Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7‐NLRP3 interaction

Article9 December 2021free access Source DataTransparent process Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interaction Qiang Li Qiang Li School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Hui Feng Hui Feng Department of Ultrasound, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Hongbo Wang Hongbo Wang Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Yinghao Wang Yinghao Wang School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China Search for more papers by this author Wenqing Mou Wenqing Mou China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Guang Xu Guang Xu Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Ping Zhang Ping Zhang Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Ruisheng Li Ruisheng Li Research Center for Clinical and Translational Medicine, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Wei Shi Wei Shi China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhilei Wang Zhilei Wang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhie Fang Zhie Fang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Lutong Ren Lutong Ren China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Yan Wang Yan Wang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Li Lin Li Lin China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xiaorong Hou Xiaorong Hou China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Wenzhang Dai Wenzhang Dai China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhiyong Li Zhiyong Li China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Ziying Wei Ziying Wei China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Tingting Liu Tingting Liu China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Jiabo Wang Jiabo Wang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Yuming Guo Yuming Guo Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Pengyan Li Pengyan Li Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xu Zhao Xu Zhao Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xiaoyan Zhan Corresponding Author Xiaoyan Zhan [email protected] orcid.org/0000-0002-2446-5967 Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xiaohe Xiao Corresponding Author Xiaohe Xiao [email protected] orcid.org/0000-0002-2836-2738 School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhaofang Bai Corresponding Author Zhaofang Bai [email protected] orcid.org/0000-0002-6208-150X Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Qiang Li Qiang Li School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Hui Feng Hui Feng Department of Ultrasound, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Hongbo Wang Hongbo Wang Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Yinghao Wang Yinghao Wang School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China Search for more papers by this author Wenqing Mou Wenqing Mou China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Guang Xu Guang Xu Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Ping Zhang Ping Zhang Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Ruisheng Li Ruisheng Li Research Center for Clinical and Translational Medicine, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Wei Shi Wei Shi China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhilei Wang Zhilei Wang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhie Fang Zhie Fang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Lutong Ren Lutong Ren China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Yan Wang Yan Wang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Li Lin Li Lin China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xiaorong Hou Xiaorong Hou China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Wenzhang Dai Wenzhang Dai China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhiyong Li Zhiyong Li China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Ziying Wei Ziying Wei China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Tingting Liu Tingting Liu China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Jiabo Wang Jiabo Wang China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Yuming Guo Yuming Guo Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Pengyan Li Pengyan Li Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xu Zhao Xu Zhao Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xiaoyan Zhan Corresponding Author Xiaoyan Zhan [email protected] orcid.org/0000-0002-2446-5967 Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Xiaohe Xiao Corresponding Author Xiaohe Xiao [email protected] orcid.org/0000-0002-2836-2738 School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Zhaofang Bai Corresponding Author Zhaofang Bai [email protected] orcid.org/0000-0002-6208-150X Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China Search for more papers by this author Author Information Qiang Li1,2,3,†, Hui Feng4,†, Hongbo Wang2,†, Yinghao Wang1,†, Wenqing Mou3, Guang Xu2,3, Ping Zhang2,3, Ruisheng Li5, Wei Shi3, Zhilei Wang3, Zhie Fang3, Lutong Ren3, Yan Wang3, Li Lin3, Xiaorong Hou3, Wenzhang Dai3, Zhiyong Li3, Ziying Wei3, Tingting Liu3, Jiabo Wang3, Yuming Guo2,3, Pengyan Li2,3, Xu Zhao2,3, Xiaoyan Zhan *,2,3, Xiaohe Xiao *,1,2,3 and Zhaofang Bai *,2,3 1School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China 2Department of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China 3China Military Institute of Chinese Materia, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China 4Department of Ultrasound, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China 5Research Center for Clinical and Translational Medicine, Fifth Medical Center of Chinese PLA General Hospital, Beijing, China † These authors contributed equally to this work *Corresponding author. E-mail: [email protected] author. E-mail: [email protected] author. E-mail: [email protected] EMBO Reports (2022)23:e53499https://doi.org/10.15252/embr.202153499 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 Figures & Info Abstract The activation of the nucleotide oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is related to the pathogenesis of a wide range of inflammatory diseases, but drugs targeting the NLRP3 inflammasome are still scarce. In the present study, we demonstrated that Licochalcone B (LicoB), a main component of the traditional medicinal herb licorice, is a specific inhibitor of the NLRP3 inflammasome. LicoB inhibits the activation of the NLRP3 inflammasome in macrophages but has no effect on the activation of AIM2 or NLRC4 inflammasome. Mechanistically, LicoB directly binds to NEK7 and inhibits the interaction between NLRP3 and NEK7, thus suppressing NLRP3 inflammasome activation. Furthermore, LicoB exhibits protective effects in mouse models of NLRP3 inflammasome-mediated diseases, including lipopolysaccharide (LPS)-induced septic shock, MSU-induced peritonitis and non-alcoholic steatohepatitis (NASH). Our findings indicate that LicoB is a specific NLRP3 inhibitor and a promising candidate for treating NLRP3 inflammasome-related diseases. Synopsis Licochalcone B, a main component of the medicinal herb licorice, specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interaction and exhibits protective effects in mouse models of NLRP3 inflammasome-mediated diseases. Licochalcone B specifically inhibits both canonical and non-canonical NLRP3 inflammasome activation. Licochalcone B blocks NLRP3-dependent ASC oligomerization. Licochalcone B binds to NEK7 and disrupts the NEK7-NLRP3 interaction. Licochalcone B effectively protects against NLRP3-mediated diseases. Introduction The nucleotide oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome, is a protein complex formed by the NLR family member NLRP3, adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1 (Mariathasan et al, 2004; Jo et al, 2016; Song et al, 2017). Upon activation, the NLRP3 inflammasome mediates the activation of caspase-1 and the subsequent cleavage of pro-interleukin (IL)-1β and pro-IL-18, leading to the release of the pro-inflammatory cytokines IL-1β and IL-18, respectively (Lim et al, 2020). Recent studies have reported that never in mitosis A (NIMA)-related kinase-7 (NEK7) can directly bind to NLRP3, and NEK7 is an essential for the activation of the NLRP3 inflammasome (He et al, 2016; Shi et al, 2016; Nozaki & Miao, 2019; Sharif et al, 2019). The NLRP3 inflammasome is the best-characterised inflammasome that can be activated by factors derived not only from pathogens but also from the environment or host; thus, its dysregulation is related to the pathogenesis of a variety of human diseases. Several mutations in the NLRP3 gene can result in spontaneous activation of the NLRP3 inflammasome, which is central to the development of cryopyrin-associated auto-inflammatory syndromes (CAPS, a rare, hereditary, auto-inflammatory disease; Broderick et al, 2015; Coll et al, 2015). In addition, the NLRP3 inflammasome also responds to some host-derived danger signals, including monosodium urate crystals (MSU), amyloid starch, cholesterol crystals, high glucose, unsaturated fatty acids, and ceramide. These risk factors may result in the development and deterioration of a variety of chronic inflammatory diseases such as gout (Dalbeth et al, 2016, 2019), neurodegenerative diseases (Wu et al, 2021), atherosclerosis (Zhuang et al, 2019), and NASH (Mridha et al, 2017; Thomas, 2017; Gaul et al, 2021). Thus, NLPR3 inflammasome has been regarded as a potential drug target for the treatment of inflammatory diseases. In recent years, some small-molecule compounds, such as Tranilast (Huang et al, 2018), oridonin (He et al, 2018), MCC950 (Coll et al, 2015, 2019; Tapia-Abellan et al, 2019), OLT1177 (Marchetti et al, 2018; Lonnemann et al, 2020), CY-09 (Jiang et al, 2017), sulforaphane (Greaney et al, 2016), cardamonin (Wang et al, 2019b), carnosol (Shi et al, 2020), dehydrocostus lactone (Chen et al, 2020), echinatin (Xu et al, 2021), and cryptotanshinone (Liu et al, 2021), have been shown to have a potential inhibitory effect on the activation of the NLRP3 inflammasome in vitro. The above compounds have been tested in animal models of human diseases and showed potential therapeutic effects. Among these compounds, MCC950 is the best-characterised NLRP3 inhibitor and was tested in a phase II clinical trial for rheumatoid arthritis, but was not developed further because its application resulted in hepatotoxicity (Mangan et al, 2018). Sulforaphane is an isothiocyanate found in broccoli sprout extracts (Fahey et al, 1997) and has been tested in humans with autism and exhibited negligible toxicity (Singh et al, 2014). Sulforaphane has been demonstrated to inhibit the NLRP1b, NLRP3, NAIP/NLRC4, and AIM2 inflammasomes independent of Nrf2 (Greaney et al, 2016), so it may not be a specific inhibitor of the NLRP3 inflammasome. OLT1177 (also known as dapansutrile) has been reported to specifically inhibit the NLRP3 inflammasome (Marchetti et al, 2018), and its safety and efficacy in the treatment of gout flares in an open-label, proof-of-concept, phase 2a trial has been demonstrated (Klück et al, 2020). Moreover, OLT1177 has been tested in a phase 1B trial and the result showed that treatment with OLT1177 for 14 days was safe and well tolerated in patients with heart failure and reduced ejection fraction (Wohlford et al, 2020). Further studies are needed to confirm the clinical potential of OLT1177. Overall, inhibitors of NLRP3 inflammasome show great potential in the treatment of NLRP3-mediated diseases. Licochalcone B (LicoB) is a flavonoid bioactive ingredient found in licorice, the age-old and widely used traditional herbal medicinal plant (Wang et al, 2015). Modern pharmacological research reports that LicoB has various biological activities (Wang et al, 2020), including anti-inflammatory (Fu et al, 2013), anti-oxidant (Fu et al, 2013) and anti-tumour (Wang et al, 2019a) effects. LicoB has been shown to protect liver cells from alcohol-induced cell damage by inhibiting cell apoptosis (Yuan et al, 2014), upregulating extracellular signal-regulated kinase (Erk)-nuclear factor erythroid related factor 2 (Nrf2) (Gao et al, 2017) and significantly inhibiting lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) production and expression of tumour necrosis factor-α (TNF-α) and monocyte chemotactic protein 1 (MCP-1) (Furusawa et al, 2009). It has been shown that LicoB can inhibit the production of IL-6, prostaglandin E2 (PGE2) and superoxide anions in the xanthine oxidase system and has a significant inhibitory effect on lipid peroxidation, and a strong scavenging effect on 2,2'-azinobis-(3-ethylbenzthiazoline-sulphonate) (ABTS) (+) radicals and 1,1-Diphenyl-2-picrylhydrazyl radical and 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) free radical activity (Haraguchi et al, 1998; Furusawa et al, 2009; Thiyagarajan et al, 2011). Although LicoB exhibits obviously beneficial effects, its underlying mechanism and direct targets remain to be elucidated. In this study, we found that LicoB is a potential and effective NLRP3 inflammasome inhibitor. Our results demonstrated that LicoB directly binds to NEK7, interfering with the interaction between NLRP3 and NEK7, resulting in the inhibition of NLRP3 inflammasome activation. Moreover, LicoB displays significant therapeutic effects in several mouse models of NLRP3-mediated diseases and may be developed as a promising candidate for the treatment of NLRP3 inflammasome-related inflammatory diseases. Results LicoB inhibits both canonical and non-canonical NLRP3 activation To identify potential candidates for the treatment of NLRP3-mediated diseases, we screened inhibitors of NLRP3 and found that LicoB could block NLRP3 inflammasome activation (Fig EV1A). To further study the effect of LicoB on the activation of the NLRP3 inflammasome (Fig 1A), we first tested the cytotoxicity of LicoB in mouse bone marrow-derived macrophages (BMDMs). Cell viability assays showed that LicoB did not exhibit any cytotoxicity at doses below 80 μM in BMDMs (Fig 1B). The BMDMs were first primed with LPS and then pre-treated with a range of LicoB concentrations before being stimulated with nigericin at a dose of 10 μM or ATP at a dose of 5 mM, to induce NLRP3 inflammasome activation. The results showed that LicoB dose-dependently inhibited caspase-1 activation or IL-1β secretion triggered by nigericin (Fig 1C–E) or ATP (Fig 1G–I) in LPS-primed BMDMs, and the half-maximal inhibitory concentration (IC50) of LicoB was approximately 18.1 μM (Fig 1E). Correspondingly, the protein levels of NLRP3 and pro-IL-1β in the whole cell lysates were not affected (Fig 1C and G). Click here to expand this figure. Figure EV1. Licochalcone B inhibits NLRP3 inflammasome activation A. BMDMs were primed with LPS for 4 h and then treated with LicoB 1 h prior to stimulation of nigericin for 45 min. Activity of caspase-1 in culture supernatants of BMDMs were shown. B, C. BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of GSDMD, pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture SN of BMDMs. Coomassie Blue staining was used as the SN loading control, while lamin B was used as the lysate loading control. D–F. PMA-primed THP-1 cells were treated with LicoB 1 h prior to stimulation with nigericin for 45 min. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC in the WCL; and activated caspase-1 (p20) and cleaved IL-1β (p17) (D) in the culture supernatants (SN) of THP-1 cells. Caspase-1 activity (E) and IL-1β secretion (F) in the SN were measured. G, H. Human PBMCs were primed with LPS for 4 h and then treated with LicoB 1 h prior to stimulation with nigericin for 45 min (G) or with ATP for 1 h (H). Caspase-1 activity in supernatant was measured. Data information: Error bars, mean ± SEM from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 and n.s.: not significant (one-way ANOVA with Dunnett's post hoc test). Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Licochalcone B (LicoB) inhibits NLRP3 inflammasome activation A. Licochalcone B (LicoB) structure. B. Cell Counting Kit 8 (CCK-8) was used to assess the viability of BMDMs treated with different doses of LicoB for 24 h. C–I. BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control. Data information: Error bars, mean ± SEM from three biological replicates. **P < 0.01, ***P < 0.001, and n.s.: not significant (one-way ANOVA with Dunnett's post hoc test). Source data are available online for this figure. Source Data for Figure 1 [embr202153499-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Meanwhile, nigericin or ATP stimulation induced lactate dehydrogenase (LDH) release (marker of cell death) and gasdermin D (GSDMD) cleavage (indicator of pyro ptosis), and the release of LDH (Fig 1F and J) or GSDMD cleavage (Fig EV1, EV2, EV3B and C) was significantly blocked by LicoB. LicoB also impaired nigericin-induced IL-1β secretion and caspase-1 cleavage in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells (Fig EV1, EV2, EV3D–F) and human peripheral blood mononuclear cells (hPBMCs) (Fig EV1, EV2, EV3G and H). In summary, LicoB effectively blocks the nigericin- or ATP-induced activation of the NLRP3 inflammasome in mouse and human immune cells. Click here to expand this figure. Figure EV2. Licochalcone B inhibits K+ Efflux-Independent NLRP3 Inflammasome Activation in BMDMs BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h prior to stimulation with imiquimod (70 μM) for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture SN of BMDMs were shown. Coomassie Blue staining was used as the SN loading control, while lamin B was used as the lysate loading control. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Licochalcone B directly binds to NEK7 but does not affect the kinase activity of NEK7 A. Cell lysates of PMA-primed THP-1 treated with nigericin or not were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed by Western blot. B. Cell lysates of PMA-primed THP-1 were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 and 1 mM). The pull-down samples and input were analysed by Western blot. C. Cell lysates of LPS-primed hPBMCs treated with nigericin or not were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed by Western blot. D. Cell lysates of LPS-primed hPBMCs were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 and 1 mM). The pull-down samples and input were analysed by Western blot. E. NEK7 was incubated with β-casein and ATP in the presence of different concentrations of LicoB. NEK7 kinase activity was measured using an ADP-based phosphatase coupled kinase assay. F. Cell lysates of LPS-primed BMDMs treated with nigericin or not were incubated with Sepharose, Sepharose-LicoA or Sepharose-LicoB. The pull-down samples and input were analysed by Western blot. G, H. Human monocytes were treated with LicoB for 1 h, prior to stimulation with LPS (200 ng/ml) for 14 h. (G) Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC in the whole cell lysate (WCL); cleaved IL-1β (p17) in the culture SN of BMDMs were shown. Coomassie Blue staining was used as the SN loading control, while lamin B was used as the lysate loading control. IL-1β secretion (H) in the supernatant were measured by ELISA. Data information: Error bars, mean ± SEM from three biological replicates. **P < 0.01, ***P < 0.001 and n.s.: not significant (one-way ANOVA with Dunnett's post hoc test). Source data are available online for this figure. Download figure Download PowerPoint To test whether LicoB has a broad-spectrum anti-NLRP3 inflammasome effect, we investigated the effect of LicoB on NLRP3 inflammasome activation induced by multiple agonists. First, the effect of LicoB on canonical NLRP3 inflammasome activation was evaluated. It was found that LicoB effectively inhibited caspase-1 activation and IL-1β secretion induced by the canonical NLRP3 inflammasome agonists (Youm et al, 2013), such as nigericin, ATP, poly(I:C) or MSU (Fig 2A–C). We then evaluated the effect of LicoB on the activation of the non-canonical NLRP3 inflammasome (Kayagaki et al, 2011, 2013, 2015). The results showed that LicoB dose-dependently inhibited caspase-1 cleavage and IL-1β secretion induced by LPS transfection in Pam3CSK4-primed BMDMs. Next, we evaluated the effect of LicoB on K+ efflux-independent NLRP3 inflammasome activation induced by imiquimod (Groß et al, 2016). The results showed that LicoB dose-dependently inhibited caspase-1 cleavage and IL-1β secretion induced by the K+ efflux-independent NLRP3 inflammasome agonist imiquimod (Fig EV2). These results indicated that LicoB is a broad-spectrum inhibitor of the NLRP3 inflammasome. Figure 2. Licochalcone B is a specific inhibitor of the NLRP3 inflammasome A–C. BMDMs were primed with LPS and then treated with LicoB (20 μΜ) for 1 h, followed by stimulation with nigericin, ATP, poly (I:C) or MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then transfected with LPS. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC in the whole cell lysate (WCL), and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured. D–F. LPS-primed BMDMs were tr