Chrysanthemum indicum extract inhibits NLRP3 and AIM2 inflammasome activation via regulating ASC phosphorylation

Accepted Manuscript Chrysanthemum indicum extract inhibits NLRP3 and AIM2 inflammasome activation via regulating ASC phosphorylation Sang-Hyeun Yu, Xiao Sun, Myong-Ki Kim, Mahbuba Akther, Jun-Hyuk Han, TaeYeon Kim, Jun Jiang, Tae-Bong Kang, Kwang-Ho Lee PII:

S0378-8741(19)30342-3

DOI:

https://doi.org/10.1016/j.jep.2019.111917

Article Number: 111917 Reference:

JEP 111917

To appear in:

Journal of Ethnopharmacology

Received Date: 24 January 2019 Revised Date:

22 April 2019

Accepted Date: 23 April 2019

Please cite this article as: Yu, S.-H., Sun, X., Kim, M.-K., Akther, M., Han, J.-H., Kim, T.-Y., Jiang, J., Kang, T.-B., Lee, K.-H., Chrysanthemum indicum extract inhibits NLRP3 and AIM2 inflammasome activation via regulating ASC phosphorylation, Journal of Ethnopharmacology (2019), doi: https:// doi.org/10.1016/j.jep.2019.111917. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Chrysanthemum indicum extract inhibits NLRP3 and AIM2 inflammasome activation

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via regulating ASC phosphorylation

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Sang-Hyeun Yua,†, Xiao Suna,†, Myong-Ki Kimb, Mahbuba Akthera, Jun-Hyuk Hana,

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Tae-Yeon Kima, Jun Jiangc, Tae-Bong Kangd, Kwang-Ho Leea,d,*

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a

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Program of Neutraceuticals Development, Konkuk University, Chungju, Korea

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b

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c

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(Ministry of Education), College of Agriculture, Yanbian University, Yanji, Jilin, China

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d

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of Inflammatory Diseases, Konkuk University, Chungju, Korea

Department of Food Science and Engineering, Seowon University, Cheongju, Korea

Key Laboratory of Natural Resource of Changbai Mountain and Functional Molecules

†

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Department of Biotechnology, College of Biomedical & Health Science, Research Institute

These authors contributed equally to this work

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Department of Applied Life Science, Graduate School, BK21 Plus Glocal Education

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*

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Science, Konkuk University, Chungju, Korea

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Tel: +82 43 840 3613; Fax: +82 43 851 5235

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E-mail address: [email protected] (K.-H. Lee)

Corresponding author: Department of Biotechnology, College of Biomedical & Health

ACCEPTED MANUSCRIPT ABSTRACT

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Ethnopharmacological relevance: Chrysanthemum indicum (C. indicum), a perennial plant,

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has long been used to treat inflammation-related disorders, such as pneumonia, hypertension,

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gastritis, and gastroenteritis.

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Aim of the study: The inhibitory effect of C. indicum extract (C.I) on inflammasome

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activation was investigated to validate its potential in treating inflammation related disorders.

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Materials and methods: LPS-primed bone marrow-derived macrophages (BMDMs) were

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used to confirm the inhibitory effect of C.I on selective inflammasome activation in vitro. A

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monosodium urate (MSU)-induced murine peritonitis model was employed to study the

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effect of C.I in vivo.

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Results: C.I inhibited activation of NLRP3 and AIM2 inflammasomes, leading to suppression

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of interleukin-1β secretion in vitro. Further, C.I regulates the phosphorylation of apoptosis-

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associated speck-like protein containing a CARD (ASC), which could be the main

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contribution to attenuate these inflammasomes activation. C.I also suppressed secretion of

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pro-inflammatory cytokines and neutrophils recruitment in MSU-induced murine peritonitis

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model.

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Conclusions: This study provides scientific evidence substantiating the traditional use of C.

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indicum in the treatment of inflammatory diseases, including gout, which is induced by

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physiologically analogous cause to MSU-induced peritonitis.

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Keywords: Chrysanthemum indicum; inflammasome; ASC phosphorylation; MSU-induced

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peritonitis; NLRP3

ACCEPTED MANUSCRIPT 1. Introduction

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Inflammasomes are large cytosolic multiprotein complexes that interact with diverse

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pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns

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(DAMPs), leading to inflammatory responses in stimulated immune cells (Davis et al., 2011;

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Martinon et al., 2002; Martinon et al., 2002). There are several receptors that interact with

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PAMPs and DAMPs, such as nucleotide-binding oligomerization domain (NOD)-like

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receptors (NLRs) family consisting of NLRP1, NLRP3, and NLRC4, and the HIN-200

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family’s absent in melanoma 2 (AIM2). These receptors react to specific stimulus sources:

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NLRP1 responds to Bacillus anthracis lethal factor; NLRP3 to various stimuli, including

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crystalline, intracellular ATP, pore-forming toxin, and several microbial pathogens; and

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NLRC4 to specific bacterial proteins, such as flagellin and AIM2 with double stranded DNAs

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(dsDNAs) (Broz and Dixit, 2016; Duncan and Canna, 2018; Duncan and Canna, 2018;

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Fernandes-Alnemri et al., 2009; Fernandes-Alnemri et al., 2009; Man et al., 2016; Man et al.,

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2016; Mariathasan et al., 2004; Mariathasan et al., 2004). Commonly when the

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inflammasomes are being activated by stimuli, activated inflammasome cleaves pro caspase-

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1 to caspase-1. In turn, caspase-1 regulates the maturation of pro-inflammatory cytokines,

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such as interleukin-1β (IL-1β) and interleukin-18 (IL-18), and induces lytic cell death termed

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as pyroptosis (Broz and Dixit, 2016; Miao et al., 2011; Miao et al., 2011; Zhou et al., 2011;

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Zhou et al., 2011). Excessive amount of IL-1β can cause various inflammatory diseases

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(Strowig et al., 2012).

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NLRP3 inflammasome is the most studied among the inflammasomes. Continuous and

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excessive activation of NLRP3 inflammasomes leads to the outbreak of acute and chronic

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inflammatory diseases, including type 2 diabetes (Vandanmagsar et al., 2011; Wen et al.,

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2011; Wen et al., 2011), gout disease (Martinon et al., 2006), Alzheimer’s disease (Heneka et

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ACCEPTED MANUSCRIPT al., 2013), bronchitis (Pauwels et al., 2011; Simpson et al., 2014; Simpson et al., 2014),

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atherosclerosis (Duewell et al., 2010), and multiple sclerosis (Shaw et al., 2010).

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AIM2 is one of the cytosolic sensors that recognizes dsDNA of microbial or host origin. The

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assembly of AIM2 forms a multiprotein complex structure, termed as AIM2 inflammasome.

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The excessive activation of AIM2 inflammasome by cytoplasmic self DNA causes the

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development of psoriasis, dermatitis, arthritis, and other autoimmune diseases (Man et al.,

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2016).

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A common feature of the activation of NLRP3 and AIM2 inflammasomes is the need for

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phosphorylation of the adapter ASC during the formation of the inflammasome complex.

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ASC is the adaptor protein that interacts with the PYD of both NLRP3 and AIM2 and the

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CARD of pro caspase-1 (Hara et al., 2013).

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Chrysanthemum indicum (C. indicum), a wild herb belonging to the chrysanthemum family

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has long been used as a traditional medicine to treat various inflammatory diseases such as

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inflammation, hypertension, and respiratory diseases in China, Japan, and Korea (Cheng et al.,

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2005). Previous reports have suggested that the extract of C. indicum exhibits anti-gout

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(Dong et al., 2017; Kong et al., 2000; Kong et al., 2000; Park and Cho, 2016; Park and Cho,

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2016), anti-inflammatory (Cheng et al., 2005; Wu et al., 2013; Wu et al., 2013; Xue et al.,

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2018; Xue et al., 2018), anti-oxidant (Dong et al., 2017; Dong et al., 2017; Kong et al., 2000),

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and anti-diabetic (Cha et al., 2018) effects.

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Although it has been reported that C. indicum exerts an anti-inflammatory effect, the detailed

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mechanism of its inhibitory effect, including anti-inflammasome activity, are unknown.

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Therefore, this study aimed to investigate and elucidate the inhibitory mechanisms of C.

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indicum on inflammasome activation.

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2. Materials and methods

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2.1. Reagents and antibodies

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ATP and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). ELISA kits for IL-

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6, Nigericin, silica crystal, z-VAD, and Lipofectamine 2000 were purchased from Invivogen

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(San Diego, CA, USA). Antibody against β-actin was purchased from Santa Cruz

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Biotechnology (San Diego, CA, USA). ELISA kit for IL-1β and antibody against IL-1β (AF-

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401-NA) were obtained from R&D systems (Minneapolis, MN, USA). Antibodies against

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NLRP3 (Cryo-2), ASC (AL177), and caspase-1 (Casper-1) were obtained from Adipogen

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(San Diego, CA, USA). Antibody against ASC phospho-specific (Tyr144) was obtained from

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ECM Bioscience (Versailles, KY). Antibody against phosphorylated c-Jun N-terminal kinase

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(JNK) (G9) was obtained from Cell Signaling Technology (Danvers, MA, USA). Cocktails of

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the protease and phosphatase inhibitor were obtained from Thermo (Rockford IL, USA).

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2.2. Plant material and extraction

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The methanol extract of flowers, leaves, and stems of C. indicum (C.I) was purchased from

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the plant extract bank at the Korea Research Institute of Bioscience and Biotechnology

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(KRIBB) in Daejeon, Korea. KRIBB collected C. indicum from Korea during the month of

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December in 2003. A voucher specimen was deposited at the herbarium of the Plant Extract

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Bank at KRIBB for future reference (KRIBB 023-004).

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2.3. Animals

ACCEPTED MANUSCRIPT Female C57BL/6 mice (22–25 g, 6 weeks old) were purchased from Orient Bio Co., Korea.

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Mice were housed in groups of five under standard conditions (temperature: 22 ± 2 °C,

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humidity: 55 ± 5%, 12 h light/dark cycle) with food and distilled water. All experiments were

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performed under the guidelines of the Konkuk University Animal Care Committee, Republic

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of Korea (Permit No, KU18201).

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2.4. Monosodium urate (MSU)-induced peritonitis mouse model and FACS analysis

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The protocol for the MSU-induced peritonitis mouse model was followed as described in our

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previous report (Han et al., 2016). The obtained peritoneal cells were stained with Ly6G (Gr-

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1, clone 1A8-Ly6g) and F4/80 (clone BM8) (eBioscience San Diego, CA, USA), and

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analyzed using a FACS Calibur (Becton Dickinson San Diego, CA, USA). The number of

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neutrophils was evaluated by multiplying the total number of cells by the Ly6G+/F4/80- cell

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ratio. The supernatant of peritoneal lavage fluid was used to evaluate levels of IL-1β and IL-

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2.5 Analysis of inflammasome activation

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BMDMs were first primed with LPS (100 ng/mL) for 3 h, then the medium was replaced

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with Opti-MEM, and the cells were pre-incubated for 1 h with C.I and inhibitors, zVAD (20

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µM) the pan-caspase or KCl (150 mM) to block potassium efflux. The employed

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concentrations of those inhibitors were referred in previous report (Sun et al., 2015). The

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cells were then stimulated with ATP (5 mM) or nigericin (10 µM) for 1 h; silica crystals (150

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µg/mL) for 3 h; and with flagellin (1.5 µg/mL) (3 h) or poly(dA:dT) (2 µg/mL) (1 h).

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ACCEPTED MANUSCRIPT 2.6. Analysis of ASC and JNK phosphorylation

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The immunoblotting of phosphorylated ASC was performed as described previously (Hoyt et

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al., 2016; Kwak et al., 2018; Kwak et al., 2018). BMDMs were seeded in 6-well plates and

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primed with LPS (100 ng/mL) for 3 h in RPMI, then incubated with nigericin (5 µM) and 1

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mM sodium orthovanadate (Na3VO4) with or without C.I for 1 h in Opti-MEM. Cell lysates

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were prepared with RIPA buffer (150 mM NaCl, 0.1% SDS, 50 mM Tris, 1.0% NP-40, and

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0.5% DOC, pH 8). The phosphorylation of ASC in lysates was detected by immunoblotting

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using the antiphospho-ASC antibody (Tyr144). To examine the role of C.I on JNK

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phosphorylation, BMDMs were seeded in 12-well plates and primed with LPS (100 ng/mL)

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for 3 h in RPMI, then incubated with nigericin (10 µM), poly(dA:dT) (2 µg/mL) with or

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without C.I for 20 min, 40 min, and 60 min in Opti-MEM. Cell lysates were prepared with

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RIPA buffer. The phosphorylation of JNK in lysates was detected by immunoblotting using

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the antiphospho-JNK antibody.

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2.7. Other experimental methods

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The experimental protocols for preparation of BMDMs, cell culture and stimulation, LDH

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assay (Koh and Choi, 1987), MTT assay (Slater et al., 1963), and ELISA assay were followed

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as described previously (Han et al., 2015; Shim et al., 2015; Shim et al., 2015). The protocols

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for the separation of Triton X-100 soluble and insoluble fractions, ASC oligomerization, and

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HPLC fingerprinting analysis were followed as described previously (Shim et al., 2013; Sun

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et al., 2015; Sun et al., 2015).

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2.8. Statistical analyses

ACCEPTED MANUSCRIPT All values are expressed as the mean ± S.E.M. (n = 3). The statistical analysis was performed

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using the student’s t test for two groups or a one-way ANOVA (Tukey post-hoc) for three or

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more multiple groups (GraphPad Software San Diego, CA, USA). For all results, a P value <

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0.05 was considered statistically significant.

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3. Results

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3.1. C.I inhibits activation of NLRP3 and AIM2 inflammasomes but not of NLRC4

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inflammasome

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BMDMs were treated with various concentrations of C.I (12.5-100 µg/mL) for 24 h. MTT

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and LDH assays were conducted to determine the cytotoxic effect of C.I. As shown in Fig.

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1A and B, C.I did not show any cytotoxicity up to 100 µg/mL concentration in BMDMs.

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Therefore, in this study, we used non-cytotoxic concentrations of C.I: 25, 50, and 100 µg/mL,

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for further experiments.

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To investigate whether C.I exerts inhibitory effect on inflammasome activation, we treated

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LPS-primed BMDMs with C.I, and then, stimulated them with different inducers for

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inflammasome activation. BMDMs were treated with nigericin, ATP, and silica crystals to

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induce NLRP3 inflammasome, with p(dA:dT) to induce AIM2 inflammasome activation, and

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with flagellin to induce NLRC4 inflammasome activation. As shown in Fig. 1C to 1F, C.I

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suppressed NLRP3 and AIM2 inflammasome-mediated secretion of cleaved IL-1β and

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caspase-1 in a dose-dependent manner without affecting the expression of inflammasome

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components. C.I also inhibited NLRP3 and AIM2 inflammasome-induced pyroptosis (Fig.

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1H). However, C.I did not suppress NLRC4 inflammasome-mediated secretion of cleaved IL-

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1β and caspase-1 (Fig. 1G). Taken together, these data suggested that C.I exerted inhibitory

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effect on both NLRP3 and AIM2 inflammasomes but not on NLRC4 inflammasome

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activation.

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ACCEPTED MANUSCRIPT 3.2 C.I inhibits both ASC speck formation and translocation of ASC

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The adaptor ASC acts as the bridge between caspase-1 and pyrin domain on both NLRP3 and

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AIM2 inflammasomes to form inflammasome complex, which forms insoluble specks (Case

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et al., 2009; Masumoto et al., 1999; Masumoto et al., 1999). Activated ASC constitutes ASC

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oligomerization and forms insoluble speck, which is one of the essential processes for the

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activation of some inflammasomes (Hara et al., 2013). To investigate whether C.I could

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inhibit ASC speck formation, LPS-primed BMDMs were treated with inflammasome

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inducers, such as nigericin or poly(dA:dT), to induce NLRP3 and AIM2 inflammasome

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activation, respectively. ASC specks were detected by immunocytochemistry. C.I inhibited

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ASC speck formation during NLRP3 and AIM2 inflammasome activation (Fig. 2A-B).

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Further, to examine the effect of C.I on ASC oligomerization, LPS-primed BMDMs were

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treated with nigericin or poly(dA:dT) to induce ASC oligomerization, followed by

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inflammasome activation. As shown in Fig. 2C, C.I significantly inhibited formation of

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nigericin or poly(dA:dT)-induced ASC oligomers, dimers as well as monomers. When the

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NLRP3 inflammasome was activated, ASC translocated from soluble fraction to insoluble

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fraction under Triton X-100 detergent lysis condition (Han et al., 2016; Shim et al., 2016;

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Shim et al., 2016; Sun et al., 2015; Sun et al., 2015). We investigated whether C.I could

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inhibit translocation of inflammasome complex components into Triton X-100 insoluble

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fraction. As shown in Fig. 2D, C.I inhibited secretion of IL-1β and cleaved caspase-1 in

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supernatant from NLRP3 or AIM2 inflammasome activated BMDMs. The levels of

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components in soluble fractions were not changed, but the level of ASC was decreased in

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insoluble fractions of NLRP3 or AIM2 inflammasome-activated BMDMs.

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ACCEPTED MANUSCRIPT Fig. 2

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3.3 C.I inhibits phosphorylation of ASC

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Phosphorylation of ASC plays an important role in formation of inflammasome complex. In

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previous reports, ASC phosphorylation was detected using sodium orthovanadate, a

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phosphatase inhibitor, in J774A.1 cells (Hoyt et al., 2016; Kwak et al., 2018; Kwak et al.,

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2018). In this study, BMDMs were primed with LPS (37 ng/mL) for 8 h and treated with

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sodium orthovanadate for 3 h to conserve phosphorylation status. C.I decreased ASC

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phosphorylation and inhibited IL-1β secretion (Fig. 3A). Furthermore, we investigated

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whether C.I can inhibit ASC phosphorylation during the inflammasome activation. LPS

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ACCEPTED MANUSCRIPT primed BMDMs were co-treated with nigericin and sodium orthovanadate for 1 h. As shown

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in Fig. 3B, C.I inhibited phosphorylation of ASC in activated NLRP3 inflammasomes.

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Previous studies with JNK knockout condition validated that phosphorylation of JNK occurs

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upstream of ASC phosphorylation (Hara et al., 2013). In these reports, C.I inhibited ASC

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phosphorylation during NLRP3 inflammasome activation. Therefore, we examined whether

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C.I could also inhibit JNK phosphorylation. LPS primed BMDMs were pretreated with either

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C.I or SP600125, the JNK inhibitor, and subsequently, with nigericin or poly(dA:dT). Both

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C.I and SP600125 suppressed secretion of matured IL-1β and cleaved caspase-1 in the

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supernatant, but C.I could not attenuate phosphorylation of JNK, unlike SP600125, during

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NLRP3 and AIM2 inflammasome activation in cell lysates (Fig. 3C and D).These results

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suggested that C.I inhibits ASC phosphorylation independently of JNK phosphorylation.

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Fig. 3

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3.4 C.I attenuates MSU induced peritonitis model

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Following the in vitro findings, which indicated that C.I inhibits NLRP3 inflammasome

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activation, we further investigated the biological effect of C.I in NLRP3 inflammasome

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activation in vivo. MSU induced mouse peritonitis model has been widely used for NLRP3

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inflammasome as an in vivo model (Martinon et al., 2006; Zhou et al., 2010; Zhou et al.,

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2010). We analyzed the recruitment of neutrophils into the peritoneal cavity and amount of

ACCEPTED MANUSCRIPT IL-1β levels in peritoneal lavage fluid as a barometer of stimulant induced inflammation. As

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shown in Fig. 4A, C.I treatment caused no significant effect on IL-6 that is secreted

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independently of the NLRP3 inflammasome activation. However, a significant decrease in

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IL-1β production in the peritoneal lavage fluid was observed after the administration of C.I

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via both intraperitoneal and oral routes, which indicates the potential inhibition of NLRP3

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inflammasome activation (Fig. 4B). Moreover, C.I was able to decrease the recruitment of

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MSU-induced total cells and Ly6G+/F4/80- neutrophils in peritonium in both intraperitoneal

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(I.P) injected model and oral administered model (Fig. 4C-E). These results suggested that

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C.I has a protective effect on MSU induced peritonitis via the inhibition of NLRP3

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inflammasome activation and concurrent IL-1β secretion.

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ACCEPTED MANUSCRIPT Fig. 4

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3.5 HPLC fingerprinting analysis of C.I

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A previous phytochemical study on C. indicum reported that the extract contained several

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compounds, such as luteolin, acacetin-7-O-rutinoside, luteolin-7-O-glucoside, apigenin-7-O-

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glucoside, 1,5-dicaffeoylqunic acid, and chlorogenic acid (Choi et al., 2016). Similarly, our

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HPLC fingerprinting analysis of C.I revealed that the major peak corresponded to is 1,5-

ACCEPTED MANUSCRIPT dicaffeolyquinic acid and minor peaks to luteolin and chlorogenic acid (Fig. 5). Previous

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studies have reported the inhibitory effect of luteolin and chlorogenic acid on NLRP3

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inflammasome activation (Shi et al., 2018; Shi et al., 2018; Zhang et al., 2018). 1,5-

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dicaffeolyquinic acid did not show any inhibitory effect on inflammasomes activation in our

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study (data not shown).

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Fig. 5

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4. Discussion

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In this investigation, C.I attenuated the secretion of cleaved IL-1β and caspase-1 in NLRP3

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and AIM2 inflammasome activated BMDMs, but not in NLRC4 inflammasome activated

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BMDMs. When the NLRP3 and AIM2 inflammasomes are activated, ASC translocates from

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Triton X-100 detergent soluble fraction to Triton X-100 insoluble fraction, and forms ASC

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oligomerization, which is observed as ASC specks (Hara et al., 2013). It suggested that ASC

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plays a key role in NLRP3 and AIM2 inflammasome activation. In our study, C.I suppressed

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ASC speck formation and the level of protein in Triton X-100 insoluble fraction. These

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ACCEPTED MANUSCRIPT results suggested that C.I attenuates NLRP3 and AIM2 inflammasomes by attenuation of

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ASC speck formation and translocation.

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In previous studies and our report, it was proposed that ASC phosphorylation is necessary for

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inflammasome complex formation (Hara et al., 2013; Hoyt et al., 2016; Hoyt et al., 2016;

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Kwak et al., 2018; Kwak et al., 2018). In this investigation, we detected a decrease in ASC

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phosphorylation in C.I pretreated group. ASC phosphorylation can be detected not only in the

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artificial condition but also in inflammasome activated condition. Therefore, we designed a

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novel protocol by co-treatment of cells through inflammasome activation and treatment with

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sodium orthovanadate. This method was applied for the first time. As a result, the decreased

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amount of ASC phosphorylation in C.I pretreated group compared to nigericin treated group

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could be clearly detected.

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JNK phosphorylation is known to be upstream of ASC phosphorylation (Hara et al., 2013).

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C.I attenuates ASC phosphorylation but does not regulate the phosphorylation of JNK.

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Therefore, we speculated that either unknown kinases could be involved between JNK and

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ASC phosphorylation or C.I could directly regulate the ASC phosphorylation by unknown

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mechanism.

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MSU crystals are well known as danger molecules which are developed after the release of

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uric acid in dying cells (Bomalaski et al., 1986). Phagocytized MSU in macrophages

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stimulate NLRP3 inflammasome to activate pro-caspase-1. Moreover, they are also well-

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known stimuli for NLRP3 inflammasome, and their accumulation in joint tissue causes gout

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arthritis via NLRP3 inflammasome (Martinon et al., 2006). The recognition of MSU crystal

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by innate immune cells, including monocytes and resident macrophages, plays an important

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role in gout and leads to excessive release of inflammatory cytokines, such as IL-1β (Chen et

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al., 2006). In our present study, the in vitro results were consistent with those of in vivo

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ACCEPTED MANUSCRIPT 299

analysis. C.I inhibited the release of IL-1β level in the fluid of peritoneal cavity of MSU-

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induced peritonitis mouse, suggesting the potential availability of C.I on treatment of gout

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arthritis.

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5. Conclusion

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This research is the first to demonstrate scientific evidence of inhibitory effect of C. indicum

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on inflammasome activation, as well as validating the potential of the traditional medicine of

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C. indicum in the treatment of inflammatory disorders, such as gout, which is induced by

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physiologically analogous cause to MSU-induced peritonitis.

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Conflict of interest statement

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The authors have declared that there is no conflict of interest.

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Author Contributions

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All authors discussed the research process, results, and implications, and all authors

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commented on the manuscript. Sang-Hyeun Yu: Experimental design and execution, data

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analysis and manuscript writing. Xiao Sun: Experimental design, data analysis, and

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manuscript writing. Mahbuba Akther, Jun-Hyuk Han, Tae-Yeon Kim: In vivo experiments.

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Jun Jiang: In vitro experiments. Myong-Ki Kim: Execution of fingerprinting analysis. Tae-

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Bong Kang: Data analysis, discussion, and manuscript review. Kwang-Ho Lee: Supervised

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the project and contributed to the experimental design, data analysis, and manuscript writing.

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ACCEPTED MANUSCRIPT Acknowledgements

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This paper was supported by Konkuk University in 2018.

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References

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knee joints of patients with asymptomatic nontophaceous gout. Arthritis. Rheum. 29, 1480-

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Broz, P., Dixit, V.M., 2016. Inflammasomes: mechanism of assembly, regulation and

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ACCEPTED MANUSCRIPT Figure captions

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Fig. 1. C.I specifically inhibited activation of NLRP3 and AIM2 inflammasomes. BMDMs

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were treated with the indicated concentrations of C.I for 24 h, and their viability and

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cytotoxicity were determined by MTT assay (A) and LDH release assay (B), respectively.

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LPS primed-BMDMs were treated with various doses of C.I for 1 h, and then, stimulated

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with nigericin for 1 h (C), ATP for 1 h (D), silica crystals (Silica) for 3 h (E), poly(dA:dT) for

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1 h (F), and flagellin for 3 h (G). Supernatants (SN) were used to detect the levels of cleaved

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IL-1β and caspase-1. Cell lysates (Lys) were used to detect inflammasome components. (H)

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LPS primed BMDMs were treated with C.I at the indicated concentrations for 1 h, and then,

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treated with inflammasome activators; supernatants were collected for LDH determination.

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The results are expressed as mean ± S.E.M (n = 3). ns: non-significant; **p < 0.01 and ***p

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< 0.001 compared with control cells.

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Fig. 2. C.I inhibited both ASC speck formation and translocation of ASC into Triton X-100

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insoluble fraction. (A) ASC specks were observed by immunofluorescent microscope. (B)

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The proportion of cells containing ASC specks are represented as histograms. (C) ASC

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oligomerization of NLRP3 and AIM2 in Triton X-100 insoluble (Pel) fractions from cell

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lysates was determined by western bolt analysis. Cleaved IL-1β was detected from culture

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supernatant (SN). Total ASC and β-actin levels in total cell lysates (Lys) were used as internal

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control. (D) LPS primed BMDMs were treated for 1 h with various doses of C.I, and then,

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stimulated with NLRP3 or AIM2 inflammasome stimulators. Inflammasome components in

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Triton X-100 soluble (Lys) or insoluble (Pel) fractions of cell lysates were detected by

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western blot using indicated antibodies. The results are expressed as mean ± S.E.M (n = 3).

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*p < 0.05 and ***p < 0.001 compared with nigericin or poly(dA:dT)-treated cells.

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Fig. 3. C.I inhibits ASC phosphorylation. (A) LPS primed BMDMs were pretreated with C.I

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for 1 h, and then, treated with sodium orthovanadate (Na3VO4) for 3 h. IL-1β level was

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detected by ELISA and phosphorylated ASC was detected by immunoblot. (B) LPS primed

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BMDMs were pretreated with C.I, followed by co-treatment with nigericin and sodium

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orthovanadate for 1 h. Phosphorylated-ASC was detected by immunoblot. LPS primed

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BMDMs were pretreated with C.I for 1 h or SP600125 for 30 min, and then, stimulated with

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nigericin (C) or poly(dA:dT) (D) for indicated times. Supernatants (SN) were used to detect

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levels of cleaved IL-1β and caspase-1. Cell lysates (Lys) were used to detect p-JNK and total

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JNK (t-JNK).

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Fig. 4. C.I attenuates MSU induced peritonitis model. C.I was intraperitoneally (i.p) injected

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into mice 12 h and 2 h before i.p injection with MSU, or C.I was orally administered (oral)

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into mice once a day for seven days before i.p injection with MSU. Peritoneal lavage fluid

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was harvested 6 h after MSU injection. Concentrations of IL-6 (A) and IL-1β (B) were

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measured by ELISA. (C) Total cells were quantified by microscope. (D) Neutrophils were

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determined by multiplying the cell numbers by the percentage of Ly6G+/F4/80- cells from

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total cells. (E) Ly6G+/F4/80- neutrophils were quantified by flow cytometry. * p < 0.05, ** p

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< 0.01, and *** p < 0.001 compared to the MSU-injected group.

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Fig. 5. HPLC fingerprinting analysis of C.I. The components of C.I were determined using an

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HPLC system.