Suppresses IL-1β Secretion and Prevents Inflammation through Inhibition of the NLRP3 Inflammasome

This information is current as Haipeng Yin, Qiang Guo, Xin Li, Tiantian Tang, Cuiling Li, of October 4, 2021. Hengxiao Wang, Yuanxin Sun, Qi Feng, Chunhong Ma, Chengjiang Gao, Fan Yi and Jun Peng J Immunol published online 16 March 2018 http://www.jimmunol.org/content/early/2018/03/15/jimmun ol.1701495 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 16, 2018, doi:10.4049/jimmunol.1701495 The Journal of Immunology

Curcumin Suppresses IL-1b Secretion and Prevents Inflammation through Inhibition of the NLRP3 Inflammasome

Haipeng Yin,*,† Qiang Guo,† Xin Li,* Tiantian Tang,‡ Cuiling Li,† Hengxiao Wang,† Yuanxin Sun,* Qi Feng,* Chunhong Ma,x Chengjiang Gao,x Fan Yi,x and Jun Peng*,{

Turmeric is traditionally used as a spice and coloring in foods. Curcumin is the primary active ingredient in the turmeric, and compelling evidence has shown that it has the ability to inhibit inflammation. However, the mechanism mediating its anti- inflammatory effects are not fully understood. We report that curcumin inhibited caspase-1 activation and IL-1b secretion through suppressing LPS priming and the inflammasome activation pathway in mouse bone marrow–derived macrophages. Downloaded from The inhibitory effect of curcumin on inflammasome activation was specific to the NLRP3, not to the NLRC4 or the AIM2 inflammasomes. Curcumin inhibited the NLRP3 inflammasome by preventing K+ efflux and disturbing the downstream events, including the efficient spatial arrangement of mitochondria, ASC oligomerization, and speckle formation. Reactive oxygen species, autophagy, sirtuin-2, or acetylated a-tubulin was ruled out as the mechanism by which curcumin inhibits the inflammasome. Importantly, in vivo data show that curcumin attenuated IL-1b secretion and prevented high-fat diet–induced insulin resistance in wide-type C57BL/6 mice but not in Nlrp3-deficient mice. Curcumin also repressed monosodium urate crystal–induced peritoneal http://www.jimmunol.org/ inflammation in vivo. Taken together, we identified curcumin as a common NLRP3 inflammasome activation inhibitor. Our findings reveal a mechanism through which curcumin represses inflammation and suggest the potential clinical use of curcumin in NLRP3-driven diseases. The Journal of Immunology, 2018, 200: 000–000.

urmeric, a common oriental spice that gives curry powder However, despite the mounting evidence of the anti-inflammatory its yellow color, is frequently used in Asian cooking, effects of curcumin, large gaps in knowledge still exist regarding its T particularly Indian, Pakistani, and Thai cooking. Curcumin, mechanisms of action. a polyphenolic compound, is the principal derived from Inflammasomes are multimeric protein complexes that orches-

the rhizomes of turmeric (1). The other two derived trate host defense mechanisms against pathogen-associated mo- by guest on October 4, 2021 from turmeric extract are desmethoxycurcumin (DMC, 15%) and lecular patterns released by infectious agents and danger-associated (BDMC, 5%) (2). Turmeric has a long molecular patterns released during noninfectious physiological history of use in Ayurvedic medicine as a treatment for inflamma- damage (5). Assembly of the inflammasome complex is initiated tory conditions. Because of its numerous biological activities and by a nucleotide-binding domain and leucine-rich repeat receptors health benefits, curcumin is of current interest, especially as an anti- or absent in melanoma 2–like receptors. NOD-like receptors and inflammatory agent (3). In particular, both in vitro and in vivo AIM2-like receptors mediate host recognition of a diverse set of studies have demonstrated that curcumin can suppress inflamma- inflammation-inducing stimuli and control the production of tion with no associated toxicities and plays a beneficial role in a highly proinflammatory cytokines IL-1b and IL-18 (6). Addi- variety of inflammatory diseases, including obesity, diabetes, car- tionally, inflammasome activation causes a rapid, proinflammatory diovascular diseases, bronchial asthma, and rheumatoid arthritis (4). form of cell death called pyroptosis (7).

*Department of Hematology, Qilu Hospital, Shandong University, Jinan 250012, analyzed the data; and J.P. designed the research studies, supervised the project, and China; †Key Laboratory for Tumor Immunology and Traditional Chinese Medicine edited the manuscript. J.P. is the guarantor of this work and, as such, had full access Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, to all the data in the study and takes responsibility for the integrity of the data and the Jinan 250062, China; ‡Institute of Immunology and the CAS Key Laboratory of accuracy of the data analysis. Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, x Address correspondence and reprint requests to Prof. Jun Peng, Department of He- University of Science and Technology of China, Hefei 230027, China; Shandong { matology, Hematology Oncology Center, Qilu Hospital of Shandong University, University School of Medicine, Jinan 250012, China; and Key Laboratory of Car- Jinan 250012, China. E-mail address: [email protected] diovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Jinan 250012, China The online version of this article contains supplemental material. ORCIDs: 0000-0001-5403-0692 (Q.G.); 0000-0003-2035-3527 (H.W.). Abbreviations used in this article: Alum, aluminum salts; APG-2, Asante Potassium Green-2; ASC, apoptosis-associated speck-like protein containing a CARD; BDMC, Received for publication October 27, 2017. Accepted for publication February 21, bisdemethoxycurcumin; BMDM, bone marrow–derived macrophage; COX-2, cyclo- 2018. oxygenase-2; DMC, desmethoxycurcumin; HFD, high-fat diet; ICP-OES, inductively This work was supported by National Natural Science Foundation of China Grants coupled plasma optical emission spectrometry; LDH, lactate dehydrogenase; DCm, 81370623, 81401314, 91442204, 81125002, and 81321061, Natural Sciences Foun- mitochondrial membrane potential; MSU, monosodium urate crystal; mtROS, mito- dation of Shandong Province Grant ZR2010HQ005, the Innovation Project of the chondrial ROS; ND, normal diet; poly(dA:dT), poly(deoxyadenylic-deoxythymidylic) Shandong Academy of Medical Sciences, and by the Project for Laureate of Taishan acid; ROS, reactive oxygen species; SIRT2, sirtuin 2; THC, tetrahydrocurcumin; TSA, Scholar (Grant ts201511075). trichostatin A; WT, wild-type.

H.Y. performed the in vitro cell tests and wrote the manuscript; H.Y., Q.G., X.L., T.T., Ó C.L., Y.S., H.W., Q.F., C.M., C.G., and F.Y. performed the experiments in mice and Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701495 2 CURCUMIN INHIBITS THE NLRP3 INFLAMMASOME

The NLRP3 inflammasome is the most characterized inflam- S. typhimurium was cultured overnight. On the following day, BMDMs masome. As an important innate immune sensor, it is activated by a were primed with LPS for 3 h, infected with the Salmonella (1:200) for 1 h, wide range of signals of pathogenic, endogenous, and environ- and then either untreated or treated with curcumin in the presence of genta- micin. Supernatants and cell lysates were collected 3 h after treatment. mental origin and consists of the NLRP3 scaffold, the apoptosis- associated speck-like protein containing a CARD (ASC), and ELISA the inflammatory protease caspase-1 (8). Given the emerging role Supernatants from cell culture or mouse sera were collected and stored of the NLRP3 inflammasomes in type 2 diabetes, obesity, and at 280˚C, then assayed for mouse IL-1b, mouse IL-18, mouse TNF-a, and other autoinflammatory diseases, identification of potential agents human IL-1b, according to the manufacturer’s instructions. and their mechanisms that control NLRP3 inflammasome deacti- Immunoblotting analysis vation may provide insights into the control of several chronic inflammatory disorders (9–11). Medium supernatants from treated macrophages were precipitated and Curcumin has been shown to inhibit the production of the proin- whole-cell lysates were prepared. The samples were separated by SDS- PAGE, transferred to polyvinylidene difluoride membranes, and hybrid- flammatory cytokine IL-1b (12). This finding prompted us to in- ized with primary Abs. The immune complexes were incubated with vestigate the possible role of curcumin in controlling inflammasome HRP-conjugated secondary Ab. The membranes were scanned with an activity. In this study, we found that curcumin suppressed inflam- LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan). mation via strong inhibition of NLRP3-dependent caspase-1 activa- Lactate dehydrogenase release assay tion and IL-1b secretion. Furthermore, curcumin attenuated the excessive IL-1b in high-fat diet (HFD)–treated wide-type (WT) mice LPS-primed BMDMs were plated at a density of 105 cells per well in but not in Nlrp3-deficient mice. Finally, curcumin prevented in- 96-well plates and treated with nigericin in the presence of curcumin. Culture supernatants were collected and assayed with a lactate dehydro- Downloaded from flammation in a monosodium urate crystal (MSU)–mediated perito- genase (LDH) cytotoxicity assay kit (Beyotime, Shanghai, China) nitis model. These findings suggest that the NLRP3 inflammasome is according to the manufacturer’s instructions. an important mediator of the anti-inflammatory actions of curcumin. Confocal microscopy Materials and Methods Macrophages were plated overnight on coverslips and stimulated as de- Mice scribed above. After stimulation, cells were washed, fixed with 4% para-

formaldehyde, and processed for immunocytochemistry. MitoSOX and http://www.jimmunol.org/ Nlrp32/2 mice were described previously (13). All mice were from a MitoTracker Deep Red staining were used according to the manufacturer’s C57BL/6 background and were housed with an alternating 12-h light/12-h instructions. Genomic DNA was stained with DAPI. Samples were imaged dark cycle. All animal experiments were performed according to the with an LSM 710 confocal laser scanning microscope (Carl Zeiss). The Guidelines for the Care and Use of Laboratory Animals and were approved fluorescence intensity in images was analyzed by ImageJ. by the Ethics Committee of Shandong University. NAD+ measurement Reagents Intracellular NAD+ levels were measured by an NAD/NADH quantifica- Curcumin, nigericin, MSU, PMA, poly(deoxyadenylic-deoxythymidylic) tion kit. The NAD+ level in treated BMDMs was divided by the NAD+ acid [poly(dA:dT)], celecoxib, 3-methyladenine, and glucose were pur- level in the untreated control BMDMs to determine the relative concen-

chased from Sigma-Aldrich (St. Louis, MO). Imiquimod (R837) and ul- tration (presented in arbitrary units). by guest on October 4, 2021 trapure LPS, MitoTracker Deep Red, and MitoSOX were obtained from + InvivoGen (San Diego, CA). DMC and BDMC were from Shifeng Bio- Intracellular K measurement logical Technology (Shanghai, China). Tetrahydrocurcumin (THC) was LPS-primed BMDMs were loaded with APG-2, a fluorescent indicator of Salmonella typhimurium + from Natural Remedies (Bangalore, India). was a intracellular K , and simultaneously incubated with or without curcumin. donated from the microbiology laboratory of the Institute of Basic Medical Cells were then stimulated with nigericin. After washing, the cells were Sciences, Shandong Academy of Medical Sciences. The Ab to human fixed with 4% paraformaldehyde and counterstained with DAPI. Confocal IL-1b was from Abcam (Cambridge, MA). Anti–human caspase-1, anti–b- microscopy analyses were performed at 488 and 405 nm. a + actin, anti–mouse tubulin, anti– -tubulin (acetyl K40), and anti–LC3 Ab Additionally, K in BMDMs was also measured with inductively cou- were from Cell Signaling Technology (Danvers, MA). The Ab against pled plasma optical emission spectrometry (ICP-OES). Cells were digested mouse IL-1b and the ELISA kits for human and mouse IL-1b, mouse in boiling nitric acid and samples were diluted to 3–8% (v/v) HNO3 with TNF-a, and IL-18 were from R&D Systems (Minneapolis, MN). Anti- + dH2O. The K was detected using an Optima 7300 DV ICP-OES. Samples mouse caspase-1 (p20) and anti-NLRP3 were from Adipogen (San and standards were run in duplicate. Diego, CA). The Abs targeting ASC, sirtuin 2 (SIRT2), and Tom20 were from PTG (Wuhan, China). Asante Potassium Green 2 (APG-2) was ASC oligomerization and speckle detection purchased from Teflabs (Austin, TX). The NAD/NADH quantification kit was from BioVision (Milpitas, CA). All tissue culture reagents were BMDMs were cultured on chamber slides overnight, then primed with LPS bought from Invitrogen. and treated with nigericin in the presence or absence of curcumin. For ASC oligomerization detection, BMDMs were seeded on six-well plates (2 3 106 Cell culture and stimulation cells per well) and treated with different stimuli. The cells were pelleted by centrifugation, resuspended in 1% Nonidet P-40 lysis buffer (0.5 ml, ice- THP-1 cells were grown in RPMI 1640 medium supplemented with 10% cold), and lysed by shearing 10 times through a 21-gauge needle. Cell FBS and 50 mM 2-ME. THP-1 cells were differentiated for 3 h with 100 nM 3 g PMA. Mouse bone marrow–derived macrophages (BMDMs) were derived lysates were then centrifuged (5000 , 10 min, 4˚C). Pellets were m from the femurs and tibias of C57BL/6 mice and cultured in DMEM washed twice with PBS, resuspended in PBS (500 l), crosslinked with medium complemented with 10% FBS in the presence of L929 culture fresh disuccinimidyl suberate (2 mM, 37˚C, 30 min), and pelleted by 3 g supernatants. centrifugation (5000 , 10 min). Crosslinked pellets were resuspended 6 in SDS sample buffer (20 ml), separated using 12% SDS-PAGE, and For inducing NLRP3 inflammasome activation, 1.0 3 10 macrophages immunoblotted using anti-mouse ASC Abs. were plated in 12-well plates overnight, then the medium was changed to Opti-MEM, cells were primed with LPS (500 ng/ml) for 3 h, and curcumin or To measure ASC speckles, cells were fixed with 4% paraformaldehyde indicated compounds were added for another hour. Cells were then stim- followed by ASC and DAPI staining. ASC speckles were quantified using ulated with MSU (150 mg/ml), aluminum salts (Alum; 300 mg/ml), and ImageJ software. At least four distinct fields were analyzed. R837 (15 mg/ml) for 6 h or with nigericin (10 mM) for 40 min. For Metabolic studies AIM2 inflammasome activation, poly(dA:dT) (0.5 mg/ml) was trans- fected using Lipofectamine according to the manufacturer’s instruc- For metabolic studies, male mice (6 wk old) were fed with normal diet (ND), tions. After 6 h, the supernatants and cell lysates were collected and HFD (D12492, Research diets), or HFD with curcumin supplementation for analyzed for caspase-1 and IL-1b activation by immunoblotting and for 12 wk. Curcumin (150 mg/kg daily) was administered by gavage during IL-1b levels by ELISA. weeks 9–12. For glucose tolerance tests, mice were fasted overnight (16 h) The Journal of Immunology 3 and were i.p. injected with glucose (1.5 mg/g body weight). Blood samples were whether curcumin specifically targets common signaling mech- drawn 0, 30, 60, 90, and 120 min after injection. For insulin tolerance tests, mice anisms in response to structurally diverse NLRP3 activators, we were fasted for 2 h and then injected with recombinant human insulin (1.5 IU/kg assessed other NLRP3 agonists. As found with nigericin, cur- body weight). Blood glucose was measured with a glucometer (Roche). At the end of each experiment, mice were euthanized. Serum cytokines were measured cumin blocked inflammasome activation by R837, Alum, and by ELISA. Liver tissues were isolated, washed in cold PBS supplemented with MSU (Fig. 2A, 2B). We further investigated the specificity of penicillin and streptomycin, and cultured in 12-well plates in Opti-MEM me- curcumin to NLRP3 as compared with other inflammasomes. dium supplemented with penicillin and streptomycin. After 24 h, supernatants The LPS-primed BMDMs were either infected with S. typhi- were collected and stored at 280˚C until analyzed. murium to activate the NLRC4 inflammasome or transfected MSU-induced peritonitis with poly(dA:dT) to activate the AIM2 inflammasome. Our data For peritonitis, mice were treated with curcumin (100 mg/kg body weight, showed that neither the NLRC4 inflammasome nor the AIM2 i.p. injection). After 1 h, peritonitis was induced by injection of MSU (3 mg inflammasome was inhibited by curcumin (Fig. 2C, 2D). We also in 200 ml of sterile PBS). Mice were euthanized 6 h later and peritoneal detected that with regard to expression of the NLRP3 inflam- cavities were washed with 10 ml of PBS. Lavage fluids were analyzed for masome components, neither NLRP3 nor ASC is modified by b IL-1 production by ELISA and for polymorphonuclear neutrophil re- curcumin (Supplemental Fig. 1). These results indicate that cruitment by flow cytometry using the neutrophil marker Ly6G-PE. Serum IL-1b was also measured. curcumin acts on a common signaling pathway specific to the NLRP3 inflammasome activated by broad proinflammatory Statistical analysis activators. Samples were analyzed using a Student t test unless indicated in the figure To investigate whether curcumin exerts the same anti- legends. Differences between treatments with p , 0.05 were considered inflammatory effects in humans, we primed PMA-differentiated statistically significant. THP-1 cells with LPS, then treated them with curcumin or vehi- Downloaded from cle for 1 h, and finally stimulated the cells with nigericin. Results Results obtained by immunoblotting indicated that the cells treated with Curcumin suppresses caspase-1 activation and IL-1b secretion curcumin showed markedly reduced processing of caspase-1 and by inhibiting both inflammasome priming and activation secretion of mature IL-1b (Fig. 2E). Taken together, these findings To test whether curcumin affects inflammasome activation, we first demonstrate that curcumin can specifically inhibit the NLRP3 examined whether curcumin could inhibit caspase-1 cleavage and inflammasome in both mice and humans. http://www.jimmunol.org/ IL-1b secretion. We pretreated LPS-primed BMDMs with curcumin All curcuminoids inhibit NLRP3 inflammasome activation for 1 h and then treated them with the NLRP3 inflammasome acti- independent of enzyme-catalyzed transformation vator nigericin. We measured both caspase-1 activation and IL-1b maturation using immunoblots that detect the enzymatically active In addition to curcumin, the other curcuminoids in turmeric are two p20 subunit of caspase-1 and the biologically active p17 form of structurally related derivatives that lack either one (DMC) or both IL-1b, respectively. Curcumin dose-dependently blocked nigericin- (BDMC) methoxy groups in the phenyl rings (Fig. 3A). We found induced cleavage of caspase-1 into p20 and the IL-1b maturation that both DMC and BDMC blocked caspase-1 cleavage, IL-1b maturation, and IL-18 secretion at similar concentrations to cur- (Fig. 1A, 1B). Similarly, curcumin suppressed the secretion of IL-18, by guest on October 4, 2021 another inflammasome-dependent cytokine (Fig. 1C). However, cur- cumin when the primed BMDMs were stimulated with nigericin cumin had no effect on TNF-a production or pro–IL-1b expression (Fig. 3B–D). (Fig. 1A, 1D). These results suggest that curcumin inhibited IL-1b THC is the main metabolite of curcumin that lacks the maturation independently of priming in LPS-primed BMDMs. a,b-unsaturated ketone groups (Fig. 3A) (15). THC pretreatment Because the inhibition of NF-kB activation and TNF-a pro- had no significant effect on nigericin-induced caspase-1 activation duction has been implicated in the anti-inflammatory activity of or IL-1b or IL-18 secretion in BMDMs (Fig. 3B–D). These results curcumin, we sought to determine whether curcumin had an im- suggest that the a,b-unsaturated ketone groups, but not the pact on LPS-induced priming for inflammasome activation (14). methoxy group, are critical for curcuminoid-mediated inflamma- BMDMs were treated with curcumin for 3 h, then primed with some inhibition. LPS for 3 h, and finally stimulated with nigericin. Curcumin Recently, it was suggested that oxidative activation should be inhibited LPS-induced pro–IL-1b expression and TNF-a pro- considered as a potential mechanism of action of curcumin and that duction (Fig. 1E, 1F). The caspase-1 activation and IL-1b secre- enzymatic oxidation of curcumin occurs by cyclooxygenase-2 tion were also blocked (Fig. 1E, 1G). The inhibition of priming (COX-2) (16). We found that inhibition of COX-2 activity by was not as strong as the inflammasome inhibition detected at celecoxib did not significantly alter the inhibitory effects of cur- similar concentrations of curcumin. These results suggest that cumin on the inflammasome (Fig. 3E, 3F). This result suggests curcumin can inhibit caspase-1 activation and IL-1b secretion by that the inhibitory effects of curcumin on IL-1b secretion are not suppressing both LPS priming and inflammasome activation. due to COX-2–modified enzymatic products. To identify the mechanisms underlying curcumin-induced Curcumin inhibits potassium efflux inflammasome suppression, we treated BMDMs with curcumin after LPS priming in the subsequent experiments. To exclude the Several molecular and cellular events have been proposed as the + possibility that curcumin inhibition of IL-1b secretion was due to trigger for NLRP3 inflammasome activation. Potassium (K ) efflux cell death, cell death was analyzed by the LDH release assay is thought to act at or upstream of NLRP3 activation and it under similar experimental conditions. Curcumin had no effect on has emerged as a common event and plays a critical role in cell viability (Fig. 1H), suggesting that the inflammasome inhi- NLRP3 activation (17). To assess whether curcumin inhibited + bition is not due to cell death. NLRP3 activation by suppressing K efflux, we incubated primed BMDMs with APG-2, a fluorescent indicator of cytosolic K+. Curcumin inhibits NLRP3 inflammasome activation in murine Consistent with recent studies, curcumin prevented the decline of macrophages and human monocytes intracellular K+ in response to incubation with the NLRP3 acti- Besides nigericin, NLRP3 inflammasome can be activated by R837 vators nigericin and MSU (Fig. 4A) (18). Results were confirmed or crystalline substances, such as Alum and MSU (8). To determine by ICP-OES (Fig. 4B, 4C). 4 CURCUMIN INHIBITS THE NLRP3 INFLAMMASOME

FIGURE 1. Curcumin suppresses caspase-1 activation and IL-1b secretion. (A) LPS-primed BMDMs were treated with curcumin (CUR) for 1 h and then stimulated with nigericin. Medium supernatants (SN) and cell extracts (Input) were analyzed for IL-1b and caspase-1 activation by immunoblotting. (B–D)

Supernatants were also analyzed by ELISA for IL-1b (B), IL-18 (C), and TNF-a (D) release (n = 3, mean 6 SEM). The p values were determined by an Downloaded from unpaired t test. ***p , 0.001. (E) BMDMs were treated with curcumin for 3 h, then primed with LPS for 3 h, and finally stimulated with nigericin. SN and Input were analyzed by immunoblotting for the indicated proteins. (F and G) Supernatants were also analyzed by ELISA for IL-1b (F) and TNF-a (G) (n = 3, mean 6 SEM). *p , 0.05, **p , 0.01, by a Student t test. (H) LDH release assay in supernatants from LPS-primed BMDMs treated with various doses of curcumin for 1 h and then stimulated with nigericin or not (n = 3, mean 6 SEM). p . 0.05, determined by ANOVA with a Tukey test. b-Actin served as a loading control in (A) and (E). All immunoblots are representative of at least three independent experiments. CUR, curcumin; mIL-1b, mature IL-1b, active form of IL-1b; P20, active subunit of caspase-1; Procasp1, procaspase1, biologically inactive; Pro–IL-1b, nonsecreted biologically inactive form of IL-1b; SN, supernatant. http://www.jimmunol.org/

In addition to K+ efflux, mitochondrial dysfunction and reactive have shown that NLRP3 inflammasome inducers cause oxygen species (ROS) have also been proposed to be important mitochondrial-associated dysfunction and that the characteristics signals responsible for NLRP3 inflammasome activation. Studies of mitochondrial damage are the production of ROS, lower by guest on October 4, 2021

FIGURE 2. Curcumin inhibits NLRP3 inflammasome activation in murine macrophages and human monocytes. (A) LPS-primed BMDMs were treated with curcumin (CUR; 40 mM) for 1 h and then stimulated with imiquimod (R837), MSU crystals, Alum, and nigericin. Medium supernatants (SN) and cell extracts (Input) were analyzed by immunoblotting for caspase-1 activation. (B) ELISA analysis of IL-1b in supernatants from LPS-primed BMDMs treated for 1 h with curcumin and then stimulated with R837, MSU, Alum, and nigericin (n = 3, mean 6 SEM). **p , 0.01, ***p , 0.001, by Student t test. (C) LPS-primed BMDMs were treated with curcumin (40 mM), then stimulated with Salmonella. IL-1b and caspase-1 activation were analyzed by immu- noblotting. (D) LPS-primed BMDMs were treated with curcumin (40 mM) and then transfected with poly(dA:dT). IL-1b and caspase-1 activation were analyzed by immunoblotting. (E) Immunoblotting analysis of caspase-1 and IL-1b activation of PMA-differentiated THP-1 cells stimulated with nigericin with the presence of curcumin. b-Actin served as a loading control. All immunoblots are representative of three independent experiments. CUR, curcumin; mIL-1b, mature IL-1b, active form of IL-1b; P20, active subunit of caspase-1; Procasp1, procaspase-1, biologically inactive; Pro–IL-1b, nonsecreted biologically inactive form of IL-1b; SN, supernatant. The Journal of Immunology 5 Downloaded from FIGURE 3. Curcuminoids’ inhibition of NLRP3 inflammasome activation is dependent on the a,b-unsaturated ketone groups and independent of COX- 2–catalyzed transformation. (A) Structures of curcumin derivatives and its main metabolite, THC. (B) LPS primed-BMDMs were treated with DMC (40 mM), BDMC (40 mM), or THC (40 mM) for 1 h and then stimulated with nigericin. Medium supernatants (SN) and cell extracts (Input) were analyzed by immunoblotting as indicated. (C and D) Supernatants were also analyzed by ELISA for IL-1b (C) or IL-18 (D) release (n = 3, mean 6 SEM). *p , 0.05, **p , 0.01, ***p , 0.001, by Student t test. (E) LPS primed-BMDMs were treated with celecoxib (50 mM, 30 min), then curcumin (40 mM), and stimulated with nigericin. Caspase-1 activation was analyzed by immunoblotting. (F) Supernatants were also analyzed by ELISA for IL-1b release (n =3, mean 6 SEM). NS, by Student t test. b-Actin served as a loading control. All immunoblots are representative of three independent experiments. CUR, http://www.jimmunol.org/ curcumin; mIL-1b, mature IL-1b, active form of IL-1b; P20, active subunit of caspase-1; Procasp1, procaspase-1, biologically inactive; Pro–IL-1b, nonsecreted biologically inactive form of IL-1b; SN, supernatant. mitochondrial membrane potential (DCm), a reduction in intra- of curcumin (Fig. 4H, 4I), ruling out a major role for autophagy in cellular NAD+, and morphological changes of mitochondria from the effects of curcumin on the inflammasome. string-shaped to dot-shaped structures (19, 20). We wanted to Our data suggest that curcumin inhibits NLRP3 inflammasome determine whether reduced mitochondrial impairment mediated activation by controlling an unknown upstream event that reduces by guest on October 4, 2021 curcumin’s effects on the NLRP3 inflammasome. First, we ex- K+ efflux from macrophages, and not by preventing mitochondrial amined mitochondrial ROS (mtROS) production by MitoSOX, a destabilization. mitochondrial superoxide indicator. Consistent with recent data, curcumin decreased mtROS production in BMDMs upon treat- Curcumin blocks mitochondrial transport and ment with nigericin (Fig. 4D) (18). In contrast, curcumin did not NLRP3-mediated ASC speckle formation show such reduction in BMDMs upon treatment with MSU. It has been suggested that ASC on mitochondria moves to the Furthermore, although mitochondrial complex I inhibitor rotenone perinuclear region and localizes together with NLRP3 on the robustly increased mtROS production (Fig. 4D), it failed to ab- endoplasmic reticulum during activation of the NLRP3 inflam- rogate the suppressive effects of curcumin on nigericin-induced masome. Moreover, this microtubule-driven spatial arrangement of NLRP3 inflammasome activation significantly (Fig. 4E), indicat- mitochondria provides a platform for complex assembly and is ing that reduction in mtROS was not a general feature of curcumin necessary for NLRP3 inflammasome activation (19, 23). Addi- on NLRP3 inflammasome inhibition. tionally, tubulin has been reported to be a target of curcumin (24). Additionally, we assessed the functional mitochondrial pool in To clarify the role of the subcellular localization of mitochondria macrophages using MitoTracker Deep Red, a fluorescent probe in curcumin’s inhibition of NLRP3, we examined the subcellular sensitive to the mitochondrial inner transmembrane potential. location of Tom20 and a-tubulin in primed BMDMs pretreated Pretreatment with curcumin decreased DCm further in LPS- with curcumin and then stimulated with nigericin. Colchicine, an primed BMDMs upon nigericin stimuli (Fig. 4F). Consistent inhibitor of tubulin polymerization, was used as a positive control, with the pattern of DCm, a lower abundance of NAD+ was ob- as it inhibits NLRP3 inflammasome activation by breaking the tained in the presence of curcumin (Fig. 4G). Furthermore, cur- microtubule structure and subsequently disturbing the mitochon- cumin did not significantly alter nigericin-induced morphological drial subcellular localization. Similar to colchicine, imaging changes of mitochondria in BMDMs; mitochondria still changed revealed that curcumin blocked the nigericin-induced trans- into dot-like shapes. These results indicate that curcumin fails to portation of mitochondria to the perinuclear region (Fig. 5A). block the nigericin-induced mitochondrial damage, ruling it out as Acetylated a-tubulin has been reported to mediate mitochon- the mechanism by which curcumin inhibits the inflammasome. drial transport to promote activation of the NLRP3 inflammasome Autophagy is a negative regulator targeting mitochondria for (23). However, curcumin promoted, rather than suppressed, the degradation or other pathways (20, 21). Furthermore, induction of accumulation of acetylated a-tubulin during nigericin-induced autophagy has also been linked to the biological effects of cur- activation of the NLRP3 inflammasome (Fig. 5B). Consistent re- cumin (22). We found that curcumin treatment did not signifi- sults were obtained in BMDMs by immunoblotting (Fig. 5C). We cantly induce autophagy in primed BMDMs, and the autophagy further detected SIRT2, a-tubulin deacetylase specifically in- inhibitor 3-methyladenine failed to abrogate the inhibitory effects volved in the process for activation of the NLRP3 inflammasome, 6 CURCUMIN INHIBITS THE NLRP3 INFLAMMASOME Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 4. Curcumin inhibits potassium efflux. (A) Intracellular potassium levels in LPS-primed BMDMs unstimulated (untreated) or stimulated with MSU (5 h) or nigericin (40 min) in the absence (Mock) or presence of curcumin (CUR) were assessed using an APG-2 (green) dye that selectively binds potassium. Blue shows nuclei. Scale bar, 50 mm. (B and C) ICP-OES was used to measure intracellular potassium (K+) levels in primed BMDMs treated with curcumin at 30, 40, and 50 mM, respectively, then stimulated with nigericin (B) or with curcumin (40 mM) followed by nigericin or MSU stimulation (C). (D) mtROS production by LPS-primed BMDMs unstimulated (untreated) or stimulated with MSU (5 h) or nigericin (40 min) in the absence (Mock) or presence of curcumin or combinations (CUR + rotenone) of rotenone (40 mM) and curcumin (40 mM) for 1 h. Cells were labeled with MitoSOX (red) and the mtROS was determined by fluorescence intensity. The relative mtROS was calculated as the fluorescence intensity in treated BMDMs divided by the fluorescence intensity in the untreated control BMDMs. The values were calculated from three independent experiments with four fields of view in each experiment. Blue shows nuclei. Scale bar, 50 mm. (E) ELISA analysis of IL-1b release in supernatants from LPS-primed BMDMs treated with combi- nations of rotenone, curcumin, and nigericin. (F) Confocal fluorescence microscopy of LPS-primed BMDMs unstimulated (untreated) or stimulated with nigericin in absence (Nigericin) or presence of curcumin (Nigericin + CUR). Cells were stained with MitoTracker Deep Red and DCm was determined by fluorescence intensity. The fluorescence intensity in treated BMDMs was divided by the fluorescence intensity in the untreated BMDMs to determine the relative DCm. The values were calculated from three independent experiments with four fields of view in each experiment. Blue shows nuclei. Scale bar, 20 mm. (G) Intracellular NAD+ in primed BMDMs with or without curcumin (40 mM), followed by nigericin. (H) Immunoblot analysis of LC3 and b-actin in cell lysates from LPS-primed BMDMs unstimulated or stimulated with nigericin. (I) ELISA analysis of IL-1b release in supernatants of LPS-primed BMDMs pretreated with 3-methyladenine (3-MA) (10 mM) for 30 min and stimulated in the presence of nigericin. n = 3. All data are representative of three independent experiments. The p values were determined by ANOVA with a Tukey post hoc test (B)ort test (C–E, G, and I). *p , 0.05, **p , 0.01, ***p , 0.001. CUR, curcumin. by immunoblotting. We found that curcumin did not alter the macrophages despite robust induction of a-tubulin acetylation in SIRT2 expression during nigericin-induced activation of the macrophages (Fig. 5B, 5D). Our data suggest that the blocking of NLRP3 inflammasome in BMDMs (Fig. 5C). Studies have sug- mitochondrial transport is independent of acetylated a-tubulin. gested that curcumin can act as a histone deacetylase inhibitor Besides caspase-1 activation and IL-1b release, an additional (25). However, we found that inhibition of histone deacetylases marker for NLRP3 inflammasome activation is the formation of using trichostatin A (TSA) did not significantly affect NLRP3 ASC nucleation-induced polymerization or oligomerization, a inflammasome activation in LPS-primed and nigericin-treated large structure localized to a detergent-insoluble fraction in The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 5. Curcumin blocks mitochondrial transport and ASC speckle formation. (A) Immunocytochemistry of the subcellular location of Tom20 (green) and a-tubulin (red) in primed BMDMs unstimulated (untreated) or stimulated with nigericin (40 min) in the absence (Nigericin) or presence of curcumin (Nigericin + CUR) or colchicine (5 mM) (Nigericin + colchicine). Blue shows nuclei. Scale bar, 20 mm. (B) Immunocytochemistry of acetylated a-tubulin (Ac-a-tubulin, green) and total a-tubulin (red) in primed BMDMs with or without curcumin (40 mM) or TSA (50 nM), and then left unstimulated or stimulated with nigericin. Blue shows nuclei. Scale bar, 20 mm. (C) Immunoblotting analysis of SIRT2 and acetylated and total a-tubulin in LPS-primed BMDMs left unstimulated or stimulated with nigericin in the presence of various doses of curcumin. b-Actin served as a loading control. (D) IL-1b in medium supernatants of primed BMDMs activated with LPS, stimulated with nigericin, and coincubated with TSA (50 nM) (n =3,mean6 SEM). NS, by t test. (E) Representative immunofluorescence images of ASC speckle formation in LPS-primed BMDMs stimulated with nigericin in the presence or absence of curcumin (40 mM) for 1 h, left unstimulated or stimulated with nigericin, and then stained for ASC (green) and DNA (with DAPI, blue). Scale bars, 50 mm, 10 mm (inset). (F) Percentage of macrophages containing ASC speckle. Quantification represents the mean of two independent experiments with five fields of view in each experiment (n = 5, mean 6 SEM). **p , 0.01, by t test. (G) Immunoblotting analysis of disuccinimidyl suberate (DSS)–cross-linked ASC in the Nonidet P-40-insoluble pellet of BMDMs that were primed with LPS and treated with curcumin, then stimulated by nigericin or MSU. b-Actin served as a loading control. All immunoblots and images are representative of three independent experiments. CUR, curcumin.

the cell that is thought to mediate caspase-1 activation (7, 26). inflammasome also by blocking mitochondrial transport, ASC Consistent with previous findings, immunofluorescent staining for polymerization, and assembly of the inflammasome. endogenous ASC in BMDMs showed that the polymerization or oligomerization of ASC into a large protein speckle at the peri- Curcumin mitigates HFD-induced insulin resistance and MSU- nuclear region was triggered upon activation with nigericin induced peritoneal inflammation by inhibition of the (Fig. 5E). Treatment of LPS-primed BMDMs with curcumin NLRP3 inflammasome markedly reduced the formation of nigericin-induced ASC Increasing evidence suggests the benefits of curcumin in the speckles (Fig. 5E, 5F). Similar results were obtained by immu- prevention and treatment of diabetes and its associated disorders noblotting; curcumin prevented nigericin-induced or MSU- (4, 27). Additionally, it has been confirmed that the NLRP3 induced ASC oligomerization in primed BMDMs (Fig. 5G). inflammasome can produce the proinflammatory cytokines and is These experiments confirm that curcumin inhibits the NLRP3 critically involved in insulin resistance in pancreatic islets and in 8 CURCUMIN INHIBITS THE NLRP3 INFLAMMASOME Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 6. Curcumin prevents insulin resistance induced by HFD by inhibiting NLRP3 inflammasome activation. (A–D)WT(n = 7 per group) or Nlrp32/2 (n = 6 per treatment) mice were fed an ND or HFD for 12 wk with or without curcumin during the last 4 wk (150 mg/kg of body weight daily). Glucose tolerance (A), insulin tolerance (B), serum IL-1b levels (C), and serum IL-18 (D) were measured as indicated. (E and F) Liver tissue was isolated and cultured for 24 h, and supernatants were analyzed by ELISA for IL-1b (E) and IL-18 (F) release. n = 6–7 per group. Values are mean 6 SEM. *p , 0.05, ***p , 0.001, by nonparametric Mann–Whitney U test (A and B) or Student t test (C–F). CUR, curcumin. adipose, liver, and kidney tissues (10, 28). We investigated whether NLRP3 inflammasome activation in vivo. Serum concentrations of curcumin can improve insulin sensitivity via inhibition of NLRP3 IL-1b and IL-18 in HFD-treated mice were higher than in ND-fed inflammasomeactivation.MicewerefedanHFDfor12wktoinduce mice, and the robust elevation was blocked by curcumin. How- the emergence of insulin resistance. To assess whether curcumin can ever, Nlrp3 deficiency prevented HFD-induced IL-1b and IL-18 reverse insulin insensitivity, we performed glucose tolerance tests and production (Fig. 6C, 6D). Importantly, Nlrp3 deficiency abrogated insulin tolerance tests in HFD-treated mice with or without curcumin curcumin inhibition of IL-1b and IL-18 production (Fig. 6C, 6D). supplementation. Following i.p. injection of glucose, plasma glucose In accord with the above results, liver tissue from HFD-treated levels in HFD-treated WT mice were significantly higher compared mice showed higher IL-1b or IL-18 production compared with with those in WT mice fed an ND (Fig. 6A). Curcumin treatment ND-treated mice. Moreover, curcumin administration and Nlrp3 reduced the glucose elevation and the mice were more glucose tol- deficiency both blocked HFD-induced IL-1b or IL-18 production erant than HFD-treated WT mice without curcumin administration. in the liver, indicating that curcumin supplementation can sup- Meanwhile, by measuring the reduction in plasma glucose after in- press metabolic stress–induced NLRP3 inflammasome activation sulin treatment, the insulin tolerance tests showed that insulin sensi- in HFD-treated mice (Fig. 6E, 6F). Taken together, these results tivitywasmarkedlyimprovedbycurcumininHFD-treatedWTmice indicate that curcumin can prevent HFD-induced insulin resis- (Fig. 6B). In contrast, in Nlrp32/2 mice, curcumin did not signifi- tance by blocking NLRP3 inflammasome activation. cantly improve glucose tolerance or insulin sensitivity (Fig. 6A, 6B). Next we verified whether delivery of curcumin can inhibit the Moreover, no significant weight changes of mice were observed after NLRP3 inflammasome in mouse models of NLRP3-driven in- treatment of curcumin (Supplemental Fig. 2). Therefore, curcumin- flammation in vivo. The NLRP3 inflammasome was activated mediated prevention of metabolic disorders may rely on inhibition of following i.p. injection of MSU crystals, resulting in an influx of the NLRP3 inflammasome. neutrophils into the peritoneum and increased secretion of IL-1b by To confirm that curcumin prevents HFD-induced insulin resistance macrophages 6 h after injection. Compared to mice given vehicle, by inhibition of NLRP3 inflammasome activation, we examined curcumin treatment inhibited MSU-induced IL-1b production and The Journal of Immunology 9 Downloaded from

FIGURE 7. Curcumin suppresses MSU-induced peritoneal inflammation in vivo. (A) Serum IL-1b in mice challenged with MSU (3 mg per mouse) and http://www.jimmunol.org/ treated with curcumin (100 mg/kg body weight, n = 7 per group). (B) IL-1b production in peritoneal lavage fluid was assessed by ELISA. n = 7 per group. (C) Ly6G+ cells in the peritoneum of mice treated with MSU and curcumin were detected by flow cytometry. Numbers above plots indicate percentage of cells stained by Ab for Ly6G. (D) Flow cytometry analysis of neutrophil numbers in the peritoneal cavity of mice i.p. injected with MSU with or without curcumin. n = 7. Data are mean 6 SEM. *p , 0.05, by Student t test. CUR, curcumin. reduced neutrophil infiltration into the peritoneum, suggesting We confirmed the ability of curcumin to suppress NLRP3 direct effects of curcumin on NLRP3-driven peritoneal inflam- inflammasome activation by various stimuli, including crys- by guest on October 4, 2021 mation in vivo (Figs. 7, 8). talline molecules such as MSU and Alum, that require phago- cytosis for activation, as well as pore-forming toxins such as Discussion nigericin (18). Also, similar to most regulators of the NLRP3 In this study, we identified curcumin as a common NLRP3 inflammasome reported, curcumin can prevent both steps to inflammasome activation inhibitor. Curcumin inhibited both LPS- inhibit the NLRP3 inflammasome. The inhibition of NF-kB priming and NLRP3 inflammasome activation pathway in mac- activity by curcumin has been discussed extensively, and hence rophages. The inhibitory effect of curcumin on inflammasome we focused our investigation of how curcumin inhibits NLRP3 activation was specific to the NLRP3 inflammasome, and it did not inflammasome activation in the present study. affect the NLRC4 or AIM2 inflammasomes. Importantly, curcumin The generation of ROS was one of the first intermediates dis- delivery could prevent HFD-induced insulin resistance and MSU- covered to be common to various stimuli-induced NLRP3 acti- induced peritoneal inflammation by inhibition of the NLRP3 vation (36). Since then, there have been many conflicting reports inflammasome in vivo. Taken together, our results demonstrate a regarding the role of ROS in this process, creating much contro- previously unrecognized mechanism through which curcumin re- versy in understanding the regulation of NLRP3 inflammasome press inflammation in diabetes and other NLRP3-driven diseases activation (37, 38). Our data reveal that the effects of curcumin (Fig. 8). vary depending on the nature of the NLRP3 activator; curcumin The NLRP3 inflammasome is activated in a two-checkpoint decreased the effects of nigericin, but enhanced the effects of activation mechanism. First, LPS or another TLR agonist in- crystalline substances, such as MSU. Therefore, mtROS is likely duces NLRP3 and pro–IL-1b synthesis via the NF-kB pathway. not the common pathway by which curcumin inhibits the NLRP3 Second, the NLRP3 inflammasome and the subsequent caspase-1 inflammasome. Moreover, these results demonstrate that only processing can be activated by nigericin, MSU crystals, or other mtROS are not enough to activate the NLRP3 inflammasome. The stimuli. Dysregulated NLRP3 inflammasome activity is associated separation of ROS between nigericin and MSU might due to the with a wide range of diseases, including diabetes and auto- redox state in cells induced by curcumin and various challenges or inflammation (10, 29, 30). Additionally, work from our laboratory has the cooperative sensitized effects between curcumin and MSU. shown that omega-3 fatty acids and the endogenous neurotransmitter Besides, lower DCm and reduction of intracellular NAD+ are also dopamine exerts anti-NLRP3 inflammasome effects (13, 31). Re- the characteristics of mitochondrial damage, which plays a critical cently, various plant-derived polyphenols have been identified as in- role in the activation of the NLRP3 inflammasome. However, our hibitors of NLRP3 inflammasome, including isoliquiritigenin, findings revealed that instead of preventing mitochondrial dam- , emodin, and epigallocatechin-3-gallate (32–35). It is well age, curcumin resulted in more damage to it, exacerbated depo- known that curcumin is one of the most studied polyphenol com- larization of DCm, and further diminished the amount of cellular pounds. How, then, does it affect NLRP3 inflammasome activation? NAD+. This suggests that maybe curcumin inhibits NLRP3 10 CURCUMIN INHIBITS THE NLRP3 INFLAMMASOME Downloaded from http://www.jimmunol.org/ FIGURE 8. Model for inhibitory efficacy of curcumin on NLRP3 inflammasome activation in inflammatory diseases. Red lines denote multistep processes of curcumin. The NLRP3 inflammasome is composed of NLRP3, ASC, and procaspase-1. It is activated by a wide range of stimuli, including pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and metabolic products. The activation of NLRP3 inflammasome and subsequent secretion of IL-1b and IL-18 cause insulin resistance and organ dysfunction, including the adipose tissue, pancreas, brain, and others, during inflammatory disorders. Curcumin could suppress NLRP3 inflammasome activation through damping potassium efflux and holding back the microtubule-driven apposition of ASC on the mitochondria to NLRP3 on the endoplasmic reticulum (ER). Additionally, curcumin also could inhibit the priming step, which leads to the transcription and translation of NLRP3 and pro–IL-1b. Thus, the ability of curcumin to block NLRP3 inflammasome would benefit diabetes and other NLRP3-driven inflammatory diseases. by guest on October 4, 2021 inflammasome activation by impairing the mitochondria in a are currently unknown, future studies are needed to further char- manner that prevents mitochondrial from responding to nigericin. acterize this pathway. In addition to mitochondrial damage, the efficient spatial ar- Proper maintenance of the delicate balance between immune rangement of mitochondria has been shown to be a limiting step in response and metabolism is crucial for health and has important NLRP3 inflammasome activation. The NLRP3 inflammasome is a implications for many pathological states such as obesity, diabetes, multiprotein complex that is composed of a sensor protein NLRP3, and other chronic noncommunicable diseases (39). Increasing the adaptor protein ASC, and the inflammatory protease caspase-1. evidence suggests that NLRP3 inflammasome functions as a ASC on mitochondria bridges NLRP3 and caspase-1 to form sensor to detect danger signals and induce downstream chronic, ternary inflammasome complexes. Mitochondria act as a platform low-grade, metabolic inflammatory signaling that contributes to to facilitate the molecular complexity of sensor and adapter in- obesity and associated disorders such as insulin resistance (10, 28, teractions that promote effective NLRP3 inflammasome activation. 40). Proinflammatory cytokines, including IL-1b and TNF-a, are Recently, microtubules have been suggested to mediate the significantly elevated in diabetes. Curcumin has been reported to transport of mitochondria to create optimal sites for activation of exert antidiabetic effects via inhibition of diabetes-induced in- the NLRP3 inflammasome (23). In the present study, we indeed creases in IL-1b, TNF-a, and NF-kB activity (4). However, some found that curcumin blocked transportation of damaged mito- animal studies and several clinical trials using TNF-a blockade chondria induced by nigericin. have failed to prevent insulin resistance, suggesting that TNF-a Additionally, the pathway has been proposed that NLRP3 ag- may not be the primary target of curcumin to exert beneficial onists lead to an abundance of acetylated a-tubulin that drives effects (41). In the current study, we present that curcumin pre- mitochondria to the perinuclear area to promote the NLRP3 vents NLRP3 inflammasome-dependent IL-1b production and inflammasome activation (23). Curcumin-mediated inhibition of HFD-induced insulin resistance in WT mice. More importantly, NLRP3 inflammasome activation appears to be independent of the these effects were abrogated in Nlrp32/2 mice. Although some pathway mentioned above because curcumin delivery enhances, other targets for curcumin, such as VEGF and PPAR-g, have also rather than inhibits, the accumulation of acetylated a-tubulin. been proposed, our data support a critical role for the inhibition of Moreover, TSA did not affect NLRP3 inflammasome activation the NLRP3 inflammasome underlying the beneficial effect of despite significant induction of tubulin acetylation. The results curcumin in inflammatory disorders, at least in the HFD-induced indicate that there is another alternative, acetylated a-tubulin– model we explored (42, 43). independent mechanism involved in the upstream of transport of During past decades, as a potential treatment for diabetes and its mitochondria that promotes NLRP3 activation. As the molecular associated complications, the function of curcumin has been in- mechanisms by which NLRP3 agonist–driven spatial arrangement vestigated and the mainstream view at present is that curcumin is of mitochondria promotes activation of the NLRP3 inflammasome safe, nontoxic, and improves most of the complications of diabetes The Journal of Immunology 11

(1–4). In contrast, other researchers claim that curcumin has cy- 11. Wen, H., D. Gris, Y. Lei, S. Jha, L. Zhang, M. T. Huang, W. J. Brickey, and totoxic effects in vitro and no significant effect on blood glucose J. P. Ting. 2011. Fatty acid-induced NLRP3-ASC inflammasome activation in- terferes with insulin signaling. Nat. Immunol. 12: 408–415. in vivo (44). Perhaps various disease models and ways of drug 12. Das, L., and M. Vinayak. 2014. Curcumin attenuates carcinogenesis by down delivery or different bioavailability at physiologically achievable regulating proinflammatory cytokine interleukin-1 (IL-1a and IL-1b) via mod- ulation of AP-1 and NF-IL6 in lymphoma bearing mice. Int. Immunopharmacol. concentrations can account for the differences among findings. We 20: 141–147. found that curcumin demonstrated a time- and dose-dependent 13. Yan, Y., W. Jiang, T. Spinetti, A. Tardivel, R. Castillo, C. Bourquin, G. Guarda, cytotoxic effect in BMDMs (data not shown), and curcumin also Z. Tian, J. Tschopp, and R. Zhou. 2013. Omega-3 fatty acids prevent inflam- mation and metabolic disorder through inhibition of NLRP3 inflammasome failed to inhibit the caspase-1–dependent pyroptotic cell death activation. Immunity 38: 1154–1163. induced by nigericin, despite this compound blocking the ASC 14. Rahimifard, M., F. Maqbool, S. Moeini-Nodeh, K. Niaz, M. Abdollahi, speckle formation. Moreover, although curcumin indeed reduced N. Braidy, S. M. Nabavi, and S. F. Nabavi. 2017. Targeting the TLR4 signaling pathway by polyphenols: a novel therapeutic strategy for neuroinflammation. NLRP3 inflammasome–dependent IL-1b production in mice, Ageing Res. Rev. 36: 11–19. curcumin did not significantly improve blood glucose, serum in- 15. Metzler, M., E. Pfeiffer, S. I. Schulz, and J. S. Dempe. 2013. Curcumin uptake sulin, cholesterol, triglyceride, low-density lipoprotein, or high- and metabolism. Biofactors 39: 14–20. 16. Griesser, M., V. Pistis, T. Suzuki, N. Tejera, D. A. Pratt, and C. Schneider. 2011. density lipoprotein (data not shown). Thus, despite a broad Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary spectrum of potentially beneficial pharmacological activities, chemopreventive agent curcumin. J. Biol. Chem. 286: 1114–1124. many questions remain regarding the fate of this compound in the 17. Mun˜oz-Planillo, R., P. Kuffa, G. Martı´nez-Colo´n, B. L. Smith, T. M. Rajendiran, and G. Nu´n˜ez. 2013. K+ efflux is the common trigger of NLRP3 inflammasome mammalian organism. More investigation is required to improve activation by bacterial toxins and particulate matter. Immunity 38: 1142–1153. our understanding of curcumin and facilitate successful translation 18. Gong, Z., J. Zhou, H. Li, Y. Gao, C. Xu, S. Zhao, Y. Chen, W. Cai, and J. Wu. to human diseases. 2015. Curcumin suppresses NLRP3 inflammasome activation and protects

against LPS-induced septic shock. Mol. Nutr. Food Res. 59: 2132–2142. Downloaded from Collectively, our findings demonstrate a direct inhibitory effect 19. Zhou, R., A. S. Yazdi, P. Menu, and J. Tschopp. 2011. A role for mitochondria in of curcumin on NLRP3 inflammasome activation in macrophages. NLRP3 inflammasome activation. [Published erratum appears in 2011 Nature Our results further show that dietary curcumin can prevent HFD- 475: 122.] Nature 469: 221–225. 20. Tanaka, A. 2010. Parkin-mediated selective mitochondrial autophagy, mitoph- induced insulin resistance via specific inhibition of the NLRP3 agy: Parkin purges damaged organelles from the vital mitochondrial network. inflammasome in vivo. It may provide a low-cost, well-tolerated FEBS Lett. 584: 1386–1392. addition to a high-calorie diet for preventing chronic, low-grade, 21. Shi, C. S., K. Shenderov, N. N. Huang, J. Kabat, M. Abu-Asab, K. A. Fitzgerald, A. Sher, and J. H. Kehrl. 2012. Activation of autophagy by inflammatory signals http://www.jimmunol.org/ metabolic inflammation. Moreover, the ability of curcumin to limits IL-1b production by targeting ubiquitinated inflammasomes for destruc- block NLRP3 inflammasome makes it an attractive new candidate tion. Nat. Immunol. 13: 255–263. 22. Han, J., X. Y. Pan, Y. Xu, Y. Xiao, Y. An, L. Tie, Y. Pan, and X. J. Li. 2012. for even more clinical applications, including diabetes, gout, Curcumin induces autophagy to protect vascular endothelial cell survival from Alzheimer disease, or other NLRP3-driven disorders. oxidative stress damage. Autophagy 8: 812–825. 23. Misawa, T., M. Takahama, T. Kozaki, H. Lee, J. Zou, T. Saitoh, and S. Akira. 2013. Microtubule-driven spatial arrangement of mitochondria promotes acti- Acknowledgments vation of the NLRP3 inflammasome. Nat. Immunol. 14: 454–460. We thank Rongbin Zhou (School of Life Sciences and Medical Center, Univer- 24. Chakraborti, S., L. Das, N. Kapoor, A. Das, V. Dwivedi, A. Poddar, sity of Science and Technology of China) for providing the Nlrp32/2 mice, and G. Chakraborti, M. Janik, G. Basu, D. Panda, et al. 2011. Curcumin recognizes a unique binding site of tubulin. J. Med. Chem. 54: 6183–6196. Peng Li (Microbiological Research Department, Institute of Basic Medicine, 25. Pham, T. X., and J. Lee. 2012. Dietary regulation of histone acetylases and by guest on October 4, 2021 Shandong Academy of Medical Sciences) for providing S. typhimurium.We deacetylases for the prevention of metabolic diseases. Nutrients 4: 1868–1886. thank Dr. Hua-Ming Yu (Instruments’ Center for Physical Science, University 26. Yu, J. W., J. Wu, Z. Zhang, P. Datta, I. Ibrahimi, S. Taniguchi, J. Sagara, of Science and Technology of China) for ICP-OES. T. Fernandes-Alnemri, and E. S. Alnemri. 2006. Cryopyrin and pyrin activate caspase-1, but not NF-kB, via ASC oligomerization. Cell Death Differ. 13: 236–249. Disclosures 27. Lu, M., N. Yin, W. Liu, X. Cui, S. Chen, and E. Wang. 2017. Curcumin ame- liorates diabetic nephropathy by suppressing NLRP3 inflammasome signaling. The authors have no financial conflicts of interest. BioMed Res. Int. 2017: 1516985. 28. Vandanmagsar, B., Y. H. Youm, A. Ravussin, J. E. Galgani, K. Stadler, R. L. Mynatt, E. Ravussin, J. M. Stephens, and V. D. Dixit. 2011. The NLRP3 References inflammasome instigates obesity-induced inflammation and insulin resistance. 1. Prasad, S., S. C. Gupta, A. K. Tyagi, and B. B. Aggarwal. 2014. Curcumin, a Nat. Med. 17: 179–188. component of golden spice: from bedside to bench and back. Biotechnol. Adv. 29. Davis, B. K., H. Wen, and J. P. Ting. 2011. The inflammasome NLRs in im- 32: 1053–1064. munity, inflammation, and associated diseases. Annu. Rev. Immunol. 29: 707– 2. Jurenka, J. S. 2009. Anti-inflammatory properties of curcumin, a major con- 735. stituent of Curcuma longa: a review of preclinical and clinical research. Altern. 30. Horng, T., and G. S. Hotamisligil. 2011. Linking the inflammasome to obesity- related disease. Nat. Med. 17: 164–165. Med. Rev. 14: 141–153. 3. Esatbeyoglu, T., P. Huebbe, I. M. Ernst, D. Chin, A. E. Wagner, and G. Rimbach. 31. Yan, Y., W. Jiang, L. Liu, X. Wang, C. Ding, Z. Tian, and R. Zhou. 2015. Do- pamine controls systemic inflammation through inhibition of NLRP3 inflam- 2012. Curcumin—from molecule to biological function. Angew. Chem. Int. Ed. masome. Cell 160: 62–73. Engl. 51: 5308–5332. 32. Honda, H., Y. Nagai, T. Matsunaga, N. Okamoto, Y. Watanabe, K. Tsuneyama, 4. Shehzad, A., G. Rehman, and Y. S. Lee. 2013. Curcumin in inflammatory dis- H. Hayashi, I. Fujii, M. Ikutani, Y. Hirai, et al. 2014. Isoliquiritigenin is a potent eases. Biofactors 39: 69–77. inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue 5. Martinon, F., K. Burns, and J. Tschopp. 2002. The inflammasome: a molecular inflammation. J. Leukoc. Biol. 96: 1087–1100. platform triggering activation of inflammatory caspases and processing of proIL- 33. Fu, Y., Y. Wang, L. Du, C. Xu, J. Cao, T. Fan, J. Liu, X. Su, S. Fan, Q. Liu, and b. Mol. Cell 10: 417–426. F. Fan. 2013. Resveratrol inhibits ionising irradiation-induced inflammation in 6. Schroder, K., and J. Tschopp. 2010. The inflammasomes. Cell 140: 821–832. MSCs by activating SIRT1 and limiting NLRP-3 inflammasome activation. Int. 7. Fernandes-Alnemri, T., J. Wu, J. W. Yu, P. Datta, B. Miller, W. Jankowski, J. Mol. Sci. 14: 14105–14118. S. Rosenberg, J. Zhang, and E. S. Alnemri. 2007. The pyroptosome: a supra- 34. Han, J. W., D. W. Shim, W. Y. Shin, K. H. Heo, S. B. Kwak, E. J. Sim, molecular assembly of ASC dimers mediating inflammatory cell death via J. H. Jeong, T. B. Kang, and K. H. Lee. 2015. Anti-inflammatory effect of caspase-1 activation. Cell Death Differ. 14: 1590–1604. emodin via attenuation of NLRP3 inflammasome activation. Int. J. Mol. Sci. 16: 8. Sutterwala, F. S., S. Haasken, and S. L. Cassel. 2014. Mechanism of NLRP3 8102–8109. inflammasome activation. Ann. N. Y. Acad. Sci. 1319: 82–95. 35. Tsai, P. Y., S. M. Ka, J. M. Chang, H. C. Chen, H. A. Shui, C. Y. Li, K. F. Hua, 9. Vande Walle, L., N. Van Opdenbosch, P. Jacques, A. Fossoul, E. Verheugen, W. L. Chang, J. J. Huang, S. S. Yang, and A. Chen. 2011. Epigallocatechin-3- P. Vogel, R. Beyaert, D. Elewaut, T. D. Kanneganti, G. van Loo, and gallate prevents lupus nephritis development in mice via enhancing the Nrf2 M. Lamkanfi. 2014. Negative regulation of the NLRP3 inflammasome by A20 pathway and inhibiting NLRP3 inflammasome activation. Free protects against arthritis. Nature 512: 69–73. Radic. Biol. Med. 51: 744–754. 10. Benetti, E., F. Chiazza, N. S. Patel, and M. Collino. 2013. The NLRP3 inflam- 36. Dostert, C., V. Pe´trilli, R. Van Bruggen, C. Steele, B. T. Mossman, and masome as a novel player of the intercellular crosstalk in metabolic disorders. J. Tschopp. 2008. Innate immune activation through Nalp3 inflammasome Mediators Inflamm. 2013: 678627. sensing of asbestos and silica. Science 320: 674–677. 12 CURCUMIN INHIBITS THE NLRP3 INFLAMMASOME

37. Abais, J. M., M. Xia, Y. Zhang, K. M. Boini, and P. L. Li. 2015. Redox regu- 41. Ferraz-Amaro, I., M. Arce-Franco, J. Mun˜iz, J. Lo´pez-Ferna´ndez, V. Herna´ndez- lation of NLRP3 inflammasomes: ROS as trigger or effector? Antioxid. Redox Herna´ndez, A. Franco, J. Quevedo, J. Martı´nez-Martı´n, and F. Dı´az-Gonza´lez. Signal. 22: 1111–1129. 2011. Systemic blockade of TNF-a does not improve insulin resistance in hu- 38. Hornung, V., F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock, mans. Horm. Metab. Res. 43: 801–808. K. A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activate 42. Zhou, H., C. S. Beevers, and S. Huang. 2011. The targets of curcumin. Curr. the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9: Drug Targets 12: 332–347. 847–856. 43. Jacob, A., R. Wu, M. Zhou, and P. Wang. 2007. Mechanism of the anti- 39. Hotamisligil, G. S. 2017. Inflammation, metaflammation and immunometabolic disorders. Nature 542: 177–185. inflammatory effect of curcumin: PPAR-g activation. PPAR Res. 2007: 40. Bugyei-Twum, A., A. Abadeh, K. Thai, Y. Zhang, M. Mitchell, G. Kabir, and 89369. K. A. Connelly. 2016. Suppression of NLRP3 inflammasome activation ame- 44. Hollborn, M., R. Chen, P. Wiedemann, A. Reichenbach, A. Bringmann, and liorates chronic kidney disease-induced cardiac fibrosis and diastolic dysfunc- L. Kohen. 2013. Cytotoxic effects of curcumin in human retinal pigment epi- tion. Sci. Rep. 6: 39551. thelial cells. PLoS One 8: e59603. Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021