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MAPK pathways or NLRP3 inflammasome activation in macrophages
International Immunopharmacology 45 (2017) 163–173
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Sinigrin inhibits production of inflammatory mediators by suppressing NF-κB/MAPK pathways or NLRP3 inflammasome activation in macrophages Hee-Weon Lee a, Chung Gi Lee a, Dong-Kwon Rhee a, Sung Hee Um b, Sukneung Pyo a,⁎ a b
School of Pharmacy, Sungkyunkwan University, Suwon, Gyunggi-do 16419, Republic of Korea Department of Molecular Cell Biology, Samsung Biomedical Research Institute, School of Medicine, Sungkyunkwan University, Suwon, Gyunggi-do 16419, Republic of Korea
a r t i c l e
i n f o
Article history: Received 29 July 2016 Received in revised form 13 January 2017 Accepted 30 January 2017 Available online xxxx Keywords: 2-Propenyl glucosinolate Inflammation NLRP3 inflammasome NF-κB/MAPK Macrophage
a b s t r a c t Sinigrin (2-propenyl glucosinolate) is found mainly in broccoli, brussels sprouts, and black mustard seeds. Recently, sinigrin has received attention for its role in disease prevention and health. This study investigated the effect of sinigrin on macrophage function, including the activity of Nod-like receptor protein 3 (NLRP3) inflammasome. In a concentration-dependent manner, sinigrin inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production and the expression of COX-2 and prostaglandin E2 (PGE2) in RAW 264.7 cells. In addition, sinigrin significantly suppressed the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 via suppression of MAPK phosphorylation and nuclear factor-kappa B (NF-κB) activity. Treatment with sinigrin decreased IL-1β and IL-18 production and concurrently suppressed NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 expression in LPS/ATP-stimulated cells, suggesting that the blocking of NLRP3 inflammasome activation prevented the production of both cytokines. Collectively, these results suggest that sinigrin has immunomodulatory effects by suppressing the production of inflammatory mediators, possibly by inhibiting the NF-κB/MAPK pathways or NLRP3 inflammasome activation. Our findings also provide evidence that the pharmacological modulation of sinigrin could have an anti-inflammatory effect. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Inflammation plays a critical role in the development and progression of various diseases. Rather than being a single process, inflammation is a complex, multifaceted phenomenon consisting of individual events that may differ in their responses to pathogens. Many bacterial components and products, including lipopolysaccharide (LPS), can initiate local inflammatory responses. The inflammatory response depends on a complicated network of cellular components, including inflammatory mediators, which contribute to the pathogenesis of various diseases. Inflammasomes are multi-subunit complexes activated by cellular infection or stress, and it has been suggested that inflammasome activation is associated with diverse inflammatory diseases [1]. In particular, the NLRP3 inflammasome plays a critical role in the development and progression of many diseases [2–4], and its activation by LPS recruits the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and pro-
⁎ Corresponding author at: School of Pharmacy, Sungkyunkwan University, Suwon City, Gyunggi-do 440-746, Republic of Korea. E-mail address: [email protected] (S. Pyo).
http://dx.doi.org/10.1016/j.intimp.2017.01.032 1567-5769/© 2017 Elsevier B.V. All rights reserved.
caspase-1, which results in the processing and secretion of pro-inflammatory cytokines such as interleukin (IL)-1β and IL-18 [5]. An important cell type in the inflammatory process is an innate, phagocyte-like macrophage. In the inflammatory response, the macrophage population increases, an event that appears to play a major role at the site of tissue inflammation [6]. Macrophages are critical for recognizing pathogens and for initiating and amplifying the inflammatory cascade, mainly through the regulation of inducible enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS), and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-6, and IL-1β [7]. These inflammatory mediators are induced by lipopolysaccharide (LPS) in macrophages and MAPK/NF-κB pathways play important roles in LPS-induced pro-inflammatory mediators and cytokines expression [8–10]. In addition, persistent pro-inflammatory macrophages contribute to the development of chronic inflammatory diseases, such as atherosclerosis, type 2 diabetes, and hay fever [11]. Overproduction of inflammatory mediators has been implicated in many manifestations of disease. Therefore, modulating macrophage-mediated inflammatory responses could potentially reduce the incidence of various inflammatory diseases. In recent years, pharmacological preparations of plants and their components have attracted considerable interest because of their ability
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to prevent and treat various chronic diseases and to improve health [12]. Many plants are a rich source of certain functional food factors and bioactive metabolites that might have immunomodulatory properties [13–15]. Sinigrin is a thioglucoside of cruciferous vegetables, including brussels sprouts and broccoli, and the seeds of black mustard. It has potent antitumor activity, and its hydrolysis products are effective against bacteria [16,17]. In addition, sinigrin has antioxidant activity and suppresses the production of nitric oxide (NO) in LPS-treated animals [18,19]. In preliminary experiments, we have shown that the exposure of macrophages to sinigrin induces significant changes in the expression pattern of proinflammatory mediator genes [20] However, its immunomodulating effects on the function of macrophages and the underlying mechanisms remain unknown. The objective of this study was to examine the effects of sinigrin on LPS-induced inflammatory responses in macrophages and the potential mechanisms involved. We found that sinigrin inhibited the LPS-induced production of pro-inflammatory mediators by suppressing the nuclear factor-kappa B/mitogen-activated protein kinase (NF-κB/MAPK) pathways or the activation of NLRP3 inflammasome. 2. Materials and methods 2.1. Chemical reagents and antibodies All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. Sinigrin hydrate was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Lipofetamine Plus, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). The reporter plasmid pGL3-NF-κB used in the luciferase assay system was obtained from Promega (Madison, WI, USA), and pCMV-β-gal was obtained from Lonza (Walkersville, MD). IL-6 and tumor necrosis factor-alpha (TNF-α) ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against inducible NO synthase (iNOS), extracellular signal-regulated kinase (ERK)1/2, phospho-ERK1/ 2, p38, phospho-p38, C-Jun N-terminal kinase (JNK), phospho-JNK, IκBα, NF-κB, NLRP3, caspase-1, IL-1, IL-18, α-tubulin, lamin A, and βactin were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.2. Cell culture The murine macrophage cell line RAW 264.7 was obtained from ATCC (Rockville, MD, USA), and the RAW 264.7 cells were cultured in DMEM (Lonza) supplemented with 2 mM of L -glutamine, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 10% heatinactivated FBS. The cells were incubated at 37 °C in a fully humidified incubator containing 5% CO2 and were subcultured twice weekly. RAW 264.7 cells were pretreated for 2 h with sinigrin, followed by treatment with LPS (1 μg/mL) for various prespecified times. The cells were harvested by centrifugation at 13,000g for 3 min at 4 °C, and the pellets were lysed by radioimmunoprecipitation assay buffer (RIPA buffer). 2.3. Assessment of cell proliferation RAW 264.7 cells were seeded at a concentration of 3 × 105 cells/well in 96-well tissue culture plates and were treated with various concentrations of sinigrin for 24 h. Cell viability was measured for 2 h by means of quantitative colorimetric assay with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which showed the mitochondrial activity of living cells, as previously described [21]. The extent of the reduction of MTT to formazan within cells was quantified by measuring the optical density at 550 nm using a microplate reader (Molecular Devices, Carlsbad, CA, USA). The blank control contained only cell culture medium, and the absorbance of untreated cultures
was set at 100%. Cell viability was expressed as a percentage of the untreated control. At least three independent experiments were performed. 2.4. Determination of NO production RAW 264.7 cells seeded at a density of 3 × 105 cells in a 96-well plate were pretreated with various doses of sinigrin (1, 10, and 100 μg/mL) for 2 h and were then stimulated with 1 μg/mL of LPS. After incubation for 24 h, the supernatant was collected from each well, and the amount of nitrite (NO− 2 ) was measured by reaction with the Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 2.5% phosphoric acid) for 10 min at room temperature in the dark [22]. Absorbance was measured at 550 nm using the aforementioned microplate reader. The NO− 2 concentration was calculated from a NaNO2 standard curve, and NO− 2 levels were indicative of the amount of NO production. 2.5. Immunoblotting RAW 264.7 cells were seeded at 2 × 106 cells/well in 60-mm cell culture plates. Cells were treated with LPS (1 μg/mL) after being treated with sinigrin, and cellular material was extracted. After the treatment, the cells were washed in ice-cold PBS and suspended in RIPA lysis buffer (0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 1% NP-40, 1 μg/mL of pepstatin, 2 μg/mL of aprotinin, 10 μg/mL of leupeptin, 100 μg/mL of phenylsulfonyl fluoride, 50 mM of Tris, pH 8.0, and 150 mM of NaCl). The cells were incubated on ice for 1 h. The supernatant was collected after centrifugation at 13,000g for 15 min at 4 °C. The protein concentration was measured using a Bradford assay kit (Bio-Rad Lab, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard. The whole lysates were separated by 8– 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene fluoride (PVDF) membrane (Pall Corporation, Westborough, MA, USA). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.2% Tween 20 (TBST) at room temperature for 1 h. Membranes were probed with primary antibody and subsequently incubated with secondary antibodies conjugated to horseradish peroxidase for 50 min. The blots were developed using an enhanced chemoluminescence kit (Intron Biotechnology, Gyeonggi-do, Korea). Each band was quantitatively determined using ImageJ software. In each experiment the density ratio represented the relative intensity of each band against that of β-actin as a control. 2.6. Enzyme-linked immunosorbent assay (ELISA) RAW 264.7 cells were seeded at 2 × 10 6 cells/well in 6-well plates. The cells were pretreated with sinigrin for 2 h, followed by the addition of LPS (1 μg/mL) to the cultures for 24 h. The culture supernatants were collected, and the TNF-α and IL-6 concentrations in the culture supernatants were determined using a DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. Samples were assessed in triplicate relative to standards supplied by the manufacturer. 2.7. RNA interference RAW 264.7 cells were plated in 6 well culture plate and transiently transfected with NLRP3 siRNA (Genolution Parmaceuticals Inc., Seoul, Korea) mixed with siRNA transfection reagent (iNtRON Biotechnology, Inc. Sungnam, Korea) according to the manufacturer's instructions. To knockdown of endogenous NLRP3, cells were transiently transfected with siRNA for 20 h in a concentration of 100 nM.
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Fig. 1. Chemical structure and effects of sinigrin on cell viability in RAW 264.7 cells. A: Chemical structure of sinigrin hydrate. B: RAW 264.7 cells were pretreated with different concentrations of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for another 24 h. Cell viability was measured by MTT assay. The results are means ± SEM of quintuplicates from a representative experiment.
2.8. Luciferase reporter activity assay RAW 264.7 cells (5 × 105 cells/mL) were plated into each well of a 6well plate. The cells were transiently co-transfected with pGL3-NF-κB, pCMV-β-gal, and pcDNA3.1 plasmids using Lipofectamine Plus according to the manufacturer's protocol. Briefly, a transfection mixture
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containing 0.5 μg of pGL3-NF-κB and 0.2 μg of pCMV-β-gal was mixed with the Lipofectamine Plus and added to the cells. After transfection for 24 h, the cells were pretreated with sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for 4 h. Each well was washed with cold PBS. Luciferase activity was measured in the cell lysates using a Luciferase Assay System according to the manufacturer's instructions (Promega).
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Fig. 2. Effects of sinigrin on LPS-induced NO and PGE2 production in RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for another 24 h. A: The amount of nitrite production in the medium was measured using the Griess reaction. B, C, and D: Expression of iNOS, PGE2, and COX-2 protein was measured by Western blot assay. The β-actin protein level was considered as an internal control. The results illustrated are from a single experiment and are representative of three separate experiments.
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Fig. 4. Effects of sinigrin on LPS-induced activation and translocation of NF-κB in RAW 264.7 cells. A: RAW 264.7 cells were transfected with a pGL3-NF-κB-Luc reporter plasmid and pCMVβ-gal, pretreated with sinigrin for 2 h, and stimulated with LPS (1 μg/mL) for 4 h. Luciferase activity in the cells was measured. The results are means ± SEM of three experiments. *p b 0.05, significantly different from the group treated with LPS. B: RAW 264.7 cells were preincubated with or without various concentrations of sinigrin for 2 h and then treated with LPS for 4 h. The whole cell lysates were analyzed by Western blot assay with anti-IκBα antibody. C: The nuclear protein levels of p65 were detected by Western blot assay to analyze the translocation of NF-κB. The β-actin protein level was considered as an internal control. D: RAW 264.7 cells were pretreated with or without sinigrin for 2 h and then stimulated (or not) with LPS for 4 h. After incubation with the NF-κB p65 primary antibody followed by FITC-labeled anti-rabbit IgG antibody, the cells were visualized by means of fluorescence microscopy. The results illustrated are from a single experiment and are representative of three separate experiments.
2.9. Immunofluorescence assay The translocation of NF-κB proteins in LPS-induced RAW 264.7 cells was determined by means of immunofluorescence microscopy. RAW 264.7 cells were grown on glass coverslips, 22 mm in diameter, at a density of 2 × 106 cells, and they were then pretreated with sinigrin (1 to 100 μg/mL) for 2 h and stimulated with LPS (1 μg/mL) for 4 h. Cells
were washed in PBS and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, and washed in PBS. Ice-cold methanol was added to the cells, which were then incubated at − 20 °C for 10 min and washed in PBS. They were permeabilized with 1% BSA/ 0.2% Triton X-100/PBS for 1 h. Thereafter, they were washed in PBS and incubated with antibody against NF-κB p65 overnight at 4 °C. After being washed in PBS, the cells were incubated for 1 h with anti-
Fig. 3. Effects of sinigrin on the production, expression, and mRNA levels of TNF-α, IL-6, IL-1β, and IL-18 in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for another 24 h. A: The levels of cytokines in the medium were measured using an ELISA kit. B: The protein levels of TNF-α, IL-6, IL-1β, and IL-18 were measured by Western blot assay. C: The mRNA expression of TNF-α, IL-6, IL-1β, and IL-18 was determined by RT-PCR. The results are means ± SEM of quintuplicates from a representative experiment. *p b 0.05, significantly different from the group treated with LPS.
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rabbit IgG-fluorescein isothiocyanate (FITC) in 1% BSA/0.05% Triton X100/PBS. The cells were washed thoroughly, and samples were mounted with glycerol/PBS (4:1) and photographed using a model BX51 fluorescence microscope (Olympus Optical, Tokyo, Japan). 2.10. Quantitative real-time polymerase chain reaction (qRT-PCR) The expression of cytokine mRNA was determined by qRT-PCR analysis. After drug treatment, the total RNA was isolated from the cultured cells using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. qRT-PCR was performed using cytokinespecific primers to identify cDNA. The cDNA was amplified in 20 μL of PCR (8 μL of cDNA solution in water, 1 μL of forward primer and reverse primer, and 10 μL of Power SYBR Green PCR Master Mix) in a quantitative real-time PCR system, and fluorescence was monitored at each cycle. The TNF-α RT-PCR primers were 5′-CCCTCACACTCAGATCATCTTCT-3′ (sense) and 5′-GCTACGACGTGGGCTACAG-3′ (antisense). The IL-6 RT-PCR primers were 5′-CCACGGCCTTCCCTACTTC-3′ (sense) and 5′-TTGGGAGTGGTATCCTCTGTGA-3′ (antisense). The IL-1β RT-PCR primers were 5′-TTGACGGACCCCAAAAGATG-3′ (sense) and 5′-TGGACAGCCCAGGTCAAAG-3′ (antisense). The IL-18 RT-PCR primers were 5′-CTGAAGAAAATGGAGACCTGGAA-3′ (sense) and 5′-TCCGTATTACTGCGGTTGTACAGT-3′ (antisense). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RT-PCR primers were 5′-TGCATCCTGCACCACCAA-3′ (sense) and 5′-TCC ACG ATG CCA AAG TTG TC-3′ (antisense). 2.11. Statistical analyses For each experiment, data were obtained in triplicate and were reported as means ± SEM. Comparisons of the means for the sinigrintreated cells and the untreated control cells were analyzed using ANOVA and Student's t-test, and significant values are represented by an asterisk (*p b 0.05).
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and D, the levels of PGE2 and COX-2 expression were markedly upregulated by LPS. However, increased PGE2 and COX-2 expression was attenuated by sinigrin in a concentration-dependent manner. Taken together, these results suggest that sinigrin regulates the function of activated macrophage by inhibiting the production of inflammatory mediators. 3.3. Effect of sinigrin on cytokine production in LPS-stimulated RAW 264.7 cells To investigate whether sinigrin influences the production of pro-inflammatory cytokines that are produced by activated macrophages in inflammatory responses, RAW 264.7 cells were pretreated with 1– 100 μg/mL of sinigrin for 2 h before stimulation with LPS for 24 h. ELISA assay showed that LPS significantly increased the release of TNF-α and IL-6, and sinigrin concentration-dependently reduced these LPS-enhanced cytokines (Fig. 3A). Moreover, in good agreement with the ELISA results, sinigrin treatment resulted in the expression of TNF-α and IL-6 in LPS-stimulated cells. In addition, the expression of IL-1β and IL-18 was inhibited by sinigrin in a concentration-dependent manner (Fig. 3B). Furthermore, we examined whether sinigrin exerts the inhibitory effect on LPS-induced cytokine expression at the level of transcription. To investigate gene transcription level of mRNA, quantitative RT-PCR analysis was performed. As shown in Fig. 3C, treatment of cells with LPS resulted in increased expression of cytokine mRNA compared to untreated cells. However, the markedly increased transcription of cytokines in LPS-treated cells was significantly suppressed by pretreatment of cells with sinigrin in a concentration-dependent manner. 3.4. Effect of sinigrin on NF-κB activation and IκBα degradation in LPS-stimulated macrophages
3. Results 3.1. Effect of sinigrin on RAW 264.7 cell viability The cytotoxic effect of sinigrin on RAW 264.7 cells was examined using the MTT assay. The cells were treated with various concentrations (1–200 μg/mL) of sinigrin in the presence or absence of LPS (1 μg/mL) for 24 h. Sinigrin did not affect the viability of cells (Fig. 1B). Thus, 1– 100 μg/mL of sinigrin were used in all subsequent experiments. 3.2. Effect of sinigrin on NO and prostaglandin E2 (PGE2) production in LPStreated cells The inhibitory effect of sinigrin on NO and PGE2 production was assessed to determine whether sinigrin possesses an anti-inflammatory effect in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pretreated with or without 1–100 μg/mL of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for 24 h. LPS treatment resulted in a significant increase in NO production, whereas the production of NO was inhibited by sinigrin in LPS-stimulated RAW 264.7 cells (Fig. 2A). Treatment with sinigrin also suppressed iNOS expression in a concentration-dependent manner (Fig. 2B). Next, we evaluated the effect of sinigrin on PGE2 and cyclooxygenase-2 (COX-2) production in LPS-stimulated cells. As shown in Fig. 2C
Since NF-κB is an essential transcription factor involved in the expression of iNOS and several cytokines mediating inflammatory responses, we examined the effect of sinigrin on NF-κB activation. RAW 264.7 cells were pretreated with 1–100 μg/mL of sinigrin for 2 h and then stimulated with LPS for 4 h. As shown in Fig. 4A, stimulation of the cells with LPS resulted in a 1.75-fold increase in luciferase activity. This increased activity was significantly inhibited by sinigrin in a concentration-dependent manner. We next determined whether sinigrin affects the degradation of IκBα. This is because NF-κB is translocated to the nucleus by the degradation of IκBα upon stimulation with LPS. As expected, stimulation with LPS caused significant degradation of IκBα in a time-dependent manner (Fig. 4B). However, LPS-induced degradation of IκBα did not occur in the cells treated with sinigrin. We also determined the effect of sinigrin on the expression and translocation of NF-κB p65. As shown in Fig. 4C, NF-κB p65 was translocated to the nucleus in RAW 264.7 cells stimulated with LPS for 4 h. However, preincubation with sinigrin significantly inhibited NF-κB p65 translocation in a concentration-dependent manner. In addition, immunofluorescence analysis showed that sinigrin blocked NF-κB p65 translocation from the cytosol to the nucleus (Fig. 4D). Collectively, these results demonstrate that sinigrin inhibits LPS-induced NF-κB activation, which could reduce the production of LPS-inducible inflammatory mediators.
Fig. 5. Effects of sinigrin on LPS-induced NLRP3 inflammasome activation and the expression of IL-1β in LPS-induced RAW 264.7 cells. A: RAW 264.7 cells were pretreated with the indicated concentration of sinigrin for 2 h and then incubated with LPS for 7 h and ATP for 1 h. B, C, and D: RAW 264.7 cells were pretreated with the indicated concentration of sinigrin for 2 h and then incubated with LPS for 23 h and ATP for 1 h. E: RAW 264.7 cells were pretreated with the indicated concentration of allyl isothiocyanate for 2 h and then incubated with LPS for 7 h and ATP for 1 h. F, G, and H: RAW 264.7 cells were pretreated with the indicated concentration of allyl isothiocyanate for 2 h and then incubated with LPS for 23 h and ATP for 1 h. The whole cell lysates were analyzed by Western blot assay. The results illustrated are from a single experiment and are representative of three separate experiments.
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Fig. 6. Sinigrin inhibits the phosphorylation of MAPKs in LPS-induced RAW 264.7 cells. A: RAW 264.7 cells were treated with LPS for several times in the absence and presence of sinigrin (100 μg/mL). B: RAW 264.7 cells were pretreated with the indicated concentration of sinigrin for 2 h and then incubated with LPS for 20 min. C: RAW 264.7 cells were pretreated with vehicle (medium) or sinigrin for 2 h in the absence or presence of the p38 MAPK inhibitor SB203580 (10 μM), the ERK1/2 inhibitor PD98059 (20 μM), and the JNK inhibitor SP600125 (10 μM) and then stimulated with LPS. The whole cell lysates were analyzed by Western blot assay. The results illustrated are from a single experiment and are representative of three separate experiments. D: RAW 264.7 cells were treated with NF-κB inhibitor Bay 11-7082 in the absence and presence of sinigrin (100 μg/mL) for 2 h and then incubated with LPS for 24 h. E: RAW 264.7 cells were transfected with siNLRP3 and treated with sinigrin (100 μg/mL) for 2 h and then incubated with LPS for 24 h.
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Fig. 6 (continued).
3.5. Effect of sinigrin on NLRP3 inflammasome activation in LPS-stimulated RAW 264.7 cells The NLRP3 inflammasome is the best characterized inflammasome activated by microbes and endogenous indicators of cell stress such as ATP, which trigger the maturation of IL-1β and IL-18 [1]. To investigate the effect of sinigrin on NLRP3 inflammasome activation, we treated the cells with LPS and 5 μM of ATP to trigger NLRP3 activation, and we measured the protein expression of NLRP3, ASC, and caspase-1 using Western blot analysis. The present data showed that sinigrin inhibited NLRP3 expression in a concentration-dependent manner (Fig. 5A). Furthermore, the level of ASC expression, which increased following LPS and ATP treatments, appeared to be decreased in sinigrin-treated cells (Fig. 5B). We also observed that sinigrin treatment suppressed the expression of caspase-1 and the production of IL-1β in LPS/ATP-treated macrophages (Fig. 5C–D). These data suggest that sinigrin inhibits IL-1β secretion by suppressing NLRP3 activation and caspase-1 expression. 3.6. Effect of allyl isothiocyanate on NLRP3 inflammasome activation in LPS-treated RAW 264.7 cells Cruciferous vegetables contain sinigrin, which is enzymatically broken down into allyl isothiocyanate (AITC) and other metabolites by the thioglucosidase enzyme myrosinase. For this reason, we examined whether the sinigrin metabolite allyl isothiocyanate affects the NLRP3 inflammasome activation and IL-1β in RAW 264.7 cells. Cells were pretreated with 1–100 μg/mL of allyl isothiocyanate for 2 h and then incubated with LPS. We examined the protein expression of NLRP3, ASC, and caspase-1. Allyl isothiocyanate inhibited the expression of these proteins in a concentration-dependent manner (Fig. 5E–G). These data indicated that allyl isothiocyanate inhibits IL-1β secretion by impairing the NLRP3 inflammasome (Fig. 5H). 3.7. Effect of sinigrin on MAP kinases in LPS-stimulated RAW 264.7 cells Since MAPKs activated by LPS have been known to play an important role in the production of inflammatory mediators and NF-κB activation, we investigated the effect of sinigrin on MAPKs activation. RAW 264.7 cells were treated with LPS for several times in the absence and presence of sinigrin. Three MAPK proteins were reached their peak in early
time and the increased phosphorylation of MAPKs was diminished by sinigrin (Fig. 6A). RAW 264.7 cells were pretreated with 1–100 μg/mL of sinigrin for 2 h before being stimulated with LPS for 20 min. As shown in Fig. 6B, LPS treatment induced p38, ERK, and JNK phosphorylation, whereas the increased phosphorylation of p38 MAPK and JNK, but not of ERK1/2, was attenuated in the presence of sinigrin. Next, to confirm whether the MAPK signaling pathway is involved in NLRP3 and pro-inflammatory cytokine expression in the LPS-stimulated RAW 264.7 cells, we investigated the effects of the p38 MAPK inhibitor SB203580, the ERK1/2 inhibitor PD98059, and the JNK inhibitor SP600125 on LPS-induced NLRP3 and pro-inflammatory cytokine expression. As shown in Fig. 6C, pretreatment of RAW 264.7 cells with inhibitors for 2 h before LPS exposure inhibited the expression of NLRP3 and pro-inflammatory cytokines upregulated by LPS. Together, these data suggest that MAPK pathways are involved in NLRP3 and pro-inflammatory cytokine gene expression and that sinigrin inhibits the production of pro-inflammatory mediators by blocking the p38 MAPK and JNK pathways. Furthermore, we examined whether NF-κB and NLRP3 signaling pathways are involved in pro-inflammatory cytokine level in the LPSstimulated RAW 264.7 cells (Fig. 6D and E). Treatment of cells with LPS resulted in increased level of cytokine compared to untreated cells. However, elevated levels of cytokines were significantly decreased by pretreatment of cells with sinigrin alone, combination of sinigrin and NF-κB inhibitor or combination of sinigrin and siNLRP3 followed by LPS. Collectively, these results demonstrate that NF-κB and NLRP3 signaling pathways are involved in pro-inflammatory cytokine gene expression and that sinigrin suppressed the level of pro-inflammatory cytokine through inhibition of the NF-κB and NLRP3 signaling pathways. 4. Discussion In the present study, we elucidated the ability and molecular mechanisms of sinigrin to inhibit the production of inflammatory mediators and NLRP3 inflammasome activation in LPS-stimulated macrophages. Although sinigrin has been reported to be an effective anti-inflammatory activity [22], its exact mechanism in the production of inflammatory mediators is still unknown. To the best knowledge of our knowledge, this is the first report describing mechanisms involved in the anti-inflammatory effects of sinigrin, which suppresses the production of
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pro-inflammatory mediators via downregulation of p38 and JNK phosphorylation, NF-κB activation, and NLRP3 inflammasome activation in activated macrophages. Macrophages are an extremely important component of the innate immune system and play a critical role in the host defense mechanism during inflammation and infection. Our data demonstrated that LPSstimulated RAW 264.7 cells strongly produce NO, PGE2, and pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IL-18. NO plays a critical role in the innate immune response and is an important inflammatory mediator that is synthesized from arginine by iNOS [23]. However, overproduction of NO in macrophages could lead to inflammation, cytotoxicity, and immune disorders [24]. Moreover, PGE2 is produced at inflammatory sites by COX-2 and has also been implicated as an important mediator of the inflammatory process [25]. It has been suggested that the induction of COX-2 activity and the subsequent production of PGE2 are closely related to NO production [26]. Thus, a reduction in PGE2 and COX-2 levels may be useful for preventing inflammation. In this study, sinigrin treatment significantly suppressed the production of TNF-α, IL-6, IL-1β, and IL-18 and inhibited NO production through the suppression of iNOS expression in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. In addition, PGE2 production was suppressed by sinigrin via the inhibition of COX-2 expression in LPS-stimulated cells. Based on these findings, sinigrin exerts anti-inflammatory activity by inhibiting the production of inflammatory mediators in LPS-treated macrophages. Several studies have shown that the inflammatory process is a physiological immune response initiated by cell and tissue damage and pathogen invasion, which is regulated mainly by the MAPK and NF-κB signaling pathways in macrophages [27,28]. Diverse stimuli, including LPS, activate MAPKs, leading to the production of inflammatory mediators, and these pathways are also involved in iNOS, COX-2 and pro-inflammatory cytokine expression such TNF-α and IL-6 in activated macrophages [8,10,29]. Thus, in this study, we examined the effect of sinigrin on the activation of MAPKs in LPS-induced RAW 264.7 cells. In agreement with previous reports, LPS treatment resulted in the activation of p38, JNK and ERK, whereas the phosphorylation of p38 and JNK was inhibited by sinigrin. These results suggest that the inhibition of LPS-induced inflammatory mediators by sinigrin is mediated by the p38 and JNK pathways but not ERK1/2 pathway. However, it has been suggested that the PI3K/Akt pathway is involved in iNOS or COX-2 gene expression in activated macrophages [30]. Therefore, it is plausible that the anti-inflammatory activity of sinigrin might be related to inhibition of PI3K/Akt activation. NF-κB is an essential transcription factor involved in the inflammatory response, and it plays an important role in the production of inflammatory mediators such as NO, PGE2, and pro-inflammatory cytokines [31–35]. In addition, it has been suggested that inducible NF-κB activation requires the nuclear translocation of p65 through the phosphorylation and degradation of IκBα to prompt transcription of pro-inflammatory genes [36,37]. In a concentration-dependent manner, sinigrin blocked activation of NF-κB through the inhibition of IκBα degradation and subsequent p65 nuclear translocation in LPS-stimulated RAW 264.7 cells, suggesting that this inhibitory mechanism is associated with the suppressive effect of sinigrin on the production of inflammatory mediators. Therefore, it appears that inhibition of iNOS, COX-2, and pro-inflammatory cytokine expression is mainly associated with suppression of NF-κB pathway. These results indicate novel findings to address the effects of sinigrin via NF-κB pathway in response to LPS. However, our data do not totally rule out the possibility that sinigrin inhibits the activity ATF-2, a transcription factor, because ATF-2 has been suggested to be involved in the expression of iNOS and COX-2 [38] and ATF-2 activity is known to be regulated by MAPK pathways [39]. In the present study, sinigrin treatment resulted in the inhibition of p38 and JNK activation induced by LPS, suggesting that sinigrin-mediated regulation of iNOS and COX-2 expression is partially mediated through suppression of the p38/JNK-ATF-2 pathway.
The inflammasome is a multi-protein complex that acts as a platform for IL-1β and IL-18 processing and production. The complex is located in the cytosol and also includes NLRP3, ASC, and caspase-1 [1,40]. Inflammasomes promote the cleavage of pro-IL-1β and pro-IL-18 into their active forms [41,42]. NLRP3 inflammasome can be activated in response to various stimuli, including ATP, crystals, danger-associated molecular patterns (DAMP), and pathogen-associated molecular patterns (PAMP) during, for example, metabolic dysregulation [7,43–47]. Extracellular ATP induces the activation of P2 receptor-mediated purinergic signaling on macrophages and promotes inflammation via NLRP3 inflammasome signaling. ATP binds to P2X7R in primed macrophages and promotes activation of NLRP3 inflammasome and the production of IL-1β and IL-18 [48,49]. In the present study, the activation of NLRP3 inflammasome was suppressed by sinigrin. Sinigrin significantly inhibited the expression of NLRP3 and ASC in a concentration-dependent manner and also suppressed caspase-1 expression, which regulates the processing of IL-1β and IL-18. In addition, we observed the anti-inflammatory effect of sinigrin in LPS-primed, ATP-stimulated RAW 264.7 cells. These results indicate that sinigrin blocked the production of IL-1β and IL-18 through NLRP3 inflammasome activation in LPSactivated macrophages. Additionally, it has been reported that NF-κB is involved in the expression of IL-1β, NLRP3 and caspase-1 and MAPK signaling play an important role in the expression of IL-1β and inflammasome components in macrophages [50–52] Based on these findings, the results of the present study suggest that sinigrin suppresses NLRP3 inflammasome and IL-1β expression and ultimately IL1β cytokine production through blocking these signal transduction pathways. The collective results demonstrated that sinigrin can suppress the production of pro-inflammatory mediators in activated macrophages, providing a possible molecular mechanism for the anti-inflammatory activity of sinigrin. Our results revealed that sinigrin is able to inhibit the production of LPS-induced inflammatory mediators via the p38, JNK, and NF-κB signaling pathways. In addition, the inhibitory effect of sinigrin could be mediated by the suppression of NLRP3 inflammasome activation. Taken together, these data indicated that sinigrin could have significant effects on the modulation of innate immune response and potential therapeutic value in preventing inflammation- and NLRP3-related inflammatory diseases. References [1] K. Schroder, J. Tschopp, The inflammasomes, Cell 140 (6) (2010) 821–832. [2] C. Dodé, N. Le Duˆ, L. Cuisset, F. Letourneur, J.-M. Berthelot, G. Vaudour, A. Meyrier, R.A. Watts, G.D. Scott, A. Nicholls, New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel mutation underlies both syndromes, Am. J. Hum. Genet. 70 (6) (2002) 1498–1506. [3] J. Feldmann, A.-M. Prieur, P. Quartier, P. Berquin, S. Certain, E. Cortis, D. TeillacHamel, A. Fischer, G. de Saint Basile, Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes, Am. J. Hum. Genet. 71 (1) (2002) 198–203. [4] H.M. Hoffman, J.L. Mueller, D.H. Broide, A.A. Wanderer, R.D. Kolodner, Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle–Wells syndrome, Nat. Genet. 29 (3) (2001) 301–305. [5] T. Strowig, J. Henao-Mejia, E. Elinav, R. Flavell, Inflammasomes in health and disease, Nature 481 (7381) (2012) 278–286. [6] T.A. Wynn, A. Chawla, J.W. Pollard, Macrophage biology in development, homeostasis and disease, Nature 496 (7446) (2013) 445–455. [7] C. Dostert, V. Pétrilli, R. Van Bruggen, C. Steele, B.T. Mossman, J. Tschopp, Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica, Science 320 (5876) (2008) 674–677. [8] G. Pearson, F. Robinson, T. Beers Gibson, B.E. Xu, M. Karandikar, K. Berman, M.H. Cobb, Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions, Endocr. Rev. 22 (2) (2001) 153–183. [9] S.J. Ajizian, B.K. English, E.A. Meals, Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-γ, J. Infect. Dis. 179 (4) (1999) 939–944. [10] T. Uto, M. Fujii, D.X. Hou, 6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Sinigrin inhibits production of inflammatory mediators by suppressing NF-κB/MAPK pathways or NLRP3 inflammasome activation in macrophages Hee-Weon Lee a, Chung Gi Lee a, Dong-Kwon Rhee a, Sung Hee Um b, Sukneung Pyo a,⁎ a b
School of Pharmacy, Sungkyunkwan University, Suwon, Gyunggi-do 16419, Republic of Korea Department of Molecular Cell Biology, Samsung Biomedical Research Institute, School of Medicine, Sungkyunkwan University, Suwon, Gyunggi-do 16419, Republic of Korea
a r t i c l e
i n f o
Article history: Received 29 July 2016 Received in revised form 13 January 2017 Accepted 30 January 2017 Available online xxxx Keywords: 2-Propenyl glucosinolate Inflammation NLRP3 inflammasome NF-κB/MAPK Macrophage
a b s t r a c t Sinigrin (2-propenyl glucosinolate) is found mainly in broccoli, brussels sprouts, and black mustard seeds. Recently, sinigrin has received attention for its role in disease prevention and health. This study investigated the effect of sinigrin on macrophage function, including the activity of Nod-like receptor protein 3 (NLRP3) inflammasome. In a concentration-dependent manner, sinigrin inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production and the expression of COX-2 and prostaglandin E2 (PGE2) in RAW 264.7 cells. In addition, sinigrin significantly suppressed the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 via suppression of MAPK phosphorylation and nuclear factor-kappa B (NF-κB) activity. Treatment with sinigrin decreased IL-1β and IL-18 production and concurrently suppressed NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 expression in LPS/ATP-stimulated cells, suggesting that the blocking of NLRP3 inflammasome activation prevented the production of both cytokines. Collectively, these results suggest that sinigrin has immunomodulatory effects by suppressing the production of inflammatory mediators, possibly by inhibiting the NF-κB/MAPK pathways or NLRP3 inflammasome activation. Our findings also provide evidence that the pharmacological modulation of sinigrin could have an anti-inflammatory effect. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Inflammation plays a critical role in the development and progression of various diseases. Rather than being a single process, inflammation is a complex, multifaceted phenomenon consisting of individual events that may differ in their responses to pathogens. Many bacterial components and products, including lipopolysaccharide (LPS), can initiate local inflammatory responses. The inflammatory response depends on a complicated network of cellular components, including inflammatory mediators, which contribute to the pathogenesis of various diseases. Inflammasomes are multi-subunit complexes activated by cellular infection or stress, and it has been suggested that inflammasome activation is associated with diverse inflammatory diseases [1]. In particular, the NLRP3 inflammasome plays a critical role in the development and progression of many diseases [2–4], and its activation by LPS recruits the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and pro-
⁎ Corresponding author at: School of Pharmacy, Sungkyunkwan University, Suwon City, Gyunggi-do 440-746, Republic of Korea. E-mail address: [email protected] (S. Pyo).
http://dx.doi.org/10.1016/j.intimp.2017.01.032 1567-5769/© 2017 Elsevier B.V. All rights reserved.
caspase-1, which results in the processing and secretion of pro-inflammatory cytokines such as interleukin (IL)-1β and IL-18 [5]. An important cell type in the inflammatory process is an innate, phagocyte-like macrophage. In the inflammatory response, the macrophage population increases, an event that appears to play a major role at the site of tissue inflammation [6]. Macrophages are critical for recognizing pathogens and for initiating and amplifying the inflammatory cascade, mainly through the regulation of inducible enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS), and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-6, and IL-1β [7]. These inflammatory mediators are induced by lipopolysaccharide (LPS) in macrophages and MAPK/NF-κB pathways play important roles in LPS-induced pro-inflammatory mediators and cytokines expression [8–10]. In addition, persistent pro-inflammatory macrophages contribute to the development of chronic inflammatory diseases, such as atherosclerosis, type 2 diabetes, and hay fever [11]. Overproduction of inflammatory mediators has been implicated in many manifestations of disease. Therefore, modulating macrophage-mediated inflammatory responses could potentially reduce the incidence of various inflammatory diseases. In recent years, pharmacological preparations of plants and their components have attracted considerable interest because of their ability
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to prevent and treat various chronic diseases and to improve health [12]. Many plants are a rich source of certain functional food factors and bioactive metabolites that might have immunomodulatory properties [13–15]. Sinigrin is a thioglucoside of cruciferous vegetables, including brussels sprouts and broccoli, and the seeds of black mustard. It has potent antitumor activity, and its hydrolysis products are effective against bacteria [16,17]. In addition, sinigrin has antioxidant activity and suppresses the production of nitric oxide (NO) in LPS-treated animals [18,19]. In preliminary experiments, we have shown that the exposure of macrophages to sinigrin induces significant changes in the expression pattern of proinflammatory mediator genes [20] However, its immunomodulating effects on the function of macrophages and the underlying mechanisms remain unknown. The objective of this study was to examine the effects of sinigrin on LPS-induced inflammatory responses in macrophages and the potential mechanisms involved. We found that sinigrin inhibited the LPS-induced production of pro-inflammatory mediators by suppressing the nuclear factor-kappa B/mitogen-activated protein kinase (NF-κB/MAPK) pathways or the activation of NLRP3 inflammasome. 2. Materials and methods 2.1. Chemical reagents and antibodies All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. Sinigrin hydrate was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Lipofetamine Plus, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). The reporter plasmid pGL3-NF-κB used in the luciferase assay system was obtained from Promega (Madison, WI, USA), and pCMV-β-gal was obtained from Lonza (Walkersville, MD). IL-6 and tumor necrosis factor-alpha (TNF-α) ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against inducible NO synthase (iNOS), extracellular signal-regulated kinase (ERK)1/2, phospho-ERK1/ 2, p38, phospho-p38, C-Jun N-terminal kinase (JNK), phospho-JNK, IκBα, NF-κB, NLRP3, caspase-1, IL-1, IL-18, α-tubulin, lamin A, and βactin were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.2. Cell culture The murine macrophage cell line RAW 264.7 was obtained from ATCC (Rockville, MD, USA), and the RAW 264.7 cells were cultured in DMEM (Lonza) supplemented with 2 mM of L -glutamine, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 10% heatinactivated FBS. The cells were incubated at 37 °C in a fully humidified incubator containing 5% CO2 and were subcultured twice weekly. RAW 264.7 cells were pretreated for 2 h with sinigrin, followed by treatment with LPS (1 μg/mL) for various prespecified times. The cells were harvested by centrifugation at 13,000g for 3 min at 4 °C, and the pellets were lysed by radioimmunoprecipitation assay buffer (RIPA buffer). 2.3. Assessment of cell proliferation RAW 264.7 cells were seeded at a concentration of 3 × 105 cells/well in 96-well tissue culture plates and were treated with various concentrations of sinigrin for 24 h. Cell viability was measured for 2 h by means of quantitative colorimetric assay with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which showed the mitochondrial activity of living cells, as previously described [21]. The extent of the reduction of MTT to formazan within cells was quantified by measuring the optical density at 550 nm using a microplate reader (Molecular Devices, Carlsbad, CA, USA). The blank control contained only cell culture medium, and the absorbance of untreated cultures
was set at 100%. Cell viability was expressed as a percentage of the untreated control. At least three independent experiments were performed. 2.4. Determination of NO production RAW 264.7 cells seeded at a density of 3 × 105 cells in a 96-well plate were pretreated with various doses of sinigrin (1, 10, and 100 μg/mL) for 2 h and were then stimulated with 1 μg/mL of LPS. After incubation for 24 h, the supernatant was collected from each well, and the amount of nitrite (NO− 2 ) was measured by reaction with the Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 2.5% phosphoric acid) for 10 min at room temperature in the dark [22]. Absorbance was measured at 550 nm using the aforementioned microplate reader. The NO− 2 concentration was calculated from a NaNO2 standard curve, and NO− 2 levels were indicative of the amount of NO production. 2.5. Immunoblotting RAW 264.7 cells were seeded at 2 × 106 cells/well in 60-mm cell culture plates. Cells were treated with LPS (1 μg/mL) after being treated with sinigrin, and cellular material was extracted. After the treatment, the cells were washed in ice-cold PBS and suspended in RIPA lysis buffer (0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 1% NP-40, 1 μg/mL of pepstatin, 2 μg/mL of aprotinin, 10 μg/mL of leupeptin, 100 μg/mL of phenylsulfonyl fluoride, 50 mM of Tris, pH 8.0, and 150 mM of NaCl). The cells were incubated on ice for 1 h. The supernatant was collected after centrifugation at 13,000g for 15 min at 4 °C. The protein concentration was measured using a Bradford assay kit (Bio-Rad Lab, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard. The whole lysates were separated by 8– 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene fluoride (PVDF) membrane (Pall Corporation, Westborough, MA, USA). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.2% Tween 20 (TBST) at room temperature for 1 h. Membranes were probed with primary antibody and subsequently incubated with secondary antibodies conjugated to horseradish peroxidase for 50 min. The blots were developed using an enhanced chemoluminescence kit (Intron Biotechnology, Gyeonggi-do, Korea). Each band was quantitatively determined using ImageJ software. In each experiment the density ratio represented the relative intensity of each band against that of β-actin as a control. 2.6. Enzyme-linked immunosorbent assay (ELISA) RAW 264.7 cells were seeded at 2 × 10 6 cells/well in 6-well plates. The cells were pretreated with sinigrin for 2 h, followed by the addition of LPS (1 μg/mL) to the cultures for 24 h. The culture supernatants were collected, and the TNF-α and IL-6 concentrations in the culture supernatants were determined using a DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. Samples were assessed in triplicate relative to standards supplied by the manufacturer. 2.7. RNA interference RAW 264.7 cells were plated in 6 well culture plate and transiently transfected with NLRP3 siRNA (Genolution Parmaceuticals Inc., Seoul, Korea) mixed with siRNA transfection reagent (iNtRON Biotechnology, Inc. Sungnam, Korea) according to the manufacturer's instructions. To knockdown of endogenous NLRP3, cells were transiently transfected with siRNA for 20 h in a concentration of 100 nM.
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Fig. 1. Chemical structure and effects of sinigrin on cell viability in RAW 264.7 cells. A: Chemical structure of sinigrin hydrate. B: RAW 264.7 cells were pretreated with different concentrations of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for another 24 h. Cell viability was measured by MTT assay. The results are means ± SEM of quintuplicates from a representative experiment.
2.8. Luciferase reporter activity assay RAW 264.7 cells (5 × 105 cells/mL) were plated into each well of a 6well plate. The cells were transiently co-transfected with pGL3-NF-κB, pCMV-β-gal, and pcDNA3.1 plasmids using Lipofectamine Plus according to the manufacturer's protocol. Briefly, a transfection mixture
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Fig. 2. Effects of sinigrin on LPS-induced NO and PGE2 production in RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for another 24 h. A: The amount of nitrite production in the medium was measured using the Griess reaction. B, C, and D: Expression of iNOS, PGE2, and COX-2 protein was measured by Western blot assay. The β-actin protein level was considered as an internal control. The results illustrated are from a single experiment and are representative of three separate experiments.
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Fig. 4. Effects of sinigrin on LPS-induced activation and translocation of NF-κB in RAW 264.7 cells. A: RAW 264.7 cells were transfected with a pGL3-NF-κB-Luc reporter plasmid and pCMVβ-gal, pretreated with sinigrin for 2 h, and stimulated with LPS (1 μg/mL) for 4 h. Luciferase activity in the cells was measured. The results are means ± SEM of three experiments. *p b 0.05, significantly different from the group treated with LPS. B: RAW 264.7 cells were preincubated with or without various concentrations of sinigrin for 2 h and then treated with LPS for 4 h. The whole cell lysates were analyzed by Western blot assay with anti-IκBα antibody. C: The nuclear protein levels of p65 were detected by Western blot assay to analyze the translocation of NF-κB. The β-actin protein level was considered as an internal control. D: RAW 264.7 cells were pretreated with or without sinigrin for 2 h and then stimulated (or not) with LPS for 4 h. After incubation with the NF-κB p65 primary antibody followed by FITC-labeled anti-rabbit IgG antibody, the cells were visualized by means of fluorescence microscopy. The results illustrated are from a single experiment and are representative of three separate experiments.
2.9. Immunofluorescence assay The translocation of NF-κB proteins in LPS-induced RAW 264.7 cells was determined by means of immunofluorescence microscopy. RAW 264.7 cells were grown on glass coverslips, 22 mm in diameter, at a density of 2 × 106 cells, and they were then pretreated with sinigrin (1 to 100 μg/mL) for 2 h and stimulated with LPS (1 μg/mL) for 4 h. Cells
were washed in PBS and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, and washed in PBS. Ice-cold methanol was added to the cells, which were then incubated at − 20 °C for 10 min and washed in PBS. They were permeabilized with 1% BSA/ 0.2% Triton X-100/PBS for 1 h. Thereafter, they were washed in PBS and incubated with antibody against NF-κB p65 overnight at 4 °C. After being washed in PBS, the cells were incubated for 1 h with anti-
Fig. 3. Effects of sinigrin on the production, expression, and mRNA levels of TNF-α, IL-6, IL-1β, and IL-18 in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for another 24 h. A: The levels of cytokines in the medium were measured using an ELISA kit. B: The protein levels of TNF-α, IL-6, IL-1β, and IL-18 were measured by Western blot assay. C: The mRNA expression of TNF-α, IL-6, IL-1β, and IL-18 was determined by RT-PCR. The results are means ± SEM of quintuplicates from a representative experiment. *p b 0.05, significantly different from the group treated with LPS.
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rabbit IgG-fluorescein isothiocyanate (FITC) in 1% BSA/0.05% Triton X100/PBS. The cells were washed thoroughly, and samples were mounted with glycerol/PBS (4:1) and photographed using a model BX51 fluorescence microscope (Olympus Optical, Tokyo, Japan). 2.10. Quantitative real-time polymerase chain reaction (qRT-PCR) The expression of cytokine mRNA was determined by qRT-PCR analysis. After drug treatment, the total RNA was isolated from the cultured cells using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. qRT-PCR was performed using cytokinespecific primers to identify cDNA. The cDNA was amplified in 20 μL of PCR (8 μL of cDNA solution in water, 1 μL of forward primer and reverse primer, and 10 μL of Power SYBR Green PCR Master Mix) in a quantitative real-time PCR system, and fluorescence was monitored at each cycle. The TNF-α RT-PCR primers were 5′-CCCTCACACTCAGATCATCTTCT-3′ (sense) and 5′-GCTACGACGTGGGCTACAG-3′ (antisense). The IL-6 RT-PCR primers were 5′-CCACGGCCTTCCCTACTTC-3′ (sense) and 5′-TTGGGAGTGGTATCCTCTGTGA-3′ (antisense). The IL-1β RT-PCR primers were 5′-TTGACGGACCCCAAAAGATG-3′ (sense) and 5′-TGGACAGCCCAGGTCAAAG-3′ (antisense). The IL-18 RT-PCR primers were 5′-CTGAAGAAAATGGAGACCTGGAA-3′ (sense) and 5′-TCCGTATTACTGCGGTTGTACAGT-3′ (antisense). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RT-PCR primers were 5′-TGCATCCTGCACCACCAA-3′ (sense) and 5′-TCC ACG ATG CCA AAG TTG TC-3′ (antisense). 2.11. Statistical analyses For each experiment, data were obtained in triplicate and were reported as means ± SEM. Comparisons of the means for the sinigrintreated cells and the untreated control cells were analyzed using ANOVA and Student's t-test, and significant values are represented by an asterisk (*p b 0.05).
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and D, the levels of PGE2 and COX-2 expression were markedly upregulated by LPS. However, increased PGE2 and COX-2 expression was attenuated by sinigrin in a concentration-dependent manner. Taken together, these results suggest that sinigrin regulates the function of activated macrophage by inhibiting the production of inflammatory mediators. 3.3. Effect of sinigrin on cytokine production in LPS-stimulated RAW 264.7 cells To investigate whether sinigrin influences the production of pro-inflammatory cytokines that are produced by activated macrophages in inflammatory responses, RAW 264.7 cells were pretreated with 1– 100 μg/mL of sinigrin for 2 h before stimulation with LPS for 24 h. ELISA assay showed that LPS significantly increased the release of TNF-α and IL-6, and sinigrin concentration-dependently reduced these LPS-enhanced cytokines (Fig. 3A). Moreover, in good agreement with the ELISA results, sinigrin treatment resulted in the expression of TNF-α and IL-6 in LPS-stimulated cells. In addition, the expression of IL-1β and IL-18 was inhibited by sinigrin in a concentration-dependent manner (Fig. 3B). Furthermore, we examined whether sinigrin exerts the inhibitory effect on LPS-induced cytokine expression at the level of transcription. To investigate gene transcription level of mRNA, quantitative RT-PCR analysis was performed. As shown in Fig. 3C, treatment of cells with LPS resulted in increased expression of cytokine mRNA compared to untreated cells. However, the markedly increased transcription of cytokines in LPS-treated cells was significantly suppressed by pretreatment of cells with sinigrin in a concentration-dependent manner. 3.4. Effect of sinigrin on NF-κB activation and IκBα degradation in LPS-stimulated macrophages
3. Results 3.1. Effect of sinigrin on RAW 264.7 cell viability The cytotoxic effect of sinigrin on RAW 264.7 cells was examined using the MTT assay. The cells were treated with various concentrations (1–200 μg/mL) of sinigrin in the presence or absence of LPS (1 μg/mL) for 24 h. Sinigrin did not affect the viability of cells (Fig. 1B). Thus, 1– 100 μg/mL of sinigrin were used in all subsequent experiments. 3.2. Effect of sinigrin on NO and prostaglandin E2 (PGE2) production in LPStreated cells The inhibitory effect of sinigrin on NO and PGE2 production was assessed to determine whether sinigrin possesses an anti-inflammatory effect in LPS-induced RAW 264.7 cells. RAW 264.7 cells were pretreated with or without 1–100 μg/mL of sinigrin for 2 h and then stimulated with LPS (1 μg/mL) for 24 h. LPS treatment resulted in a significant increase in NO production, whereas the production of NO was inhibited by sinigrin in LPS-stimulated RAW 264.7 cells (Fig. 2A). Treatment with sinigrin also suppressed iNOS expression in a concentration-dependent manner (Fig. 2B). Next, we evaluated the effect of sinigrin on PGE2 and cyclooxygenase-2 (COX-2) production in LPS-stimulated cells. As shown in Fig. 2C
Since NF-κB is an essential transcription factor involved in the expression of iNOS and several cytokines mediating inflammatory responses, we examined the effect of sinigrin on NF-κB activation. RAW 264.7 cells were pretreated with 1–100 μg/mL of sinigrin for 2 h and then stimulated with LPS for 4 h. As shown in Fig. 4A, stimulation of the cells with LPS resulted in a 1.75-fold increase in luciferase activity. This increased activity was significantly inhibited by sinigrin in a concentration-dependent manner. We next determined whether sinigrin affects the degradation of IκBα. This is because NF-κB is translocated to the nucleus by the degradation of IκBα upon stimulation with LPS. As expected, stimulation with LPS caused significant degradation of IκBα in a time-dependent manner (Fig. 4B). However, LPS-induced degradation of IκBα did not occur in the cells treated with sinigrin. We also determined the effect of sinigrin on the expression and translocation of NF-κB p65. As shown in Fig. 4C, NF-κB p65 was translocated to the nucleus in RAW 264.7 cells stimulated with LPS for 4 h. However, preincubation with sinigrin significantly inhibited NF-κB p65 translocation in a concentration-dependent manner. In addition, immunofluorescence analysis showed that sinigrin blocked NF-κB p65 translocation from the cytosol to the nucleus (Fig. 4D). Collectively, these results demonstrate that sinigrin inhibits LPS-induced NF-κB activation, which could reduce the production of LPS-inducible inflammatory mediators.
Fig. 5. Effects of sinigrin on LPS-induced NLRP3 inflammasome activation and the expression of IL-1β in LPS-induced RAW 264.7 cells. A: RAW 264.7 cells were pretreated with the indicated concentration of sinigrin for 2 h and then incubated with LPS for 7 h and ATP for 1 h. B, C, and D: RAW 264.7 cells were pretreated with the indicated concentration of sinigrin for 2 h and then incubated with LPS for 23 h and ATP for 1 h. E: RAW 264.7 cells were pretreated with the indicated concentration of allyl isothiocyanate for 2 h and then incubated with LPS for 7 h and ATP for 1 h. F, G, and H: RAW 264.7 cells were pretreated with the indicated concentration of allyl isothiocyanate for 2 h and then incubated with LPS for 23 h and ATP for 1 h. The whole cell lysates were analyzed by Western blot assay. The results illustrated are from a single experiment and are representative of three separate experiments.
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Fig. 6. Sinigrin inhibits the phosphorylation of MAPKs in LPS-induced RAW 264.7 cells. A: RAW 264.7 cells were treated with LPS for several times in the absence and presence of sinigrin (100 μg/mL). B: RAW 264.7 cells were pretreated with the indicated concentration of sinigrin for 2 h and then incubated with LPS for 20 min. C: RAW 264.7 cells were pretreated with vehicle (medium) or sinigrin for 2 h in the absence or presence of the p38 MAPK inhibitor SB203580 (10 μM), the ERK1/2 inhibitor PD98059 (20 μM), and the JNK inhibitor SP600125 (10 μM) and then stimulated with LPS. The whole cell lysates were analyzed by Western blot assay. The results illustrated are from a single experiment and are representative of three separate experiments. D: RAW 264.7 cells were treated with NF-κB inhibitor Bay 11-7082 in the absence and presence of sinigrin (100 μg/mL) for 2 h and then incubated with LPS for 24 h. E: RAW 264.7 cells were transfected with siNLRP3 and treated with sinigrin (100 μg/mL) for 2 h and then incubated with LPS for 24 h.
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Fig. 6 (continued).
3.5. Effect of sinigrin on NLRP3 inflammasome activation in LPS-stimulated RAW 264.7 cells The NLRP3 inflammasome is the best characterized inflammasome activated by microbes and endogenous indicators of cell stress such as ATP, which trigger the maturation of IL-1β and IL-18 [1]. To investigate the effect of sinigrin on NLRP3 inflammasome activation, we treated the cells with LPS and 5 μM of ATP to trigger NLRP3 activation, and we measured the protein expression of NLRP3, ASC, and caspase-1 using Western blot analysis. The present data showed that sinigrin inhibited NLRP3 expression in a concentration-dependent manner (Fig. 5A). Furthermore, the level of ASC expression, which increased following LPS and ATP treatments, appeared to be decreased in sinigrin-treated cells (Fig. 5B). We also observed that sinigrin treatment suppressed the expression of caspase-1 and the production of IL-1β in LPS/ATP-treated macrophages (Fig. 5C–D). These data suggest that sinigrin inhibits IL-1β secretion by suppressing NLRP3 activation and caspase-1 expression. 3.6. Effect of allyl isothiocyanate on NLRP3 inflammasome activation in LPS-treated RAW 264.7 cells Cruciferous vegetables contain sinigrin, which is enzymatically broken down into allyl isothiocyanate (AITC) and other metabolites by the thioglucosidase enzyme myrosinase. For this reason, we examined whether the sinigrin metabolite allyl isothiocyanate affects the NLRP3 inflammasome activation and IL-1β in RAW 264.7 cells. Cells were pretreated with 1–100 μg/mL of allyl isothiocyanate for 2 h and then incubated with LPS. We examined the protein expression of NLRP3, ASC, and caspase-1. Allyl isothiocyanate inhibited the expression of these proteins in a concentration-dependent manner (Fig. 5E–G). These data indicated that allyl isothiocyanate inhibits IL-1β secretion by impairing the NLRP3 inflammasome (Fig. 5H). 3.7. Effect of sinigrin on MAP kinases in LPS-stimulated RAW 264.7 cells Since MAPKs activated by LPS have been known to play an important role in the production of inflammatory mediators and NF-κB activation, we investigated the effect of sinigrin on MAPKs activation. RAW 264.7 cells were treated with LPS for several times in the absence and presence of sinigrin. Three MAPK proteins were reached their peak in early
time and the increased phosphorylation of MAPKs was diminished by sinigrin (Fig. 6A). RAW 264.7 cells were pretreated with 1–100 μg/mL of sinigrin for 2 h before being stimulated with LPS for 20 min. As shown in Fig. 6B, LPS treatment induced p38, ERK, and JNK phosphorylation, whereas the increased phosphorylation of p38 MAPK and JNK, but not of ERK1/2, was attenuated in the presence of sinigrin. Next, to confirm whether the MAPK signaling pathway is involved in NLRP3 and pro-inflammatory cytokine expression in the LPS-stimulated RAW 264.7 cells, we investigated the effects of the p38 MAPK inhibitor SB203580, the ERK1/2 inhibitor PD98059, and the JNK inhibitor SP600125 on LPS-induced NLRP3 and pro-inflammatory cytokine expression. As shown in Fig. 6C, pretreatment of RAW 264.7 cells with inhibitors for 2 h before LPS exposure inhibited the expression of NLRP3 and pro-inflammatory cytokines upregulated by LPS. Together, these data suggest that MAPK pathways are involved in NLRP3 and pro-inflammatory cytokine gene expression and that sinigrin inhibits the production of pro-inflammatory mediators by blocking the p38 MAPK and JNK pathways. Furthermore, we examined whether NF-κB and NLRP3 signaling pathways are involved in pro-inflammatory cytokine level in the LPSstimulated RAW 264.7 cells (Fig. 6D and E). Treatment of cells with LPS resulted in increased level of cytokine compared to untreated cells. However, elevated levels of cytokines were significantly decreased by pretreatment of cells with sinigrin alone, combination of sinigrin and NF-κB inhibitor or combination of sinigrin and siNLRP3 followed by LPS. Collectively, these results demonstrate that NF-κB and NLRP3 signaling pathways are involved in pro-inflammatory cytokine gene expression and that sinigrin suppressed the level of pro-inflammatory cytokine through inhibition of the NF-κB and NLRP3 signaling pathways. 4. Discussion In the present study, we elucidated the ability and molecular mechanisms of sinigrin to inhibit the production of inflammatory mediators and NLRP3 inflammasome activation in LPS-stimulated macrophages. Although sinigrin has been reported to be an effective anti-inflammatory activity [22], its exact mechanism in the production of inflammatory mediators is still unknown. To the best knowledge of our knowledge, this is the first report describing mechanisms involved in the anti-inflammatory effects of sinigrin, which suppresses the production of
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pro-inflammatory mediators via downregulation of p38 and JNK phosphorylation, NF-κB activation, and NLRP3 inflammasome activation in activated macrophages. Macrophages are an extremely important component of the innate immune system and play a critical role in the host defense mechanism during inflammation and infection. Our data demonstrated that LPSstimulated RAW 264.7 cells strongly produce NO, PGE2, and pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IL-18. NO plays a critical role in the innate immune response and is an important inflammatory mediator that is synthesized from arginine by iNOS [23]. However, overproduction of NO in macrophages could lead to inflammation, cytotoxicity, and immune disorders [24]. Moreover, PGE2 is produced at inflammatory sites by COX-2 and has also been implicated as an important mediator of the inflammatory process [25]. It has been suggested that the induction of COX-2 activity and the subsequent production of PGE2 are closely related to NO production [26]. Thus, a reduction in PGE2 and COX-2 levels may be useful for preventing inflammation. In this study, sinigrin treatment significantly suppressed the production of TNF-α, IL-6, IL-1β, and IL-18 and inhibited NO production through the suppression of iNOS expression in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. In addition, PGE2 production was suppressed by sinigrin via the inhibition of COX-2 expression in LPS-stimulated cells. Based on these findings, sinigrin exerts anti-inflammatory activity by inhibiting the production of inflammatory mediators in LPS-treated macrophages. Several studies have shown that the inflammatory process is a physiological immune response initiated by cell and tissue damage and pathogen invasion, which is regulated mainly by the MAPK and NF-κB signaling pathways in macrophages [27,28]. Diverse stimuli, including LPS, activate MAPKs, leading to the production of inflammatory mediators, and these pathways are also involved in iNOS, COX-2 and pro-inflammatory cytokine expression such TNF-α and IL-6 in activated macrophages [8,10,29]. Thus, in this study, we examined the effect of sinigrin on the activation of MAPKs in LPS-induced RAW 264.7 cells. In agreement with previous reports, LPS treatment resulted in the activation of p38, JNK and ERK, whereas the phosphorylation of p38 and JNK was inhibited by sinigrin. These results suggest that the inhibition of LPS-induced inflammatory mediators by sinigrin is mediated by the p38 and JNK pathways but not ERK1/2 pathway. However, it has been suggested that the PI3K/Akt pathway is involved in iNOS or COX-2 gene expression in activated macrophages [30]. Therefore, it is plausible that the anti-inflammatory activity of sinigrin might be related to inhibition of PI3K/Akt activation. NF-κB is an essential transcription factor involved in the inflammatory response, and it plays an important role in the production of inflammatory mediators such as NO, PGE2, and pro-inflammatory cytokines [31–35]. In addition, it has been suggested that inducible NF-κB activation requires the nuclear translocation of p65 through the phosphorylation and degradation of IκBα to prompt transcription of pro-inflammatory genes [36,37]. In a concentration-dependent manner, sinigrin blocked activation of NF-κB through the inhibition of IκBα degradation and subsequent p65 nuclear translocation in LPS-stimulated RAW 264.7 cells, suggesting that this inhibitory mechanism is associated with the suppressive effect of sinigrin on the production of inflammatory mediators. Therefore, it appears that inhibition of iNOS, COX-2, and pro-inflammatory cytokine expression is mainly associated with suppression of NF-κB pathway. These results indicate novel findings to address the effects of sinigrin via NF-κB pathway in response to LPS. However, our data do not totally rule out the possibility that sinigrin inhibits the activity ATF-2, a transcription factor, because ATF-2 has been suggested to be involved in the expression of iNOS and COX-2 [38] and ATF-2 activity is known to be regulated by MAPK pathways [39]. In the present study, sinigrin treatment resulted in the inhibition of p38 and JNK activation induced by LPS, suggesting that sinigrin-mediated regulation of iNOS and COX-2 expression is partially mediated through suppression of the p38/JNK-ATF-2 pathway.
The inflammasome is a multi-protein complex that acts as a platform for IL-1β and IL-18 processing and production. The complex is located in the cytosol and also includes NLRP3, ASC, and caspase-1 [1,40]. Inflammasomes promote the cleavage of pro-IL-1β and pro-IL-18 into their active forms [41,42]. NLRP3 inflammasome can be activated in response to various stimuli, including ATP, crystals, danger-associated molecular patterns (DAMP), and pathogen-associated molecular patterns (PAMP) during, for example, metabolic dysregulation [7,43–47]. Extracellular ATP induces the activation of P2 receptor-mediated purinergic signaling on macrophages and promotes inflammation via NLRP3 inflammasome signaling. ATP binds to P2X7R in primed macrophages and promotes activation of NLRP3 inflammasome and the production of IL-1β and IL-18 [48,49]. In the present study, the activation of NLRP3 inflammasome was suppressed by sinigrin. Sinigrin significantly inhibited the expression of NLRP3 and ASC in a concentration-dependent manner and also suppressed caspase-1 expression, which regulates the processing of IL-1β and IL-18. In addition, we observed the anti-inflammatory effect of sinigrin in LPS-primed, ATP-stimulated RAW 264.7 cells. These results indicate that sinigrin blocked the production of IL-1β and IL-18 through NLRP3 inflammasome activation in LPSactivated macrophages. Additionally, it has been reported that NF-κB is involved in the expression of IL-1β, NLRP3 and caspase-1 and MAPK signaling play an important role in the expression of IL-1β and inflammasome components in macrophages [50–52] Based on these findings, the results of the present study suggest that sinigrin suppresses NLRP3 inflammasome and IL-1β expression and ultimately IL1β cytokine production through blocking these signal transduction pathways. The collective results demonstrated that sinigrin can suppress the production of pro-inflammatory mediators in activated macrophages, providing a possible molecular mechanism for the anti-inflammatory activity of sinigrin. Our results revealed that sinigrin is able to inhibit the production of LPS-induced inflammatory mediators via the p38, JNK, and NF-κB signaling pathways. In addition, the inhibitory effect of sinigrin could be mediated by the suppression of NLRP3 inflammasome activation. Taken together, these data indicated that sinigrin could have significant effects on the modulation of innate immune response and potential therapeutic value in preventing inflammation- and NLRP3-related inflammatory diseases. References [1] K. Schroder, J. Tschopp, The inflammasomes, Cell 140 (6) (2010) 821–832. [2] C. Dodé, N. Le Duˆ, L. Cuisset, F. Letourneur, J.-M. Berthelot, G. Vaudour, A. Meyrier, R.A. Watts, G.D. Scott, A. Nicholls, New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel mutation underlies both syndromes, Am. J. Hum. Genet. 70 (6) (2002) 1498–1506. [3] J. Feldmann, A.-M. Prieur, P. Quartier, P. Berquin, S. Certain, E. Cortis, D. TeillacHamel, A. Fischer, G. de Saint Basile, Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes, Am. J. Hum. Genet. 71 (1) (2002) 198–203. [4] H.M. Hoffman, J.L. Mueller, D.H. Broide, A.A. Wanderer, R.D. Kolodner, Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle–Wells syndrome, Nat. Genet. 29 (3) (2001) 301–305. [5] T. Strowig, J. Henao-Mejia, E. Elinav, R. Flavell, Inflammasomes in health and disease, Nature 481 (7381) (2012) 278–286. [6] T.A. Wynn, A. Chawla, J.W. Pollard, Macrophage biology in development, homeostasis and disease, Nature 496 (7446) (2013) 445–455. [7] C. Dostert, V. Pétrilli, R. Van Bruggen, C. Steele, B.T. Mossman, J. Tschopp, Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica, Science 320 (5876) (2008) 674–677. [8] G. Pearson, F. Robinson, T. Beers Gibson, B.E. Xu, M. Karandikar, K. Berman, M.H. Cobb, Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions, Endocr. Rev. 22 (2) (2001) 153–183. [9] S.J. Ajizian, B.K. English, E.A. Meals, Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-γ, J. Infect. Dis. 179 (4) (1999) 939–944. [10] T. Uto, M. Fujii, D.X. Hou, 6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-
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[11] [12] [13]
[14]
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[20]
[21]
[22]
[23] [24]
[25]
[26]
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