression of NLRP3 and NEK7 genes compared to controls (p < 0.05 for both). There was a significant correlation between NEK7 and NLRP3 mRNA expression for all subjects (r ¼ 0.798, p < 0.001) (Fig. 1B). However, a close correlation between the two mRNA expressions was maintained in patients with gout (r ¼ 0.822, p ¼ 0.012). Human U937 macrophages stimulated by either LPS (10 ng/ml) or MSU crystals (0.1 mg/ml) did not induce significantly higher expression in NLRP3 and NEK7 genes than controls (p > 0.01 of both) (Fig. 1C). Macrophages stimulated with both LPS and MSU crystals enhanced mRNA expression of NLRP3 and NEK7 genes compared to controls (p < 0.001 for both). 3.2. Effect of artemisinin on uric acid-induced NF-kB pathway and NLRP3 inflammasome We investigated whether uric acid inflammation is mediated through NF-kB signal pathway and NLRP3 inflammasome and evaluated the effect of artemisinin on these signaling pathways. First, we noted that treatment with LPS and MSU crystals activated NF-kB signal pathway, showing phosphorylation of IkBa and p65 in U937 cells (Fig. 2A). Artemisinin treatment (1, 10, and 100 mM) markedly suppressed phosphorylation of IkBa and p65 in dose dependent manner. Considering the effect of artemisinin on NLRP3 inflammasome, enhanced production of NLRP3, caspase-1, and IL1b proteins under stimulation of LPS and MSU crystals was significantly attenuated by treatment with artemisinin in a dose dependent manner (Fig. 2B). Enhanced NLRP3, caspase-1, and IL-1b mRNA expression in human macrophages stimulated with LPS (10 ng/ml) and MSU crystals (0.1 mg/ml) was significantly inhibited by treatment with 100 mM of artemisinin but not with 1 mM and 10 mM of artemisinin (Fig. 2C). 3.3. Effect of artemisinin on interaction between NLRP3 and NEK7 We assessed whether interaction between NLRP3 and NEK7 is an essential component in NLRP3 inflammasome activation under stimulation with uric acid. In in vitro study using U937 cells, stimulation with LPS and MSU crystals induced endogenous interaction between NLRP3 and NEK7, which was suppressed by treatment with artemisinin (1, 10, and 100 mM) (Fig. 3A and B). Artemisinin also sequentially inhibited assembly of NLRP3 with ASC and caspase-1 in a dose-dependent manner (Fig. 3B). As shown in Fig. 3C, artemisinin (100 mM but not 1 mM and 10 mM) inhibited NEK7 m RNA expression in U937 cells treated with LPS (10 ng/ml) and MSU crystals (0.1 mg/ml). In immunofluorescence staining to assess the effect of artemisinin on NEK7 in macrophages, LPS and MSU crystals showed that intracellular NEK7 expression, compared to control cells, was significantly blocked by artemisinin (Fig. 3D). NEK7 is dependent on intracellular Kþ efflux under stimulation by cell danger signals. We evaluated whether artemisinin affected intracellular Kþ level in uric acid-induced inflammation (Fig. 3E). Stimulation with LPS and MSU crystals caused a decrease in Kþ level, which was blocked by
Thickness of foot and ankle swelling in mice induced by intraarticular injection with MSU crystals (0.5 mg/ml) was markedly increased compared to control mice and was significantly inhibited by artemisinin (50 mg/kg and/or 100 mg/kg) (Fig. 4A). Pretreatment with artemisinin (50 mg/kg and 100 mg/kg) inhibited phosphorylation of IkBa and p65 in synovial tissue extracted from MSU crystal-induced mice (Fig. 4B). In addition, activation of IL-1b and caspase-1 was significantly suppressed in synovial tissue of mice treated with 50 mg/kg and 100 mg/kg of artemisinin (Fig. 4C). Similarly, mRNA expression of IL-1b and caspase-1 in spleen tissue extracted from MSU crystal-induced arthritis mice was significantly inhibited by pretreatment with artemisinin (Fig. 4D). IP analysis showed that endogenous interaction of NLRP3 with NEK7 was markedly inhibited in synovial tissue of mice pretreated with artemisinin (Fig. 4E). The interaction of NLRP3 with ASC and caspase-1 was also significantly blocked by artemisinin treatment. The mRNA expression of NEK7 and NLRP3 in spleen tissue from MSU crystal-induced arthritis mice was significantly attenuated by pretreatment with artemisinin (Fig. 4F). 4. Discussion The NLRP3 inflammasome is a multiple protein complex that induces activation of pro-inflammatory cytokines, IL-1b and IL-18, and ultimately releases these cytokines and plays a crucial role in innate immunity in response to endogenous or exogenous pathogens and pathogenic microbes [6,7]. The NLRP3 inflammasome is found to be associated with the pathogenesis of various inflammatory and autoimmune diseases [18]. Therefore, possible treatment for these diseases have been suggested by identifying therapeutic candidates such as miR-223, parthenolide, MCC950, and tranilast that block activation of NLRP3 inflammasome through regulation of targets including inhibition of caspase-1, ASC oligomerization, and NLRP3 oligomerization [19e22]. In this study, we found that artemisinin inhibits NLRP3 inflammasome activation through down-regulating interaction between NLRP3 and NEK7. NEK7 is involved in NLRP3 oligomerization and activates the NLRP3 inflammasome using danger signals such as nigericin, ATP, and gramicidin [8]. NLRP3 inflammasome activators can induce the NLRP3-NKE7 interaction that is dependent on Kþ efflux. NEK7 binding to the leucine-rich repeat (LRR) domain of NLRP3 is required for NLRP3 inflammasome [9]. IL-1b production under stimulation with NLRP3 inducers such as nigericin, ATP, and gramicidin was significantly reduced in NEK7/ BMDMs compared to NEK7þ/þ BMDMs [8]. LPS-primed Nek7Cu/Cu macrophages treated with inducers of NLRP3 inflammasome such as ATP and alum significantly attenuated IL-1b production but not that of flagellin or poly (dA:dT), stimulators of the NLRC4 and AIM2 inflammasomes [9], which is consistent with the results of other studies [8]. In this study, increased NEK7 gene expression and protein production and subsequent activation of NLRP3 inflammasome were noted in LPS-primed macrophages stimulated with MSU crystals. These results suggest that NEK7 is an important mediator of the mechanism of NLRP3 inflammasome activation, and that it could also provide a potent therapeutic target for NLRP3 inflammasome-mediated inflammation. The NLRP3 inflammasome is an important pathogenic mechanism in diverse inflammatory disorders such as cryopyrinassociated periodic syndromes, gout, atherosclerosis, and type II
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Fig. 1. NLRP3 and NEK7 mRNA expression is increased in uric acid-induced condition. A) Comparison of NLRP3 and NEK7 mRNA expression from PBMCs between gout (n ¼ 8) and healthy controls (n ¼ 8). *p < 0.05 versus controls. B) Correlation between NLRP3 and NEK7 mRNA expression was analyzed in total subjects (n ¼ 16) and only gout (n ¼ 8). C) RTqPCR analysis of NLRP3 and NEK mRNA expression was measured in macrophages treated with LPS alone, MSU crystal alone, and LPS-primed MSU crystal. *p < 0.05 versus nonstimulated macrophages. Data are expressed as mean ± SEM. The images are representative of three independent experiments.
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Fig. 2. Artemisinin attenuates activation of NF-kB pathway and NLRP3 inflammasome in uric acid-induced inflammation. A) Effect of artemisinin on phosphorylation of IkBa and p65 (component of NF-kB) in LPS-primed macrophages treated with MSU crystals. B) Effect of artemisinin on NLRP3 expression and activation of caspase-1 and IL-1b in LPS-primed macrophages treated with MSU crystals. C) RT-qPCR analysis of NLRP3, caspase-1, and IL-1b mRNA expression in macrophages costimulated with LPS and MSU crystals. *p < 0.01, ** p < 0.05, and ***p < 0.001 versus LPS and MSU crystal-stimulated macrophages without artemisinin treatment. Data are expressed as mean ± SEM. The images are representative of three independent experiments.
diabetes mellitus [7,23]. Uric acid is also a potent stimulator in development of gouty arthritis pathogenically associated with NLRP3 inflammasome activation [14,15]. Based on the crucial role of NEK7 in NLRP3 inflammasome activation, He et al. demonstrated that uric acid reduced activation of caspase-1 and IL-1b release in NEK7/ BMDMs compared to NEK7þ/þ BMDMs [8]. The in vivo study showed that intraperitoneal stimulation with monosodium urate crystals promoted an NEK7-dependent inflammatory response and IL-1b production, which were associated with higher peritoneal exudate cells, neutrophils, or monocyte/macrophage recruitment in NEK7þ/þ mice rather than NEK7þ/Cu or NEK7Cu/Cu Cuties mutant mice [9]. In our study, NEK7 gene expression in PBMC of patients with gout was significantly increased compared with the PBMC of healthy controls, together with NLRP3 gene expression. In addition, increased NEK7 protein production was consistently observed in foot and ankle joint tissue from MSU crystal-induced arthritis model. These results indicate that NEK7 is involved in the pathogenesis of gouty arthritis. Several studies recently demonstrated that anti-inflammatory effect of artemisinin was associated with tight regulation of the NLRP3 inflammasome [10e13]. Activation of the NLRP3 inflammasome is a two-step signal model that involves priming (signal 1) and activation (signal 2). At the priming step, microbial or
endogenous danger stimuli lead to upregulation of NLRP3 and proIL-1b expression by activating the NF-kB transcription factor. Shi et al. showed the inhibitory effect of artemisinin on NF-kB activity and NLRP3 inflammasome activation in a neuroinflammation model using APPswe/PS1dE9 transgenic mice [12]. In another study, administration of artemisinin (100 mg/kg) for 16 weeks reduced tubulointerstitial inflammation and fibrosis in 5/6 subtotal nephrectomy rats [13]. The renal protective effect of artemisinin was found to be dependent on downregulation of NF-kB activity and NLRP3 inflammasome activation. We also consistently observed that artemisinin blocked the NF-kB signal pathway, showing reduced phosphorylation of IkBa and p65 in LPSpretreated macrophages stimulated by MSU crystals. In the same conditions, both IL-1b and NLRP3 gene expression in macrophages treated with MSU crystals was also markedly attenuated by artemisinin treatment. Other inhibitory mechanisms of artemisinin have been proposed at the step of assembly and activation in the NLRP3 inflammasome. Artemisinin suppressed NLRP3 inflammasome activation stimulated by danger signals such as ATP, nigericin, and silica through inhibition of ASC oligomerization and speck formation in LPS-primed BMDMs [10]. Here, artemisinin inhibited the effects on the NF-kB pathway and NLRP3 expression and reduced the assembly of NLRP3 inflammasome components during
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Fig. 3. Artemisinin suppresses the role of NEK7 in activation of MSU crystal-induced NLRP3 inflammasome. A) Effect of artemisinin on interaction of NLRP3 and NEK7 in LPS-primed U937 macrophages stimulated with MSU crystals. B) Effect of artemisinin on interaction of ASC and caspase-1 with NLRP3 in the uric acid-induced condition. C) RT-qPCR analysis for effect of artemisinin on NEK7 m RNA expression in LPS-primed U937 cells stimulated with MSU crystals. *p < 0.05 versus LPS and MSU crystal-stimulated macrophages without artemisinin treatment. D) Immunofluorescent staining for intracellular NEK7 expression with and without pretreatment with artemisinin for 24 h. E) Analysis of intracellular Kþ level in MSU crystal-stimulated U937 macrophages with and without artemisinin. *p < 0.05 and **p < 0.01 versus LPS and MSU crystal-stimulated macrophages without artemisinin treatment.
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Fig. 4. Artemisinin improves MSU crystal-induced arthritis. A) C57BL/6 male mice were treated with a) saline, b) MSU crystals (0.5 mg/ml for 24 h), c) artemisinin (50 mg/kg for 2 h), and d) artemisinin (100 mg/kg for 2 h). Thickness of foot and paw swelling was measured in experimental mice. *p < 0.05 and **p < 0.01 versus mice without artemisinin treatment. B) Effect of artemisinin on phosphorylation of IkBa and p65 (component of NF-kB) in synovial tissue of MSU crystal-induced arthritis mice. C) Effect of artemisinin on activation of caspase-1 and IL-1b in synovial tissue of MSU crystal-induced arthritis mice. D) RT-qPCR analysis for effect of artemisinin on caspase-1 and IL-1b mRNA expression in spleen tissue of MSU crystal-induced arthritis mice. *p < 0.05 and **p < 0.01 versus mice without artemisinin treatment. E) Interaction of NLRP3 with NEK7, ASC, and caspase-1 in synovial tissue of arthritis mice was determined by immunoprecipitation assay. F) RT-qPCR analysis for effect of artemisinin on NEK7 and NLRP3 mRNA expression in spleen tissue of MSU crystalinduced arthritis mice. *p < 0.05 and **p < 0.01 versus mice without artemisinin treatment.
NLRP3 inflammasome activation. The intracellular Kþ efflux (or lower intracellular Kþ concentration) in response to most NLRP3 inducers is a crucial mechanism for NLRP3 inflammasome activation [10,24,25]. High extracellular Kþ concentration attenuated inflammasome activation, which was supported by evidence that ASC expression was significantly blocked by extracellular KCl treatment to LPS-primed BMDMs under ATP, nigerisin, and silica stimulation [10]. Because NEK7 is required for NLRP3 inflammasome activation downstream of the Kþ efflux [8], the role of NEK7 is also affected by intracellular Kþ level via the Kþ efflux. Lower intracellular Kþ concentration enhanced NEK7 and NLRP3 interactions in NLRP3 inflammasome activation. In this study, MSU crystal stimulation in LPS-primed macrophages led to decreased intracellular Kþ concentration, which was reversed by artemisinin treatment. There is currently little research on the mechanism of intracellular Kþ increase induced artemisinin. However, inhibition of the Kþ efflux of artemisinin may be attributed to indirect anti-inflammatory effects rather than direct effects of the drug. This suggests that the inhibitory effect of artemisinin on intracellular Kþ efflux can be deduced to block NLRP3 inflammasome activation. Although NEK7 is a potent therapeutic target in activation of the NLRP3 inflammasome, there are limited potent pharmaceutical
agents that specifically control NEK7. Recently, Moraes et al. demonstrated that GSK-3 Inhibitor XIII suppressed the activity of hNEK7 through its non-ATP-competitive inhibition by approximately 46.3% [26]. Recently, He et al. demonstrated that oridonin, the main extract from Rabdosia Rubescens, specifically blocked NLRP3 inflammasome activation through cysteine 279 binding of the NACHT domain of NLRP3 and inhibition of the NLRP3-NEK7 interaction [27]. Although there are diverse anti-inflammatory effects associated with artemisinin, the action mechanism on NEK7 has not been determined. We found that NEK7-NLRP3 interaction was prominently noted in MSU crystal-stimulated macrophages and the uric acid-induced arthritis mice model and was significantly blocked by artemisinin treatment. Based on these observations, artemisinin also shows anti-inflammatory effects through regulation of NEK7 expression or activity. In conclusion, artemisinin downregulates NEK7-NLRP3 interaction in LPS-primed macrophages treated with MSU crystals and MSU crystal-induced arthritis mice and attenuates the intracellular Kþ efflux in the NLRP3 inflammasome process. The results of this study provide evidence that artemisinin may be a potent therapeutic agent for blocking NLRP3 inflammasome activation in uric acid-induced inflammation.
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Funding source This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2019R1F1A1061098). This work was supported by the grant of Research Institute of Medical Science, Catholic University of Daegu (2019). Disclosure statement The authors do not have an actual or potential conflict of interest or not get interest of any kind to declare with regards to this work. Acknowledgement None. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.087. References [1] S. Krishna, L. Bustamante, R.K. Haynes, H.M. Staines, Artemisinins: their growing importance in medicine, Trends Pharmacol. Sci. 29 (2008) 520e527. [2] W.E. Ho, H.Y. Peh, T.K. Chan, W.S. Wong, Artemisinins: pharmacological actions beyond anti-malarial, Pharmacol. Ther. 142 (2014) 126e139. [3] Y. Wu, W. Tang, J. Zuo, Development of artemisinin drugs in the treatment of autoimmune Diseases, Sci. Bull. 61 (2016) 37e41. [4] J.X. Wang, W. Tang, R. Zhou, J. Wan, L.P. Shi, Y. Zhang, Y.F. Yang, Y. Li, J.P. Zuo, The new water-soluble artemisinin derivative SM905 ameliorates collageninduced arthritis by suppression of inflammatory and Th17 responses, Br. J. Pharmacol. 153 (2008) 1303e1310. [5] W.D. Li, Y.J. Dong, Y.Y. Tu, Z.B. Lin, Dihydroarteannuin ameliorates lupus symptom of BXSB mice by inhibiting production of TNF-a and blocking the signaling pathway NF-kB translocation, Int. Immunopharmacol. 12 (2006) 1243e1250. ~ ez, Mechanism and regulation of NLRP3 inflammasome [6] Y. He, H. Hara, G. Nún activation, Trends Biochem. Sci. 41 (2016) 1012e1021. [7] V.A. Rathinam, K.A. Fitzgerald, Inflammasome complexes: emerging mechanisms and effector functions, Cell 165 (2016) 792e800. ~ ez, NEK7 is an essential mediator of [8] Y. He, M.Y. Zeng, D. Yang, B. Motro, G. Nún NLRP3 activation downstream of potassium efflux, Nature 530 (2016) 354e357. [9] H. Shi, Y. Wang, X. Li, X. Zhan, M. Tang, M. Fina, L. Su, D. Pratt, C.H. Bu, S. Hildebrand, S. Lyon, L. Scott, J. Quan, Q. Sun, J. Russell, S. Arnett, P. Jurek, D. Chen, V.V. Kravchenko, J.C. Mathison, E.M. Moresco, N.L. Monson, R.J. Ulevitch, B. Beutler, NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component, Nat. Immunol.
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