Tranilast directly targets NLRP 3 to treat inflammasome‐driven diseases

Research Article12 March 2018Open Access Source DataTransparent process Tranilast directly targets NLRP3 to treat inflammasome-driven diseases Yi Huang Yi Huang Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, China Search for more papers by this author Hua Jiang Hua Jiang Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Search for more papers by this author Yun Chen Yun Chen State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China Search for more papers by this author Xiaqiong Wang Xiaqiong Wang Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Search for more papers by this author Yanqing Yang Yanqing Yang Department of Clinical Laboratory, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China Search for more papers by this author Jinhui Tao Jinhui Tao Department of Rheumatology & Immunology, The First Affiliated Hospital of University of Science and Technology of China, Hefei, Anhui, China Search for more papers by this author Xianming Deng Xianming Deng State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China Search for more papers by this author Gaolin Liang Gaolin Liang CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, China Search for more papers by this author Huafeng Zhang Huafeng Zhang orcid.org/0000-0001-9923-7531 Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, China Search for more papers by this author Wei Jiang Corresponding Author Wei Jiang [email protected] orcid.org/0000-0001-8327-3079 Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Search for more papers by this author Rongbin Zhou Corresponding Author Rongbin Zhou [email protected] orcid.org/0000-0002-3351-6420 Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, China Search for more papers by this author Yi Huang Yi Huang Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, China Search for more papers by this author Hua Jiang Hua Jiang Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Search for more papers by this author Yun Chen Yun Chen State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China Search for more papers by this author Xiaqiong Wang Xiaqiong Wang Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Search for more papers by this author Yanqing Yang Yanqing Yang Department of Clinical Laboratory, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China Search for more papers by this author Jinhui Tao Jinhui Tao Department of Rheumatology & Immunology, The First Affiliated Hospital of University of Science and Technology of China, Hefei, Anhui, China Search for more papers by this author Xianming Deng Xianming Deng State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China Search for more papers by this author Gaolin Liang Gaolin Liang CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, China Search for more papers by this author Huafeng Zhang Huafeng Zhang orcid.org/0000-0001-9923-7531 Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, China Search for more papers by this author Wei Jiang Corresponding Author Wei Jiang [email protected] orcid.org/0000-0001-8327-3079 Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Search for more papers by this author Rongbin Zhou Corresponding Author Rongbin Zhou [email protected] orcid.org/0000-0002-3351-6420 Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, China Search for more papers by this author Author Information Yi Huang1,2,‡, Hua Jiang1,‡, Yun Chen3, Xiaqiong Wang1, Yanqing Yang4, Jinhui Tao5, Xianming Deng3, Gaolin Liang6, Huafeng Zhang1,2, Wei Jiang *,1 and Rongbin Zhou *,1,2 1Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China 2Innovation Center for Cell Signaling Network, University of Science and Technology of China, Hefei, China 3State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China 4Department of Clinical Laboratory, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China 5Department of Rheumatology & Immunology, The First Affiliated Hospital of University of Science and Technology of China, Hefei, Anhui, China 6CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86-551-63600125; E-mail: [email protected] *Corresponding author. Tel: +86-551-63600302; Fax: +86-551-63600831; E-mail: [email protected] EMBO Mol Med (2018)10:e8689https://doi.org/10.15252/emmm.201708689 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The dysregulation of NLRP3 inflammasome can cause uncontrolled inflammation and drive the development of a wide variety of human diseases, but the medications targeting NLRP3 inflammasome are not available in clinic. Here, we show that tranilast (TR), an old anti-allergic clinical drug, is a direct NLRP3 inhibitor. TR inhibits NLRP3 inflammasome activation in macrophages, but has no effects on AIM2 or NLRC4 inflammasome activation. Mechanismly, TR directly binds to the NACHT domain of NLRP3 and suppresses the assembly of NLRP3 inflammasome by blocking NLRP3 oligomerization. In vivo experiments show that TR has remarkable preventive or therapeutic effects on the mouse models of NLRP3 inflammasome-related human diseases, including gouty arthritis, cryopyrin-associated autoinflammatory syndromes, and type 2 diabetes. Furthermore, TR is active ex vivo for synovial fluid mononuclear cells from patients with gout. Thus, our study identifies the old drug TR as a direct NLRP3 inhibitor and provides a potentially practical pharmacological approach for treating NLRP3-driven diseases. Synopsis Tranilast (TR), an anti-allergic clinical drug, is here reported as a NLRP3 inflammasome inhibitor with beneficial effects for NLRP3-driven diseases. By direct binding to NLRP3, it inhibits its oligomerization and subsequent inflammasome assembly, caspase-1 activation and IL-1β production. TR specifically inhibits NLRP3 inflammasome activation in both human and mouse cells. TR binds to NLRP3 and inhibits its oligomerization and inflammasome complex formation. TR has remarkable preventive or therapeutic effects on the mouse models of NLRP3-driven diseases. Introduction NLRP3 inflammasome is a protein complex formed by NOD-like receptor (NLR) family member NLRP3, adaptor protein ASC, and caspase-1 (Martinon et al, 2009; Davis et al, 2011; Jo et al, 2016). A variety of factors derived from not only pathogen, but also environment or host, can activate NLRP3 inflammasome to result in pyroptosis and the release of secretion of several proinflammatory cytokines, such as IL-1β or IL-18 (Chen et al, 2009). The dysregulation of NLRP3 inflammasome has been reported to be involved in the pathogenesis of several human diseases. Mutations in NLRP3 gene can result in spontaneous NLRP3 inflammasome activation and are associated with cryopyrin-associated autoinflammatory syndromes (CAPS), which are a group of rare, inherited, autoinflammatory diseases (Broderick et al, 2015). In addition, NLRP3 inflammasome can sense some host-derived “danger signals”, including monosodium urate crystals (MSU), cholesterol crystals, amyloid-β aggregates, unsaturated fatty acids, high glucose, and ceramide, to promote chronic inflammation and contribute to the development of human complex diseases, including gout, atherosclerosis, neurodegenerative diseases, and type 2 diabetes (T2D) (Martinon et al, 2006; Duewell et al, 2010; Masters et al, 2010; Zhou et al, 2010; Wen et al, 2011; Heneka et al, 2012; Lamkanfi & Dixit, 2012). Thus, NLPR3 inflammasome has been regarded as a potential drug target for the treatment of inflammatory diseases. In recent years, a few small-molecule compounds have shown potential inhibitory effects on NLRP3 inflammasome activation in vitro, including dimethyl sulfoxide (DMSO), 3,4-methylenedioxy-β-nitrostyrene, glyburide, parthenolide, sulforaphane, isoliquiritigenin, MCC950, β-hydroxybutyrate, flufenamic acid, and mefenamic acid (Lamkanfi et al, 2009; Juliana et al, 2010; Yan et al, 2013; Ahn et al, 2014; He et al, 2014; Honda et al, 2014; Coll et al, 2015; Youm et al, 2015; Daniels et al, 2016; Yang et al, 2016). Among them, several compounds have been tested in animal models of human diseases. Sulforaphane and isoliquiritigenin have been shown to alleviate high-fat diet (HFD)-induced metabolic disorders in mice (Honda et al, 2014; Yang et al, 2016). MCC950 and β-hydroxybutyrate have shown beneficial effects in mice models of CAPS (Coll et al, 2015; Youm et al, 2015). MCC950, flufenamic acid, and mefenamic acid have been shown to delay the development of AD in mice model (Daniels et al, 2016; Dempsey et al, 2017). Although the beneficial effects of these compounds on NLRP3-driven diseases in animal models are promising, none of them have been tested in clinic. Thus, it is urgent to develop NLRP3 inflammasome inhibitors with high safety for clinical trials. Tranilast (N-[3′,4′-dimethoxycinnamoyl]-anthranilic acid, TR) is an analog of a tryptophan metabolite and has been reported to have an inhibitory effect on homologous passive cutaneous anaphylaxis because it can inhibit IgE-induced histamine release from mast cells (Azuma et al, 1976; Koda et al, 1976; Platten et al, 2005). Subsequently, TR has been clinically used in the treatment of a variety of inflammatory diseases, including bronchial asthma, atypical dermatitis, allergic conjunctivitis, and hypertrophic scars (Darakhshan & Pour, 2015). Moreover, TR is a relatively safe drug and is well tolerated by most patients, at doses of up to 600 mg/day over a period of months (Darakhshan & Pour, 2015). Although TR has shown favorable pharmacological effects against inflammatory diseases, the molecular mechanisms and the direct target of its anti-inflammatory activity are still unknown. In this study, we showed that TR directly bound to NLRP3 and inhibited NLRP3 inflammasome assembly and the subsequent caspase-1 activation and IL-1β production. More importantly, TR could prevent or treat NLRP3-dependent inflammatory diseases in mice models and was also active ex vivo for samples from patients with gout. Results TR specifically inhibits NLRP3 inflammasome activation in macrophages To confirm the inhibitory effects of TR on inflammasome activation, we first examined whether TR inhibited caspase-1 cleavage and IL-1β secretion. We indeed observed that TR treatment blocked nigericin-induced caspase-1 cleavage, IL-1β secretion, and pyroptosis (Fig 1A and B, and Appendix Fig S1A). It has been reported that TR can inhibit cytokine-induced NF-κB activation (Oh et al, 2010), we then examined whether TR inhibited NLRP3 inflammasome activation via regulating the expression of NF-κB-dependent NLRP3 or pro-IL-1β expression. When BMDMs were stimulated with TR for 30 min after 3-h LPS treatment, TR had no effect on LPS-induced NLRP3, pro-IL-1β expression, TNF-α, or IL-6 production (Fig 1C and D, and Appendix Fig S1B–D), suggesting that TR-induced NLRP3 inflammasome inhibition was not caused by the downregulation of NLRP3 or pro-IL-1β expression at this condition. In contrast, when BMDMs were stimulated with TR for 30 min before 3-h LPS treatment, TR inhibited LPS-induced pro-IL-1β expression and IL-6 production, but had minimal effects on NLRP3 expression and TNF-α production (Appendix Fig S1B–D). These results suggest that TR can suppress both NLRP3 inflammasome activation and pro-IL-1β expression. In order to clarify the mechanisms underlying TR-induced inflammasome inhibition, we stimulated BMDMs with TR after 3-h LPS treatment in the later experiments. The observed inhibitory effects of TR on IL-1β secretion were also confirmed in human THP-1 macrophages (Fig 1E). Taken together, these results indicate that TR has the potential to inhibit caspase-1 activation and IL-1β secretion. Figure 1. TR specifically inhibits NLRP3 inflammasome activation A. Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs treated with various doses (above lanes) of TR and then stimulated with nigericin, and immunoblot analysis of the precursors of IL-1β (pro-IL-1β) and caspase-1 (pro-casp1) in lysates of those cells (Input). B–D. ELISA of IL-1β (B), TNF-α (C) and IL-6 (D) in supernatants from LPS-primed BMDMs treated with various doses (above lanes) of TR and then stimulated with nigericin. E. Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in supernatants from PMA-differentiated THP-1 cells treated with various doses (above lanes) of TR and then stimulated with nigericin. F. Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs treated with TR (100 μM) and then stimulated with MSU, nigericin, ATP, Alum, and immunoblot analysis of the precursors of IL-1β (pro-IL-1β) and caspase-1 (pro-casp1) in lysates of those cells (Input). G. ELISA of IL-1β in culture supernatants (SN) of LPS-primed BMDMs treated with TR (100 μM) and then stimulated with MSU, nigericin, ATP, Alum. H. Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of Pam3-primed BMDMs treated with various doses (above lanes) of TR and then stimulated with cytosolic LPS (cLPS), and immunoblot analysis of the precursors of IL-1β (pro-IL-1β) and caspase-1 (pro-casp1) in lysates of those cells (Input). I. ELISA of IL-1β in culture supernatants (SN) of Pam3-primed BMDMs treated with various doses (above lanes) of TR and then stimulated with cytosolic LPS (cLPS). Data information: Data are from three independent experiments with biological duplicates in each (B–D, G, I; mean and s.e.m. of n = 6) or are representative of at least three independent experiments (A, E, F, H). Statistics were analyzed using an unpaired Student's t-test: **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [emmm201708689-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint In addition to nigericin, NLRP3 inflammasome can be activated by both pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), including aluminum salts (Alum), ATP, monosodium urate crystals (MSU), and cytosolic LPS (cLPS) (Davis et al, 2011; Kayagaki et al, 2011). To determine whether TR was a common inhibitor for NLPR3 inflammasome, we examined other NLRP3 agonists. Pretreatment with TR inhibited caspase-1 cleavage and IL-1β secretion triggered by all examined agonists, including MSU, Alum, ATP, and cLPS (Fig 1F–I), similar to nigericin. Interestingly, TR could not block cLPS-induced gasdermin D (Gsdmd) activation and pyroptosis, suggesting that TR targets the downstream of caspase-11 to inhibit non-canonical NLRP3 activation (Appendix Fig S1E and F). These results indicate that TR is a potent and broad inhibitor for the activation of both canonical and non-canonical NLRP3 inflammasome activation. We also tested whether TR could inhibit other inflammasomes, such as NLRC4 and AIM2 inflammasome. The results showed that TR had no effect on NLRC4 or AIM2 inflammasome activation, which were triggered by Salmonella typhimurium (Salmonella) infection or poly A:T transfection, respectively (Appendix Fig S2A and B). Taken together, these results demonstrate that TR can specifically inhibit NLRP3 inflammasome activation and the subsequent IL-1β production. TR has no effects on upstream signaling of NLRP3, but inhibits the assembly of NLRP3 inflammasome We then investigated how TR inhibited NLRP3 inflammasome activation. Although TR has been reported to exert different biological effects, the targets of TR have not been validated. TR has been proposed to inhibit transient receptor potential cation channel subfamily V member 2 (TRPV2) (Zhang et al, 2012), and we then examined whether TR blocked NLRP3 inflammasome via inhibition of TRPV2. The results showed that knockdown of TRPV2 in BMDMs had no effect on nigericin-induced NLRP3 inflammasome activation (Appendix Fig S3A and B), suggesting TRPV2 is not involved in TR-induced NLRP3 inflammasome inhibition. Previous study has shown that TR can inhibit the activity of hematopoietic prostaglandin D2 synthase (HPGDS) (Ikai et al, 1989), but our results showed that HPGDS was not involved in NLRP3 inflammasome activation (Appendix Fig S3C and D). During NLRP3 inflammasome activation, ASC oligomerization is a critical step for the subsequent caspase-1 activation (Lu et al, 2014; Dick et al, 2016). Consistent with the inhibitory effects of TR on caspase-1 activation and IL-1β production, TR remarkably suppressed nigericin-induced ASC oligomerization (Fig 2A). These results suggest that TR acts upstream of caspase-1 and ASC oligomerization to inhibit NLRP3 inflammasome activation. We then studied whether TR affected potassium efflux, which is an upstream signaling event of NLRP3 activation (Petrilli et al, 2007; Munoz-Planillo et al, 2013). Nigericin treatment could result in dramatically decrease in intracellular potassium, but this effect was not suppressed by TR (Appendix Fig S4A), suggesting that TR has no effect on potassium efflux during NLRP3 inflammasome activation. Mitochondrial damage, represented as mitochondria fission, clustering, and ROS production, is another upstream signaling event of NLRP3 activation (Zhou et al, 2011). Nigericin-induced mitochondrial damage and ROS production were normal in BMDMs pretreated with TR (Appendix Fig S4B), indicating TR does not affect mitochondrial damage in NLRP3 inflammasome activation. In addition, chloride efflux has been proposed as another upstream signaling event for NLRP3 activation (Daniels et al, 2016; Tang et al, 2017), but TR could not block nigericin-induced decrease in intracellular chloride in BMDMs (Appendix Fig S4C), suggesting that TR has no effect on the chloride efflux upstream of NLRP3 activation. Thus, these results suggest that TR acts downstream of potassium efflux, mitochondrial damage, and chloride efflux to inhibit NLRP3 activation. Figure 2. TR suppresses the assembly of NLRP3 inflammasome Immunoblot analysis of ASC oligomerization in lysates of BMDMs treated with various doses (above lanes) of TR for 30 min and then stimulated with nigericin for another 30 min. Immunoprecipitation (IP) and immunoblot analysis of the interaction of endogenous NLRP3 and NEK7 in LPS-primed BMDMs treated with various doses (above lanes) of TR for 30 min and then stimulated with nigericin for another 20 min. IP and immunoblot analysis of the interaction of endogenous NLRP3 and ASC in LPS-primed BMDMs treated with various doses (above lanes) of TR for 30 min and then stimulated with nigericin for another 20 min. Data information: Data are representative of two or three independent experiments. Source data are available online for this figure. Source Data for Figure 2 [emmm201708689-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Next, we assessed whether TR inhibited the assembly of NLRP3 inflammasome. Recently, NEK7 has been proposed as an essential component of NLRP3 inflammasome and NEK7–NLRP3 interaction is important for NLRP3 oligomerization and inflammasome assembly (He et al, 2016; Schmid-Burgk et al, 2016; Shi et al, 2016). Consistent with these previous studies, nigericin treatment promoted the endogenous interaction between NEK7 and NLRP3, which could not be suppressed by TR treatment (Fig 2B). Another critical step for NLRP3 inflammasome assembly is the recruitment of ASC to NLRP3 (Martinon et al, 2009; Davis et al, 2011). We next determined whether TR could impact on NLRP3–ASC interaction and found that pretreatment with TR inhibited the endogenous interaction between NLRP3 and ASC in nigericin-treated macrophages (Fig 2C). Thus, these results indicate that TR blocks NLRP3-ASC complex formation to inhibit NLRP3 inflammasome activation. TR directly binds to NLRP3 and inhibits its oligomerization Since TR inhibited NLRP3-ASC complex formation, we next investigated whether TR bound to these proteins. A synthesized biotinylated analog of TR (biotin-TR) was used as an affinity reagent and incubated with cell lysates of LPS-primed BMDMs or PMA-differentiated THP-1 cells. Compound was pulled down with streptavidin beads, and NLRP3 component proteins were detected by immunoblot analysis. The data showed that NLRP3, but not ASC or NEK-7, was pulled down by biotin-TR (Fig 3A and B). To determine whether TR interacts with NLRP3 directly, purified recombinant NLRP3 protein could be pulled down with biotin-TR (Fig 3C and Appendix Fig S5A), confirming that TR directly interacts with NLRP3. We next studied whether TR bound to other innate immune sensors. Flag-tagged NLRP3, NOD1, NOD2, NLRP1, or NLRC4 were overexpressed in HEK-293T cells, and the cell lysates were then incubated with biotin-TR. The results showed that only NLRP3 could be pulled down (Fig 3D), suggesting that TR specifically binds with NLRP3. NLRP3 contains three functional domains, LRR, NACHT, and PYD. We then studied which domain was responsible for the binding between NLRP3 and TR, and the results showed that only NACHT domain of NLRP3 bound TR (Fig 3E). These results indicate that TR directly binds to the NACHT domain of NLRP3. Figure 3. TR directly binds to NLRP3 and inhibits its oligomerization A, B. Cell lysates of LPS-primed BMDMs (A) or PMA-differentiated THP-1. (B) Cells were incubated with different concentrations of biotin-TR for 1 h, which were then pulled down with streptavidin beads. C. Purified recombinant NLRP3 protein was incubated with different concentrations of biotin-TR for 1 h, which were then pulled down with streptavidin beads. D, E. Cell lysates from HEK-293T cells transfected with Flag-tagged NLRP3, NOD1, NOD2, NLRP1, NLRC4, NLRP3-LRR, NLPR3-NACHT, or NLRP3-PYD constructs were incubated with different concentrations of biotin-TR for 1 h, which were then pulled down with streptavidin beads. F. Immunoprecipitation (IP) and immunoblot analysis of the interaction of Flag-NLRP3 and VSV-NLRP3 in the lysates of HEK-293T cells. TR was added at 8 h post-transfection. G. Immunoblot analysis of NLRP3 by SDD-AGE or SDS–PAGE assay in BMDMs treated with TR for 30 min and then left stimulated with nigericin for 20 min. Data information: Data are representative of two or three independent experiments. Source data are available online for this figure. Source Data for Figure 3 [emmm201708689-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint We then investigated how the binding of TR to NACHT inhibited the assembly of NLRP3 inflammasome. The NACHT domain is important for NLRP3 oligomerization, which is a critical step for the assembly of NLRP3 inflammasome (Martinon et al, 2009; Davis et al, 2011). We then investigated whether TR could prevent the direct NLRP3–NLRP3 interaction. HEK-293T cells were transfected with Flag-NLRP3 and VSV-NLRP3 and treated with TR, and then, a co-immunoprecipitation assay was performed. The results showed that the direct NLRP3–NLRP3 interaction was also suppressed by TR treatment (Fig 3F). In contrast, the direct interaction between NLRP3 and ASC was not altered (Appendix Fig S5B). The effect of TR on the endogenous NLRP3 oligomerization was confirmed using semi-denaturing detergent agarose gel electrophoresis (SDD–AGE) (Fig 3G), a method for detecting large protein oligomers in studying prions (Alberti et al, 2009; Hou et al, 2011). Since the ATPase activity of NACHT domain of NLRP3 is critical for NLRP3 oligomerization (Duncan et al, 2007), we then evaluated whether TR inhibited the ATPase of recombinant NLRP3 protein and found that TR had no effect on NLRP3 ATPase activity (Appendix Fig S5C), suggesting that TR blocks NLRP3 oligomerization via an ATPase-independent manner. Thus, these results indicate TR can directly bind to the NACHT domain of NLRP3 and inhibit NLRP3 oligomerization by blocking direct NLRP3–NLRP3 interaction. TR inhibits NLRP3 activation in vivo and has beneficial effects in mouse models of gouty arthritis and CAPS Since TR inhibits NLRP3 inflammasome activation in vitro, we then determined to analyze the activity of TR in vivo. Intraperitoneal injection of MSU elicited an NLRP3-dependent peritonitis characterized by IL-1β production and massive neutrophil influx (Martinon et al, 2006). Consistent with the role of TR in macrophages, TR treatment in vivo efficiently suppressed MSU-induced IL-1β production and neutrophil influx (Fig 4A and B). We also compared the anti-inflammasome activity of TR with MCC950, which is a selective inhibitor for NLRP3 inflammasome (Coll et al, 2015). Although the in vitro inhibitory activity of TR on MSU-induced IL-1β secretion was around 400 times less potent than MCC950 (Appendix Fig S6A), its in vivo activity on MSU-induced peritonitis was only around 5–10 times less potent than MCC950 (Appendix Fig S6B and C). The deposition of MSU is the major cause for the development of arthritis in the patients with gout (McQueen et al, 2012). Delivery of MSU to the joints of mice leads to NLRP3 inflammasome-dependent inflammation and pathology (Reber et al, 2014). Consistent with this, MSU injection induced acute joint swelling, which could be alleviated by Nlrp3 deficiency or oral TR treatment (Fig 4C). TR also suppressed MSU-induced NLRP3-dependent