Tetrachlorobenzoquinone Stimulates NLRP3 Inflammasome-Mediated

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Tetrachlorobenzoquinone stimulates NLRP3 inflammasome-mediated posttranslational activation and secretion of IL-1# in endothelial HUVEC cell line Xiaomin Xia, Qiong Shi, Xiufang Song, Juanli Fu, Zixuan Liu, Yawen Wang, Yuxin Wang, Chuanyang Su, Erqun Song, and Yang Song Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00021 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on February 28, 2016

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Chemical Research in Toxicology

Tetrachlorobenzoquinone stimulates NLRP3 inflammasome-mediated post-translational activation and secretion of IL-1β in endothelial HUVEC cell line

Xiaomin Xia, Qiong Shi, Xiufang Song, Juanli Fu, Zixuan Liu, Yawen Wang, Yuxin Wang, Chuanyang Su, Erqun Song, Yang Song*

Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing, People’s Republic of China, 400715

*Corresponding author: College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing, People’s Republic of China, 400715. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail addresses: [email protected] or [email protected]

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TABLE OF CONTENTS

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ABSTRACT Our previous studies suggested the pro-inflammatory activities of tetrachlorobenzoquinone (TCBQ), however, its toxic mechanism towards vascular endothelial cell has not been characterized. Although TCBQstimulated interleukin-1beta (IL-1β) expression has been identified, whether TCBQ regulates post-translational IL-1β activation is current unknown. By using human umbilical vein endothelial cells (HUVEC), we discovered that TCBQ not only promotes NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) components [comprising of NLRP3, adaptor molecule apoptosis-associated speck like protein containing a caspase activation and recruitment domain (ASC) and pro-caspase 1] expression, but also participates in the prime of NLRP3 inflammasome. The activation of NLRP3 inflammasome result in the maturation and the release of IL-1β. Further experiments showed that K+ efflux, reactive oxygen species (ROS) production and mitochondrial DNA damage may involve in NLRP3 inflammasome activation by TCBQ stimulation. Moreover, TCBQ downregulates the ubiquitination of NLRP3 and further facilitates the activation of NLRP3 inflammasome. These results proposed that NLRP3/IL-1β signaling pathway play an important role in TCBQinduced endothelial pro-inflammatory responses, which may point to potential therapeutic approaches against TCBQ toxicity.

KEYWORDS: Tetrachlorobenzoquinone; inflammation; IL-1β; NLRP3; caspase 1; ROS

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INTRODUCTION Pentachlorophenol (PCP) was extensively used as wood preservative in the last century, which was banned in most of countries since its apparent toxicities were noticed. PCP has been listed as one of the prior pollutants by the US Environmental Protection Agency.1 Although PCP is a stable and persistent compound, its metabolic activation play an important role in PCP’s toxicity. One of the presumptive metabolic pathway starting with an oxidative dehalogenation mediated by cytochrome P450 (CYP450) to yield an active metabolite tetrachlorobenzoquinone (TCBQ).2 Our recent works highlighted TCBQ-induced inflammatory response through the releases of proinflammatory cytokines. For instance, mice administrated with TCBQ showed increased inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB) levels in the liver.3 Consistent with these data, TCBQ exhibits similar effect by inducing oxidative stress-mediated inflammatory responses via NF-κB activation in PC12 cells.4 Although in vivo and in vitro pro-inflammatory potential of TCBQ has been suggested, the signaling pathway leading to the pro-inflammatory cytokine IL-1β production and secretion has not been well determined. IL-1β is one of the members of the IL-1 cytokine superfamily, which play an important role in inducing a pro-inflammatory gene expressions in various cell types.5, 6 The abnormal IL-1β expression has been implicated in a range of diseases and conditions, thus, IL-1β become an ideal target for clinical intervention of inflammatory diseases.7 In general, IL-1β activity regulation can be orchestrated through transcriptionaldependent and -independent mechanisms. Specifically, transcriptional regulation of IL-1β means the production of pro-IL-1β mRNA, and transcriptional-independent mechanism involves the cleavage of pro-IL-1β and the release of mature IL-1β.8,

9

Recently, we demonstrated that TCBQ induces pro-inflammatory responses,

including the upregulation of protein and mRNA expressions of IL-1β,4 and these events communicated through ROS-mediated inhibitor of NF-κB kinase (IKK)/inhibitor of NF-κB (IκB)/NF-κB, following signaling from 4


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pattern-recognition receptors (PRRs) such as toll-like receptors (TLRs). Meanwhile, TCBQ-mediated damage was alleviated by the silence of TLR4 or myeloid differentiation primary response protein (MyD88) gene. These results indicated that TCBQ regulates IL-1β in a transcriptional-dependent manner, however, whether TCBQ controls the transcriptional-independent regulation of IL-1β needs further investigation. The function of IL-1β need to be proteolytically activated through caspase 1, which cleavage IL-1β. Caspase 1 activation is regulated by the assembly of the inflammasome, which consists of nucleotide binding oligomerization domain containing (NOD)-like receptor family, pyrin domain-containing protein 3 (NLRP3), adaptor molecule apoptosis-associated speck like protein containing a caspase activation and recruitment domain (ASC), and caspase 1. With the stimulation of danger signals, NLRP3 interacts with pro-caspase 1 via ASC, then leads to the tandem cleavage of caspase 1 and IL-1β, and matured IL-1β release from the cells.10 For this matter, the priming of NLRP3 inflammasome is essential for IL-1β maturation and secretion. Indeed, ASC-, NLRP3- or caspase 1-deficient mice showed defect in the production of mature IL-1β, which are more resistant to pro-inflammatory stimulus.11, 12 Recently, the activations of NLRP3 inflammasome in which stimulated by chemicals and nanoparticles have been extensively characterized.13, 14 Moreover, numerous works have suggested that ROS derived from complex I and complex III of the mitochondrial respiratory chain are the common integrator for the activation of NLRP3 inflammasome.15, 16 Since ROS generation has been proposed in the redox-cycling of TCBQ and its corresponding reduced form tetrachlorohydroquinone,17 and increasing evidence suggested that ROS provide great contribution on the initiation and progression of atherosclerosis in vascular endothelial cells.18, 19 Thus, we hypothesize the involvement of NLRP3 inflammasome activation upon TCBQ exposure in vascular endothelial cells.

MATERIALS AND METHODS

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Reagents. TCBQ was purchased from Aladdin Reagent Database Inc. (Shanghai, China). NLRP3, ASC, caspase 1, vascular cell adhesion molecule 1 (VCAM-1) and E-selectin antibodies were purchased from Bioss Biotech Co. Ltd (Beijing, China). Intercellular adhesion molecules 1 (ICAM-1) antibody was purchased from Proteintech (Wuhan, China). IL-1β and β-actin antibodies and goat anti-rabbit IgG-HRP-conjugated secondary antibody were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). B-cell lymphoma-2 (Bcl-2), Bcl-2antagonist/killer 1 (Bak) and Bcl-2 associated X protein (Bax) antibodies were obtained from Ruiying Biological (Suzhou, China). N-acetylcysteine (NAC), apocynin (APO) and cycloheximide were purchased from Sigma-Aldrich Co. LLC. (Shanghai, China). Mito-TEMPO, PR-619, Z-VAD-FMK and Ac-YVAD-CHO were purchased from Santa Cruz Biotechnology Co. Ltd. (Shanghai, China). Cell culture. Human umbilical vein endothelial cell (HUVEC) was purchased from KeyGEN Biotech. Co., Ltd. (Nanjing, China). HUVEC (7×105 cells) were grown in 5 mL RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator with 95% air and 5% CO2. Cells were treated with TCBQ, or pretreated with 60 mM KCl (K+ efflux inhibitor), 5 mM NAC, 200 µM APO, 500 µM Mito-TEMPO, 10 µg/ml cycloheximide, 10 µM PR-619, 10 µM Z-VADFMK and 10 µM Ac-YVAD-CHO for 1 hour before TCBQ stimulation. Culture supernatant sample preparation. Culture supernatants were precipitated by adding an equal volume of methanol and 0.25 volumes of chloroform. The mixtures were vortexed and centrifuged for 10 min at 20,000 g. Upper phase was discarded and 500 µL methanol was added in the interphase. This mixture was centrifuged at 20,000 g for 10 min and the protein pellet was dried at 55°C, then resuspended in Nu-PAGE loading buffer and boiled at 97°C for 5 min. Samples were stored for SDS-PAGE analysis. Western blotting. HUVECs were lysed by RIPA lysis buffer (50 mM Tris, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 0.1% SDS) with protease inhibitor mixture. Protein concentrations were determined using the Bradford protein assay kit. Proteins were separated by 10% or 12.5% SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in 50 mM Tris-buffered saline 6


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containing 0.1% Tween 20 (TBST) at 37°C for 1.5 h and were then incubated appropriate primary antibodies overnight at 4°C. After washing in TBST three times, the membranes were incubated with secondary antibody at 37°C for 2 h. The membranes were washed three times with TBST and visualized with BeyoECL Star (Beyotime Biotechnology, Shanghai, China). Representative blots were chosen from at least three independent experiments. Real-time quantitative PCR (RT-qPCR). Total RNA was extracted from HUVECs using High Pure RNA Isolation Kit (Bioteke Corporation, Beijing, China) according to the manufacturer’s instructions. cDNA was synthesized with random primers in Transcriptor First Strand cDNA Synthesis Kit (Roche, Shanghai, China). qPCR was performed with primers specific for IL-1β (forward: 5'-TTACAGTGGCAATGAGGATGAC-3', reverse: 5'-GTCGGAGATTCGTAGCTGGAT-3'), for NLRP3 (forward; 5'-GATCTTCGCTGCGATCAACAG3',

reverse:

5'-CGTGCATTATCTGAACCCCAC-3'),

for

caspase

1

(forward;

5'-

TTTCCGCAAGGTTCGATTTTCA-3', reverse: 5'-GGCATCTGCGCTCTACCATC-3'), and for β-actin (forward;

5'-TCCTCCCTGGAGAAGAGCTAC-3',

reverse:

5'-TCCTGCTTGCTGATCCACAT-3').

The

amount of mRNA expressions of IL-1β, NLRP3 or caspase 1 were normalized to that of β-actin. RNA interference. HUVECs were grown up to 30% confluence. Briefly, cells were transfected with signal silence-negative control or NLRP3 siRNA (siNLRP3) at 50 nM concentration using siRNA-MateTM (Shanghai GenePharma Co. Ltd) for 48 h, according to the manufacturer’s instructions. The sequences for siRNA targeting were as follows: NLRP3 sense, 5'-GGUUUGGAAUUAGACAACTT-3' and NLRP3 antisense, 5'GUUGUCUAAUUCCAACACCTT-3'. After stimulating with TCBQ for 6 h, cells were subjected to further analysis. Co-immunoprecipitation (Co-IP). To assess the protein composition and association of proteins in the inflammasome, HUVECs (8×106 cells) were lysed in 800 µL of RIPA lysis buffer with protease inhibitor mixture. Approximately 500 µg of cell lysates were immuno-precipitated with 2 µg of NLRP3 or ASC antibodies using Protein A+G Agarose (Beyotime Biotechnology, Shanghai, China). The mixture was incubated 7



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at 4°C overnight, and agarose was pelleted by centrifugation at 2,500 g for 10 min. The pelleted agarose was washed five times in PBS, resuspended in loading buffer, heated at 97°C for 5 min and resolved by Western immunoblotting using antibodies against ASC, caspase 1 or NLRP3. Ubiquitination. NLRP3 ubiquitination was assayed by immunoprecipitation with NLRP3 antibody using Protein A+G agarose, followed by immunoblotting with ubiquitin antibody. Briefly, cells (7×106) were lysed in 600 µL of denaturation buffer [50 mM Tris-HCl (pH 7.5), 1% SDS, 150 mM NaCl and 10 mM Nethylmaleimide] and then heated at 97°C for 10 min, followed by dilution with 10 volumes of binding buffer [50 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40, 150 mM NaCl and 10 mM N-ethylmaleimide]. The diluted lysates were immunoprecipitated with anti-NLRP3 and Protein A+G agarose at 4°C for 4 h. The agarose were washed five times with binding buffer, followed by elution with loading buffer and fractionation on 8% SDSPAGE. qPCR for mitochondrial DNA (mtDNA) damage. Quantitative PCR technique was used to assess mtDNA damage as previous described.20,

21

Briefly, mtDNA and nuclear DNA were isolated using the

TIANamp Genomic DNA kit (TIANGEN, Beijing, China). A fragment of the mtDNA or nuclear DNA was amplified with specific primers. The relative amplification of the 10 kb product was calculated by the comparative Ct method, DNA lesion frequencies were calculated using Poisson transformation. Statistical analysis. The data were generated from at least three independent experiments and expressed as the means ± SD. Analyses were done by using SPSS statistical 19.0 software. Significant differences among multiple groups were examined using two-way ANOVA followed by an unpaired Student’s t test and a value of p < 0.05 was considered significant.

RESULTS AND DISCUSSION Effect of TCBQ on the expression of IL-1β and adhesion molecules. Among various pro-inflammatory cytokines, IL-1β play an indispensable role in innate and adaptive immunological responses.22 The assistance of 8


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phagocytes, epithelial and endothelial cells activation and T-lymphocytes polarization are the bright side of IL1β, however, the prolonged production of IL-1β can be deterious.23 We previous demonstrated that TCBQ stimulation upregulated IL-1β expression in PC12 cells.4 However, there is no information regarding to the effect of TCBQ on IL-1β maturation and secretion. To decipher this, we first investigated whether TCBQ stimulation could induce IL-1β secretion. Clearly, the present of mature IL-1β (molecule weight 17 kDa) in the supernatants from TCBQ-stimulated HUVECs suggested the secretion of IL-1β, Fig 1A and 1B. Meanwhile, TCBQ induced the expression of pre- IL-1β (molecule weight 31 kDa) in both concentration and timedependent manners. Interestingly, we also found TCBQ upregulated IL-1β at transcriptional level, Fig 1C and 1D. The expressions of a diverse family of cellular adhesion molecules, such as ICAM-1, VCAM-1 and Eselectin, play a key role in the development of inflammation-related diseases, e.g., atherosclerosis.24 Our result indicated the elevation of ICAM-1, VCAM-1 and E-selectin in HUVECs dependent upon time and dose, Fig 1E and 1F. These findings implicated the association of TCBQ exposure with vascular endothelial cell inflammation. Effect of TCBQ on the expression of NLRP3 complex components and the priming of NLRP3 inflammasome. IL-1β maturation requires pro-IL-1β cleavage by caspase 1, follow up the construction of a large protein complex, NLRP3 inflammasome which comprising NLRP3, ASC and pro-caspase 1.25 Thus, we next investigated TCBQ-induced NLRP3 inflammasome activation in HUVECs. The levels of inflammasome components, i.e., NLRP3, ASC and pro-caspase 1 (molecule weight 45 kDa) were analyzed by Western blotting, Fig 2A and 2B. Furthermore, the activated form of caspase 1 (molecule weight 20 kDa) in the supernatants was also detected. We confirmed the upregulation of NLRP3, ASC and caspase 1 by TCBQ treatment. Likewise, RT-qPCR assay indicated TCBQ increased NLRP3 and caspase 1 expressions at transcriptional level, Fig 2C. Although the induction of NLRP3 expression is a critical feature, the priming of NLRP3 inflammasome is necessary for pro-IL-1β cleavage.26 In order to confirm the upstream mechanism by which TCBQ induced 9



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caspase 1 activation, we examined the effect of TCBQ on the priming of NLRP3 inflammasome. First, NLRP3 (or ASC) antibody, bound to Protein A+G immobilized beads was used to capture cellular NLRP3 (or ASC) protein from whole cell lysates. After washing the precipitates, captured proteins were separated by SDS-PAGE and transferred to blotting membranes, which were overlaid with corresponding antibodies. The blotting results demonstrated when recover equal amounts of NLRP3 (or ASC) from lysates, other components in NLRP3 inflammasome were increased in TCBQ-treated group, compare with the control group, Fig 3A and 3B. Taking together, our data suggested NLRP3 inflammasome activation is responsible for TCBQ-induced IL-1β secretion in HUVECs. The inhibition of NLRP3 components by siRNA or inhibitors lead to reduced NLRP3 inflammasome construction and downstream cytokines production. To further confirm the essentiality of NLRP3 on TCBQ-induced IL-1β secretion, we used siRNA to silence NLRP3 in HUVECs. In contrast to scrambled siRNA group, siRNA-NLRP3 treated cells failed to show caspase 1 activation and IL-1β secretion, Fig 4A. Consistently, ICAM-1, VCAM-1 and E-selectin were all inhibited by siRNA-NLRP3 treatment. NLRP3 inflammasome activation resulted in the recruitment of caspase 1, the key component of the inflammasome complex responsible for processing of IL-1β maturation.27 Here, silencing of pro-caspase 1 with pan-caspase inhibitor (Z-VAD-FMK) or caspase 1 inhibitor Ac-YVAD-CHO abolished IL-1β, ICAM-1, VCAM-1 and Eselectin expressions, and IL-1β secretion, Fig 4B. This data advocated that TCBQ-induced NLRP3 expression is necessary but not sufficient, meanwhile the priming of NLRP3 inflammasome is essential, for caspase 1 activation and IL-1β release. Effect of TCBQ on K+ efflux may contributed to IL-1β secretion from HUVECs. A few distinct upstream signals have been proposed to explain the activation of the NLRP3 inflammasome, including K+ efflux.28 K+ efflux prompted by exogenous stimulus is crucial for trigger caspase 1 activation.29, 30 To test the importance of K+ efflux on TCBQ-induced IL-1β secretion, we treated HUVECs with 60 mM extracellular K+ by two different methods. In agreement with previous result, the formation of NLRP3 inflammasome and the 10


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release of caspase 1 and IL-1β stimulated by TCBQ were significantly blocked by the introduction of extracellular K+ (Fig 5), further support that intracellular K+ depletion is a common trigger of NALP3 activation.29, 31 Effect of TCBQ on IL-1β secretion from HUVECs requires upstream signals including ROS production and mtDNA damage. ROS positively regulate NLRP3 inflammasome activation, as well as proIL-1β expression.10 More specifically, ROS derived from NADPH oxidase involved in the NLRP3 inflammasome activation stage.32 In parallel with the contribution of ROS on NLRP3 activation, antioxidants or ROS-scavengers inhibit NLRP3 expression or activity.33, 34 Here, TCBQ-induced caspase 1 activation and proIL-1β expression were blocked by the pretreatment of APO (NADPH oxidase inhibitor) or NAC (total ROS scavenger), Fig 6. APO inhibits the NADPH oxidase activity by blocking the translocation of p47 subunit of NADPH oxidase to cytosolic subunit, which in turn decreased ROS production.35 From this point of view, APO is also considered as an antioxidant. Previous study also suggested mitochondria-derived ROS are important in the control of NLRP3 inflammasome activation.15, 36, 37 Given the fact that mtDNA damage frequently occurs in the vessel wall in human atherosclerosis, we speculate that TCBQ exposure causes mtDNA damage in HUVECs. A wellstablished quantitative PCR analysis for mtDNA adducts was used to assess mtDNA damage, which based on the premise that DNA template contains a lesion, such as oxidized bases, single- and double-strand breaks, and abasic sites, will stop the polymerase chain reaction. Therefore, the difference in qPCR amplification reflects template copy number or DNA quality directly, which is a useful indicator to assess the global DNA damage. Indeed, TCBQ treatment increases lesion frequency both concentration- and time-dependently, Fig 7A and 7B. Consistently, the increased expressions of Bak and Bax and the decreased expression of Bcl-2 suggested mitochondrial-related apoptosis (Fig 7C and 7D), which further verified the link between mtDNA damage and NLRP3 inflammasome activation.38 As we estimated, Mito-TEMPO (mitochondrial ROS inhibitor) ameliorated NLRP3 inflammasome formation and IL-1β release, as well as the expression of adhesion molecules, Fig 7E. 11



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Mitochondrial ROS induce the translocation of NLRP3 to mitochondria-associated endoplasmic reticulum membranes, then recruit ASC adaptor for the priming of inflammasome.16, 39 However, the role of ROS on NLRP3 inflammasome is still incompatible. Liao et al illustrated that the inhibition of NADPH oxidase by diphenyliodonium or APO suppressed LPS-induced increases in NLRP3 and pro-IL-1β expression,32 whilst van de Veerdonk et al proposed that diphenyliodonium was unable to inhibit NLRP3 inflammasome activation in LPS+nigericin-activated macrophages.40 Of note, NAC has better capacity than APO or Mito-TEMPO in the inhibition of TCBQ-induced IL-1β release, Fig 6 and Fig 7E. In contrast to APO and Mito-TEMPO, which only prevent NADPH oxidase- and mitochondria-derived ROS production, NAC is a universal scavenger of ROS regardless of the source. Together, these results indicated that TCBQ-mediated ROS production from NADPH oxidase- or mitochondrial-independent sources contributes to NLRP3 inflammasome activation. TCBQ promotes rapid non-transcriptional priming of NLRP3 inflammasome. Interestingly, ROS inhibition does not directly block the activation of NLRP3 inflammasome, but shuts down the priming step of NLRP3 inflammasome.33 Although the upregulation of NLRP3 expression by TCBQ acts as a priming signal, a significant elevation of NLRP3 needs a minimum of 60 min of TCBQ incubation. However, 5 min of TCBQ incubation induced the mature and the release of caspase 1 and IL-1β, even without the increase expression of NLRP3, Fig 8A. Furthermore, the pretreatment with protein synthesis inhibitor cycloheximide failed to prevent NLRP3 inflammasome prime at 5 min in TCBQ-stimulated HUVECs, based on the observation of increased caspase 1 and IL-1β secretion. These results suggested that TCBQ initiate NLRP3 inflammasome prime through a non-transcriptional mechanism even at relative low NLRP3 level, which do not require new NLRP3 synthesis.41 However, at the time point of 60 min, this effect was completely abrogated by cycloheximide. In supporting this, RT-qPCR analysis showed that the transcriptional activation of NLRP3 does not occur at 5 min, but at a longer period of 60 min, Fig 8B. The prolonged treatment of cycloheximide (60 min) leads to a downregulation of NLRP3 expression, which is responsible for inhibition of caspase 1 activation. This result

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suggested that a de novo synthesis of NLRP3 is necessary for the maintaining NLRP3 inflammasome activity, which leads us to explore the degradation of NLRP3. TCBQ triggers the deubiquitination of NLRP3, which facilitates its activation. Ubiquitin-mediated protein degradation represents the most ubiquitous mechanism for the regulation of protein availability and activity, which is carried out by ubiquitin proteasome system.42 Juliana et al immunoprecipitated NLRP3 and followed by blotting ubiquitin revealed that NLRP3 is ubiquitinated.41 To investigate whether TCBQ promotes the priming of NLRP3 inflammasome by regulating its ubiquitination, we further measured the ubiquitination status of NLRP3. As shown in Fig 9A, TCBQ induced significant deubiquitination of NLRP3. In contrast, treatment with general deubiquitinating enzyme inhibitors PR-619 reversed TCBQ-induced deubiquitination of NLRP3, indicating the deubiquitination of NLRP3 is critical for its prime. In addition, when deubiquitination of NLRP3 was inhibited by PR-619, caspase 1 activation and IL-1β secretion were both reduced, Fig 9B. As ROS are important for the priming of NLRP3, we further tested whether antioxidants inhibited deubiquitination of NLRP3. Indeed, the pretreatment of NAC, APO or Mito-TEMPO considerably inhibited this effect, which suggest TCBQ-induced ROS play a role on the deubiquitination of NLRP3, Fig 9A.

CONCLUSION In the current study, our work discovered a novel mechanism for TCBQ-induced inflammatory reflection by activating NLRP3 inflammasome. The results indicated that the use of reagents, such as NADPH oxidase inhibitors, antioxidants, caspase inhibitors or K+ efflux inhibitors, might be a promising strategy in the clinical anti-inflammatory therapeutics to against environmental pollutants-induced injuries.

AUTHOR INFORMATION *Corresponding author

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Phone: +86-23-68251503. Fax: +86-23-68251225. E-mail addresses: [email protected] or [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding This work is supported by National Natural Science Foundation of China (21477098 and 21575118), Science and Technology Talent Cultivation Project of Chongqing (cstc2014kjrc-qnrc00001) and Fundamental Research Funds for the Central Universities (XDJK2015A017 and XDJK2016A004).

Notes The authors declare no competing financial interest.

ABBREVIATIONS APO, apocynin; ASC, adaptor molecule apoptosis-associated speck like protein containing a caspase activation and recruitment domain; Bak, Bcl-2-antagonist/killer 1; Bax, Bcl-2 associated X protein; Bcl-2, Bcell lymphoma-2; COX-2, cyclooxygenase-2; CYP450, cytochrome P450; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecules 1; IκB, inhibitor of NF-κB; IKK, inhibitor of NFκB kinase; IL-1β, interleukin-1beta; iNOS, inducible nitric oxide synthase; MyD88, myeloid differentiation primary response protein; NAC, N-acetyl-L-cysteine; NF-κB, nuclear factor-kappa B; NLRP3, NOD-like receptor family, pyrin domain-containing protein 3; NOD, nucleotide binding oligomerization domain containing; PCP, pentachlorophenol; PRRs, pattern-recognition receptors; RT-qPCR, Real-time Quantitative 14


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PCR; ROS, reactive oxygen species; siRNA, small interfering RNA; RAGE, receptor for advanced glycation end-products; TCBQ, tetrachlorobenzoquinone; TLRs, toll-like receptors; TNF-α, tumor necrosis factor-alpha; VCAM-1, vascular cell adhesion molecule 1 REFERENCES (1)

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Figures Figure 1. TCBQ upregulates the expression of IL-1β and adhesion molecules in a (A) dose- and (B) timedependent manner. IL-1β in the supernatants and cell lysates were measured by Western blotting assay. sup, desalinized culture supernatants of HUVECs. (C) and (D) The level of mRNA IL-1β was determined by RTqPCR, and the data (mean ± standard deviation) are shown as a ratio of IL-1β compared with β-actin (fold change). (E) and (F) VCAM-1, ICAM-1 and E-selectin protein levels were determined in HUVECs by Western blotting assay. The β-actin protein level was considered as an internal control.

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Figure 2. TCBQ induces NLRP3, ASC and caspase1 protein expressions in a (A) dose- and (B) timedependent manner. Proteins in the supernatants and cell lysates were measured by Western blotting assay. sup, desalinized culture supernatants of HUVECs. The β-actin protein level was considered as an internal control. (C) TCBQ induces the mRNA level of NLRP3 and caspase 1 mRNA. Data (mean ± standard deviation) are shown as a ratio of IL-1β compared with β-actin (fold change).

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Figure 3. TCBQ promotes NLRP3 inflammasome priming. HUVECs were incubated with TCBQ (5, 10 or 20 µM) for 6 h. Interaction between ASC and NLRP3 was analyzed by co-immunoprecipitation assay. Cell lysates were immunoprecipitated with (A) NLRP3 or (B) ASC antibody, respectively. Then, precipitated proteins were analyzed by SDS-PAGE with (A) ASC or (B) NLRP3 and caspase 1 antibodies.

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Figure 4. Inhibition of NLRP3 components by siRNA or inhibitor leads to reduced NLRP3 inflammasome construction and downstream cytokines production. (A) Cells were transfected with NLRP3 siRNA for 48 h, then the cells were treated with 20 µM TCBQ for 6 h. Representative Western blotting images indicated siRNA treatment diminished caspase 1 and IL-1β secretion and the expressions of ICAM-1, VCAM-1, E-selectin. (B) Cells were pretreated with either 10 µM Z-VAD-FMK (pan-caspase inhibitor) or 10 µM AcYVAD-CHO (caspase 1 inhibitor) for 1 h. Then, IL-1β and caspase 1 secretion and NLRP3, pro-caspase 1, ICAM-1, VCAM-1 and E-selectin were detected. sup, desalinized culture supernatants of HUVECs. The βactin protein level was considered as an internal control.

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Figure 5. The inhibition of TCBQ-induced NLRP3 inflammasome activation by K+ inhibitor. Cells were incubated with 60 mM KCl, and then the secretions of IL-1β and caspase 1, the expressions of NLRP3, procaspase 1 and pro-IL-1β from HUVECs were detected by Western blotting assay. The β-actin protein level was ①

considered as an internal control. : Medium was added 60 mM KCl for 30 min after replacing the medium ②

given 20 µM TCBQ for 6 h. : Medium was added 60 mM KCl for 30 min after directly given 20 µM TCBQ for 6 h.

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Figure 6. The inhibition of TCBQ-induced NLRP3 inflammasome activation by ROS scavenger or inhibitor. Cells were pretreated with 200 µM APO or 5 mM NAC for 1 h, then cells were treated with 20 µM of TCBQ for 6 h. Then the secretions of IL-1β and caspase 1, the expressions of NLRP3, pro-caspase 1, ICAM1, VCAM-1 and E-selectin from HUVECs were detected by Western blotting assay. sup, desalinized culture supernatants of HUVECs. The β-actin protein level was considered as an internal control.

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Figure 7. TCBQ induces mtDNA damage which promoted NLRP3 inflammasome activation. (A) TCBQ induces mtDNA damage in a dose-dependent manner. HUVECs were treated with 5, 10 or 20 µM TCBQ for 6 h. (B) TCBQ induces mtDNA damage in a time-dependent manner. Cells were treated with 20 µM of TCBQ for 1, 3 or 6 h. mtDNA adducts were analyzed by qPCR assay and presented as lesion frequency, which reflect the extent of mtDNA damage. (C) TCBQ modulates mitochondrial-driven apoptosis-related gene expressions in a dose-dependent manner. HUVECs were treated with 5, 10 or 20 µM TCBQ for 6 h. (D) TCBQ modulates mitochondrial-driven apoptosis-related gene expressions in a time-dependent manner. (E) Cells were pretreated with 500 µM Mito-TEMPO for 1 h, then, cells were treated with 20 µM of TCBQ for 6 h. The secretions of IL1β and caspase 1, the expressions of NLRP3, pro-caspase 1 and pro-IL-1β from HUVECs were detected by Western blotting assay. sup, desalinized culture supernatants of HUVECs. The β-actin protein level was considered as an internal control.

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Figure 8. TCBQ promotes rapid non-transcriptional priming of NLRP3 inflammasome. (A) Cells were pretreated with 10 µg/ml cycloheximide for 1 h, then cells were treated with 20 µM of TCBQ for 5 min or 60 min. Immunoblots of NLRP3, pro-caspase 1 and pro-IL-β in cell lysates and caspase 1 and IL-1β in supernatants of HUVECs were determined by Western blotting assay. sup, desalinized culture supernatants of HUVECs. The β-actin protein level was considered as an internal control. (B) Cells stimulated with TCBQ for 5 min or 60 min, the levels of NLRP3 mRNA were detected by qPCR assay.

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Figure 9. TCBQ triggers the deubiquitination of NLRP3. (A) Cells were pretreated with 10 µM PR-619, 200 µM APO, 5 mM NAC or 500 µM Mito-TEMPO for 1 h, then, cells were treated with 20 µM of TCBQ for 5 min. Interaction between ubiquitin and NLRP3 was analyzed by co-immunoprecipitation assay. Cell lysates were immunoprecipitated with NLRP3 antibody, then, precipitated proteins were analyzed by SDS-PAGE with ubiquitin antibody. (B) Cells were pretreated with 10 µM PR-619 for 1 h, then, cells were treated with 20 µM of TCBQ for 1 h. Then, the secretions of IL-1β and caspase 1, the expressions of pro-caspase 1 and pro-IL-1β from HUVECs was detected by Western blotting assay. sup, desalinized culture supernatants of HUVECs. The β-actin protein level was considered as an internal control.

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Tetrachlorobenzoquinone Stimulates NLRP3 Inflammasome-Mediated

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