Immunobiology 219 (2014) 315–322
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Dimethyl sulfoxide inhibits NLRP3 inﬂammasome activation Huijeong Ahn a , Jeeyoung Kim a , Eui-Bae Jeung b , Geun-Shik Lee a,∗ a
College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 30 July 2013 Received in revised form 16 October 2013 Accepted 14 November 2013 Available online 22 November 2013 Keywords: Cytokine DMSO Inﬂammasome Interleukin-1 Macrophages NLRP3
a b s t r a c t Dimethyl sulfoxide (DMSO) is an amphipathic molecule that is commonly/widely used as a solvent for biological compounds. In addition, DMSO has been studied as a medication for the treatment of inﬂammation, cystitis, and arthritis. Based on the anti-inﬂammatory characteristics of DMSO, we elucidated the effects of DMSO on activation of inﬂammasomes, which are cytoplasmic multi-protein complexes that mediate the maturation of interleukin (IL)-1␤ by activating caspase-1 (Casp1). In the present study, we prove that DMSO attenuated IL-1␤ maturation, Casp1 activity, and ASC pyroptosome formation via NLRP3 inﬂammasome activators. Further, NLRC4 and AIM2 inﬂammasome activity were not affected, suggesting that DMSO is a selective inhibitor of the NLRP3 inﬂammasomes. The anti-inﬂammatory effect of DMSO was further conﬁrmed in animal, LPS-endotoxin sepsis and inﬂammatory bowel disease models. In addition, DMSO inhibited LPS-mediating IL-1s transcription. Taken together, DMSO shows anti-inﬂammatory characteristics, attenuates NLRP3 inﬂammasome activation, and mediates inhibition of IL-1s transcription. Crown Copyright © 2013 Published by Elsevier GmbH. All rights reserved.
Introduction Dimethyl sulfoxide (DMSO, (CH3 )2 SO) is an amphipathic molecule with a highly polar domain and two apolar groups, making it soluble in both aqueous and organic media (Santos et al., 2003). Due to its physical and chemical properties, DMSO is a very efﬁcient solvent for water-insoluble compounds and is a hydrogen-bound disrupter. Although the biological effects of DMSO have not been fully identiﬁed, it is commonly adopted as a solvent of pharmacological studies. DMSO was previously reported to increase drug permeability and transport across tissue membranes using animal models (Jacob and Herschler, 1986). Thus, DMSO is popularly used as a vehicle for drug therapy for treatment of various diseases, including dermatological disorders, amyloidosis, gastrointestinal diseases, traumatic brain edema, musculoskeletal disorders, pulmonary adenocarcinoma, rheumatologic diseases, and schizophrenia (Santos et al., 2003). DMSO has been reported as a hydroxyl radical scavenger that improves mitochondrial oxidative phosphorylation and neutralizes the cytotoxic effects of free radicals formed upon ischemia in mitochondria (Colucci et al., 2008). DMSO has also been studied as a treatment for inﬂammation, cystitis, and arthritis (Kloesch et al., 2011).
∗ 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]
DMSO exhibits anti-inﬂammatory properties by reducing NF-B activation in association with decreased secretion and/or mRNA expression of pro-inﬂammatory mediators such as interleukin (IL)1␤ (Hollebeeck et al., 2011). However, there are conﬂicting reports about the effect of DMSO on IL-1␤ production (Xing and Remick, 2005). Inﬂammation mediated by the innate immune system as the ﬁrst line of host defense against pathogenic microbial or pathogen infection is an acute response that limits tissue damage and harm in the body (Schroder et al., 2010). Membrane-associated Toll-like receptors (TLR), c-type lectin receptors (CLR), cytosolic nucleotide-binding domain and leucine-rich repeat receptors (NLRs), retinoic acid-inducible gene (RIG)-like helicase (RLH) receptors, and absent in melanoma (AIM)-like receptors comprise the ﬁve main pattern recognition receptor families. TLR activation triggers different signaling cascades that result in NF-B activation and the synthesis of various pro-inﬂammatory mediators and cytokines (Medzhitov, 2001). One pro-inﬂammatory cytokine, IL1␤, is an important inﬂammatory mediator generated at sites of injury or immunological challenge that coordinates programs as diverse as cellular recruitment to sites of infection or injury as well as the regulation of sleep, appetite, and body temperature (Dinarello et al., 2010). However, dysregulation and chronic inﬂammation may result in secondary damage and immune pathology to the host. IL-1␤ activity is rigorously controlled at the levels of expression, maturation, and secretion. Pro-inﬂammatory stimuli induce expression of the inactive pro-from of IL-1␤, but its maturation and release are controlled by inﬂammasomes.
0171-2985/$ – see front matter. Crown Copyright © 2013 Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.imbio.2013.11.003
H. Ahn et al. / Immunobiology 219 (2014) 315–322
Inﬂammasomes are multiprotein complexes that operate as platforms for the activation of caspase-1 and are composed of one of several NLR and PYHIN proteins, including NLRP1, NLRP3, NLRC4, and AIM2 (Rathinam et al., 2012). Inﬂammasomes are sensors of endogenous or exogenous pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), which govern cleavage of effector pro-inﬂammatory cytokines such as pro-IL-1␤ and pro-IL-18. Inﬂammasomes have been found to regulate other important aspects of inﬂammation and tissue repair such as pyroptosis, a form of cell death (Lamkanﬁ and Dixit, 2011; Lamkanﬁ, 2011). Mutation of just one inﬂammasomeassociating gene has been shown to induce auto-inﬂammatory disease (McDermott et al., 1999). Inﬂammasome activation requires two steps, priming and activation. During the priming step, TLR stimuli induce production of pro-IL-1␤ and inﬂammasome components such as NLRP3 (Lee et al., 2012). In the activation step, inﬂammasome activators facilitate assembly of inﬂammasome complexes to induce self-cleavage of caspase-1. Although the effect of DMSO on IL-1␤ production has been progressively studied, controversial results have been reported (Xing and Remick, 2005). In this study, we investigated the effects of DMSO on the activation of well-described inﬂammasomes such as NLRP3, NLRC4, and AIM2 using bone marrow-derived macrophages (BMDMs) and animal models.
(Sigma–Aldrich Co.) were collected for further analysis. For cytotoxicity assay, BMDMs (10,000 cells/well) were plated in a 96-well plate (SPL life science Co.) and allowed to be attached for 3 h. The BMDMs were treated with the indicated dosage of DMSO for 1 h and replaced with fresh media (RPMI 1640 containing 10% FBS and antibiotics) followed by further incubation for 14 h. The cytotoxicity was measured by Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc. MD, USA) as manufacture’s protocol.
Materials and methods
Differentiation of bone marrow-derived macrophages (BMDMs)
S. typhimurium (Sun and Hahn, 2012) were kindly provided from Prof. Tae-Wook Han (Kangwon National University, Gangwon-do, Republic of Korea). Salmonella were grown on Luria-Bertani (LB, Laboratories Conda, Madrid, Spain) agar plates for 18 h at 37 ◦ C. A single colony was transferred to LB broth, after which the culture was incubated for 18 h with shaking at 37 ◦ C. The cultured bacteria were serially diluted and plated on the agar plates to calculate colony-forming units (cfu). To elucidate the effect of DMSO on bacterial growth, Salmonella (1.5 × 104 cfu) were growth in LB broth containing PBS, DMSO, or gentamycin for 18 h at 37 ◦ C, followed by calculation of cfu. For Salmonella host invasion assay, BMDMs (1 × 106 cells per well) were plated in 12-well plates and inoculated with Salmonella (2 × 107 cfu) in DMEM containing 10% FBS for 1 h. The plate was then washed with Gentamycin (50 g/ml; Komipharm International Co., Ltd., Gyeonggi-do, Republic of Korea) containing PBS to eliminate extracellular Salmonella and then plated on an LB plate to calculate cfu.
Unless otherwise indicated, all materials for cell culture were obtained from PAA (GE Healthcare Bio-Sciences Co., NJ, USA). BMDMs were obtained by differentiating bone marrow progenitors from the tibia and femur bones using L929 cell-conditioned medium (LCCM) as a source of granulocyte/macrophage colonystimulating factor (Englen et al., 1995). The progenitors were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 30% LCCM, 100 U/ml of penicillin, and 100 g/ml of streptomycin. Cells were seeded in non-tissue culture-treated Petri dishes (SPL Life Science Co., Gyeonggi-do, Republic of Korea) and incubated at 37 ◦ C in a 5% CO2 atmosphere for 7 days. Cell treatments BMDMs (1.0 × 106 cells per well) were plated in 12-well plates (SPL Life Science Co.) and primed with 10 g/ml of lipopolysaccharide (LPS; Sigma–Aldrich Co., MO, USA) in RPMI 1640 containing 10% FBS and antibiotics for 3 h. After LPS priming, BMDMs were subjected to the following activation step for 1 or 3 h. For NLRP3 inﬂammasome activation, the medium was replaced with RPMI 1640 supplemented with ATP (3.5 mM; InvivoGen, CA, USA) for 1 h, nigericin (NG, 40 M; Tocris Bioscience, Bristol, UK) for 1 h, calcium pyrophosphate dihydrate (CPPD; 200 g/ml; InvivoGen) for 1 h, CaCl2 (1 mM; Biosesang, Seoul, Republic of Korea) for 1 h, aluminum potassium sulfate (Alum; 200 g/ml; Daejeung Chemicals & Materials Co., Gyeonggi-do, Republic of Korea) for 3 h. For NLRC4 inﬂammasome activation, the media were exchanged with Salmonella typhimurium (OD600: 1.2, 0.5%) or puriﬁed ﬂagellin (500 ng/ml; InvivoGen) with Lipofectamin 10 ml/ml (Invitrogen, CA, USA) and collected after 1 h. For AIM2 inﬂammasome activation, LPS-primed BMDMs were supplemented with 2 g/ml of dsDNA containing 4 l/ml of Lipofectamine 2000 (Invitrogen) for 1 h. To determine the inhibitory effect of DMSO on inﬂammasome activation, DMSO (Daejeung Chemicals & Materials Co.) was co-treated with the above activators. Cellular supernatant (Sup), lysate (Lys), and cross-linked pellets (Pellet) with suberic acid bis
Western blot analysis Sup, Lys, and Pellet samples were separated by SDS-PAGE (10% or 16%) and blotted onto a polyvinylidene diﬂuoride membrane (Pall Co., NY, USA). Immunoblots were probed overnight at 4 ◦ C with anti-mouse IL-1␤ antibody (AF-401-NA, R&D Systems, MN, USA), anti-caspase-1 p20 antibody (06-503, EMD Millipore Co, MA, USA), anti-ASC antibody (sc-22514, Santa Cruz Biotechnology, CA, USA), anti-caspase-1 antibody (sc-622, Santa Cruz Biotechnology), or anti-actin antibody (sc-1615, Santa Cruz Biotechnology). The membranes were further probed with HRP-conjugated 2nd antisera (sc-2020 or sc-2004, Santa Cruz Biotechnology) and visualized by Power-Opti ECLTM solution (BioNote Co., Gyeonggi-do, Republic of Korea) and a cooled CCD camera System (AE-9150, EZ-Capture II, ATTO technology, Tokyo, Japan).
Animal experiments C57BL/6 mice (6- to 8-weeks-old) were purchased from Orient Co. (Seongnam, Republic of Korea) or Narabio Co. (Seoul, Republic of Korea). The animals were housed at 24 ± 1 ◦ C under 50% humidity and a 12-h light/dark cycle. 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 (ACUCC; approval no. KIACUC-12-0009). For LPS-induced endotoxin shock, mice were intraperitoneally (ip) pre-treated with vehicle (200 l of saline) or 10% DMSO at 1 h and 1 day before LPS (20 mg/kg) ip injection. The animals were observed every 8 h for 3 days. To induce inﬂammatory bowel disease (IBD) by dextran sulfate sodium (DSS), mice drank tap water only or water containing 3% DSS (MWs = 40 kDa; MP Biomedicals, CA, USA) for 7 days. The mice were then ip injected with 10% DMSO in 200 l of saline on days 0, 2, 4, 6, and 8. Body weights were recorded daily. For the Salmonella-mediated lethality study, mice were ip injected with vehicle (200 l of saline) or 10% DMSO at
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1 h and 1 day before oral S. typhimurium (3 × 106 cfu per mouse) administration. Lethality was monitored every 8 h for 9 days.
NM 007393) 5 -AGC CAT GTA CGT AGC CAT CC-3 and 5 -CTC TCA GCT GTG GTG GTG AA-3 . Statistical analyses
RNA extraction and real-time PCR For RNA extraction, BMDMs (2.0 × 106 cells per well) were plated in 6-well plates (SPL Life Science Co.) and treated 10 ng/ml of LPS with the indicated DMSO for 3 h. Total RNA was extracted using Trizol (Invitrogen) and reverse-transcribed to ﬁrst-stand complementary DNA (cDNA) using random primer (9-mer) and m M-MLV reverse transcripatase (Invitrogen) according to the manufacturer’s protocols. Transcripts were quantitated using SyberGreen (Applied Biosystems, Carlsbad, CA, USA) and the Eco Real-Time PCR system (Illumina, San Diego, CA, USA). Quantitation was normalized with beta-actin (Actb). IL-1␤ (Il1b; Genebank ID: NM 008361) primers 5 -CCC AAG CAA TAC CCA AAG AA-3 and 5 -GCT TGT GCT CTG CTT GTG AG-3 ; IL-1␣ (Il1a; NM 010554) 5 -CCG ACC TCA TTT TCT TCT GG-3 and 5 -GTG CAC CCG ACT TTG TTC TT-3 ; IL-6 (Il6; NM 031168) 5 -CCG GAG AGG AGA CTT CAC AG-3 and 5 -TCC ACG ATT TCC CAG AGA AC-3 ; TNF-␣ (Tnfa; NM 013693) 5 -ACG GCA TGG ATC TCA AAG AC-3 and 5 -GTG GGT GAG GAG CAC GTA GT-3 ; IL-10 (Il10; NM 010548) 5 -GCC TGG CTC AGC ACT GCT AT-3 and 5 GAA GGC AGT CCG CAG CTC TA-3 ; IL-12␣ (Il12a; NM 001159424) 5 -GCT ATC TGA GCT CCG CCT GA-3 and 5 -AGG AGC TTA AGG CCC ACC AG-3 ; IL-1RN (Il1rn; NM 001039701) 5 -GCC TGA TCA CTC TGG CCA TC-3 and 5 -AGG CCA GCC AAC AGA CTG AG-3 ; ␤-actin (Actb;
Statistical analyses were performed with a t-test for the two groups, or one-way ANOVA for multiple groups, and survival analysis for lethality test using GraphPad Prism (GraphPad Software, San Diego, CA). Results DMSO inhibits inﬂammasome activation To elucidate the effects of DMSO on inﬂammasome activation, we ﬁrstly treated various dosages of DMSO to LPS-primed BMDMs with/without ATP, a speciﬁc activator of the NLRP3 inﬂammasome. As shown in Fig. 1A, DMSO alone did not induce IL-1␤ (p17, active form) production nor alter pro-IL-1␤ secretion in the cell supernatant (Sup). In addition, any cytotoxicity of BMDMs was not detected by DMSO treatment (up to 10%) based on the cytotoxicity assay (Fig. 1B), and unchanged proIL-1␤ and actin expressions. However, DMSO co-treatment with ATP promoted reduction of IL-1␤ secretion in a dose-dependent manner. Active IL-1␤ was no longer present in the lysate (Lys), indicating that DMSO inhibited IL-1␤ production but not secretion. We further treated the other NLRP3 inﬂammasome
Fig. 1. Effect of DMSO on inﬂammasome activation. BMDMs (1 × 106 cells per well) were primed by LPS (10 g/ml) in RPMI medium containing 10% FBS and antibiotics for 3 h. (A) Cells were supplied with RPMI medium in the present with DMSO with/without ATP (3.5 mM) and were analyzed for IL-1 ␤ secretion by immunoblotting. (B) The cytotoxicity of DMSO was measured after applying the indicated dosage of DMSO on BMDMs for 1 h like to the inﬂammasome activating step. Triton X-100 (0.01%, Triton) suggested by the manufacturer presents complete cell death. Triton treated group sets as 0% of the survival rate and non-treated group sets as 100%. Data represent the mean ± s.e.m of three independent experiments, each performed in triplicate. (C) LPS-primed BMDMs were treated with NG (40 M), CPPD (200 g/ml), and CaCl2 (1 mM) for 1 h, or Alum (200 g/ml) for 3 h in the present of DMSO. (D) LPS-primed BMDMs were activated in the present of DMOS with Salmonella (OD600: 1.2, 0.5%) or 2 g/ml of dsDNA with 4 l/ml of Lipofectamine 2000 for 1 h. Cell culture supernatants (Sup) and cell lysates (Lys) were analyzed by immunoblotting as indicated.
H. Ahn et al. / Immunobiology 219 (2014) 315–322
Fig. 2. DMSO inhibits NLRP3 inﬂammasome activation. (A) LPS-primed BMDMs were co-treated with NLRP3 inﬂammasome activators, ATP or nigericin (NG), as well as the indicated percentages of DMSO. Cell culture supernatants (Sup), cell lysates (Lys), and cross-linked pellets (Pellet) from whole-cell lysates were analyzed by immunoblotting as indicated. (B) LPS-primed BMDMs (1.5 × 106 cells per reaction) were further incubated with NG for 5 min and then subjected to Casp-1 activity assay using YVAD-pNA. Data represent the mean ± s.e.m for three independent experiments. (C) Mice (n = 10 per each group) were intraperitoneally (ip) injected with saline (200 l) with or without 10% DMSO at 1 h before LPS (20 mg/kg) administration. Survival rates were observed at the indicated times. (D) Mice (n = 10 per each group) were treated with 3% DSS for the indicated period (DSS) and then ip injected with 10% DMSO in saline (200 l) at the indicated time (arrows). Body weights were monitored daily for 11 days. Data represent the mean ± s.d. for ﬁve mice per group. *P < 0.05 vs. DSS only.
activators, such as nigericin (NG), calcium pyrophosphate dihydrate (CPPD), calcium chloride (CaCl2 ), and aluminum potassium sulfate (Alum), to LPS-primed BMDMs in the presence of DMSO to conﬁrm the inhibitory effect of DMSO on NLRP3 inﬂammasome activation. DMSO attenuated mature IL-1␤ production in NG, CPPD, CaCl2 , and Alum-treated macrophages, whereas the inhibitory potency of DMSO on NLRP3 inﬂammasome activation varied (Fig. 1C). To conﬁrm the inhibitory potencies of DMSO on the NLRC4 and AIM2 inﬂammasomes, we co-treated DMSO to Salmonella-infected and dsDNA-transfected BMDMs, respectively (Fig. 1D). Salmonella-mediated IL-1␤ maturation via the NLRC4 inﬂammasome was attenuated by DMSO, similar to the NLRP3 inﬂammasome result. However, dsDNA-induced IL-1␤ maturation via the AIM2 inﬂammasome was not altered by DMSO, suggesting that DMSO only inhibited the NLRP3 and NLRC4 inﬂammasomes. Hence, we conclude that DMSO could be a candidate for the selective inhibition of the NLRP3 and NLRC4 inﬂammasomes.
DMSO inhibits NLRP3 inﬂammasome Inﬂammasome activation involves the production of activated caspase-1 (Casp1; p10 and p20) as well as the induction of pyroptosis (Lamkanﬁ and Dixit, 2012). To determine the role of DMSO in these two phenomena of inﬂammasome activation, we observed the effects of DMSO on secretion of Casp1 (p20) in the cell supernatant as well as on formation of the ASC pyroptosome in the insoluble pellet after cross-linking with disuccinimidyl suberate (Fig. 2A). Secretion of Casp1 was attenuated by DMSO in the presence of ATP or NG, although pro-Casp1 was evenly observed in the lysate. In addition, DMSO reduced formation of the ASC pyroptosome by NLRP3 inﬂammasome activators. Thus, DMSO inhibited Casp1 secretion and ASC pyroptosis followed by NLRP3 inﬂammasome activation, similar to the results on IL-1␤ secretion. In addition, we tested Casp1 activity by incubating YVAD-pNA, a Casp1 substrate, in BMDM lysate containing NG with or without DMSO (Fig. 2B). Casp1
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activity was induced by NG alone, whereas activities were inhibited by DMSO in a dose-dependent manner. Taken together, we propose that DMSO blocked activation of the NLRP3 inﬂammasome. In the literature, the susceptibility of LPS-induced endotoxin shock is reduced in NLRP3 gene-depleted mice when compared with intact mice (Mariathasan et al., 2006). In addition, loss of body weight in an inﬂammatory bowel disease (IBD) model was shown to be less severe in NLRP3-deﬁcient mice (Bauer et al., 2010). In this study, we elucidated the protective effects of DMSO in these animal models. As shown in Fig. 2C, mice treated with LPS alone presented a 30% of survival rate at 32 h after post-injection, whereas 70% of mice were alive in the DMSO-treated group at the same time point. Finally, DMSO-treated mice showed 60% survival by the end of the experiment. In the IBD model, mice that received water containing dextran sulfate sodium (DSS) presented 13.5% body weight loss, whereas DMSO-injected mice treated with DSS-containing water (DSS + DMSO) showed only 6.7% body weight loss at the same time point (Fig. 2D). Those mice treated with tap water or DMSO only showed no body weight loss. Thus, DMSO ameliorated the severity of NLRP3 inﬂammasome-mediated disease symptoms in animals, which is similar to our result that DMSO inhibited IL-1␤ maturation in BMDMs. DMSO does not directly inhibit NLRC4 inﬂammasome DMSO not only inhibited IL-1␤ maturation via the NLRP3 inﬂammasome but also attenuated IL-1␤ production via the NLRC4 inﬂammasome, as seen in Fig. 1C. For more precise analysis of inﬂammasome activation, we elucidated formation of the ASC pyroptosome in the presence of DMSO with Salmonella for NLRC4 inﬂammasome activation or dsDNA for AIM2 inﬂammasome activation (Fig. 3A). As expected, DMSO attenuated formation of the ASC pyroptosome in the presence of Salmonella, similar to the results on IL-1␤ secretion. However, DMSO did not reduce formation of the ASC pyroptosome via AIM2 inﬂammasome activation. We further elucidated the inhibitory effect of DMSO on NLRC4 inﬂammasome activation in animals. Mice were treated with DMSO before oral administration of Salmonella, and the survival rate was observed (Fig. 3B). Although the infected mouse group treated with DMSO presented a bit higher survival rate than the group without DMSO treatment, there was no signiﬁcant different between them. This result demonstrates that DMSO had no protective effect in mice against oral Salmonella infection. To elucidate the difference between in vivo and in vitro data, we further tested whether or not DMSO has a bactericidal effect on S. typhimurium, an activator of the NLRC4 inﬂammasome in the current study (Fig. 3C). DMSO did not show any bactericidal effect, whereas the antibiotic gentamycin completely blocked growth of Salmonella. Salmonella need to invade the cytoplasm for activation of inﬂammasomes due to their identity as cytosolic pattern recognition receptors. Thus, we elucidated the invasion of Salmonella into BMDMs as well as its intracellular growth rate in the presence of DMSO. After 1 h of inoculation of Salmonella to BMDMs, the number of intracellular bacteria was signiﬁcantly reduced by DMSO, which implies that DMSO may have attenuated the penetrating property of Salmonella (Fig. 3D). In addition, we elucidated the effect of DMSO on NLRC4 inﬂammasome activation using the transfection of puriﬁed ﬂagellin into BMDMs (Fig. 3E). Flagellin-mediated IL-1␤ secretion did not changed by DMSO. This result demonstrates that DMSO inhibited Salmonella-mediated IL-1␤ secretion by attenuating Salmonella invasion. DMSO reduces transcriptional levels of IL-1s and its antagonist DMSO has been studied as an anti-inﬂammatory medication due to its reported role as a free radical scavenger (Colucci et al.,
2008). Indeed, several studies have suggested that DMSO reduces the expression of pro-inﬂammatory cytokines via inhibition of NF-B signaling (Xing and Remick, 2005). However, there are conﬂicting reports about its effect on cytokine expression (Xing and Remick, 2005). Hence, we tested the effects of DMSO on the transcriptional levels of cytokines in BMDMs with or without LPS stimulation. DMSO dose-dependently reduced the expression of pro-inﬂammatory cytokines (IL-1␣, pro-IL-1␤, and IL-6) in BMDMs stimulated by LPS, although DMSO alone did not induce any pro-inﬂammatory cytokines (Fig. 4A–D). DMSO also attenuated the mRNA expression of IL-1RN (IL-1 receptor antagonist) (Fig. 4C). However, DMSO did not alter transcriptional levels of other cytokines, including TNF-␣, IL-12A, and IL-10 (Fig. 4F–H). Collectively, DMSO inhibited the transcription of IL-1s, IL-6, and IL-RN. Discussion Using in vitro and in vivo approaches, we provide evidence that DMSO plays an inhibitory role in NLRP3 inﬂammasome activation. Indeed, the anti-inﬂammatory properties of DMSO have been progressively studied (Loppnow and Libby, 1990; Reimann et al., 1994; Xaus et al., 2000), although there has been no investigation into inﬂammasome activation producing active IL-1␤. In this study, we elucidated the effect of DMSO on IL-1␤ production in the context of two distinct signals: the ﬁrst signal leads to transcriptional upregulation and synthesis of pro-IL-1␤ while the second signal leads to IL-1␤ maturation and secretion through inﬂammasome activation. In the ﬁrst signal step, DMSO down-regulated pro-IL-1␤ transcription, which may have attenuated secretion of IL-1␤ due to an insufﬁcient amount of substrate for inﬂammasome activation. In the second signal step, DMSO inhibited IL-1␤ maturation and secretion even though cells were treated with the ﬁrst signal. Thus, DMSO has dual properties that inhibit IL-1␤ production. In addition, the anti-inﬂammatory role of DMSO was conﬁrmed using animal models. Although we have not fully deﬁned the inhibitory mechanism of DMSO on inﬂammasome activation in the current study, we can speculate based on previous reports. DMSO is a well-known antioxidant that acts as a free radical scavenger (Santos et al., 2003; Phillis et al., 1998). Various models of NLRP3 inﬂammasome activation have been proposed, and the concept of reactive oxygen species (ROS) being upstream of NLRP3 activation has gained particular attention (Schroder et al., 2010). Genetic and pharmacological inhibition of NADPH oxidase-dependent ROS production is suggested to be upstream of NLRP3 inﬂammasome activation (Dostert et al., 2008). In addition, inhibitors of ROS production or ROS scavengers have been shown to strongly inhibit NLRP3 inﬂammasome activation (Cassel et al., 2008). Indeed, in line with the notion that mitochondria constitute the biggest source of cellular ROS, it was subsequently shown that mitochondria are the site of ROS production during NLRP3 inﬂammasome activation (Nakahira et al., 2011; Zhou et al., 2011). In this same context, it has also been demonstrated that inhibitors of mitochondrial ROS production (Nakahira et al., 2011) as well as knockdown of mitochondrial respiration mediated by reduced expression of voltage-dependent anion channels (Zhou et al., 2011) downregulate NLRP3-mediated inﬂammasome activation. Based on these ﬁndings, we suggest that DMSO is an ROS scavenger resulting in inhibition of NLRP3 inﬂammasome activation. ROS also activates pro-inﬂammatory gene expression in various innate immune signaling pathways (van de van de Veerdonk et al., 2010; Bulua et al., 2011). Bacterial LPS is commonly adopted as the ﬁrst signal to induce pro-IL-1␤. LPS regulates several macrophage functions (Hsu and Twu, 2000; van Lenten and Fogelman, 1992), including the induction of inﬂammatory cytokines such as IL-1,
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Fig. 3. Effect of DMSO on Salmonella mediating NLRC4 inﬂammasome activation. (A) LPS-primed BMDMs were co-treated with NLRC4 or AIM2 inﬂammasome activators, Salmonella, or dsDNA with Lipofectamine 2000 as well as the indicated percentages of DMSO. Cell culture supernatants (Sup), cell lysates (Lys), and cross-linked pellets (Pellet) from whole-cell lysates were analyzed by immunoblotting as indicated. (B) 8-weekold female mice (C57BL/6, n = 10 per each group) were intraperitoneally (ip) injected with saline (200 l) with or without 10% DMSO at 1 h before Salmonella administration. Survival rates were observed at the indicated times. (C) Bactericidal effect of DMSO was measured in the presence of the indicated percentages of DMSO. Data represent the mean ± s.e.m for three independent experiments. (D) Invasion of Salmonella into BMDMs was analyzed in the presence of the indicated percentages of DMSO. Data represent cfu for six independent experiments. (E) LPS-primed BMDMs were transfected the puriﬁed ﬂagellin in the present of DMSO and IL-1␤ secretion were analyzed.
IL-6, and TNF (Loppnow and Libby, 1990; Reimann et al., 1994; Xaus et al., 2000; Zhu et al., 2000). After LPS interacts with the TLR4 complex, macrophages produce reactive oxygen intermediates (Mendez et al., 1995) while nuclear factor (NF)-B activation is triggered, resulting in IL-1␤ production by macrophages (Xing and Remick, 2005). Further, DMSO has an inhibitory effect on NF-B activation (Chang et al., 1999). Hence, DMSO inhibits proinﬂammatory cytokine expression via ROS scavenging and NF-B inhibition, as seen in Fig. 4. Recently, we and others have reported that an increase in intracellular calcium triggers NLRP3 inﬂammasome activation (Lee et al., 2012; Murakami et al., 2012; Rossol et al., 2012). That is, inﬂammasome activators in the second signal step induce intracellular calcium ions, thereby facilitating formation of inﬂammasome complexes. DMSO has been reported to reduce the intracellular calcium concentration following exposure to hypoxic conditions and ionophore-induced calcium loading in several cell types (Santos et al., 2003). Although the precise mechanism by which DMSO prevents intracellular calcium induction has not been elucidated, it could explain how DMSO inhibits NLRP3 inﬂammasome activation. The explanation for the inhibitory effect of DMSO on NLRC4 inﬂammasome activation could involve inhibition of Salmonella host cell invasion by DMSO (Antunes et al., 2010). As shown in Fig. 3C, the invasion of Salmonella into BMDMs was attenuated by DMSO, although we could not explain the molecular aspects. The invasion of host cells by Salmonella is controlled by a complex cascade of regulatory proteins. One of the major players in the regulation of invasion-related gene expression is the OmpR/ToxRtype transcriptional regulator HilA (Lee et al., 1992). DMSO is reduced to dimethyl sulﬁde (DMS) during anaerobic growth, and
DMS strongly inhibits HilA gene expression, which may support our results (Antunes et al., 2010). We conclude that DMSO is an inhibitor of NLRP3 inﬂammasome activation in the current study. However, DMSO is a commonly adopted solvent and cryoprotectant in biological research. Indeed, several inﬂammasome activators such as nigericin, m-M3FBS, R528, Rotenone, and thapsigargin, have been dissolved in DMSO to activate inﬂammasomes (Lee et al., 2012; Zhou et al., 2011). In addition, DMSO is also used as a solvent for inhibitors of inﬂammasome activation such as BAPTA-AM (Murakami et al., 2012). Nevertheless, DMSO as a solvent does not interrupt inﬂammasome activation or inhibition as its ﬁnal concentration is less than 1%. Thus, we suggest that a greater than 2% concentration of DMSO would have inhibitory effects on inﬂammasome activation and may present anti-inﬂammatory properties. Indeed, less than 10% of DMSO is well-adopted for cryopreservation and do not induce any cytotoxicity (Da Violante et al., 2002). Although the biological and pharmacological effects of DMSO have not been clearly deﬁned, it is used extensively in a variety of ﬁelds. Due to its anti-inﬂammatory and ROS scavenger activities, DMSO is popularly used as a vehicle in drug therapy for various diseases, including dermatological disorders, amyloidosis, gastrointestinal diseases, traumatic brain edema, mausculoskeletal disorders, pulmonary adenocarcinoma, rheumatologic disease, schizophrenia, and Alzhimer’s diseases (Santos et al., 2003). On the other hand, several undesirable side effects stemming from the use of DMSO have been reported, namely cardiac problems and a garlic-like breath odor and taste in mice (Santos et al., 2003). In this study, we suggest an anti-inﬂammatory property for DMSO
H. Ahn et al. / Immunobiology 219 (2014) 315–322
Fig. 4. DMSO inhibits pro-inﬂammatory cytokine expressions. BMDMs (2 × 106 cells per well for RNA [A, C, D, E, F, G, and H] and 1 × 106 cells per well for protein [B]) were treated with LPS (10 ng/ml; w/LPS) supplemented with the indicated percentages of DMSO in RPMI medium containing 10% FBS and antibiotics for 3 h. Total RNA (1 g) was isolated and cDNA synthesized using MMLV reverse transcriptase as described in Material and Methods. Relative levels of the indicated transcripts were analyzed using SYBR green-based quantitative real-time PCR. Data represent the mean ± s.e.m for three independent experiments. *P < 0.01 vs. LPS only.
H. Ahn et al. / Immunobiology 219 (2014) 315–322
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