Cytokine 71 (2015) 223–231
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Methylsulfonylmethane inhibits NLRP3 inflammasome activation Huijeong Ahn a, Jeeyoung Kim a, Min-Jae Lee a, Young Jin Kim b, Young-Wook Cho c, Geun-Shik Lee a,⇑ a
College of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea Korea Food Research Institute, Songnam, Kyeonggi 463-746, Republic of Korea c Korean Basic Science Institute, Chuncheon 200-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 1 July 2014 Received in revised form 31 October 2014 Accepted 3 November 2014
Keywords: MSM DMSO Inflammasome Interleukin-1beta Macrophages
a b s t r a c t s Methylsulfonylmethane (MSM) is an organosulfur compound and the health benefits associated with MSM include inflammation. Although MSM has been shown to have various physiological effects, no study has yet focused on inflammasome activation. The inflammasome is a multiprotein complex that serves as a platform for caspase 1-dependent proteolytic maturation and secretion of interleukin-1b (IL-1b). In this study, we tested the effect of MSM on inflammasome activation using mouse and human macrophages. In our results, MSM significantly attenuated NLRP3 inflammasome activation in lipopolysaccharide-primed macrophages, although it had no effect on NLCR4 or AIM2 inflammasome activation. Extracts of MSM-enriched vegetables presented the same inhibitory effect on NLRP3 inflammasome activation as MSM. MSM also attenuated the transcriptional expression of IL-1a, IL-1b, IL-6, and NLRP3. Taken together, these results show that MSM has anti-inflammatory characteristics, interrupts NLRP3 inflammasome activation, and inhibits pro-cytokine expression. We further confirmed the intracellular mechanism of MSM in relation to NLRP3 inflammasome activation, followed by comparison with that of DMSO. Both chemicals showed a synergic effect on anti-NLRP3 activation and attenuated production of mitochondrial reactive oxygen species (ROS). Thus, MSM is a selective inhibitor of NLRP3 inflammasome activation and can be developed as a supplement to control several metabolic disorders. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The inflammasome is a multi-protein complex that operates as a platform for interleukin (IL)-1b processing [1–3]. IL-1b is an important mediator of the inflammatory response and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis [4]. The activity of IL-1b is rigorously controlled by its expression, maturation, and secretion, with increased production of IL-1b resulting in secondary damage as well as numerous different auto-inflammatory syndromes. Specifically, IL-1b is first produced without a leader sequence in a pro-form (pro-IL-1b), which itself is produced and released according to transcriptional and post-transcriptional processes. However, maturation and release of IL-1b are controlled by inflammasomes. The basic components of inflammasomes include a NOD-like receptor (NLR) and PYHIN protein, which recognizes danger signals or ligands, as well as pro-caspase 1, which is central to inflammasome activation [1]. In addition to these basic components, other factors could also be ⇑ Corresponding author at: College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chucheon 200-701, Republic of Korea. Tel.: +82 33 250 8683; fax: +82 33 244 2367. E-mail address: [email protected] (G.-S. Lee). http://dx.doi.org/10.1016/j.cyto.2014.11.001 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
involved in the assembly or activity of inflammasomes, depending on the type of cell and stimulus [1]. Such factors include the adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), which is an essential component of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes but not of the more complex NLR Family, caspase recruitment domain (CARD) domain-containing 4 (NLRC4) inflammasome [1]. In recent decades, inflammasomes have been shown to play a role in several pathogenic and metabolic diseases, and thus its regulation has been suggested as a new therapeutic target for metabolic diseases [5]. Methylsulfonylmethane ((CH3)2SO2, MSM), also known as dimethylsulfone (DMSO2), is an organic sulfur-containing compound that is the first oxidized metabolite of dimethylsulfoxide ((CH3)2 SO, DMSO). MSM occurs naturally in some primitive plants and animals, including humans [6]. MSM is also considered as a precursor to the synthesis of methionine and cysteine, which are sulfurcontaining amino acids, and acts as a source of sulfur [7]. MSM has been extensively used as a dietary supplement due to its potential to improve human health as well as reduce arthritic and rheumatic pain [8]. Although MSM has not been approved for medical use by any government, its health benefits have been claimed and studied previously. Specifically, MSM has been shown to protect against
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inflammation, prostacyclin synthesis, and atherosclerosis through a salutary effect on eicosanoid metabolism, as well as promote free radical-scavenging activity in vitro [8]. MSM and DMSO have different physical properties. MSM is a white crystalline solid at standard temperature and pressure, whereas DMSO is a liquid under standard conditions. DMSO is also a highly polar aprotic solvent, miscible in water, and an excellent ligand. MSM is less reactive than DMSO, as the S-atom of the sulfone is already in its highest oxidation state. Indeed oxidation of sulfoxide produces sulfone, both under laboratory conditions and metabolically. DMSO is reported to have numerous beneficial effects, including relief of muscle and joint pain, reduction of inflammation, healing of skin ulcers, and a long list of other benefits [9]. Recently, we revealed that DMSO inhibits NLRP3 inflammasome activity [10]. In this study, we assessed the effects of MSM on inflammasome activation and pro-inflammatory cytokine production. In addition, dietary MSM-enriched sources were investigated for control of inflammasome activation. Finally, we confirmed the intracellular mechanism of MSM in relation to NLRP3 inflammasome activation, followed by comparison with that of DMSO. 2. Materials and methods 2.1. Cell culture For bone marrow-derived macrophages (BMDMs), bone marrow cells were obtained by flushing tibia and femur bones from C57BL/6 mice (6–12-weeks-old; Narabio Co., Seoul, Republic of Korea) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; WELGENE Inc. Daegu, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Corning cellgro, Manassas, VA, USA) and penicillin and streptomycin solution (Corning cellgro) in the presence of L929 cell-conditioned medium containing granulocyte/ macrophage colony-stimulating factor [11]. The bone marrow cells were cultured in petri dishes (SPL Life Science Co., Gyeonggi-do, Republic of Korea) at 37 °C in a 5% CO2 atmosphere for 7 days. THP-1 cells were obtained from Korean Cell Line Bank (KCLB No. 40202; Seoul, Republic of Korea) and maintained in RPMI 1640 medium (WELGENE Inc.) containing 10% FBS and penicillin and streptomycin solution at 37 °C in a 5% CO2 atmosphere. THP-1 cells were differentiated into macrophage-like cells using phorbol 12-myristate acetate (100 nM, PMA; Cat. #tlrl-pma, Invivogen, CA, USA) for 72 h. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Kangwon National University (ACUC; approval no. KIACUC-12-0009). 2.2. Cell treatments BMDMs (1.0 106 cells per well) or PMA-differentiated THP-1 cells (1.0 106 cells per well) were plated in 12-well plates (SPL Life Science Co.) and primed with lipopolysaccharide (1 lg/ml, LPS; #L4130, Sigma–Aldrich Co., MO, USA) in RPMI 1640 containing 10% FBS and antibiotics for 3 h. After LPS priming, cells were replaced by RPMI 1640 containing the following inflammasome activators: adenosine triphosphate (2 mM, ATP; #tlrl-atp InvivoGen) for 1 h, nigericin (40 lM, NG; #4312 Tocris Bioscience, Bristol, UK) for 1 h, aluminum potassium sulfate (200 lg/ml, Alum; #10394404, Daejung Chemicals & Metals Co., Gyeonggi-do, Republic of Korea) for 3 h, double-stranded DNA (1 lg/ml, dsDNA) with jetPRIME™ (2 ll/ml, Polyplus-transfection Inc., Illkirch, France) for 1 h, Listeria monocytogenes (10%, [350MOI], OD600: 1.2) for 3 h, flagellin (0.5 mg/ml; #tlrl-stfla, InvivoGen) with Lipofectamine 2000 (10 ll/ml, Invitrogen, CA, USA) for 1 h, Salmonella typhimurium
(1%, [35MOI], OD600: 1.2) for 1 h, and rotenone (80 lM; #sc203242, Santa Cruz Biotechnology, CA, USA) for 6 h. To test the inhibitory effect of MSM (Alfa Aesar, MA, USA) on inflammasome activation, DMSO (Daejung Chemicals & Metals Co.), vegetable extracts (Korea Food Research Institutes, Gyeonggi-do, Republic of Korea), and diphenyleneiodonium (50 lM, DPI; #0504, Tocris Bioscience) were co-treated with the above activators. To determine the effect of MSM on translational expression of pro-IL-1b or TNF a, BMDMs (1.0 106 cells per well) were treated with LPS (10 ng/ ml) for 3 h. After cell lysis, cellular supernatant (Sup), lysate (Lys), and cross-linked pellets (Pellet) with suberic acid bis (Sigma– Aldrich Co.) were collected for further analysis.
2.3. Western blot analysis Sup and Lys samples were separated by SDS-PAGE (10% or 16%). Separated proteins were transferred onto a polyvinylidene difluoride membrane (PVDF; Pall Co., NY, USA), blocked with 3% skim milk, and probed overnight at 4 °C with anti-mouse IL-1b antibody (#AF-401-NA, R&D Systems, MN, USA), anti-human IL-1b antibody (#AF-201-NA, R&D Systems), anti-caspase-1 p20 antibody (#06503, EMD Millipore Co, MA, USA), anti-caspase-1 antibody (#sc622, Santa Cruz Biotechnology), anti-TNF a antibody (#sc-1351, Santa Cruz Biotechnology), or anti-actin antibody (#sc-1615, Santa Cruz Biotechnology). Membranes were further probed with HRPconjugated 2nd anti-sera (#sc-2020 or #sc-2004, Santa Cruz Biotechnology) and visualized by Power-Opti ECL™ solution (BioNote Co., Gyeonggi-do, Republic of Korea) and cooled CCD camera System (AE-9150 EZ-Capture II, ATTO Technology, Tokyo, Japan).
2.4. Bacterial growth and invasion assay Salmonella typhimurium (Salmonella) and Listeria monocytogenes (Listeria) were obtained from the Korean Culture Center of Microorganisms (KCCM; Seoul, Republic of Korea). Salmonella and Listeria strains were grown on Luria-Bertani (LB, Laboratories Conda, Madrid, Spain) and Brain Heart Infusion (BHI, Laboratories Conda) broth for 18 h with shaking at 37 °C. The cultured media (2%) were transferred to fresh LB or BHI broth and further incubated for 3–4 h with shaking at 37 °C. Bacteria host invasion assay was performed as previously described [11]. Briefly, BMDMs (1.0 105 cells per well, 96-well plate) were incubated with RPMI 1640 containing 10% FBS in the presence of Salmonella (1% [35MOI], OD600: 1.2) for 1 h or Listeria (10% [350MOI], OD600: 1.2) for 3 h. Cells were washed with PBS containing gentamycin (50 lg/ml; Komipharm International Co., Ltd., Gyeonggi-do, Republic of Korea) or penicillin and streptomycin (1000 U/ml and 1 mg/ml, Corning cellgro) to eliminate extracellular bacteria, followed by plating on a LB or BHI plate to calculate colony-forming units (cfu).
2.5. Cytotoxity assay BMDMs (10,000 cells/well) were plated in a 96-well plate (SPL Life Science Co.) and allowed to attach overnight. BMDMs were then treated for 1 h with the indicated dosage of chemicals, which were replaced with fresh media (RPMI 1640 containing 10% FBS and antibiotics), followed by further incubation for 3 h. Cytotoxicity was measured by a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc. MD, USA) per the manufacturer’s protocol. Triton -100 (0.01%, Triton; Daejung Chemicals & Metals Co.) was used as a negative control for complete cell death. The Triton-treated group was used to determine 0% survival rate while the nontreated group was used as 100%.
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2.6. RNA extraction and real-time qPCR
3. Results
For RNA extraction, BMDMs (2.0 106 cells per well) were plated in 6-well plates (SPL Life Science Co.) and primed with LPS (10 ng/ml) in RPMI 1640 containing 10% FBS and antibiotics for 1 h. Total RNA was extracted using Trizol (Invitrogen) and reverse-transcribed to first-stand complementary DNA (cDNA) using a M-MLV cDNA Synthesis kit (Enzynomics, Daejeon, Republic of Korea). Transcripts were quantitated using SYBR Green (TOPreal™ qPCR 2X PreMIX, Enzynomics) and an Eco Real-Time PCR system (Illumina, San Diego, CA, USA). Quantitation was normalized with b-actin (Actb). IL-1b (Il1b; Genebank ID: NM_008361) primers 50 -CCC AAG CAA TAC CCA AAG AA-30 and 50 -GCT TGT GCT CTG CTT GTG AG-30 ; IL-1a (Il1a; NM_010554) 50 -CCG ACC TCA TTT TCT TCT GG-30 and 50 -GTG CAC CCG ACT TTG TTC TT-30 ; IL-6 (Il6; NM_031168) 50 -CCG GAG AGG AGA CTT CAC AG-30 and 50 -TCC ACG ATT TCC CAG AGA AC-30 ; TNF-a (Tnfa; NM_013693) 50 -ACG GCA TGG ATC TCA AAG AC-30 and 50 -GTG GGT GAG GAG CAC GTA GT-30 ; NLRP3 (Nlrp3; NM_145827) 50 -CAG GCG AGA CCT CTG GGA AA-30 and 50 -CCC AGC AAA CCC ATC CAC TC-30 ; b-actin (Actb; NM_007393) 50 -AGC CAT GTA CGT AGC CAT CC-30 and 50 -CTC TCA GCT GTG GTG GTG AA-30 .
3.1. MSM inhibits NLRP3 inflammasome activation
2.7. IL-1b detection using ELISA To quantitate the secreted IL-1b, the cell culture supernatants of BMDMs were measured by mouse IL-1beta/IL-1F2 Quantikine ELISA Kit (R&D Systems). The ELISA plates were readout using a microplate reader (Bio-Red, CA, USA).
2.8. Extracts of MSM-enriched vegetables Garlic, onion, chive or wild chive (each 1 kg) were extracted with 1000 ml of ethanol (99.99% v/v, Daejung Chemicals & Materials Co., Gyeonggi-do, Republic of Korea) at RT for overnight. The ethanol extraction was filtrated with filter paper (No. 1, Advantec MFS, Inc., CA, USA) and evaporated ethanol in vacuo at 55–65 °C. The ethanol-free extraction was further filtrated with filter paper (No. 2, Advantec MFS, Inc.) and adjusted pH to pH 7.4. The solution were dried at 105 °C and dissolved in distilled water (100 mg/ml).
2.9. Biochemical assay For assay mitochondrial reactive oxygen species (ROS), BMDMs (1.25 105 cells per well) plated in 96-well black plates (SPL Life Science Co.) were incubated with MitoSOX™ Red mitochondrial superoxide indicator (2.5 lM, #M36008, Invitrogen) for 30 min at 37 °C. The cells were treated rotenone (160 lM) in the present of DMSO (2%), MSM (2%), or DPI (100 lM) for 6 h at 37 °C. The plated were readout using a plate reader (510/580 nm, SpectraMaxÒ M2e, Molecular Devices, CA, USA). For assay caspase-1 activity, human recombinant caspase-1 (1 unit/rx, #1081, BioVison Inc., CA. USA) was incubated with YVAD-pNA, a substrate of caspase-1, in the present of MSM or ZVAD-FMK (#FMK001, R&D Systems). The activity of caspase-1 was measured by caspase-1/ICE Colorimetric Assay Kit (#K111, BioVison Inc) according to the manufacturer’s protocol.
2.10. Statistical analyses Statistical analyses were performed with one-way ANOVA for multiple groups using GraphPad Prism (GraphPad Software, San Diego, CA, USA). P value is indicated in the figure.
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To assess the effect of MSM on NLRP3 inflammasome activation, we treated human or murine macrophages with various dosages of MSM along with NLRP3 inflammasome triggers (ATP, NG, or Alum) and then measured secreted and mature caspase 1 (Casp1) and/or IL-1b in cellular supernatants as readouts of inflammasome activation. In LPS-primed mouse BMDMs, MSM dose-dependently attenuated secretion of Casp1 and IL-1b resulting from ATP-, NG-, or Alum-mediated NLRP3 inflammasome activation (Fig. 1A). The inhibitory effect of MSM on NG-mediated IL-1b secretion was further confirmed by ELISA (Fig. 1B). In addition, we were observed the other readout of inflammasome activation, the formation of Asc pyroptosome (Fig. 1C). Like the secretion of Casp1 and IL-1b, MSM attenuated ATP- or NG-mediated Asc pyroptosis. Similar to murine macrophages, MSM also blocked IL-1b secretion mediated by NLRP3 inflammasome activation in PMA-stimulated and LPSprimed THP-1 cells originated from human monocytes (Fig. 1D). We further tested whether MSM inhibited the Casp1 activity (Fig. 1E). As result, the activity of human recombinant Casp1 did not change by MSM co-treatment while the activity was attenuated by pan-caspase inhibitor, Z-VAD-FMK. We also tested the cytotoxicity of MSM using BMDMs (Fig. 1F). Cytotoxicity was observed up to 8% MSM treatment. Taken together, MSM was shown to act as a new NLRP3 inflammasome inhibitor.
3.2. MSM does not block AIM2 or NLRC4 inflammasome activation We further investigated the effect of MSM on activation of other inflammasomes such as AIM2 or NLRC4. AIM2 inflammasome is activated by cytosolic double-stranded DNA (dsDNA) derived from hosts or pathogens [12]. In the present study, we transfected dsDNA into BMDMs (Fig. 2A) and observed Casp1 as well as IL-1b secretion in the presence of increasing dosages of MSM. Casp1 and IL-1b secretion resulting from dsDNA-mediated AIM2 inflammasome activation was unaltered by MSM. We further inoculated Listeria monocytogenes (Listeria) into BMDMs for AIM2 inflammasome activation [13]. Listeria-mediated IL-1b secretion was inhibited by MSM (Fig. 2B). To investigate this discrepancy regarding the effect of MSM on AIM2 inflammasome activation triggered by dsDNA and Listeria, we measured the invasion rate of Listeria into BMDMs since this event precedes AIM2 inflammasome activation [14]. Interestingly, MSM dose-dependently reduced the invasion rate of Listeria (Fig. 2C), indicating that MSM had no effect on AIM2 inflammasome activation. The NLRC4 inflammasome is activated by gram-negative bacteria possessing type III or IV secretion systems, including Salmonella typhimurium, Shigella flexneri, Legionella pneumophila, and Pseudomonas aeruginosa [15]. To activate the NLRC4 inflammasome, we transfected flagellin or inoculated Salmonella typhimurium (Salmonella) into macrophages with/without MSM and observed Casp1 and IL-1b secretion. Similar to the AIM2 inflammasome, MSM did not alter flagellin induced-NLRC4 inflammasome activation (Fig. 2D and 2E), whereas it strongly attenuated Salmonella-mediated IL-1b secretion (Fig. 2F). To elucidate this discrepancy, we again tested the invasion rate of Salmonella using BMDMs. The number of intracellular Salmonella was significantly reduced by MSM (Fig. 2G), which combined with the result of the Listeria invasion experiment implies that MSM may have attenuated bacterial penetration. We additionally elucidated the bactericidal effect of MSM on Salmonella and Listeria, but MSM did not change the bacterial growth (data not shown). Thus, MSM did not regulate AIM2 or NLRC4 inflammasome activation.
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Fig. 1. Effect of MSM on NLRP3 inflammasome activation. (A) BMDMs (1 106 cells per well) were primed by LPS (1 lg/ml) in RPMI medium containing 10% FBS and antibiotics for 3 h. Cells were supplied with RPMI medium in the presence of the indicated concentrations of MSM with ATP (2 mM), NG (40 lM), or Alum (200 lg/ml), after which they were analyzed for Casp1 and IL-1b secretion by immunoblotting. (B) Secreted IL-1b was quantitated by an ELISA based assay kit and presented with bar graphs. (C) The secretion of IL-1b and the formation of Asc pyroptosome were analyzed by immunoblotting as indicated. (D) THP-1 cells were differentiated into macrophage-like cells by PMA (100 nM) treatment for 72 h and primed with LPS (1 lg/ml) for 3 h. LPS-primed THP-1 cells were treated with the indicated dosages of MSM in the presence of ATP (2 mM), NG (40 lM), or Alum (200 lg/ml), after which IL-1b secretion was detected by immunoblotting. (E) The effect of MSM of Casp1 activity was measured in the present of MSM or Z-VAD-FMK (Z-VAD, a pan-caspase inhibitor). (F) Cytotoxicity of MSM was measured after applying the indicated dosages of MSM to BMDMs. Triton -100 (0.01%, Triton) was used as a negative control for complete cell death. The Triton-treated group was used to determine 0% survival rate while the non-treated group was used as 100%. Data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate. Cell culture supernatants (Sup), cell lysates (Lys) and cross-linked pellets (Pellet) from whole-cell lysates were analyzed by immunoblotting as indicated. All immunoblot data shown are representative of at least two independent experiments.
3.3. MSM suppress pro-inflammatory cytokine expression MSM has been reported as an anti-inflammatory molecule due to its inhibition of NF-jB signaling mediating pro-inflammatory cytokine expression in LPS-stimulated RAW264.7 cells [16]. Here, we investigated whether or not MSM inhibits expression of proIL-1b and NLRP3, which is regulated by NF-jB signaling [17], in LPS-primed BMDMs. As shown in Fig. 3A, MSM dose-dependently inhibited the translational expression of both pro-IL-1b and TNF a. In addition, MSM reduced the mRNA transcription levels of IL-1a (Fig. 3B), pro-IL-1b (Fig. 3C), IL-6 (Fig. 3D), and NLRP3 (Fig. 3E). Thus, MSM not only blocked the NLRP3 inflammasome at the activation step but also down-regulated pro-IL-1b and NLRP3 expression at the priming step.
3.4. MSM-enriched vegetables inhibit NLRP3 inflammasome activation MSM is a form of organic nutritional sulfur found in dairy products such as fruits, grains, meats, and vegetables. Especially, plants of the genus Allium contain substantial amounts of MSM [18]. Esculent plants in the genus Allium include garlic (Allium
scorodorpasum), onion (Allium cepa), chive (Allium tuberosum), and wild chive (Allium monanthum). These plants form solitary or clustered bulbs and are strongly aromatic, with a characteristic odor when crushed. We further tested the effects of extracts of MSM-enrich plants on NLRP3 inflammasome activation (Fig. 4). IL-1b secretion mediated by NLRP3 inflammasome activators such as ATP or NG in BMDMs was attenuated by all of the extracts, although the effective concentrations varied. Further, the extracts did not show any cytotoxicity in the BMDMs. Taken together, MSM-enriched vegetables could be suggested as candidates for the inhibition of NLRP3 inflammasome activation.
3.5. MSM and DMSO inhibit NLRP3 inflammasome activation via an ROS pathway MSM is chemically related to dimethyl sulfoxide (DMSO), which has one less oxygen atom than MSM (Fig. 5A). These two molecules share several pharmacological effects due to their similar chemical properties [19]. Previously, we reported that DMSO inhibits NLRP3 inflammasome activation [10]. Here, we investigated the synergic inhibitory activity of DMSO and MSM on NLRP3 inflammasome
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Fig. 2. Effect of MSM on AIM2 or NLRC4 inflammasome activation. AIM2 inflammasome in LPS-primed BMDMs were activated by dsDNA (1 lg/ml) with transfection agent for 1 h (A) or Listeria monocytogenes (Listeria; 10%, OD600: 1.2) for 3 h (B), after which Casp1 or IL-1b secretion was detected by immunoblotting. (C) BMDMs were inoculated with Listeria in the presence of MSM for 3 h, after which the number of intracellular bacteria (colony forming unit, cfu) was calculated. cfu of the Listeria-inoculated group was set as 100%, and data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate. NLRC4 inflammasome in LPS-primed BMDMs was activated by flagellin (0.5 mg/ml) transfection for 1 h (D, E) or Salmonella typhimurium (Salmonella; 1%, OD600: 1.2) for 1 h (F), after which Casp1 or IL-1b secretion was detected by immunoblotting or ELISA. (G) BMDMs were inoculated with Salmonella in the presence of MSM for 1 h, after which the number of intracellular bacteria was calculated. cfu of the Salmonella-inoculated group was set as 100%, and data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate.
activation (Fig. 5B). When BMDMs were treated with a given concentration of DMSO along with various MSM concentrations, and vice versa, we observed similar reductions in IL-1b production depending on the accumulated concentrations of DMSO and MSM. In addition, the sum concentration of DMSO and MSM inhibited IL-1b secretions in dose-dependently (Fig. 5C). Thus, the inhibitory effects of both MSM and DMSO on NLRP3 inflammasome activation could have occurred through the same intracellular mechanism. Next, we determined the intracellular inhibitory mechanisms of both MSM and DMSO on NLRP3 inflammasome activation. MSM and DMSO have been studied as free radical scavengers previously [20]. Various models for NLRP3 inflammasome activation have been proposed, with the concept of upstream reactive oxygen species (ROS) production in relation to NLRP3 activation gaining particular
attention [21,22]. To confirm whether or not MSM and DMSO inhibit NLRP3 inflammasome activation via mitochondrial ROS generation, we firstly confirmed the effect of DPI, an inhibitor of cytosolic and mitochondrial ROS production [23], on NLRP3 inflammasome activation (Fig. 5D). As expected, DPI inhibited ATP- or NG-derived IL-1b secretion. Next, we applied rotenone, which activates the NLRP3 inflammasome via mitocondrial ROS [22], in the presence of DMSO or MSM to LPS-primed BMDMs (Fig. 5E). DMSO and MSM inhibited rotenone-mediated IL-1b secretion, similar to DPI. In addition, we measured the rotenone-induced mitocondrial ROS generation in the present of MSM, DMSO, or DPI (Fig. 5G). As expected, MSM or DMSO blocked the mitocondrial ROS. Thus, MSM as well as DMSO inhibited NLRP3 inflammasome activation by blocking mitochondrial ROS generation.
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Fig. 3. Effect of MSM on expression of pro-inflammatory cytokines and NLRP3. BMDMs (1 106 cells per well for immunoblotting and 2 106 cells per well for qPCR) were treated with the indicated concentrations of MSM with/without LPS (10 ng/ml). (A) effects of MSM on LPS-mediated pro-IL-1b and TNF a translation were detected by immunoblotting. Transcriptional levels of IL-1a (B), pro-IL-1b (C), IL-6 (D), and NLRP3 (E) were analyzed using SYBR green-based quantitative real-time PCR. Data represent the mean ± s.e.m. for three independent experiments.
4. Discussion In this study, we confirmed MSM as a selective NLRP3 inflammasome inhibitor. Although the anti-inflammatory properties of MSM have been studied before, no report has considered the effect of MSM on inflammasome activation producing the active form of IL-1b. To confirm the inhibitory role of MSM in inflammation, we tested the effect of MSM on two distinct steps of inflammation. In the first step known as priming, NLRP3, a sensing protein of inflammasomes, along with pro-IL-1b, a substrate of inflammasome activation, are transcriptionally up-regulated by toll-like receptor ligands or cytokines through the NF-jB signaling pathway. The second step triggers inflammasome assembly, leading to activation of Casp1, which proteolytically cleaves the precursor form of IL-1b [24]. In our results, MSM attenuated NF-jB signalingdependent NLRP3 and pro-IL-1b expression in the priming step, resulting in insufficient inflammasome activation. In the second (activation) step, MSM inhibited IL-1b secretion resulted from NLRP3 inflammasome activation even though cells were treated independently with the first step’s signals. We further confirmed that MSM attenuated NLRP3 inflammasome activation through inhibition of mitochondrial ROS production, similar to DMSO. In addition, extracts of MSM-enriched vegetables presented the same inhibitory effects on NLRP3 inflammasome activation as MSM. Thus, MSM possesses dual anti-inflammatory properties:
down-regulation of pro-inflammatory cytokines and inhibition of NLRP3 inflammasome activation. The anti-inflammatory effect of MSM has been progressively reported. In LPS-treated RAW264.7 cells, MSM has been shown to reduce the levels of IL-6 and TNF-a in addition to down-regulating NF-jB signaling [16]. Another study showed that MSM can ameliorate experimental colitis by reducing the level of IL-1b [25]. MSM pre-treatment also was shown to protect against hepatic liver injury by reducing TNF-a and IL-6 levels [26]. In addition, MSM intake is effective for treating inflammatory diseases such as osteoarthritis, seasonal allergic rhinitis, and intestinal cystitis [8,16,27,28]. Especially, MSM has been suggested as a dietary supplement for the treatment of osteoarthritis and is believed to be non-toxic [20,28]. Osteoarthritis is a progressive joint disease characterized by cartilage degeneration, subchondral bone remodeling, and inflammation of the synovial membrane. The pro-inflammatory cytokine IL-1b has been identified as one of the key mediators of osteoarthritis produced by activated synovial macrophages, resulting in inflammatory and destructive responses [29,30]. It has been reported that mice lacking inflammasome components are protected against osteoarthritis and NLRP3 inflammasome activation, which plays a pivotal role in the development of osteoarthritis [29]. Based the above evidence, MSM might be a candidate for the treatment of osteoarthritis based on its ability to attenuate the expression of pro-inflammatory cytokines and block NLRP3 inflammasome activation.
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Fig. 4. Effects of extracts of MSM-enriched vegetables on NLRP3 inflammasome activation. Extracts of garlic (A), onion (C), chive (E), and wild chive (G) were applied to LPSprimed BMDMs in the presence of the NLRP3 activators ATP or NG. IL-1b secretion was observed by immunoblotting. Cytotoxicity of garlic (B), onion (D), chive (F), or wild chive (H) in BMDMs was measured. The Triton-treated group (0.001%, Triton -100) was used to determine 0% survival rate while the non-treated group was used as 100%. Data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate.
MSM and DMSO are very similar sulfur compounds. The ability and health benefits of MSM are almost identical to those of DMSO, with only slight differences [31]. Sulfur compounds are known to be effective hydroxyl radical scavengers [20]. Since MSM and DMSO both improve mitochondrial oxidative phosphorylation, it has been suggested that they can neutralize the cytotoxic effects of hydroxyl radicals in mitochondria themselves [28,32,33]. Several intracellular models of NLRP3 inflammasome activation have been reported, and ROS generation seems to be a unifying factor as it consistently mediates NLRP3 activation in response to several stimuli [21]. Inhibition of NADPH oxidase-dependent ROS production has been previously suggested to occur upstream of NLRP3 inflammasome activation, and accumulation of ROS-producing mitochondria by rotenone, a pharmacological inhibitor of the mitochondrial electron transport chain, was recently shown to trigger NLRP3 inflammasome activation [22]. Thus rotenone, which inhibits the transfer of electrons from iron–sulfur centers in complex I to ubiquinone, promotes mitochondrial ROS-mediated NLRP3 inflammasome activation [22]. The above evidence combined with our current data support the claim that MSM as well as DMSO attenuate IL-1b maturation through NLRP3 inflammasome activation by inhibiting mitochondrial ROS production. Induction of chronic inflammation by inflammasomes plays an essential role in the pathogenesis of several metabolic and
aged-related diseases such as type 2 diabetes, obesity, gout, and cardiovascular diseases [34]. Thus, regulation of inflammasome activity has received wide attention, and elucidation of inflammasome activation should shed light on the development of new therapeutic regimens [34]. In type 2 diabetes, the NLRP3 inflammasome activated by stimuli such as islet amyloid polypeptide promotes IL-1b secretion, which itself induces insulin resistance in insulin target cells and reduces insulin production in b cells [34,35]. In atherosclerotic disease, cholesterol crystals turn on pro-inflammatory cytokine expression via NF-jB signaling and activate the NLRP3 inflammasome, resulting in robust IL-1b release mediating arterial inflammation [36]. Metabolic byproducts of obesity activate inflammasomes in both adipocytes and adipose-associated innate immune cells that produce inflammasome-associated cytokines, thereby influencing the outcome of obesity [37]. In gouty arthritis, monosodium urate activates NF-jB signaling, similar to the priming step of inflammasome activation, and induces NLRP3 inflammasome activation for production of IL-1b [38]. Each disease induces NF-jB signaling to produce pro-inflammatory cytokines and NLRP3 as well as simultaneously triggers NLRP3 inflammasome activation. Thus, we suggest MSM as a therapeutic agent to control metabolic disorders due to its ability to effectively attenuate the expression of pro-inflammatory cytokines as well as activate the NLRP3 inflammasome in the current study.
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E
F
Fig. 5. Effect of MSM and DMSO on mitochondrial ROS-mediated inflammasome activation. (A) Chemical structures of MSM and DMSO were adopted from PubChem (http:// pubchem.ncbi.nlm.nih.gov). (B and C) BMDMs were primed with LPS (1 lg/ml) for 3 h, after which NLRP3 inflammasome activation was carried out with ATP (2 mM for 1 h) in the presence of DMSO or MSM as indicated. IL-1b secretion was measured by immunoblotting. (D) NLRP3 inflammasome activation in LPS-primed BMDMs was carried out with ATP (2 mM for 1 h), NG (40 lM for 1 h), or rotenone (80 lM for 6 h) in the presence of DPI (50 lM) to inhibit ROS production. IL-1b secretion was measured as a readout of inflammasome activation. (E) LPS-primed BMDMs were treated with rotenone (80 lM for 6 h) with/without DMSO or MSM as indicated. IL-1b secretion was detected by immunoblotting. (F) BMDMs were incubated with MitoSoxTM (2.5 lM) for 30 min and treated with rotenone (160 l) for 6 h to induce mitochondrial ROS generation in the present of MSM (2%), DMSO (2%), or DPI (100 lM). Bar graph represents the mean ± s.e.m. of three independent experiments, each performed in triplicate ‘ + ’; treatment as indicated; ‘’, non-treatment.
Acknowledgments This work was supported by the Basic Science Research Program (2012R1A1A1001645) and Radiation Technology R&D program (2012M2A2A6035733) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning, and by a 2013 Research Grant from Kangwon National University (No. 120131488).
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Methylsulfonylmethane inhibits NLRP3 inflammasome activation Huijeong Ahn a, Jeeyoung Kim a, Min-Jae Lee a, Young Jin Kim b, Young-Wook Cho c, Geun-Shik Lee a,⇑ a
College of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea Korea Food Research Institute, Songnam, Kyeonggi 463-746, Republic of Korea c Korean Basic Science Institute, Chuncheon 200-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 1 July 2014 Received in revised form 31 October 2014 Accepted 3 November 2014
Keywords: MSM DMSO Inflammasome Interleukin-1beta Macrophages
a b s t r a c t s Methylsulfonylmethane (MSM) is an organosulfur compound and the health benefits associated with MSM include inflammation. Although MSM has been shown to have various physiological effects, no study has yet focused on inflammasome activation. The inflammasome is a multiprotein complex that serves as a platform for caspase 1-dependent proteolytic maturation and secretion of interleukin-1b (IL-1b). In this study, we tested the effect of MSM on inflammasome activation using mouse and human macrophages. In our results, MSM significantly attenuated NLRP3 inflammasome activation in lipopolysaccharide-primed macrophages, although it had no effect on NLCR4 or AIM2 inflammasome activation. Extracts of MSM-enriched vegetables presented the same inhibitory effect on NLRP3 inflammasome activation as MSM. MSM also attenuated the transcriptional expression of IL-1a, IL-1b, IL-6, and NLRP3. Taken together, these results show that MSM has anti-inflammatory characteristics, interrupts NLRP3 inflammasome activation, and inhibits pro-cytokine expression. We further confirmed the intracellular mechanism of MSM in relation to NLRP3 inflammasome activation, followed by comparison with that of DMSO. Both chemicals showed a synergic effect on anti-NLRP3 activation and attenuated production of mitochondrial reactive oxygen species (ROS). Thus, MSM is a selective inhibitor of NLRP3 inflammasome activation and can be developed as a supplement to control several metabolic disorders. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The inflammasome is a multi-protein complex that operates as a platform for interleukin (IL)-1b processing [1–3]. IL-1b is an important mediator of the inflammatory response and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis [4]. The activity of IL-1b is rigorously controlled by its expression, maturation, and secretion, with increased production of IL-1b resulting in secondary damage as well as numerous different auto-inflammatory syndromes. Specifically, IL-1b is first produced without a leader sequence in a pro-form (pro-IL-1b), which itself is produced and released according to transcriptional and post-transcriptional processes. However, maturation and release of IL-1b are controlled by inflammasomes. The basic components of inflammasomes include a NOD-like receptor (NLR) and PYHIN protein, which recognizes danger signals or ligands, as well as pro-caspase 1, which is central to inflammasome activation [1]. In addition to these basic components, other factors could also be ⇑ Corresponding author at: College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chucheon 200-701, Republic of Korea. Tel.: +82 33 250 8683; fax: +82 33 244 2367. E-mail address: [email protected] (G.-S. Lee). http://dx.doi.org/10.1016/j.cyto.2014.11.001 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
involved in the assembly or activity of inflammasomes, depending on the type of cell and stimulus [1]. Such factors include the adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), which is an essential component of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes but not of the more complex NLR Family, caspase recruitment domain (CARD) domain-containing 4 (NLRC4) inflammasome [1]. In recent decades, inflammasomes have been shown to play a role in several pathogenic and metabolic diseases, and thus its regulation has been suggested as a new therapeutic target for metabolic diseases [5]. Methylsulfonylmethane ((CH3)2SO2, MSM), also known as dimethylsulfone (DMSO2), is an organic sulfur-containing compound that is the first oxidized metabolite of dimethylsulfoxide ((CH3)2 SO, DMSO). MSM occurs naturally in some primitive plants and animals, including humans [6]. MSM is also considered as a precursor to the synthesis of methionine and cysteine, which are sulfurcontaining amino acids, and acts as a source of sulfur [7]. MSM has been extensively used as a dietary supplement due to its potential to improve human health as well as reduce arthritic and rheumatic pain [8]. Although MSM has not been approved for medical use by any government, its health benefits have been claimed and studied previously. Specifically, MSM has been shown to protect against
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inflammation, prostacyclin synthesis, and atherosclerosis through a salutary effect on eicosanoid metabolism, as well as promote free radical-scavenging activity in vitro [8]. MSM and DMSO have different physical properties. MSM is a white crystalline solid at standard temperature and pressure, whereas DMSO is a liquid under standard conditions. DMSO is also a highly polar aprotic solvent, miscible in water, and an excellent ligand. MSM is less reactive than DMSO, as the S-atom of the sulfone is already in its highest oxidation state. Indeed oxidation of sulfoxide produces sulfone, both under laboratory conditions and metabolically. DMSO is reported to have numerous beneficial effects, including relief of muscle and joint pain, reduction of inflammation, healing of skin ulcers, and a long list of other benefits [9]. Recently, we revealed that DMSO inhibits NLRP3 inflammasome activity [10]. In this study, we assessed the effects of MSM on inflammasome activation and pro-inflammatory cytokine production. In addition, dietary MSM-enriched sources were investigated for control of inflammasome activation. Finally, we confirmed the intracellular mechanism of MSM in relation to NLRP3 inflammasome activation, followed by comparison with that of DMSO. 2. Materials and methods 2.1. Cell culture For bone marrow-derived macrophages (BMDMs), bone marrow cells were obtained by flushing tibia and femur bones from C57BL/6 mice (6–12-weeks-old; Narabio Co., Seoul, Republic of Korea) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; WELGENE Inc. Daegu, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Corning cellgro, Manassas, VA, USA) and penicillin and streptomycin solution (Corning cellgro) in the presence of L929 cell-conditioned medium containing granulocyte/ macrophage colony-stimulating factor [11]. The bone marrow cells were cultured in petri dishes (SPL Life Science Co., Gyeonggi-do, Republic of Korea) at 37 °C in a 5% CO2 atmosphere for 7 days. THP-1 cells were obtained from Korean Cell Line Bank (KCLB No. 40202; Seoul, Republic of Korea) and maintained in RPMI 1640 medium (WELGENE Inc.) containing 10% FBS and penicillin and streptomycin solution at 37 °C in a 5% CO2 atmosphere. THP-1 cells were differentiated into macrophage-like cells using phorbol 12-myristate acetate (100 nM, PMA; Cat. #tlrl-pma, Invivogen, CA, USA) for 72 h. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Kangwon National University (ACUC; approval no. KIACUC-12-0009). 2.2. Cell treatments BMDMs (1.0 106 cells per well) or PMA-differentiated THP-1 cells (1.0 106 cells per well) were plated in 12-well plates (SPL Life Science Co.) and primed with lipopolysaccharide (1 lg/ml, LPS; #L4130, Sigma–Aldrich Co., MO, USA) in RPMI 1640 containing 10% FBS and antibiotics for 3 h. After LPS priming, cells were replaced by RPMI 1640 containing the following inflammasome activators: adenosine triphosphate (2 mM, ATP; #tlrl-atp InvivoGen) for 1 h, nigericin (40 lM, NG; #4312 Tocris Bioscience, Bristol, UK) for 1 h, aluminum potassium sulfate (200 lg/ml, Alum; #10394404, Daejung Chemicals & Metals Co., Gyeonggi-do, Republic of Korea) for 3 h, double-stranded DNA (1 lg/ml, dsDNA) with jetPRIME™ (2 ll/ml, Polyplus-transfection Inc., Illkirch, France) for 1 h, Listeria monocytogenes (10%, [350MOI], OD600: 1.2) for 3 h, flagellin (0.5 mg/ml; #tlrl-stfla, InvivoGen) with Lipofectamine 2000 (10 ll/ml, Invitrogen, CA, USA) for 1 h, Salmonella typhimurium
(1%, [35MOI], OD600: 1.2) for 1 h, and rotenone (80 lM; #sc203242, Santa Cruz Biotechnology, CA, USA) for 6 h. To test the inhibitory effect of MSM (Alfa Aesar, MA, USA) on inflammasome activation, DMSO (Daejung Chemicals & Metals Co.), vegetable extracts (Korea Food Research Institutes, Gyeonggi-do, Republic of Korea), and diphenyleneiodonium (50 lM, DPI; #0504, Tocris Bioscience) were co-treated with the above activators. To determine the effect of MSM on translational expression of pro-IL-1b or TNF a, BMDMs (1.0 106 cells per well) were treated with LPS (10 ng/ ml) for 3 h. After cell lysis, cellular supernatant (Sup), lysate (Lys), and cross-linked pellets (Pellet) with suberic acid bis (Sigma– Aldrich Co.) were collected for further analysis.
2.3. Western blot analysis Sup and Lys samples were separated by SDS-PAGE (10% or 16%). Separated proteins were transferred onto a polyvinylidene difluoride membrane (PVDF; Pall Co., NY, USA), blocked with 3% skim milk, and probed overnight at 4 °C with anti-mouse IL-1b antibody (#AF-401-NA, R&D Systems, MN, USA), anti-human IL-1b antibody (#AF-201-NA, R&D Systems), anti-caspase-1 p20 antibody (#06503, EMD Millipore Co, MA, USA), anti-caspase-1 antibody (#sc622, Santa Cruz Biotechnology), anti-TNF a antibody (#sc-1351, Santa Cruz Biotechnology), or anti-actin antibody (#sc-1615, Santa Cruz Biotechnology). Membranes were further probed with HRPconjugated 2nd anti-sera (#sc-2020 or #sc-2004, Santa Cruz Biotechnology) and visualized by Power-Opti ECL™ solution (BioNote Co., Gyeonggi-do, Republic of Korea) and cooled CCD camera System (AE-9150 EZ-Capture II, ATTO Technology, Tokyo, Japan).
2.4. Bacterial growth and invasion assay Salmonella typhimurium (Salmonella) and Listeria monocytogenes (Listeria) were obtained from the Korean Culture Center of Microorganisms (KCCM; Seoul, Republic of Korea). Salmonella and Listeria strains were grown on Luria-Bertani (LB, Laboratories Conda, Madrid, Spain) and Brain Heart Infusion (BHI, Laboratories Conda) broth for 18 h with shaking at 37 °C. The cultured media (2%) were transferred to fresh LB or BHI broth and further incubated for 3–4 h with shaking at 37 °C. Bacteria host invasion assay was performed as previously described [11]. Briefly, BMDMs (1.0 105 cells per well, 96-well plate) were incubated with RPMI 1640 containing 10% FBS in the presence of Salmonella (1% [35MOI], OD600: 1.2) for 1 h or Listeria (10% [350MOI], OD600: 1.2) for 3 h. Cells were washed with PBS containing gentamycin (50 lg/ml; Komipharm International Co., Ltd., Gyeonggi-do, Republic of Korea) or penicillin and streptomycin (1000 U/ml and 1 mg/ml, Corning cellgro) to eliminate extracellular bacteria, followed by plating on a LB or BHI plate to calculate colony-forming units (cfu).
2.5. Cytotoxity assay BMDMs (10,000 cells/well) were plated in a 96-well plate (SPL Life Science Co.) and allowed to attach overnight. BMDMs were then treated for 1 h with the indicated dosage of chemicals, which were replaced with fresh media (RPMI 1640 containing 10% FBS and antibiotics), followed by further incubation for 3 h. Cytotoxicity was measured by a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc. MD, USA) per the manufacturer’s protocol. Triton -100 (0.01%, Triton; Daejung Chemicals & Metals Co.) was used as a negative control for complete cell death. The Triton-treated group was used to determine 0% survival rate while the nontreated group was used as 100%.
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2.6. RNA extraction and real-time qPCR
3. Results
For RNA extraction, BMDMs (2.0 106 cells per well) were plated in 6-well plates (SPL Life Science Co.) and primed with LPS (10 ng/ml) in RPMI 1640 containing 10% FBS and antibiotics for 1 h. Total RNA was extracted using Trizol (Invitrogen) and reverse-transcribed to first-stand complementary DNA (cDNA) using a M-MLV cDNA Synthesis kit (Enzynomics, Daejeon, Republic of Korea). Transcripts were quantitated using SYBR Green (TOPreal™ qPCR 2X PreMIX, Enzynomics) and an Eco Real-Time PCR system (Illumina, San Diego, CA, USA). Quantitation was normalized with b-actin (Actb). IL-1b (Il1b; Genebank ID: NM_008361) primers 50 -CCC AAG CAA TAC CCA AAG AA-30 and 50 -GCT TGT GCT CTG CTT GTG AG-30 ; IL-1a (Il1a; NM_010554) 50 -CCG ACC TCA TTT TCT TCT GG-30 and 50 -GTG CAC CCG ACT TTG TTC TT-30 ; IL-6 (Il6; NM_031168) 50 -CCG GAG AGG AGA CTT CAC AG-30 and 50 -TCC ACG ATT TCC CAG AGA AC-30 ; TNF-a (Tnfa; NM_013693) 50 -ACG GCA TGG ATC TCA AAG AC-30 and 50 -GTG GGT GAG GAG CAC GTA GT-30 ; NLRP3 (Nlrp3; NM_145827) 50 -CAG GCG AGA CCT CTG GGA AA-30 and 50 -CCC AGC AAA CCC ATC CAC TC-30 ; b-actin (Actb; NM_007393) 50 -AGC CAT GTA CGT AGC CAT CC-30 and 50 -CTC TCA GCT GTG GTG GTG AA-30 .
3.1. MSM inhibits NLRP3 inflammasome activation
2.7. IL-1b detection using ELISA To quantitate the secreted IL-1b, the cell culture supernatants of BMDMs were measured by mouse IL-1beta/IL-1F2 Quantikine ELISA Kit (R&D Systems). The ELISA plates were readout using a microplate reader (Bio-Red, CA, USA).
2.8. Extracts of MSM-enriched vegetables Garlic, onion, chive or wild chive (each 1 kg) were extracted with 1000 ml of ethanol (99.99% v/v, Daejung Chemicals & Materials Co., Gyeonggi-do, Republic of Korea) at RT for overnight. The ethanol extraction was filtrated with filter paper (No. 1, Advantec MFS, Inc., CA, USA) and evaporated ethanol in vacuo at 55–65 °C. The ethanol-free extraction was further filtrated with filter paper (No. 2, Advantec MFS, Inc.) and adjusted pH to pH 7.4. The solution were dried at 105 °C and dissolved in distilled water (100 mg/ml).
2.9. Biochemical assay For assay mitochondrial reactive oxygen species (ROS), BMDMs (1.25 105 cells per well) plated in 96-well black plates (SPL Life Science Co.) were incubated with MitoSOX™ Red mitochondrial superoxide indicator (2.5 lM, #M36008, Invitrogen) for 30 min at 37 °C. The cells were treated rotenone (160 lM) in the present of DMSO (2%), MSM (2%), or DPI (100 lM) for 6 h at 37 °C. The plated were readout using a plate reader (510/580 nm, SpectraMaxÒ M2e, Molecular Devices, CA, USA). For assay caspase-1 activity, human recombinant caspase-1 (1 unit/rx, #1081, BioVison Inc., CA. USA) was incubated with YVAD-pNA, a substrate of caspase-1, in the present of MSM or ZVAD-FMK (#FMK001, R&D Systems). The activity of caspase-1 was measured by caspase-1/ICE Colorimetric Assay Kit (#K111, BioVison Inc) according to the manufacturer’s protocol.
2.10. Statistical analyses Statistical analyses were performed with one-way ANOVA for multiple groups using GraphPad Prism (GraphPad Software, San Diego, CA, USA). P value is indicated in the figure.
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To assess the effect of MSM on NLRP3 inflammasome activation, we treated human or murine macrophages with various dosages of MSM along with NLRP3 inflammasome triggers (ATP, NG, or Alum) and then measured secreted and mature caspase 1 (Casp1) and/or IL-1b in cellular supernatants as readouts of inflammasome activation. In LPS-primed mouse BMDMs, MSM dose-dependently attenuated secretion of Casp1 and IL-1b resulting from ATP-, NG-, or Alum-mediated NLRP3 inflammasome activation (Fig. 1A). The inhibitory effect of MSM on NG-mediated IL-1b secretion was further confirmed by ELISA (Fig. 1B). In addition, we were observed the other readout of inflammasome activation, the formation of Asc pyroptosome (Fig. 1C). Like the secretion of Casp1 and IL-1b, MSM attenuated ATP- or NG-mediated Asc pyroptosis. Similar to murine macrophages, MSM also blocked IL-1b secretion mediated by NLRP3 inflammasome activation in PMA-stimulated and LPSprimed THP-1 cells originated from human monocytes (Fig. 1D). We further tested whether MSM inhibited the Casp1 activity (Fig. 1E). As result, the activity of human recombinant Casp1 did not change by MSM co-treatment while the activity was attenuated by pan-caspase inhibitor, Z-VAD-FMK. We also tested the cytotoxicity of MSM using BMDMs (Fig. 1F). Cytotoxicity was observed up to 8% MSM treatment. Taken together, MSM was shown to act as a new NLRP3 inflammasome inhibitor.
3.2. MSM does not block AIM2 or NLRC4 inflammasome activation We further investigated the effect of MSM on activation of other inflammasomes such as AIM2 or NLRC4. AIM2 inflammasome is activated by cytosolic double-stranded DNA (dsDNA) derived from hosts or pathogens [12]. In the present study, we transfected dsDNA into BMDMs (Fig. 2A) and observed Casp1 as well as IL-1b secretion in the presence of increasing dosages of MSM. Casp1 and IL-1b secretion resulting from dsDNA-mediated AIM2 inflammasome activation was unaltered by MSM. We further inoculated Listeria monocytogenes (Listeria) into BMDMs for AIM2 inflammasome activation [13]. Listeria-mediated IL-1b secretion was inhibited by MSM (Fig. 2B). To investigate this discrepancy regarding the effect of MSM on AIM2 inflammasome activation triggered by dsDNA and Listeria, we measured the invasion rate of Listeria into BMDMs since this event precedes AIM2 inflammasome activation [14]. Interestingly, MSM dose-dependently reduced the invasion rate of Listeria (Fig. 2C), indicating that MSM had no effect on AIM2 inflammasome activation. The NLRC4 inflammasome is activated by gram-negative bacteria possessing type III or IV secretion systems, including Salmonella typhimurium, Shigella flexneri, Legionella pneumophila, and Pseudomonas aeruginosa [15]. To activate the NLRC4 inflammasome, we transfected flagellin or inoculated Salmonella typhimurium (Salmonella) into macrophages with/without MSM and observed Casp1 and IL-1b secretion. Similar to the AIM2 inflammasome, MSM did not alter flagellin induced-NLRC4 inflammasome activation (Fig. 2D and 2E), whereas it strongly attenuated Salmonella-mediated IL-1b secretion (Fig. 2F). To elucidate this discrepancy, we again tested the invasion rate of Salmonella using BMDMs. The number of intracellular Salmonella was significantly reduced by MSM (Fig. 2G), which combined with the result of the Listeria invasion experiment implies that MSM may have attenuated bacterial penetration. We additionally elucidated the bactericidal effect of MSM on Salmonella and Listeria, but MSM did not change the bacterial growth (data not shown). Thus, MSM did not regulate AIM2 or NLRC4 inflammasome activation.
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Fig. 1. Effect of MSM on NLRP3 inflammasome activation. (A) BMDMs (1 106 cells per well) were primed by LPS (1 lg/ml) in RPMI medium containing 10% FBS and antibiotics for 3 h. Cells were supplied with RPMI medium in the presence of the indicated concentrations of MSM with ATP (2 mM), NG (40 lM), or Alum (200 lg/ml), after which they were analyzed for Casp1 and IL-1b secretion by immunoblotting. (B) Secreted IL-1b was quantitated by an ELISA based assay kit and presented with bar graphs. (C) The secretion of IL-1b and the formation of Asc pyroptosome were analyzed by immunoblotting as indicated. (D) THP-1 cells were differentiated into macrophage-like cells by PMA (100 nM) treatment for 72 h and primed with LPS (1 lg/ml) for 3 h. LPS-primed THP-1 cells were treated with the indicated dosages of MSM in the presence of ATP (2 mM), NG (40 lM), or Alum (200 lg/ml), after which IL-1b secretion was detected by immunoblotting. (E) The effect of MSM of Casp1 activity was measured in the present of MSM or Z-VAD-FMK (Z-VAD, a pan-caspase inhibitor). (F) Cytotoxicity of MSM was measured after applying the indicated dosages of MSM to BMDMs. Triton -100 (0.01%, Triton) was used as a negative control for complete cell death. The Triton-treated group was used to determine 0% survival rate while the non-treated group was used as 100%. Data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate. Cell culture supernatants (Sup), cell lysates (Lys) and cross-linked pellets (Pellet) from whole-cell lysates were analyzed by immunoblotting as indicated. All immunoblot data shown are representative of at least two independent experiments.
3.3. MSM suppress pro-inflammatory cytokine expression MSM has been reported as an anti-inflammatory molecule due to its inhibition of NF-jB signaling mediating pro-inflammatory cytokine expression in LPS-stimulated RAW264.7 cells [16]. Here, we investigated whether or not MSM inhibits expression of proIL-1b and NLRP3, which is regulated by NF-jB signaling [17], in LPS-primed BMDMs. As shown in Fig. 3A, MSM dose-dependently inhibited the translational expression of both pro-IL-1b and TNF a. In addition, MSM reduced the mRNA transcription levels of IL-1a (Fig. 3B), pro-IL-1b (Fig. 3C), IL-6 (Fig. 3D), and NLRP3 (Fig. 3E). Thus, MSM not only blocked the NLRP3 inflammasome at the activation step but also down-regulated pro-IL-1b and NLRP3 expression at the priming step.
3.4. MSM-enriched vegetables inhibit NLRP3 inflammasome activation MSM is a form of organic nutritional sulfur found in dairy products such as fruits, grains, meats, and vegetables. Especially, plants of the genus Allium contain substantial amounts of MSM [18]. Esculent plants in the genus Allium include garlic (Allium
scorodorpasum), onion (Allium cepa), chive (Allium tuberosum), and wild chive (Allium monanthum). These plants form solitary or clustered bulbs and are strongly aromatic, with a characteristic odor when crushed. We further tested the effects of extracts of MSM-enrich plants on NLRP3 inflammasome activation (Fig. 4). IL-1b secretion mediated by NLRP3 inflammasome activators such as ATP or NG in BMDMs was attenuated by all of the extracts, although the effective concentrations varied. Further, the extracts did not show any cytotoxicity in the BMDMs. Taken together, MSM-enriched vegetables could be suggested as candidates for the inhibition of NLRP3 inflammasome activation.
3.5. MSM and DMSO inhibit NLRP3 inflammasome activation via an ROS pathway MSM is chemically related to dimethyl sulfoxide (DMSO), which has one less oxygen atom than MSM (Fig. 5A). These two molecules share several pharmacological effects due to their similar chemical properties [19]. Previously, we reported that DMSO inhibits NLRP3 inflammasome activation [10]. Here, we investigated the synergic inhibitory activity of DMSO and MSM on NLRP3 inflammasome
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Fig. 2. Effect of MSM on AIM2 or NLRC4 inflammasome activation. AIM2 inflammasome in LPS-primed BMDMs were activated by dsDNA (1 lg/ml) with transfection agent for 1 h (A) or Listeria monocytogenes (Listeria; 10%, OD600: 1.2) for 3 h (B), after which Casp1 or IL-1b secretion was detected by immunoblotting. (C) BMDMs were inoculated with Listeria in the presence of MSM for 3 h, after which the number of intracellular bacteria (colony forming unit, cfu) was calculated. cfu of the Listeria-inoculated group was set as 100%, and data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate. NLRC4 inflammasome in LPS-primed BMDMs was activated by flagellin (0.5 mg/ml) transfection for 1 h (D, E) or Salmonella typhimurium (Salmonella; 1%, OD600: 1.2) for 1 h (F), after which Casp1 or IL-1b secretion was detected by immunoblotting or ELISA. (G) BMDMs were inoculated with Salmonella in the presence of MSM for 1 h, after which the number of intracellular bacteria was calculated. cfu of the Salmonella-inoculated group was set as 100%, and data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate.
activation (Fig. 5B). When BMDMs were treated with a given concentration of DMSO along with various MSM concentrations, and vice versa, we observed similar reductions in IL-1b production depending on the accumulated concentrations of DMSO and MSM. In addition, the sum concentration of DMSO and MSM inhibited IL-1b secretions in dose-dependently (Fig. 5C). Thus, the inhibitory effects of both MSM and DMSO on NLRP3 inflammasome activation could have occurred through the same intracellular mechanism. Next, we determined the intracellular inhibitory mechanisms of both MSM and DMSO on NLRP3 inflammasome activation. MSM and DMSO have been studied as free radical scavengers previously [20]. Various models for NLRP3 inflammasome activation have been proposed, with the concept of upstream reactive oxygen species (ROS) production in relation to NLRP3 activation gaining particular
attention [21,22]. To confirm whether or not MSM and DMSO inhibit NLRP3 inflammasome activation via mitochondrial ROS generation, we firstly confirmed the effect of DPI, an inhibitor of cytosolic and mitochondrial ROS production [23], on NLRP3 inflammasome activation (Fig. 5D). As expected, DPI inhibited ATP- or NG-derived IL-1b secretion. Next, we applied rotenone, which activates the NLRP3 inflammasome via mitocondrial ROS [22], in the presence of DMSO or MSM to LPS-primed BMDMs (Fig. 5E). DMSO and MSM inhibited rotenone-mediated IL-1b secretion, similar to DPI. In addition, we measured the rotenone-induced mitocondrial ROS generation in the present of MSM, DMSO, or DPI (Fig. 5G). As expected, MSM or DMSO blocked the mitocondrial ROS. Thus, MSM as well as DMSO inhibited NLRP3 inflammasome activation by blocking mitochondrial ROS generation.
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Fig. 3. Effect of MSM on expression of pro-inflammatory cytokines and NLRP3. BMDMs (1 106 cells per well for immunoblotting and 2 106 cells per well for qPCR) were treated with the indicated concentrations of MSM with/without LPS (10 ng/ml). (A) effects of MSM on LPS-mediated pro-IL-1b and TNF a translation were detected by immunoblotting. Transcriptional levels of IL-1a (B), pro-IL-1b (C), IL-6 (D), and NLRP3 (E) were analyzed using SYBR green-based quantitative real-time PCR. Data represent the mean ± s.e.m. for three independent experiments.
4. Discussion In this study, we confirmed MSM as a selective NLRP3 inflammasome inhibitor. Although the anti-inflammatory properties of MSM have been studied before, no report has considered the effect of MSM on inflammasome activation producing the active form of IL-1b. To confirm the inhibitory role of MSM in inflammation, we tested the effect of MSM on two distinct steps of inflammation. In the first step known as priming, NLRP3, a sensing protein of inflammasomes, along with pro-IL-1b, a substrate of inflammasome activation, are transcriptionally up-regulated by toll-like receptor ligands or cytokines through the NF-jB signaling pathway. The second step triggers inflammasome assembly, leading to activation of Casp1, which proteolytically cleaves the precursor form of IL-1b [24]. In our results, MSM attenuated NF-jB signalingdependent NLRP3 and pro-IL-1b expression in the priming step, resulting in insufficient inflammasome activation. In the second (activation) step, MSM inhibited IL-1b secretion resulted from NLRP3 inflammasome activation even though cells were treated independently with the first step’s signals. We further confirmed that MSM attenuated NLRP3 inflammasome activation through inhibition of mitochondrial ROS production, similar to DMSO. In addition, extracts of MSM-enriched vegetables presented the same inhibitory effects on NLRP3 inflammasome activation as MSM. Thus, MSM possesses dual anti-inflammatory properties:
down-regulation of pro-inflammatory cytokines and inhibition of NLRP3 inflammasome activation. The anti-inflammatory effect of MSM has been progressively reported. In LPS-treated RAW264.7 cells, MSM has been shown to reduce the levels of IL-6 and TNF-a in addition to down-regulating NF-jB signaling [16]. Another study showed that MSM can ameliorate experimental colitis by reducing the level of IL-1b [25]. MSM pre-treatment also was shown to protect against hepatic liver injury by reducing TNF-a and IL-6 levels [26]. In addition, MSM intake is effective for treating inflammatory diseases such as osteoarthritis, seasonal allergic rhinitis, and intestinal cystitis [8,16,27,28]. Especially, MSM has been suggested as a dietary supplement for the treatment of osteoarthritis and is believed to be non-toxic [20,28]. Osteoarthritis is a progressive joint disease characterized by cartilage degeneration, subchondral bone remodeling, and inflammation of the synovial membrane. The pro-inflammatory cytokine IL-1b has been identified as one of the key mediators of osteoarthritis produced by activated synovial macrophages, resulting in inflammatory and destructive responses [29,30]. It has been reported that mice lacking inflammasome components are protected against osteoarthritis and NLRP3 inflammasome activation, which plays a pivotal role in the development of osteoarthritis [29]. Based the above evidence, MSM might be a candidate for the treatment of osteoarthritis based on its ability to attenuate the expression of pro-inflammatory cytokines and block NLRP3 inflammasome activation.
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Fig. 4. Effects of extracts of MSM-enriched vegetables on NLRP3 inflammasome activation. Extracts of garlic (A), onion (C), chive (E), and wild chive (G) were applied to LPSprimed BMDMs in the presence of the NLRP3 activators ATP or NG. IL-1b secretion was observed by immunoblotting. Cytotoxicity of garlic (B), onion (D), chive (F), or wild chive (H) in BMDMs was measured. The Triton-treated group (0.001%, Triton -100) was used to determine 0% survival rate while the non-treated group was used as 100%. Data represent the mean ± s.e.m. of three independent experiments, each performed in triplicate.
MSM and DMSO are very similar sulfur compounds. The ability and health benefits of MSM are almost identical to those of DMSO, with only slight differences [31]. Sulfur compounds are known to be effective hydroxyl radical scavengers [20]. Since MSM and DMSO both improve mitochondrial oxidative phosphorylation, it has been suggested that they can neutralize the cytotoxic effects of hydroxyl radicals in mitochondria themselves [28,32,33]. Several intracellular models of NLRP3 inflammasome activation have been reported, and ROS generation seems to be a unifying factor as it consistently mediates NLRP3 activation in response to several stimuli [21]. Inhibition of NADPH oxidase-dependent ROS production has been previously suggested to occur upstream of NLRP3 inflammasome activation, and accumulation of ROS-producing mitochondria by rotenone, a pharmacological inhibitor of the mitochondrial electron transport chain, was recently shown to trigger NLRP3 inflammasome activation [22]. Thus rotenone, which inhibits the transfer of electrons from iron–sulfur centers in complex I to ubiquinone, promotes mitochondrial ROS-mediated NLRP3 inflammasome activation [22]. The above evidence combined with our current data support the claim that MSM as well as DMSO attenuate IL-1b maturation through NLRP3 inflammasome activation by inhibiting mitochondrial ROS production. Induction of chronic inflammation by inflammasomes plays an essential role in the pathogenesis of several metabolic and
aged-related diseases such as type 2 diabetes, obesity, gout, and cardiovascular diseases [34]. Thus, regulation of inflammasome activity has received wide attention, and elucidation of inflammasome activation should shed light on the development of new therapeutic regimens [34]. In type 2 diabetes, the NLRP3 inflammasome activated by stimuli such as islet amyloid polypeptide promotes IL-1b secretion, which itself induces insulin resistance in insulin target cells and reduces insulin production in b cells [34,35]. In atherosclerotic disease, cholesterol crystals turn on pro-inflammatory cytokine expression via NF-jB signaling and activate the NLRP3 inflammasome, resulting in robust IL-1b release mediating arterial inflammation [36]. Metabolic byproducts of obesity activate inflammasomes in both adipocytes and adipose-associated innate immune cells that produce inflammasome-associated cytokines, thereby influencing the outcome of obesity [37]. In gouty arthritis, monosodium urate activates NF-jB signaling, similar to the priming step of inflammasome activation, and induces NLRP3 inflammasome activation for production of IL-1b [38]. Each disease induces NF-jB signaling to produce pro-inflammatory cytokines and NLRP3 as well as simultaneously triggers NLRP3 inflammasome activation. Thus, we suggest MSM as a therapeutic agent to control metabolic disorders due to its ability to effectively attenuate the expression of pro-inflammatory cytokines as well as activate the NLRP3 inflammasome in the current study.
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E
F
Fig. 5. Effect of MSM and DMSO on mitochondrial ROS-mediated inflammasome activation. (A) Chemical structures of MSM and DMSO were adopted from PubChem (http:// pubchem.ncbi.nlm.nih.gov). (B and C) BMDMs were primed with LPS (1 lg/ml) for 3 h, after which NLRP3 inflammasome activation was carried out with ATP (2 mM for 1 h) in the presence of DMSO or MSM as indicated. IL-1b secretion was measured by immunoblotting. (D) NLRP3 inflammasome activation in LPS-primed BMDMs was carried out with ATP (2 mM for 1 h), NG (40 lM for 1 h), or rotenone (80 lM for 6 h) in the presence of DPI (50 lM) to inhibit ROS production. IL-1b secretion was measured as a readout of inflammasome activation. (E) LPS-primed BMDMs were treated with rotenone (80 lM for 6 h) with/without DMSO or MSM as indicated. IL-1b secretion was detected by immunoblotting. (F) BMDMs were incubated with MitoSoxTM (2.5 lM) for 30 min and treated with rotenone (160 l) for 6 h to induce mitochondrial ROS generation in the present of MSM (2%), DMSO (2%), or DPI (100 lM). Bar graph represents the mean ± s.e.m. of three independent experiments, each performed in triplicate ‘ + ’; treatment as indicated; ‘’, non-treatment.
Acknowledgments This work was supported by the Basic Science Research Program (2012R1A1A1001645) and Radiation Technology R&D program (2012M2A2A6035733) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning, and by a 2013 Research Grant from Kangwon National University (No. 120131488).
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