Tripartite-Motif Protein 30 Negatively Regulates NLRP3 Activation by Modulating Reactive Oxygen Species Production This information is current as of September 29, 2021. Yu Hu, Kairui Mao, Yan Zeng, Shuzhen Chen, Zhiyun Tao, Chen Yang, Shuhui Sun, Xiaodong Wu, Guangxun Meng and Bing Sun J Immunol 2010; 185:7699-7705; Prepublished online 3 November 2010; Downloaded from doi: 10.4049/jimmunol.1001099 http://www.jimmunol.org/content/185/12/7699 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/11/03/jimmunol.100109 Material 9.DC1 References This article cites 40 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/185/12/7699.full#ref-list-1

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Tripartite-Motif Protein 30 Negatively Regulates NLRP3 Inflammasome Activation by Modulating Reactive Oxygen Species Production

Yu Hu,*,1 Kairui Mao,*,1 Yan Zeng,† Shuzhen Chen,* Zhiyun Tao,* Chen Yang,* Shuhui Sun,‡ Xiaodong Wu,* Guangxun Meng,† and Bing Sun*,†

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is critical for -1 activation and the proteolytic pro- cessing of pro–IL-1b. However, the mechanism that regulates NLRP3 inflammasome activation remains unclear. In this paper, we demonstrate that tripartite-motif protein 30 (TRIM30) negatively regulates NLRP3 inflammasome activation. After stimulation with ATP, an agonist of the NLRP3 inflammasome, knockdown of TRIM30 enhanced caspase-1 activation and increased pro-

duction of IL-1b in both J774 cells and bone marrow-derived macrophages. Similarly with ATP, knockdown of TRIM30 increased Downloaded from caspase-1 activation and IL-1b production triggered by other NLRP3 inflammasome agonists, including nigericin, monosodium urate, and silica. Production of reactive oxygen species was increased in TRIM30 knockdown cells, and its increase was required for enhanced NLRP3 inflammasome activation, because antioxidant treatment blocked excess IL-1b production. Conversely, overexpression of TRIM30 attenuated reactive oxygen species production and NLRP3 inflammasome activation. Finally, in a crystal-induced NLRP3 inflammasome-dependent peritonitis model, monosodium urate-induced flux and IL-1b production was reduced significantly in TRIM30 transgenic mice as compared with that in their nontransgenic littermates. Taken http://www.jimmunol.org/ together, our results indicate that TRIM30 is a negative regulator of NLRP3 inflammasome activation and provide insights into the role of TRIM30 in maintaining inflammatory responses. The Journal of Immunology, 2010, 185: 7699–7705.

he innate immune system is the first line of defense against including TLRs (2–4), RIG-I–like receptors (5, 6), and NLRs pathogens. The response to pathogens (such as bacteria, (nucleotide-binding domain and leucine-rich repeat containing fungi, viruses, etc.) in humans depends upon the ability gene family) (7), are proteins used by nearly all organisms to

T by guest on September 29, 2021 to mount immune responses. Pattern recognition receptors (1), identify pathogen-associated molecular patterns. Once activated, the innate immune system initiates the inflammatory response by secreting and chemokines, inducing the expression of *Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science and adhesion and costimulatory molecules to recruit immune cells to †Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, the site of infection and to trigger the adaptive immune response. ‡ Institute Pasteur of Shanghai, Chinese Academy of Sciences; and Fudan University The central role of the NLR family in the immune system has School of Medicine, Shanghai, China become increasingly appreciated in recent years (8, 9). Upon ac- 1Y.H. and K.M. contributed equally to this work. tivation, NLRs, including NLRP1, NLRP3, and NLRC4, recruit Received for publication April 7, 2010. Accepted for publication October 3, 2010. adaptor ASC and effector caspase-1 to form large cytoplasmic This work was supported by grants from the National Natural Science Foundation of China complexes, defined as the inflammasome (10). The inflammasome (30623003, 30721065, 90713044, 30801011, 30870126, 31030029, and 30950002) and a Chinese Academy of Sciences Project (KSCX2-YW-R-161), grants from the 973 Key regulates the activation of caspase-1 and subsequent cleavage of Project (2006CB504300 and 2007CB512404), the 863 Key Project (2006AA02A247), the proinflammatory mediator, IL-1b, as well as IL-18 precursors grants from the National Science and Technology Major Project (2008ZX10002-014, 2008ZX10004-002, and 2009ZX10004-105), a grant from the National Ministry of Science into their functional forms, which then are released from the cell. and Technology (20072714), grants from the Technology Commission of Shanghai Mu- One of the best characterized inflammasomes is the NLRP3 in- nicipality (088014199, 08431903001, 08431903004, 2008ZX10206, and 08DZ2291703), flammasome. NLRP3-mediated inflammasome activation occurs a grant from the Sino-Germany Center on Severe Acute Respiratory Syndrome Project (GZ238(202/11)), and grants from Li Kha Shing Foundation, the European Union Project in response to diverse molecular entities, including bacteria (11), (SP5B-CT-2006-044161), and the E-institutes of Shanghai Universities Immunology Di- viruses (12, 13), fungi (14), components of dying cells (11), vision. crystal particles (15–18), and DNA (19). Address correspondence and reprint requests to Dr. Bing Sun, Laboratory of Molec- Normal activation of the NLRP3 inflammasome contributes to ular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai host defense. However, excessive activation of the NLRP3 sig- 200031, China. E-mail address: [email protected] naling pathway leads to the pathogenesis of a spectrum of auto- The online version of this article contains supplemental material. inflammatory diseases (20, 21). Mutations in the human NLRP3 Abbreviations used in this paper: BMDM, bone marrow-derived macrophage; CA, gene are associated with various autoinflammatory disorders, such C35A; CT, cycle threshold; DPI, diphenyleneiodonium; I:C, poly-IC; L+A, LPS as Muckle-Wells syndrome, familial cold autoinflammatory syn- +ATP; NAC, N-acetyl-L-cysteine; ND, not detected; NLRP3, NLR family, pyrin domain-containing 3; poly-IC, polyinosinic-polycytidylic acid; ROS, reactive oxygen drome, and chronic infantile neurologic cutaneous and articular species; SC, siCon; siCon, control siRNA; siRNA, short interfering RNA; siTRIM30, syndrome. The NLRP3 gene-targeted mice harboring mutations TRIM30 siRNA; ST, siTRIM30; Sup, supernatant; T30, TRIM30; TRIM30, tripartite- mimicking those causing disease in humans show very severe motif protein 30; WCL, whole-cell lysate; WT, wild-type. phenotypes, including delayed growth, increased mortality, and Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 development of severe cutaneous lesions associated with the ac- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001099 7700 INHIBITION OF NLRP3 INFLAMMASOME ACTIVATION BY TRIM30 cumulation of inflammation markers in these tissues (22, 23). murine leukemia virus reverse transcriptase (Invitrogen) were used for re- Therefore, NLRP3 inflammasome signaling must be regulated verse transcription of purified RNA. All of the gene transcripts were quan- 3 tightly to maintain immune balance. tified by real-time PCR with 2 SYBR Green qPCR Master Mix and a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, Previous work in our laboratory has demonstrated that tripartite- CA). The relative fold induction was calculated by the 22DDcycle threshold motif protein 30 (TRIM30) is involved in endotoxic tolerance (24). (CT) method. The sequences of primers for real-time PCR analysis are as Overexpression of TRIM30 in vivo increased the survival of mice follows: IL-1b, forward, 59-CAACTGCACTACAGGCTCCG-39, reverse, 9 9 a 9 after i.p. challenge with LPS. Moreover, the critical role of the 5 -CGACAGCACGAGGCTTTTTT-3 ; TNF- , forward, 5 -AGTGTCA- GAAGCTCCTGTGGC-39, reverse, 59-TGAGGCAGTATTCAAGCCTCC- NLRP3 inflammasome in LPS-induced endotoxic shock has been 39; IL-6, forward, 59-TTCCATCCAGTTGCCTTCTTG-39, reverse, 59-GA- documented by numerous reports in recent years (11, 25, 26). AGGCCGTGGTTGTCACC-39; IP-10, forward, 59-GGGCCAGTGAGAA- Mice with a deficiency in any component of the NLRP3 inflam- TGAGGG-39, reverse, 59-GCTCGCAGGGATGATTTCAA-39; IFN-b, for- masome, including NLRP3, ASC, or caspase-1, are resistant to ward, 59-CCACCACAGCCCTCTCCATCAACTAT-39, reverse, 59-CAAG- 9 b 9 LPS-induced endotoxic shock. Thus, we infer that TRIM30 might TGGAGAGCAGTTGAGGACATC-3 ; -actin, forward, 5 -GTACGACC- AGAGGCATACAGG-39, reverse, 59-GATGACGATATCGCTGCGCTG-39. be involved in the regulation of NLRP3 inflammasome activation. Knockdown of TRIM30 increases ATP-induced caspase-1 activa- ROS detection b tion and IL-1 production in both J774 cells and bone marrow- Intracellular ROS was measured with the ROS-specific fluorescent probe derived macrophages (BMDMs). More importantly, we show that CM-H2DCFDA (10 mM; Molecular Probes, Eugene, OR). J774 cells were TRIM30 negatively regulates the IL-1b secretion evoked by many loaded for 15 min with 10 mM CM-H2DCFDA, washed twice with PBS, other NLRP3 inflammasome agonists. Upon TRIM30 knockdown, and exposed to ATP for 30 min, nigericin for 30 min, or MSU for 3 h. The level of fluorescence was determined by flow cytometry. we observed an increase in reactive oxygen species (ROS) pro- Downloaded from duction triggered by LPS plus ATP. Inhibition of ROS by anti- Immunoblot analysis b oxidant treatment diminished the amplified IL-1 production upon For immunoblot analysis, whole-cell lysates were separated via SDS-PAGE, TRIM30 knockdown, suggesting that enhanced IL-1b production transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), hy- is mainly due to excess ROS production. Our results collectively bridized to various Abs, followed by HRP-labeled anti-mouse IgG (HAF demonstrate the role of TRIM30 in regulating the NLRP3-mediated 007; R&D Systems) or anti-rabbit IgG (4050-05; Southern Biotechnology Associates, Birmingham, AL), and visualized with SuperSignal West Pico inflammatory response. http://www.jimmunol.org/ Chemiluminescent Substrate (Pierce, Rockford, IL). For caspase-1 p10 detection, blots were incubated with rabbit polyclonal Ab to mouse caspase-1 p10 (sc-514; Santa Cruz Biotechnology, Santa Cruz, CA) and Materials and Methods visualized with SuperSignal West Dura Extended Duration Substrate Mouse strains and reagents (34075; Pierce). 2/2 C57BL/6, IL-1R1 mice were obtained from The Jackson Laboratory Retrovirus preparation and infection (Bar Harbor, ME). TRIM30 transgenic mice were generated as described previously (24); nontransgenic littermates served as controls for the trans- Retrovirus was prepared as reported previously (27). Briefly, the cDNAs of genic mice. All of the mice were maintained in specific pathogen-free fa- TRIM30 and ring dead mutation with substitution of the cysteine residue at cilities at the Care Facility of the Chinese Academy of Sciences. position 35 with alanine (C35A [CA]) of TRIM30 were cloned into MSCV- Animal care and use were in compliance with institutional guidelines. IRES-CD4 and transfected together with pCMV/VSV-G and pKF3-RSV/ by guest on September 29, 2021 All of the reagents were from Sigma-Aldrich (St. Louis, MO) unless Gag-Pol into 293T cells using Lipofectamine (Invitrogen). After 24 h, the stated otherwise. Polyinosinic-polycytidylic acid (poly-IC) and CpG were medium was replaced with DMEM containing 10% FBS. Forty-eight hours from Invivogen (San Diego, CA). Anti-NLRP3 was obtained from Alexis after transfection, the retroviral supernatants were harvested, supplemented (Lausen, Switzerland; 804-880-C100). Monosodium urate (MSU) was with 6 mg/ml polybrene, and used to infect J774 cells. Plates were centri- prepared as in a previous report (16). IL-1b, TNF-a, and IL-6 ELISA kits fuged at 2000 rpm for 90 min at room temperature. After 12 h of incubation were obtained from R&D Systems (Minneapolis, MN), and IL-18 ELISA at 37˚C, viruses were removed and cells were recultured in fresh medium. kits were obtained from MBL International (Woburn, MA). In vivo peritonitis model Cells Mice were injected i.p. with 1 mg MSU in 200 ml PBS; control mice re- J774 or HEK 293T cells were maintained in humidified 5% CO2 at 37˚C ceived PBS. Mice were sacrificed 6 h after injection. Peritoneal cells were in DMEM supplemented with 10% (v/v) FBS, penicillin (100 U/ml), and obtained with 10 ml PBS with 1% (v/v) FBS. Recovered fluid was pelleted streptomycin (100 U/ml). Lipofectamine (Invitrogen) was used for tran- by centrifugation, and total cells were counted. Subsequently, cells were sient transfection of HEK293T cells. stained for neutrophil surface marker Gr-1 and detected using flow cy- BMDMs were prepared as follows: bone marrow cells were flushed from tometry. The number of was calculated as total cells multiplied the femurs and tibias of C57BL/6 mice and subsequently depleted of by the percentage of Gr-1–positive cells. When IL-1b was measured, 0.6 red cells with ammonium chloride. Cells were cultured at 2 3 106 cells per ml PBS with 1% (v/v) FBS was used, and the concentration of IL-1b was well in 24-well plates in DMEM supplemented with 20 ng/ml murine determined in the supernatant of peritoneal lavage fluid. M-CSF. Nonadherent cells were removed carefully, and fresh medium was added every 2 d. On day 6, cells were collected for further experiments. Statistical analysis Short interfering RNA synthesis and transfection Data are presented as the mean 6 SD from three independent experiments. Statistical comparisons between different treatments were performed by Short interfering RNA (siRNA) synthesis and transfection were done as unpaired Student t test, where p , 0.05 was considered statistically sig- reported previously (24). nificant and p , 0.01 was highly significant. In vitro stimulation of macrophages Results J774 cells or BMDMs were stimulated as follows. Briefly, cells were Knockdown of endogenous TRIM30 enhances IL-1b secretion stimulated with TLR agonists and then pulsed with 5 mM ATP for 30 min, 20 mM nigericin for 30 min, 250 mg/ml MSU for 3 h, or 250 mg/ml silica and caspase-1 activation in J774 cells for 3 h. For pharmacological assessments, N-acetyl-L-cysteine (NAC; 10 On the basis of our previous work indicating that TRIM30 is capable mM) or diphenyleneiodonium (DPI; 12.5mM) was added to the cell culture 15 min before the end of LPS stimulation. of increasing the survival rate in mice suffering endotoxic shock (24), we wonder whether TRIM30 is able to modulate NLRP3 inflam- Reverse transcription and quantitative real-time PCR masome activation. To address this question, a mouse macrophage Total RNAwas extracted from J774 cells with TRIzol (Invitrogen) according cell line, J774, was selected, and the effect of TRIM30 on NLRP3 to the manufacturer’s instructions. Oligo(dT) priming and M-Moloney inflammasome activation was tested. Initially, we detected NLRP3 The Journal of Immunology 7701 inflammasome activation in J774 cells treated with LPS priming from the enhanced mRNA level of pro-IL-1b; thus, we detected the and after ATP stimulation by measuring caspase-1 activation and mRNA level of the cytokines. The data showed that knockdown of IL-1b secretion. As shown in Fig. 1A, ATP induced caspase-1 ac- TRIM30 slightly increased the levels of IL-1b mRNA by LPS sti- tivation (determined by immunoblotting of the p10 subunit of ma- mulation (∼1.5times)buthadnoinfluenceonthelevelsofTNF-a, ture caspase-1) and IL-1b secretion in LPS-primed J774 cells. We IL-6, IP-10, and IFN-b (Fig. 1D). However, we did observe the great noticed that priming of macrophages with LPS for 4–6 h induced enhancement of caspase-1 activation and much more significant the highest levels of caspase-1 activation and IL-1b secretion. As secretion of IL-1b protein (∼4 times) when TRIM30 was silenced, recently reported (28), the cells became gradually refractory to ATP suggesting that IL-1b production was enhanced largely at the post- stimulation when the time of LPS priming was extended (Fig. 1A). transcriptional level in TRIM30 knockdown cells. These data in- It was interesting to observe that the expression of TRIM30 reached dicate that knockdown of TRIM30 enhances IL-1b secretion the highest level at 6–8 h after LPS priming, the time that the through the regulation of caspase-1–mediated pro-IL-1b processing. caspase-1 activation began to decrease. Thus, we assumed that the b enhanced TRIM30 expression might result in the depression of Knockdown of TRIM30 enhances IL-1 secretion and NLRP3 inflammasome activation. To test this hypothesis, we de- caspase-1 activation in BMDMs pleted endogenous TRIM30 expression in J774 cells by TRIM30- We next address the physiological role of TRIM30 in primary cells. specific siRNA. As expected, TRIM30 siRNA (siTRIM30) re- BMDMs were transfected with the indicated siRNA, and then IL-1b duced TRIM30 protein efficiently as compared with control siRNA secretion and caspase-1 activation were assessed after stimulation. (siCon). Meanwhile, caspase-1 activation was enhanced greatly In response to LPS plus ATP stimulation, IL-1b and IL-18 secretion after LPS plus ATP stimulation (Fig. 1B), indicating that TRIM30 were increased significantly in TRIM30 knockdown BMDMs as Downloaded from negatively regulated NLRP3 inflammasome activation. To further compared with that in control cells (Fig. 2A). In contrast, the pro- confirm this, IL-1b secretion was measured in J774 cells in which duction of TNF-a and IL-6 was not altered (Fig. 2B). As expected, TRIM30 had been silenced. The results showed that in response caspase-1 activation was enhanced in TRIM30 knockdown cells to LPS plus ATP secretion of IL-1b was increased significantly in after LPS plus ATP stimulation (Fig. 2C). Collectively, we dem- the siTRIM30-transfected cells as compared with that in siCon- onstrate that the knockdown of TRIM30 enhances NLRP3 in-

transfected cells. In contrast, production of TNF-a and IL-6 was not flammasome activation in both J774 cells and primary BMDMs. http://www.jimmunol.org/ altered (Fig. 1C). Also, NLRP3, a LPS-inducible protein and lim- b iting factor for robust inflammasome activation (29), was not altered TRIM30 knockdown enhances IL-1 secretion and caspase-1 in the siTRIM30-transfected cells as compared with that in siCon- activation induced by various stimuli transfected cells (Fig. 1B), implying that knockdown of TRIM30 It is well documented that the production of mature IL-1b requires did not interfere with LPS-induced NF-kB activation under these two distinct signals (30, 31), TLR priming, and inflammasome experimental conditions. Increased secretion of IL-1b may result activation. We have demonstrated that TRIM30 is induced by by guest on September 29, 2021

FIGURE 1. Knockdown of TRIM30 enhances IL-1b secretion in response to LPS plus ATP in J774 cells. J774 cells were pretreated with or without LPS (1 mg/ml) for the indicated time period (A)or6h(B, C) and then were stimulated further with medium or 5 mM ATP for 30 min (A–C). A, ELISA of IL-1b secretion in supernatant and immunoblot analysis of whole-cell lysates with anti–caspase-1, anti-TRIM30, or anti–b-actin (loading control). B, Immunoblot analysis with anti–caspase-1, anti-TRIM30, anti-NLRP3, or anti–b-actin of the whole-cell lysate (WCL) and supernatant (Sup) of J774 cells transfected with siCon or siTRIM30 after stimulation. C, ELISA of IL-1b, TNF-a, and IL-6 in supernatant of J774 cells transfected with siCon or siTRIM30 after stimulation. D, siCon- or siTRIM30-transfected J774 cells were treated with LPS (1 mg/ml) for 4 h. Expression levels of IL-1b, TNF-a, IL-6, IP-10, and IFN-b were quantified by real-time PCR. The relative fold induction was calculated by the 22DDCT method. Data are representative of three experiments. Error bars indicate mean 6 SD between duplicates. Statistical significance was determined by the Student t test. *p , 0.05. L+A, LPS+ATP; ND, not detected; SC, siCon; ST, siTRIM30. 7702 INHIBITION OF NLRP3 INFLAMMASOME ACTIVATION BY TRIM30

TRIM30 has no interaction with the components of the NLRP3 inflammasome Because TRIM30 was seen to be a negative regulator of NLRP3 inflammasome activation, we then investigated its mechanism. Various proteins that interfere with inflammasome assembly and the processing of pro-IL-1b have been identified (32–34). They function as endogenous dominant negative proteins by preventing the recruitment of ASC or caspase-1 into the inflammasome. Therefore, we assumed that TRIM30 might regulate NLRP3 inflammasome activation by interfering with NLRP3 inflamma- some assembly. To test this hypothesis, a coimmunoprecipita- tion assay was performed. As shown in Supplemental Fig. 1A, TRIM30 did not interact with either ASC or caspase-1. As a FIGURE 2. TRIM30 knockdown enhances IL-1b secretion in response positive control, TAK1 was able to coimmunoprecipitate with to LPS plus ATP in BMDMs. BMDMs were transfected with siCon or TRIM30. In addition, we noted that TRIM30 did not interact with siTRIM30, pretreated with LPS for 6 h, and then pulsed with medium or NLRP3 or NLRC4 (Supplemental Fig. 1B). These data indicate ATP for 30min. A and B, ELISA of IL-1b and IL-18 (A) and TNF-a and that TRIM30 does not regulate NLRP3 inflammasome activation IL-6 (B) in supernatant. C, Immunoblot analysis of WCL and Sup of by blocking the interaction between components of the NLRP3 b Downloaded from BMDMs with anti–caspase-1, anti-TRIM30, or anti– -actin. Data are inflammasome. representative of three experiments. Error bars indicate mean 6 SD be- tween duplicates. Statistical significance was determined by the Student t TRIM30 regulates NLRP3 inflammasome activation by , test. *p 0.05. modulating ROS production ROS is believed to be a common NLRP3 activator (9). It is rea- LPS, CpG, and poly-IC stimulation (24). Thus, we tested the sonable to ask whether TRIM30 influences ROS production, potential role of TRIM30 in IL-1b secretion triggered by different which in turn regulates NLRP3 inflammasome activation. We first http://www.jimmunol.org/ TLR agonists plus ATP. As seen with LPS plus ATP stimulation, measured the level of ROS production in macrophages transfected IL-1b secretion and caspase-1 activation in response to CpG plus with siCon or siTRIM30 by loading with CM-H2DCFDA. As ATP stimulation were enhanced under TRIM30 knockdown con- predicted, ROS fluorescence was increased more significantly ditions (Fig. 3A). However, no IL-1b secretion or caspase-1 ac- after LPS plus ATP stimulation in TRIM30 knockdown cells than tivation was detected after poly-IC plus ATP stimulation (Fig. 3A). that of the controls (Fig. 4A). Moreover, TRIM30 knockdown Next, we determined whether TRIM30 had regulatory effects potentiated ROS accumulation by MSU and nigericin (Supple- on other NLRP3 inflammasome agonists. As shown in Fig. 3B, mental Fig. 2). In contrast, the levels of ROS with no stimulation b remained unchanged in the same conditions (Fig. 4A). It was re- similar to ATP, a significant increase in IL-1 secretion and by guest on September 29, 2021 caspase-1 activation was observed after LPS plus MSU, silica, or ported that enhanced ROS production results in elevated IL-1b nigericin stimulation. These data demonstrate that TRIM30 is production (35). To further address whether ROS was involved in a general regulator of NLRP3 inflammasome activation, which may target a common signaling pathway required for NLRP3 inflammasome activation.

FIGURE 3. Knockdown of TRIM30 enhances IL-1b secretion induced by various stimuli. A, ELISA of IL-1b in supernatant and immunoblot FIGURE 4. Knockdown of TRIM30 increases levels of cellular ROS. A, analysis of cell lysates with anti–caspase-1 of J774 cells transfected with Levels of ROS in siCon- or siTRIM30-transfected J774 cells were ana- siCon or siTRIM30, pretreated with LPS (1 mg/ml), CpG (10 mg/ml), or lyzed by CM-H2DCFDA labeling. Data in bar graphs (right panel) rep- poly-IC (25 mg/ml) for 6 h, and then stimulated with 5 mM ATP for resent mean 6 SD of the percentage of CM-H2DCFDA–positive cells. B 30 min. B, ELISA of IL-1b in supernatant and immunoblot analysis of and C, ELISA of IL-1b and TNF-a in supernatant of J774 cells transfected cell lysates with anti–caspase-1 of J774 cells transfected with siCon or with siCon or siTRIM30, pretreated with LPS (1 mg/ml) for 6 h, and then siTRIM30, pretreated with LPS (1 mg/ml) for 6 h, and then stimulated with stimulated with 5 mM ATP for 30 min. Cells were pretreated with or 5 mM ATP or 20 mM nigericin for 30 min or 250 mg/ml MSU or 250 mg/ without 10 mM NAC (B) or 12.5 mM DPI (C) for 15 min before ATP ml silica for 3 h. Data are representative of three experiments. Error bars stimulation. Data are representative of at least two experiments. Error bars indicate mean 6 SD between duplicates. Statistical significance was de- indicate mean 6 SD between duplicates. Statistical significance was de- termined by the Student t test. *p , 0.05. I:C, poly-IC. termined by the Student t test. *p , 0.05. The Journal of Immunology 7703 Downloaded from

FIGURE 5. Overexpression of TRIM30 in vitro attenuates ROS production and NLRP3 inflammasome activation. A, Schematics of retroviral vector construction. B–D, J774 cells were transduced with either control (CD4) or TRIM30-expressing retrovirus, pretreated with medium or LPS (1 mg/ml) for 1

or 2 h, and then stimulated with 5 mM ATP for 30 min. B, Levels of ROS were analyzed by CM-H2DCFDA labeling. C, Immunoblot analysis of whole-cell http://www.jimmunol.org/ lysates of J774 cells with anti–caspase-1, anti-TRIM30, anti-NLRP3, or anti–b-actin. D, ELISA of IL-1b, TNF-a, and IL-6 in supernatant. E–G, J774 cells were transduced with a retrovirus expressing either control (CD4), TRIM30, or TRIM30 (CA), pretreated with medium or LPS (1 mg/ml) for 2 h, and then stimulated with 5 mM ATP for 30 min. E, Immunoblot analysis of whole-cell lysates of J774 cells with anti–caspase-1, anti-TRIM30, and anti–b-actin. F, ELISA of IL-1b in supernatants. G, Levels of ROS were analyzed by CM-H2DCFDA labeling. Data are representative of three experiments. Error bars indicate mean 6 SD between duplicates. Statistical significance was determined by the Student t test. *p , 0.05. T30, TRIM30; CA, C35A. elevated IL-1b production, siCon- or siTRIM30-transfected J774 dead mutation with substitution of the cysteine residue at position cells were treated with an antioxidant, NAC, before ATP stimu- 35 with alanine (CA) of TRIM30 that had no NF-kB inhibitory lation. As shown in Fig. 4B, knockdown of TRIM30 caused a ability (24). As shown in Fig. 5E and 5F, overexpression of by guest on September 29, 2021 marked increase in IL-1b secretion, and this effect was abolished TRIM30 (CA) resulted in a significant reduction of NLRP3 when ROS production was disrupted by NAC treatment. Mean- inflammasome-mediated caspase-1 activation and IL-1b secretion, while, TNF-a production was not affected by NAC treatment (Fig. which was efficient as overexpression of TRIM30. In contrast, 4B). Similarly, the treatment of macrophages with another anti- overexpression of TRIM30 (CA) did not inhibit the production of oxidant, DPI, reduced the difference in IL-1b secretion between TNF-a and IL-6 (data not shown). These data indicated that the siCon and siTRIM30 groups (Fig. 4C). These data demonstrate ring domain of TRIM30 was not essential for the inhibitory ef- that ROS are required for the enhancement of IL-1b secretion in fect of TRIM30 on NLRP3 inflammasome activation. Also, over- TRIM30-silenced macrophages. Taken together, knockdown of expression of TRIM30 (CA) significantly inhibited ROS production TRIM30 increases the level of ROS, which promotes NLRP3 (Fig. 5G), which suggested that ROS but not NF-kB should be the inflammasome activation.

Overexpression of TRIM30 in vitro attenuates ROS production and NLRP3 inflammasome activation Because knockdown of TRIM30 enhanced NLRP3 inflammasome activation, it was assumed that overexpression of TRIM30 would reciprocally reduce NLRP3 inflammasome activation. To examine this possibility, we constructed a bicistronic TRIM30-expressing retrovirus (Fig. 5A) and infected J774 cells. Transduction of TRIM30 dramatically increased the expression of TRIM30 pro- tein (Fig. 5C) and concomitantly led to a decrease in the level of ROS production (Fig. 5B). As a result, overexpression of TRIM30 FIGURE 6. Overexpression of TRIM30 in vivo inhibits MSU-induced resulted in a significant reduction of NLRP3 inflammasome- acute inflammation. A, Neutrophil influx in peritoneal lavage fluid from 2/2 mediated caspase-1 activation (Fig. 5C) and subsequent IL-1b wild-type (WT) and IL-1R1 mice 6 h after i.p. challenge with MSU; secretion (Fig. 5D). Meanwhile, the expression level of NLRP3 mice challenged with PBS served as negative controls (n = 4 for each group). B and C, Neutrophil influx (B) and IL-1b concentration (C)in was not altered by overexpression of TRIM30 (Fig. 5C). These peritoneal lavage fluid from littermates and TRIM30 transgenic mice 6 h results indicate that the upregulated TRIM30 is able to inhibit after i.p. challenge with MSU; mice challenged with PBS served as neg- the outcome of NLRP3 inflammasome activation. However, over- ative controls. (PBS n = 4 for each group; MSU n = 6–8 for each group). expression of TRIM30 also reduced the production of TNF-a and Data are representative of at least two experiments. Error bars indicate IL-6 (Fig. 5D). To separate the inhibition of NF-kB and inhibition mean 6 SD between duplicates. Statistical significance was determined by of the NLRP3 inflammasome by TRIM30, we used a ring domain the Student t test. *p , 0.05; **p , 0.01. 7704 INHIBITION OF NLRP3 INFLAMMASOME ACTIVATION BY TRIM30 major target for the regulation of the NLRP3 inflammasome by Our data showed that TRIM30 was a general modulator of TRIM30. Collectively, these data indicate that overexpression of NLRP3 inflammasome activation; therefore, TRIM30 probably TRIM30 inhibits NLRP3 inflammasome activation by attenuating targets a common signaling pathway required for NLRP3 in- ROS production. flammasome activation. Various proteins have been reported to inhibit caspase-1 activation by interfering with inflammasome TRIM30 negatively regulates activation of the NLRP3 assembly. For instance, a family of small proteins that are com- inflammasome in vivo posed of either a CARD or a PYD only emerged as important To examine whether TRIM30 is involved in the inflammatory inflammasome regulators. These CARD-only proteins and PYD- response in vivo, we used the MSU-induced peritoneal inflam- only proteins function as endogenous dominant negative proteins matory model, which is NLRP3 inflammasome-dependent (16, 36). by preventing the recruitment of ASC or caspase-1 into the in- MSU crystals trigger acute inflammation with the hallmark of an flammasome (32). The SPRY–CARD domain interaction medi- infiltration of neutrophils at the site of crystal deposition in vivo ates the association of several TRIM proteins with their partners, (37). We injected MSU into the peritoneal cavity of IL-1R1– for example, association of TRIM20 (pyrin) with caspase-1 or deficient or wild-type mice and evaluated the acute inflammatory TRIM25 with RIG-I (34, 38). Therefore, it appears possible that response. Consistent with previous studies, MSU injection caused TRIM30 may regulate inflammasome activation by means of in- a marked infiltration of neutrophils into the peritoneal cavity, terfering inflammasome assembly. However, we found no asso- whereas the infiltration was impaired severely in IL-1R1–deficient ciation between TRIM30 and ASC or caspase-1, regardless of the mice (Fig. 6A), indicating that NLRP3 inflammasome-mediated existence of SPRY (TRIM30) and CARD (ASC and caspase-1)

IL-1b production is a prerequisite for MSU-induced acute in- domains (Supplemental Fig. 1). This led to looking for additional Downloaded from flammation. Therefore, we next determined the role of TRIM30 in mechanisms by which the TRIM30 might regulate NLRP3 in- the inflammatory response to MSU. Compared with nontransge- flammasome activation. nic littermates, TRIM30 transgenic mice experienced a significant ROS are believed to be a common NLRP3 activator (9). In our reduction in neutrophil influx (Fig. 6B) and IL-1b production (Fig. experiments, we found that ROS production was enhanced upon 6C) induced by MSU injection. Similar to MSU, a reduction of the knockdown of TRIM30. The antioxidant treatment blocked excess

neutrophil influx was observed in TRIM30 transgenic mice after silica IL-1b secretion induced by knockdown of TRIM30 (Fig. 4B,4C), http://www.jimmunol.org/ injection (Supplemental Fig. 3). These results indicate that TRIM30 indicating that enhanced ROS production was required for the negatively regulates NLRP3 inflammation activation in vivo. amplification of NLRP3 inflammasome activation. Conversely, overexpression of TRIM30 inhibited ROS production, leading to Discussion impaired IL-1b secretion and caspase-1 activation. Our data col- Our data demonstrate the negative role of TRIM30 in the regulation lectively suggested that TRIM30 negatively regulated NLRP3 of NLRP3 inflammasome activation in vitro and in vivo. Knock- inflammasome activation by modulating ROS production. How- down of endogenous TRIM30 increased NLRP3 inflammasome- ever, the mechanism for the regulation of ROS production by mediated caspase-1 activation and subsequent production of IL- TRIM30 remains unknown because the source of ROS for in- 1b and IL-18, which result from the excess production of ROS. flammasome activation is still uncharacterized (39). ROS can be by guest on September 29, 2021 Conversely, overexpression of TRIM30 attenuated ROS produc- generated in several sources, including mitochondria, NADPH oxi- tion and NLRP3 inflammasome activation. We further exploited the dase, 5-lipoxygenase, and other (40), whereas the specific function of TRIM30 using transgenic mice and showed that over- role for each source in NLRP3 inflammasome activation is unclear. expression of TRIM30 in vivo inhibited the NLRP3-mediated in- It has been reported that the absence of autophagy results in en- flammatory response. hanced ROS production, which in turn amplifies caspase-1 acti- In this study, the production of TNF-a and IL-6 remains un- vation (35). Due to the lysosomal localization of TRIM30 and the changed in TRIM30 knockdown cells compared with that in con- dominant role of the lysosome in autophagosome maturation, the trol cells by a single LPS stimulation. This is consistent with regulation of TRIM30 based on ROS production may be related to the previous work in our laboratory. Shi et al. (24) found that the autophagy likewise. difference between TRIM30 knockdown and control cells in the In conclusion, our study demonstrates that TRIM30 is induced production of TNF-a was only detected by secondary stimulation by TLR stimulation and in turn can restrict NLRP3 inflamma- with LPS. Moreover, previous work in our laboratory found that some activation. These findings may be helpful in understanding the ring domain of TRIM30 was required for TRIM30-mediated the precise regulation of NLRP3 inflammation activation and ad- degradation of TAB2/3 and inhibition of NF-kB. Thus, we tested vancing the development of drugs for inflammatory diseases. the involvement of the ring-mediated function of TRIM30 in NLRP3 inflammasome activation by using a ring dead mutation Acknowledgments (CA) of TRIM30 with substitution of the cysteine residue at po- We thank Prof. Ju¨rg Tschopp, Genhong Cheng, Youhai H. Chen, and Chen sition 35 of the ring domain with alanine (24). Our data showed Wang for helpful comments on this paper and Dr. Sheri Skinner and that overexpression of TRIM30 (CA) decreased NLRP3 inflam- Dangsheng Li for reviewing the manuscript. masome-mediated caspase-1 activation and IL-1b secretion, which was similar with overexpression of TRIM30 (Fig. 5E,5F). Also, overexpression of TRIM30 (CA) significantly inhibited ROS pro- Disclosures duction (Fig. 5G), suggesting that ROS but not NF-kB should be the The authors have no financial conflicts of interest. major target for the regulation of the NLRP3 inflammasome by TRIM30.However,thelevelofIL-1b mRNA was increased slightly References in TRIM30 knockdown cells after LPS stimulation (Fig. 1D). The 1. Palm, N. W., and R. Medzhitov. 2009. Pattern recognition receptors and control unchanged expression of TNF-a andIL-6(inbothmRNAand of adaptive immunity. Immunol. Rev. 227: 221–233. protein levels, Fig. 1C,1D) in TRIM30 knockdown cells implied 2. O’Neill, L. A., and A. G. Bowie. 2007. The family of five: TIR-domain- containing adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7: that there may be an unknown mechanism for regulating the level of 353–364. IL-1b mRNA besides NF-kB activation. 3. Kawai, T., and S. Akira. 2007. TLR signaling. Semin. Immunol. 19: 24–32. The Journal of Immunology 7705

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