Polychlorinated biphenyl quinone induces caspase 1-mediated

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Polychlorinated biphenyl quinone induces caspase 1mediated pyroptosis through the induction of proinflammatory HMGB1-TLR4-NLRP3-GSDMD signal axis Wenjing Dong, Qiushuang Zhu, Bingwei Yang, Qi Qin, Yawen Wang, Xiaomin Xia, Xiaokang Zhu, Zixuan Liu, Erqun Song, and Yang Song Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00376 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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Chemical Research in Toxicology

Polychlorinated biphenyl quinone induces caspase 1-mediated pyroptosis through the induction of pro-inflammatory HMGB1-TLR4-NLRP3-GSDMD signal axis Wenjing Dong, Qiushuang Zhu, Bingwei Yang, Qi Qin, Yawen Wang, Xiaomin Xia, Xiaokang Zhu, Zixuan Liu, 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: [email protected]

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Table of Content


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ABSTRACT Polychlorinated biphenyls (PCBs) are one of the most refractory environmental pollutants. Because of their ubiquitous existence in the biological systems (including human body), it is important to investigate their toxic behavior. Our previous findings demonstrated that a high reactive metabolite of PCB, namely PCB29-pQ, causes several programmed cell death (PCD), such as intrinsic/extrinsic apoptosis and autophagic cell death. The mechanistic study suggested the toxic actions of PCB29-pQ is largely related to its reactive oxygen species (ROS)-generation ability. Pyroptosis is a caspase 1-mediated pro-inflammatory PCD which was discovered recently. The aim of this study is to seek the linkage between pyroptosis and PCB29-pQ exposures. We first confirmed that PCB29-pQ stimulates Hela cells to produce excess amounts of ROS. Then, we found PCB29-pQ activates NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome that mediates caspase 1 activation. The activated caspase 1 (cleaved caspase 1) promotes gasdermin D (GSDMD) cleavage and translocation, which facilitates the release of intracellular inflammatory substances by forming membrane hole, ultimately leading cells to pyroptosis. PCB29-pQ-induced high-mobility group box 1 (HMGB1) release and subsequent binding to its receptors [toll like receptor 2 (TLR2), TLR4, TLR9 and receptor for advanced glycation end products (RAGE)] are essential for the activation of NLRP3 inflammasome. The current study revealed pyroptosis as a new death mode induced by PCB29-pQ, which enrich the understanding of PCBs-induced toxicity and help to prevent the toxic effects of residual PCBs in the environment. KEYWORDS: PCBs; pyroptosis; HMGB1; TLR4; NLRP3 inflammasome



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INTRODUCTION Polychlorinated biphenyls (PCBs) are a large group of persistent industrial contaminants, which was banned in 1970s. However, due to their stable and migratory properties, large quantities of PCB residues are still globally distributed. Accumulating investigations confirmed the adverse effects of PCBs on human beings, such as immunotoxicity,1 neurotoxicity,2 reproductive dysfunction3 and potential cancer risks.4 A recent study discovered hundreds of unrecognized halogenated contaminants in polar bear serum, including PCB sulfates (SO4-PCBs) and hydroxyl PCB sulfates (HO-SO4-PCBs).5 Some of the metabolites were reported for the first time either in polar bears or in any species. These new finding of PCB metabolites implied an underestimated PCB exposure and corresponding toxic risks. Pyroptosis is an inflammatory form of program cell death (PCD).6 Pyroptosis-stimulated pro-inflammatory caspases, i.e., caspase 1, 4, 5 and 11, contribute significantly to immune defense. Caspase 1, 4, 5 and 11 mediate gasdermin D (GSDMD) cleavage and interleukin-1beta (IL-1β, the key inflammatory cytokine) maturation. GSDMD was cleaved into gasdermin-N domain (GSDMD-NT) and gasdermin-C domain (GSDMD-CT). The GSDMD-NT executes pyroptosis via its pore-forming activity, which requires for IL-1β secretion. Excessive secretion of IL-1β (and its family member IL-18) cause widespread tissue damage and even serious acute and chronic inflammatory human diseases.7 The effect of caspase family proteins on pyroptosis have been extensively summarized.8-10 Caspase 1 is activated by a variety of canonical inflammatory ligands, e.g., NOD-like receptor pyrin domain-containing 3 (NLRP3)/apoptosis associated speck like protein containing a CARD (ASC),11 whereas caspase 4, 5 and 11 are directly activate by bacterial lipopolysaccharide via a


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noncanonical pathway.12 Interestingly, all above mentioned caspase family proteins activate pyroptosis. Due to its distinctive feature, NLRP3 is the most versatile and clinically significant inflammasome.7 ASC, an adaptor protein of NLRP3, comprises a caspase recruitment domain and a pyrin domain.13 Upon activation, NLRP3 associates with ASC and caspase 1 to form an inflammasome complex, which cleave pro-IL-1β to mature IL-1β and eventually induce pyroptosis.14 It has been proved that reactive oxygen species (ROS) is one of the trigger factor for NLRP3 activation.15 High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern molecule (DAMP), which can be actively secreted by immune cells or passively secreted by damaged or dead cells to act as an extracellular signal. HMGB1 binds to its receptors [such as toll like receptors (TLR) and receptor for advanced glycation end products (RAGE)] and functions as a proinflammatory mediator which contributes importantly to the pathogenesis of inflammatory disease. HMGB1/TLRs complex stimulate inflammasome formation governed by NLRP3 and ASC, and active caspase 1-mediated pyroptosis. ROS are one of the important mediators in the controlling of HMGB1 release. On the contrary, exogenous HMGB1 could be involved in a synergistic cycle of enhanced ROS generation.16 These reports demonstrated that ROS and HMGB1 are mutual upstream molecules and therefore promote the occurrence of pyroptosis in oxidative stressed, pro-inflammatory occasions. Our previous studies illustrated that PCB29-pQ brings various detrimental effects mediated by ROS, such as DNA damage,17-19 endoplasmic reticulum (ER) stress20, apoptosis21 and autophagy.22 However, to prevent the toxic effects caused by PCB29-pQ, other adaptive responses may apply and need further investigation. In this study, PCB29-pQ was proved to induce pyroptosis in Hela cells and ROS induces activation of NLRP3 inflammatory response, NLRP3 inflammasome activates



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caspase 1 and induces pyroptosis in Hela cells. Upon PCB29-pQ stimulation, caspase 1 cleaved GSDMD to GSDMD-NT and GSDMD-CT. The GSDMD-NT form cell membrane pores to disrupt cell membrane integrity. Consequently, the binding of extracellular HMGB1 to TLR4 receptor on the cell membrane further activates the NLRP3 inflammasome, which ultimately exacerbates cell pyroptosis. These results indicate that targeting oxidative stress and inflammasome may represent new therapeutic strategies to limit PCB-induced pyroptotic cell death.

MATEIALS AND METHODS Reagents 2, 3, 5-Trichloro-6-phenyl-[1, 4]-benzoquinone (PCB29-pQ) was synthesized as previously described.23 Cell counting kit 8 (CCK-8) was purchased from Genview (Shanghai, China). Propidium iodide (PI) was purchased from Solarbio (Beijing, China). Annexin V-FITC/PI apoptosis analysis kit, TLR2, TLR4 and TLR9 primary antibodies were purchased from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). RAGE, NLRP3 and ASC primary antibodies were obtained from Wanlei bio. (Shenyang, China). Lactate dehydrogenase (LDH) assay kit-WST® was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Ac-YVAD-CMK and Z-VAD-FMK were both supplied by MedChemExpress (Shanghai, China). N-Acetyl cysteine (NAC) and 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). HMGB1, caspase 1, IL-1β and GSDMD primary antibodies were supplied by Santa

Cruz

Biotechnology

(USA).

β-Actin

primary

antibody

and

goat

anti-rabbit

IgG-HRP-conjugated secondary antibody were obtained from Sangon Biotech Co. Ltd (Shanghai, China). Other chemicals were of the highest grade commercially available and used without further


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purification. Cell culture Hela cells, purchased from Army Medical University (Chongqing, China), were cultured in DMEM (Gibco Invitrogen Co., USA) joined with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml). Cells were incubated at 37℃ in a humidified atmosphere containing 5% CO2. Viability assay The cell viability was detected by CCK-8 assay. Hela cells (104 cell/ well) were seeded onto 96-well plates, cultured on 37℃ for 24 h. Then, cells were exposed to PCB29-pQ, Ac-YVAD-CMK, Z-VAD-FMK or NAC for an additional 24 hours. CCK-8 was added to each well and a microplate reader (ELx800, BioTek Instruments Inc., USA) was used to detect the absorbance value at 450 nm. A ratio to the control was used to express the cell viability. ROS detection ROS released from Hela cells was detected by a ROS-specific fluorescent dye DCFH-DA. The cells were treated with PCB29-pQ or NAC for 24 h and collected to incubate with DCFH-DA-containing (10 μM) medium at 37°C for 20 min. The intensity of fluorescence was detected by flow cytometry. Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay Hela cells were seed to 48-well plates for 24 h and incubated with PCB29-pQ for another 24 h. Cells were washed in PBS for three times. TUNEL assay was performed according to the manufacturer’s instructions. The fluorescent images were obtained by a reversed fluorescent microscope (OLYMPUS IX71).



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LDH release assay Hela cells were seeded onto 6-well plate and cultured for 24 hours to adhere. PCB29-pQ, Ac-YVAD-CMK, Z-VAD-FMK or NAC were added to each well and incubated at 37℃ for 24 h. The supernatants of each well were collected to detect the absorbance at 450 nm by the microplate reader. PI staining Hela cells treated with PCB29-pQ, NAC, Ac-YVAD-CMK or Z-VAD-FMK were collected to incubate with PI (10 nM) for 20 min. The fluorescence intensities were detected by flow cytometry. The red fluorescent images of PI staining were captured with inverted fluorescence microscope. Caspase 1/PI double staining assay Caspase 1/PI double staining assay was used to detect the rate of pyroptosis. Hela cells pretreated with PCB29-pQ were collected, and incubated with 50 nM PI for 15 min in the darkness. After washing for three times, caspase 1 antibody was further introduced. The percentage of caspase 1/PI double-positive cells were determined by flow cytometry. RNA interference Scramble siRNA and the siRNAs targeting GSDMD, NLRP3 and TLR4 were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai) and their sequences were listed in Table 1. To knockdown the endogenous GSDMD, NLRP3 and TLR4, Hela cells were transfected with corresponding siRNAs for 48 h at a final concentration of 50 nM and scramble siRNA was used as control. Western blotting The pretreated Hela cells were lysed by RIPA buffer and protease inhibitor before use.


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Supernatants of the pretreated cells were collected, concentrated and precipitated by CCl3/CH3OH v/v= (1:4). Membrane and cytosol protein extraction kit was used to collect the membrane protein. BCA protein assay kit was used to quantify the protein concentrations. The proteins were resolved with SDS-PAGE and transferred to nitrocellulose membrane. The 5% non-fat milk in Tris-buffered saline/Tween 20 (TBST) was supplied to block these membranes, and the membranes were probed with primary antibody over night at 4℃. After washing membranes three times with TBST, the membranes were incubated at 37℃ for 1.5 h with the HRP-conjugated secondary antibody. After the final washes, the densitometric analysis of immunoblots was performed by ImageJ software. Immunofluorescence staining Hela cells were seeded onto confocal plates and treated with PCB29-pQ for 24 h. After washing three times with PBS, cells were fixed and ruptured with 4% paraformaldehyde containing 3% sucrose for 20 min. The 10% non-fat milk in PBS was used to block cells, and primary antibodies were added to these cells over night at 4℃, then, the cells were incubated with Alexa Fluor 488 (1:250 dilution) for 2 h at room temperature. Besides, nuclei were stained with DAPI (5 μg/mL) for 10 min. After washing five times with PBS, cells were analyzed using a confocal microscope (Nikon, N-SIM E). Live cell imaging The cell death process was videoed by live cell imaging system (Olympus, Japan). Hela cells were seeded on glass-bottomed confocal plates and treated with PCB29-pQ (10 μM). The plates were placed on the workstation and the cell morphology was observed under a bright field for 24 h. The video was representative of at least three randomly selected fields. Statistical analysis



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All data were expressed as mean ± standard deviations (SD). P < 0.05 was considered statistically significant. The statistical significance of the differences was evaluated by a one-way ANOVA and followed by least significance difference (LSD) multiple comparison tests.

RESULTS AND DISCUSSION PCB29-pQ induces cytotoxicity of Hela cells through ROS-dependent manner Hela cells were exposed to PCB29-pQ at different concentrations to investigate its cytotoxicity. As CCK-8 assay indicated, exposure to PCB29-pQ led to gradual decreases in cell viability, P

Polychlorinated biphenyl quinone induces caspase 1-mediated

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