Britannin as a novel NLRP3 inhibitor, suppresses inflammasome activation in macrophages and alleviates NLRP3-related diseases in mice

Abstract

Overactivation of the NLRP3 inflammasomes induces production of pro-inflammatory cytokines and drives pathological processes. Pharmacological inhibition of NLRP3 is an explicit strategy for the treatment of inflammatory diseases. Thus far no drug specifically targeting NLRP3 has been approved by the FDA for clinical use. This study was aimed to discover novel NLRP3 inhibitors that could suppress NLRP3-mediated pyroptosis. We screened 95 natural products from our in-house library for their inhibitory activity on IL-1β secretion in LPS + ATP-challenged BMDMs, found that Britannin exerted the most potent inhibitory effect with an IC₅₀ value of 3.630 µM. We showed that Britannin (1, 5, 10 µM) dose-dependently inhibited secretion of the cleaved Caspase-1 (p20) and the mature IL-1β, and suppressed NLRP3-mediated pyroptosis in both murine and human macrophages. We demonstrated that Britannin specifically inhibited the activation step of NLRP3 inflammasome in BMDMs via interrupting the assembly step, especially the interaction between NLRP3 and NEK7. We revealed that Britannin directly bound to NLRP3 NACHT domain at Arg335 and Gly271. Moreover, Britannin suppressed NLRP3 activation in an ATPase-independent way, suggesting it as a lead compound for design and development of novel NLRP3 inhibitors. In mouse models of MSU-induced gouty arthritis and LPS-induced acute lung injury (ALI), administration of Britannin (20 mg/kg, i.p.) significantly alleviated NLRP3-mediated inflammation; the therapeutic effects of Britannin were dismissed by NLRP3 knockout. In conclusion, Britannin is an effective natural NLRP3 inhibitor and a potential lead compound for the development of drugs targeting NLRP3.

Introduction

The NLRP3 inflammasome is a protein complex that is widely found in the cytoplasm of cells including nonimmune cells and immune cells [1]. When NLRP3, which is an inflammasome sensor, is activated by factors such as pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and metabolism-associated molecular patterns (MAMPs), the inflammasome subsequently assembles with ASC and pro-Caspase-1 [2–4]. Then, mature Caspase-1 cleaves the inactive pro-inflammatory cytokine precursors, such as IL-1β and IL-18, into the mature forms. Mature Caspase-1 further proteolytically cleaves gasdermin D (GSDMD) to induce pyroptosis, which is a form of programmed cell death [3]. Furthermore, never in mitosis A-related kinase-7 (NEK7) has been characterized as a license of NLRP3 inflammasome activation by interacting with NLRP3 [5]. Over the past decades, many studies have shown that the NLRP3 inflammasome is overactivated in several pathological conditions (e.g., sepsis [6], type 2 diabetes [7], and atherosclerosis [8]), leading to increased production of pro-inflammatory cytokines and even inflammatory storms. Pharmacological inhibition of the NLRP3 inflammasome has shown favorable therapeutic effects on some diseases. OLT1177, a small molecular inhibitor of NLRP3, has exerted therapeutic effects on many NLRP3-driven diseases, such as gout, Alzheimer’s disease, and COVID-19 [9, 10]. Costunolide has been reported to be a covalent inhibitor of NLRP3 that treats gout and ulcerative colitis [11]. However, no NLRP3 inflammasome-related drugs have yet been approved for clinical use by the FDA. Thus, the discovery of novel NLRP3-targeted inhibitors is of great practical value for the clinical treatment of NLRP3-related diseases.

Traditional Chinese medicine has shown good therapeutic effects against acute inflammatory diseases [12]. Natural products extracted from traditional Chinese medicine are the material basis by which traditional Chinese medicines exert pharmacological effects. Britannin is a natural sesquiterpene lactone isolated from Inula japonica Thunb., which was used to alleviate cough and transform phlegm in ancient China. Recent studies have shown Britannin’s great pharmacologic anticancer effects, such as triple-negative breast carcinoma [13] and pancreatic cancer [14]. Britannin has also been reported to exert anti-inflammatory effects against asthma [15]. However, the specific mechanisms by which Britannin inhibits inflammation and its therapeutic role in a wide range of diseases are still unclear.

In this study, Britannin was identified as an innate inhibitor that effectively targeted NLRP3. Britannin suppressed activation of the NLRP3 inflammasome in an NF-κB-independent manner and inhibited assembly of the NLRP3 inflammasome by directly binding to the NACHT domain of NLRP3. It was also found that Britannin efficiently alleviated NLRP3-related diseases in mouse models, such as sepsis-induced acute lung injury (ALI) and gouty arthritis. Collectively, we demonstrated that Britannin was a potent inhibitor of NLRP3 and that it could be a lead compound for further development of novel NLRP3-targeted drugs.

Materials and methods

Reagents

Britannin was bought from Abphyto Biotechnology Co., Ltd. (Chengdu, China). Lipopolysaccharides (LPS, #L2880), adenosine triphosphate (ATP, #A3377), and aluminum potassium sulfate dodecahydrate (Alum, #237086) were purchased from Sigma (St. Louis, MO, USA). MitoTracker (#C1049B) and Protein A + G agarose (#P2012) were supplied by Beyotime (Shanghai, China). MitoSOX (#M36008) was obtained from Thermo Scientific (Carlsbad, CA, USA). Anti-NLRP3 (#AG-20B-0014) and anti-mouse Caspase-1 (#AG-20B-0042) antibodies were purchased from Adipogen (San Diego, CA, USA). Anti-NEK7 (#ab133514), anti-human Caspase-1 (#ab179515), and anti-GSDMD (#ab209845) antibodies were obtained from Abcam (Cambridge, UK). Anti-mouse IL-1β (#AF-401-NA) antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-human IL-1β (#AF5103) antibody was obtained from Affinity Biosciences (Jiangsu, China). Anti-Flag (#20543-1-AP), anti-HA (#66006-2-Ig), and anti-β-Actin (#66009-1-Ig) antibodies were purchased from Proteintech (Rosemont, IL, USA).

Cells

Bone marrow-derived macrophages (BMDMs) were obtained from 8-10-week-old C57BL/6 mice according to a previous study [16]. To stimulate inflammasomes, 1 × 10⁶ BMDMs were plated in 6-well plates, and then primed with LPS (500 ng/mL) for 3 h the following morning. Then, the cells were treated with Britannin for 30 min, and challenged with various NLRP3 activators (2.5 mM ATP for 30 min, 10 µM nigericin for 30 min, 300 µg/mL Alum for 2 h, or 300 µg/mL MSU for 6 h). For Caspase-11-related noncanonical NLRP3 inflammasome activation, Pam3CSK4-primed BMDMs were transfected with 1.5 μg/mL LPS via Lipofectamine 2000 for 16 h. For AIM2 inflammasome activation, the cells were transfected with 0.5 µg/mL poly(dA:dT) via Lipofectamine 2000 for 4 h. For NLRP1 inflammasome activation, LPS-primed BMDMs were treated with 10 µM Val-boroPro for 24 h. For NLRC4 inflammasome activation, LPS-primed BMDMs were challenged with Salmonella typhimurium for 0.5 h. After that, the cells were incubated with gentamicin for 4 h.

L929 mouse fibroblasts, HEK-293T cells, and THP-1 cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology. To stimulate the NLRP3 inflammasome in THP-1 cells, the cells were plated in 6-well plates, incubated with PMA (100 ng/mL) overnight, and then treated with different doses of Britannin for 30 min the following morning. After that, the cells were stimulated with LPS (500 ng/mL) for 3 h and then 10 µM nigericin for 30 min.

Animal experiments

All experimental animal protocols were approved by the Hangzhou Medical College Animal Policy and Welfare Committee (approved No.: 2021-039). 8–10-week-old male C57BL/6 mice and NLRP3 knock-out (Nlrp3^KO) mice (strain No. T010873) were purchased from GemPharmatech Co., Ltd (Jiangsu, China). The mice were housed in cages with ad libitum access to food and water. In addition, the mice were housed in a specific pathogen-free environment at 20–25 °C under 50%–60% humidity with 12 h/12 h light/dark cycles. All the experiments were performed and analyzed by random researchers.

For the gouty arthritis model, 10-week-old Nlrp3^KO and wild-type littermate control mice were randomly assigned to each group (n  =  6 each group), and then Britannin (20 mg/kg, dissolved in PBS containing 5% DMSO) or vehicle control was injected i.p. once a day for a total of three times. Then MSU (0.5 mg per foot, suspended in 20 µL PBS) or vehicle control was injected s.c. into the footpad to establish the gouty arthritis model. The footpad thicknesses were measured by vernier caliper at 1, 2, 4, and 6 h. After 6 h, the mice were narcotized and sacrificed. The footpad tissues were collected and incubated in Opti-MEM for 1 h at 37 °C. The IL-1β levels in the supernatants were tested using ELISA. The footpad tissues were then used for hematoxylin and eosin (H&E) staining.

For the septic ALI model, 10-week-old Nlrp3^KO and wild-type littermate control mice were randomly assigned to each group (n  =  6 each group). After being injected with 20 mg/kg Britannin for three consecutive days, the mice were injected i.p. with 10 mg/kg LPS or vehicle to establish the septic ALI model. After 12 h, the mice were narcotized and sacrificed. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue samples were collected. Equal portions of lung tissue were weighed immediately, and then were dried at 60 °C for 48 h before being weighed again to evaluate the levels of pulmonary edema. BALF was centrifuged at 1000 × g for 5 min at 4 °C to separate the cells and supernatant. The cells were counted, neutrophils were determined using Wright-Giemsa staining kit, and the protein concentration of the supernatants was measured using a Bradford assay. IL-1β and TNF-α levels in BALF and serum were tested by enzyme linked immunosorbent assay (ELISA). The same lung sections were collected for H&E staining. The severity of lung tissue injury was judged as previously described [17].

ELISA for cytokines examination

The supernatants from cell culture, footpad tissues culture, and the BALF were used to detect the cytokines levels as the manufacturer’s instruction, including human IL-1β (#88-7064-86, Thermo Scientific), mouse IL-1β (#88-7013-77), and mouse TNF-α (#88-7324-76).

Lactate dehydrogenase (LDH) assay

LPS-challenged BMDMs were incubated with different doses of Britannin for 30 min, and then activated with ATP for 1 h. The release of LDH in the cell supernatant was detected by an LDH assay kit (#C0016, Beyotime) according to the manufacturer’s instruction.

Intracellular potassium detection

BMDMs were stimulated and treated with different doses of Britannin as described before. After being washed three times with 0.9% normal saline, the cells were lysed with ultrapure water for 20 min at 37 °C. After the samples were collected and centrifuged, potassium concentrations in the supernatants were detected by Cobas-c-311 Automatic Biochemistry Analyzer (Roche, Germany).

Intracellular chloride detection

BMDMs (5 × 10⁵ per well) were evenly plated in a 12-well plate overnight. Drug administration and inflammasome stimulation were performed at different times the next day. After being washed with PBS three times, the cells were lysed with ultrapure water for 20 min at 37 °C. N-(methoxycarbonyl methyl)-6-methoxyquinolinium bromide (MQAE, #S1082; Beyotime) (5 μmol/L) was added to the supernatants after centrifugation, and the fluorescence intensity was measured using SpectraMax iD3 (Molecular Devices, San Jose, CA, USA).

Fluorescence staining

To analyze mitochondrial reactive oxygen species (ROS) production and mitochondrial damage, BMDMs (1 × 10⁵ per dish) were plated in glass-bottom cell culture dishes, stimulated and treated with Britannin (10 μM) the following day. The cells were stained with 5 μM MitoSOX or 20 nM MitoTracker for 30 min. After being washed, fixed, and counterstained with DAPI, the cells were observed using a fluorescence microscope.

For the ASC speck assay, BMDMs (5 × 10⁵ per dish) were plated in glass-bottom cell culture dishes and incubated overnight. The following day, the cells were stimulated and treated with Britannin (10 μM). Then the cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton X-100 and incubated with ASC antibodies overnight. The following day, the cells were incubated with a fluorescent secondary antibody for 1 h. After being counterstained with DAPI, the cells were observed using a fluorescence microscope.

Western blotting and coimmunoprecipitation

Cells or tissues were lysed in lysis buffer containing protease-phosphatase inhibitor mixtures. Total protein was collected and measured using the Bradford assay before resuspended in SDS sample loading buffer. The samples were separated by SDS‒PAGE and transferred to PVDF membranes. Then, the membranes were blocked in 5% non-fat milk for 1.5 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. The following day, the membranes were incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Then, the protein bands were detected by ECL reagents.

In the coimmunoprecipitation assay, cell lysates were prepared as described above before being incubated with decoy antibodies overnight at 4 °C. Then, they were immunoprecipitated with protein A + G agarose beads at 4 °C for 4 h. After being washed with PBS five times, the immunoprecipitation samples were subjected to immunoblotting to detect the coprecipitated proteins. Total lysates were also subjected to Western blot analysis as an input control.

ASC oligomerization assay

BMDMs were lysed in lysis buffer (0.5% Triton X-100 with protease inhibitor) after inflammasome stimulation and drug treatment. After centrifugation, the sediments were resuspended in 2 mM suberic acid bis (3-sulfo-N-hydroxysuccinimide ester) sodium salt (BS3, #S855494; Macklin, China) and incubated for 30 min. Then, the precipitates were collected and analyzed by Western blotting.

Drug affinity responsive target stability (DARTS) assay

The lysates of LPS-treated BMDMs and transfected HEK-293T cells were incubated with 10 μM Britannin overnight. The protease pronase (25 ng of enzyme per μg of protein) was added and incubated with lysates for 4 min at room temperature. Then, the reaction was stopped by adding SDS loading buffer and heated at 100 °C for 10 min. The samples were analyzed by Western blotting.

Biolayer interferometry (BLI) assay

To determine the binding affinity between Britannin and the NLRP3 NACHT domain, the recombinant protein rhNLRP3-NACHT was biotinylated using the ReadiLink Protein Biotinylation Kit (#G-MM-IGT; Bomeida, Jiangsu, China) and immobilized onto Super Streptavidin Biosensors (#18-5057; ForteBio, San Francisco, CA, USA). The association and dissociation of the protein and different doses of Britannin were monitored for 100 s. All measurements were determined by subtracting the blank reference baseline. The data were analyzed by fitting the kinetic data using a 1:1 binding model using ForteBio’s Data Analysis Software version 11.1 software (ForteBio).

NLRP3 ATPase activity assay

NLRP3 ATPase activity was detected by an ADP-Glo Kinase Assay Kit (#V6930; Promega, Madison, WI, USA) as described previously [11]. Briefly, human NLRP3 was immunoprecipitated from plasmid-transfected HEK-293T cells incubated with different concentrations of Britannin for 40 min, after which ultrapure ATP was added to the reaction buffer and incubated at 37 °C for 40 min. The amount of ATP converted into ADP was determined as luminescent ATP.

Molecular docking

The human NLRP3 crystal structure (PDB ID: 6NPY) was obtained from the Protein Data Bank. Molecular docking of NLRP3 with Britannin was performed using AutoDock 4.2.6, and AutoDock Tools 1.5.6 software was used to generate docking input files. A 60 × 60 × 60-point grid box with 0.375 Å spacing between the grid points was used. AutoGrid was used to create affinity maps of the protein. One hundred Lamarckian genetic algorithm run with default parameter settings. Then, the interactions between the protein and the ligand in the complex were analyzed.

Statistical analysis

The data are presented as the means ± standard errors (SEM) and were visualized using GraphPad Prism 8 software (GraphPad Prism, San Diego, CA, USA). The statistical significance of differences between groups was determined using Student’s t test. One or two-way ANOVA followed by Tukey’s post hoc test was used to compare more than two groups of data. Differences were considered statistically significant at P < 0.05.

Results

Britannin suppressed NLRP3-mediated pyroptosis

To identify novel natural inhibitors that can effectively suppress NLRP3-mediated pyroptosis, we conducted a screening process to measure the activity of potential inhibitors our in-house library against IL-1β secretion by LPS + ATP-challenged BMDMs, which is widely recognized as a classical approach for activating the NLRP3 inflammasome (Fig. 1a). Surprisingly, 10 μM Britannin (Fig. 1b) exerted the greatest inhibitory effects on IL-1β among the 95 natural products, and the inhibition rate exceeded 80%. After the cells were treated with different concentrations of Britannin for 12 h, the safe range was determined to be less than 20 µM, as shown by cell survival (over 85%), indicating that Britannin exhibits good safety (Fig. 1c). Consequently, we verified the inhibitory effects of Britannin on the NLRP3 inflammasome. As shown in Fig. 1d, e, Britannin inhibited the secretion of cleaved Caspase-1 (p20) and mature IL-1β in a dose-dependent manner (1, 5, and 10 µM), suggesting that Britannin suppressed LPS + ATP-induced NLRP3 inflammasome in BMDMs. Additionally, a significantly low dose of Britannin inhibited IL-1β secretion by LPS + ATP-challenged BMDMs (IC₅₀ = 3.630 µM, Supplementary Fig. S1a). Furthermore, Britannin inhibited the release of LDH by BMDMs, indicating that Britannin prevented cell death (Fig. 1f). The cleavage of gasdermin D (GSDMD), which is responsible for executing pyroptosis, was inhibited by Britannin (Fig. 1g). In addition, Britannin inhibited the release of IL-1β and the cleavage of p20 induced by cytosolic LPS, suggesting that Britannin suppressed activation of Caspase-11-related noncanonical NLRP3 inflammasome (Supplementary Fig. S1b, c). These results suggest that the natural product Britannin inhibits pyroptosis by suppressing the NLRP3 inflammasome.

Fig. 1. Britannin suppressed NLRP3-mediated pyroptosis.

a BMDMs were primed with LPS for 3 h and stimulated with ATP for 0.5 h after treatment with 10 μM natural products (95 compounds) for 0.5 h, ELISA of IL-1β levels in culture supernatant (SN). The green point is Britannin. b Structure of Britannin. c BMDMs were treated with different concentrations of Britannin for 12 h, and the cell viability (compared to DMSO treatment) was determined using CCK-8 assay. d–g BMDMs were stimulated with 500 ng/mL LPS for 3 h, then were treated with or without Britannin at different doses for 0.5 h, and then were challenged with 2.5 mM ATP for 0.5 h. Western blotting assay for IL-1β and Caspase-1 (p20) levels in culture SN and pro-IL-1β, pro-Caspase-1 (pro-Casp-1), NLRP3, and β-Actin in lysates (LYS) of BMDMs (d). IL-1β production was assessed using ELISA in SN (e). n = 3. f Detection of the LDH release in SN of LPS-primed BMDMs treated with different concentrations of Britannin for 0.5 h and then stimulated with ATP for 1 h. n = 3. g Gasdermin D (GSDMD) and cleaved GSDMD (GSDMD NT) in LYS of LPS + ATP-challenged BMDMs with or without different doses of Britannin were detected by Western blotting assay. n = 3. Data are presented as the mean ± SEM, **P < 0.01, ***P < 0.001, significantly different; ns not significant, Bri Britannin.

Britannin specifically inhibited activation of the NLRP3 inflammasome

Next, the effects of Britannin on human cells were assessed. As shown in Fig. 2a, b, Britannin significantly suppressed the inflammatory response via the NLRP3 inflammasome in THP-1 cells. Similarly, Britannin robustly inhibited the activation of NLRP3 by other stimuli via different pathways, such as nigericin (Fig. 2c, d), Alum (Supplementary Fig. S2a, b), and MSU (Supplementary Fig. S2c, d). These results suggest that Britannin is a broad inhibitor of the NLRP3 inflammasome in BMDMs. To investigate the selectivity of Britannin for different kinds of inflammasomes, we assessed the inhibitory effect of Britannin on AIM2, NLRP1, and NLRC4 inflammasomes. Interestingly, Britannin had no effect on double-stranded DNA (for the AIM2 inflammasome), Val-boroPro (for the NLRP1 inflammasome), or S. typhimurium (for the NLRC4 inflammasome)-induced p20 cleavage and IL-1β secretion in BMDMs, indicating that Britannin is a specific inhibitor of the NLRP3 inflammasome (Fig. 2e, f, and Supplementary Fig. S3a–d).

Fig. 2. Britannin specifically inhibited activation of the NLRP3 inflammasome.

a, b PMA- differentiated THP-1 cells were treated with Britannin in different doses for 30 min and then were stimulated with 500 ng/mL LPS for 3 h and then 10 µM nigericin for 30 min. ELISA of IL-1β levels in culture SN (a). Western blotting assay for IL-1β and Caspase-1 (p10) levels in culture SN and pro-IL-1β, pro-Casp-1, NLRP3, and β-Actin in LYS (b). n = 3. c, d BMDMs were stimulated with 500 ng/mL LPS for 3 h, then were treated with or without Britannin at different doses for 0.5 h, and then were challenged with 10 µM nigericin for 0.5 h. IL-1β production was assessed using ELISA in SN (c). Western blotting assay for IL-1β and p20 levels in culture SN and pro-IL-1β, pro-Casp-1, NLRP3, and β-Actin in LYS (d). n = 3. e, f LPS-primed BMDMs were treated with or without Britannin for 0.5 h, and then were challenged with 0.5 µg/mL poly(dA:dT) for 4 h. IL-1β production was assessed using ELISA in SN (e). Western blotting assay for IL-1β and p20 levels in culture SN and pro-IL-1β, pro-Casp-1, and β-Actin in LYS (f). n = 3. g, h BMDMs were treated with different doses of Britannin before or after LPS challenge. After treatment, the cells were stimulated with ATP for 0.5 h. IL-1β production was assessed using ELISA in SN (g). Western blotting assay for IL-1β and p20 levels in culture SN and pro-IL-1β, pro-Casp-1, NLRP3, ASC and β-Actin in LYS (h). n = 3. Data are presented as the mean ± SEM, **P < 0.01, ***P < 0.001, significantly different; ns not significant, Bri Britannin.

Britannin has been reported to have anti-inflammatory effects on RAW 264.7 cells via NF-κB and MAPK signaling [18]. Thus, we used different administration models to determine whether Britannin inhibited NLRP3 inflammasome activation via NF-κB signaling in BMDMs. The “Britannin before LPS” model focused on the inhibitory effect of NF-κB signaling, and the “Britannin after LPS” model focused on the inflammasome activation step. As shown in Fig. 2g, h, the “before” and “after” models showed strong inhibition of p20 cleavage and IL-1β secretion. Unlike the latter, the “before” model showed significant suppression of pro-IL-1β and NLRP3 levels in cells, indicating that in “before” model, Britannin exerts its anti-inflammasome effect by decreasing the expression of inflammasome components. We also explored whether Britannin could suppress NF-κB signaling during LPS + ATP challenge in BMDMs, and the results showed that Britannin had no inhibitory effects on the level of phosphorylated-p65 or the release of TNF-α (Supplementary Fig. S3e, f). These data show that Britannin inhibits activation of the NLRP3 inflammasome in an NF-κB-independent manner.

Britannin inhibited inflammasome assembly via the interaction between NLRP3 and NEK7

Since Britannin was shown to be a specific inhibitor of activation of the NLRP3 inflammasome, we further explored how Britannin affected the NLRP3 inflammasome (Fig. 3a, b). We observed that Britannin failed to affect potassium and chloride efflux induced by ATP in BMDMs. Britannin also did no affect on mitochondrial reactive oxygen species production or mitochondrial damage in BMDMs (Fig. 3c). These results suggest that Britannin do not influence inflammasome activation through these upstream events.

Fig. 3. Britannin inhibited the inflammasome assembly via the interaction between NLRP3 and NEK7.

a LPS-primed BMDMs were treated with or without Britannin for 0.5 h, and then were challenged with 2.5 mM ATP for 0.5 h. Potassium concentrations in the BMDMs were detected by Automatic Biochemistry Analyzer. n = 3. b LPS-primed BMDMs were treated with or without Britannin for 0.5 h, and then were challenged with ATP at different times. The cells lysates were stained with MQAE, and then the fluorescence intensity was measured using multi-function measuring instrument. n = 3. c LPS-primed BMDMs were treated with or without Britannin for 0.5 h, and then were challenged with ATP for 0.5 h. BMDMs were stained with MitoSOX or MitoTracker, and then were observed using fluorescence microscope. The representative graphics are shown. Original magnification 200×. n = 3. d LPS-primed BMDMs were treated with or without Britannin for 0.5 h, following ATP challenge. Western blotting analysis of ASC oligomerization in 0.5% Triton X-100 lysis buffer (pellets). n = 3. e Immunofluorescence assay of ASC speck in LPS-primed BMDMs treated with different doses of Britannin for 0.5 h and then stimulated with ATP for 0.5 h. Original magnification 400×. n = 3. Co-immunoprecipitation (Co-IP) with NEK7 antibody (f) or ASC antibody (g). Western blotting analysis to evaluate the NLRP3-NEK7 or NLRP3-ASC interactions in LPS + ATP-challenged BMDMs treated with or without Britannin. n = 3. h Co-IP with Flag antibody and Western blotting analysis to evaluate the NLRP3-NEK7 interactions in HEK-293T cells transfected with the high expression plasmid and treated with Britannin for 16 h. n = 3. Data are presented as the mean ± SEM, ns not significant, Bri Britannin.

ASC is the adaptor of the inflammasome, and ASC speck formation participates in the assembly of the NLRP3 inflammasome, which is followed by Caspase-1 cleavage [3]. We found that Britannin suppressed ASC oligomerization in response to stimuli that activate NLRP3 in BMDMs (Fig. 3d). The effect of Britannin on ASC specks in BMDMs was also examined by immunofluorescence assays. The fluorescent spots representing ASC specks were suppressed by Britannin treatment (Fig. 3e). Consequently, we focused on the assembly of inflammasome components. We validated the inhibitory effect of Britannin on the interactions between the components. Interestingly, Britannin inhibited the interactions of NEK7-NLRP3 and ASC-NLRP3 in BMDMs, suggesting that Britannin inhibited assembly of inflammasome (Fig. 3f, g). To determine the exact step that Britannin affected, we constructed tagged plasmids of the components and simulated the interaction between each component in HEK-293T cells. Conversely, unlike NLRP3-NEK7 compound, Britannin did not hinder the interaction between ASC and NLRP3 in plasmids-transformed HEK-293T cells (Fig. 3h and Supplementary Fig. S4). These results suggest that Britannin suppresses activation of the NLRP3 inflammasome by impairing the assembly step, especially the interaction between NEK7 and NLRP3. Thus, we deduced that Britannin could directly bind to a certain component of the NLRP3 inflammasome.

Britannin directly binds to the NACHT domain of NLRP3

To investigate the specific protein to which Britannin binds to inhibited inflammasome activity, we performed a drug affinity responsive target stability (DARTS) assay on inflammasome components in BMDMs. As shown in Fig. 4a, NLRP3 but not NEK7 or ASC, exhibited enhanced preservation through its interaction with Britannin during pronase-driven proteolysis. Furthermore, we verified the result in plasmid-transfected HEK-293T cells. As expected, Britannin protected NLRP3, suggesting that its anti-inflammasome activity was mediated by directly binding to NLRP3 (Fig. 4b–d). NLRP3 consists of three domains, an amino-terminal pyrin (PYD) domain, a central nucleotide-binding and oligomerization (NACHT) domain, and a C-terminal leucine-rich repeat (LRR) domain. We constructed plasmids encoding each of these domains, and performed the DARTS assay on plasmid-transfected HEK-293T cells to investigate which domain Britannin specificly acted on. The results showed that Britannin significantly bound to the NACHT domain, which mediates the oligomerization of NLRP3 via its ATPase activity (Fig. 4e). Therefore, we prepared recombinant protein of human NACHT (rhNACHT), and performed the biolayer interferometry (BLI) assay to determine the affinity between Britannin and NACHT. As expected, Britannin showed great binding effect for rhNACHT, and the equilibrium dissociation constant (K_D) was 6.82 × 10^-5 M (Fig. 4f).

Fig. 4. Britannin directly binds to the NACHT domain of NLRP3.

a DARTS assay of NLRP3, NEK7, and ASC in LPS-primed BMDMs analyzed by Western blotting. n = 3. DARTS assay of Flag-NLRP3 (b), Flag-NEK7 (c), and Flag -ASC (d) in HEK-293T cells transfected with plasmids analyzed by Western blotting. n = 3. e DARTS assay of Flag-LRR domain, Flag-NACHT domain, and Flag-PYD domain in HEK-293T cells transfected with plasmids analyzed by Western blotting. n = 3. f The binding affinity of Britannin binding to rhNLRP3-NACHT was detected by BLI assay. g, h Molecular docking analysis of Britannin bound to NLRP3. Britannin (yellow, sticks model) in the binding site of NLRP3 (green, cartoon, PDB ID: 6NPY) (g). The hydrogen bonds formed between Britannin and Arg335 and Gly271 (green, sticks) (h). i DARTS assay of HA-NLRP3 mutant in HEK-293T cells transfected with plasmids analyzed by Western blotting. n = 3. j ATPase activity assay for NLRP3 in the presence of different concentrations of Britannin. n = 3. Data are presented as the mean ± SEM, ns not significant, Bri Britannin.

To determine of the specific site at which Britannin binds to NACHT, we simulated a docking model in which Britannin binds to NLRP3. The data showed that Arg335 and Gly271 had hydrogen bonds that bound to the drug, suggesting that the two sites play important roles in the process by which Britannin suppresses the inflammasome (Fig. 4g, h). To verify the molecular docking results, we constructed the site-mutated plasmids and performed the DARTS assay. Similar to the former results, the protective effect of Britannin disappeared from the NLRP3 G271A and R335A mutants in the DRATS assay, confirming that Britannin bound to Arg335 and Gly271 in NLRP3 NACHT domain (Fig. 4i). In light of the fact that the NACHT domain can bind with ATP and mediates the ATP-driven oligomerization of NLRP3, we further investigate the potential effect of Britannin on NLRP3 ATPase activity. As shown in Fig. 4j, Britannin did not alter the ATPase activity of NLRP3, suggesting that Britannin suppresses NLRP3 activation in an ATPase-independent manner.

Britannin prevented gouty arthritis by suppressing NLRP3 inflammasome activation

The overactivation of the NLRP3 inflammasome plays a crucial role in the pathology of acute gouty arthritis, which has been confirmed in human studies [19]. Therefore, we constructed an MSU-induced gouty arthritis model in wild-type (WT) mice to evaluate the therapeutic efficacy of Britannin in vivo. As expected, plantar injection of MSU induced footpad swelling in mouse model, and Britannin treatment significantly suppressed the changes in footpad joint size (Fig. 5a). Following successful modeling, we evaluated the levels of NLRP3-mediated inflammation in footpad tissues. First, we examined inflammatory cell infiltration in footpad tissues via H&E staining. The results showed that Britannin treatment alleviated the migration of inflammatory cells in MSU-challenged mice (Fig. 5b). Subsequently, the activation of NLRP3, which is characterized by the presence of the inflammatory cytokines IL-1β and Caspase-1, was analyzed. As shown in Fig. 5c, d, Britannin markedly inhibited the secretion of IL-1β and the activation of Caspase-1, demonstrating that Britannin prevented gouty arthritis through anti-inflammatory effects. To confirm the target protein of Britannin, gouty arthritis was also established in Nlrp3^KO mice. Consistent with previous studies, NLRP3 deficiency led to partial remission in joint swelling, inflammatory cell infiltration, cytokine secretion, and Caspase-1 activation (Fig. 5a–d). Moreover, the additional therapeutic effects of Britannin on MSU-challenged Nlrp3^KO mice were absent, indicating that Britannin relieves the gouty arthritis by inhibiting NLRP3 activation.

Fig. 5. Britannin prevented gouty arthritis by suppressing NLRP3 inflammasome activation.

a–d 10-week-old Nlrp3^KO and wild-type littermate control mice were injected i.p. with 20 mg/kg Britannin or vehicle control three times at an interval of a day. Then MSU or vehicle control was injected s.c. into the footpad to establish the gouty arthritis model. The footpad thicknesses were measured by vernier caliper (a). Representative H&E-stained histological footpad tissues sections (b). Original magnification 100×. ELISA of IL-1β in the joint culture SN (c). Activity of caspase-1 in the joint culture SN (d). Data are presented as the mean ± SEM, n = 6 per group. ***P < 0.001, significantly different; ns not significant, Bri Britannin.

Britannin inhibited NLRP3 activation to protect against acute lung injury

Next, we investigated the effect of Britannin on acute lung injury (ALI) in a mouse model. Excessive NLRP3 inflammasome activation could dysregulate inflammation, leading to multiple organ failure and death during the acute phase of the disease [20]. LPS was used to induce ALI in WT mice, and Britannin administration suppressed pulmonary edema, breakdown of the pneumatic-blood barrier, and cell infiltration in lung tissues (Fig. 6a–c). The lung injury scores of H&E–stained sections (Fig. 6d and Supplementary Fig. S5) and neutrophil influx (Fig. 6e), which were used to illustrate pathological injury and inflammatory cell infiltration in lung tissues, were ameliorated by Britannin treatment. However, the therapeutic effects of Britannin on the progressions of this disease were not observed in LPS-challenged Nlrp3^KO mice (Fig. 6a–e and Supplementary Fig. S5). Next, we examined NLRP3-related cytokines in BALF. The results showed that Britannin suppressed IL-1β levels in the BALF of WT mice, and the pharmacological effects were not observed in LPS-challenged Nlrp3^KO mice treated with Britannin (Fig. 6f). Furthermore, we examined systemic infection in ALI mice. Evidence showed that Britannin treatment reduced IL-1β but not TNF-α levels in serum (Fig. 6g, h). In summary, these data show that Britannin ameliorates lung injury and systemic infection in septic mice in an NLRP3-dependent manner.

Fig. 6. Britannin inhibited NLRP3 activation to protect against ALI.

a–h 10-week-old Nlrp3^KO and wild-type littermate control mice were injected i.p. with 20 mg/kg Britannin for three consecutive days, then were injected i.p. with 10 mg/kg LPS or vehicle for establishing septic ALI model. After 12 h, the mice were narcotized and sacrificed. Panels showing lung wet/dry ratio (a), amount of protein (b) and numbers of total cells (c) in BALF. Representative H&E-stained histological lung tissue sections (d). Original magnification 200×. Neutrophils in BALF (e). IL-1β in BALF were tested using ELISA (f). IL-1β (g) and TNF-α (h) in serum were tested using ELISA. Data are presented as the mean ± SEM, n = 6 per group. **P < 0.01, ***P < 0.001, significantly different; ns not significant, Bri Britannin.

Discussion

In this study, based on the screening of our natural product library, we identified Britannin as a potential traditional Chinese medicine drug candidate with anti-inflammatory effects. Britannin inhibited NLRP3 inflammasome activation and NLRP3-mediated pyroptosis in murine cells and human cells. Mechanistically, Britannin is a specific NLRP3 targeting inhibitor which showed excellent inhibitory effects on IL-1β with an IC₅₀ value of 3.6 µM in BMDMs. Britannin suppressed assembly of the NLRP3 inflammasome by inhibiting the formation of the complex of NLRP3 and NEK7. Furthermore, we found that Britannin directly bound to the NACHT domain of NLRP3 in an ATPase-independent manner and that Arg335 and Gly271 might be the critical residues in NLRP3 that mediate the effects of Britannin, as shown by molecular docking. In vivo studies showed that Britannin relieved NLRP3-mediated gouty arthritis. Moreover, Britannin showed a remarkable therapeutic effect on ALI and systemic infection in septic mice. Accordingly, these findings identified a novel natural inhibitor of NLRP3 and provided a new lead compound for the treatment of NLRP3-mediated inflammatory diseases.

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are pattern recognition receptors (PRRs) in the cytoplasm [21]. Most NLRs participate in the formation of inflammasomes, and the NLRP3 inflammasome is the most well-characterized. NLRP3 is associated with a wide range of diseases [22]. It has been well established that the NLRP3 inflammasome plays an important role in inflammatory diseases, such as diabetes and colitis [22]. In atherogenesis, the NLRP3 inflammasome is activated by crystalline cholesterol, and NLRP3-deficient mice show reduced levels of atherosclerotic lesions and IL-1β secretion [8]. Similarly, pharmacological inhibition of NLRP3 is effective in treating atherogenesis [23]. MSU crystals, which cause gout flares, are NLRP3 agonist that promote the IL-1β secretion and Capase-1 cleavage [24]. Zhong et al. demonstrated that MSU crystals and other particulate matters could induce different forms of necrosis including pyroptosis and necroptosis [25]. Indeed, we observed that NLRP3 knockout was partly, rather than completely, alleviated gouty arthritis, suggesting that MSU-induced NLRP3 activation is an important signaling pathway but not the only contributor to gouty arthritis. In chronic or acute inflammation, the release of proinflammatory cytokines, especially IL-1β, causes disease progression. In vivo studies have shown that the NLRP3 inflammasome is a considerable target in metabolic aseptic inflammation, such as gouty arthritis, and infectious inflammation. We noticed that noncanonical Caspase-11 inflammasome may also play an important role in the pathology of ALI [26]. High-dose LPS might be sufficient to trigger the noncanonical Caspase-11 inflammasome in vivo. Likewise, the NLRC4 inflammasome is clearly important for the clearance of intracellular bacterial infection in vitro [27]. It was found that Salmonella typhimurium promoted Caspase-1 activation and IL-1β secretion through the NLRC4 inflammasome in macrophages [28]. The AIM2 inflammasome, recognizes the double-stranded DNA of multiple bacterial and viral pathogens, and is mainly activated in infectious diseases [29]. Recently, a new role of AIM2 in cardiovascular disease has been discovered [30]. Therefore, inflammasomes might be novel targets in diseases in different areas. Further study of NLRs and the components in inflammasomes will be performed.

Pharmacological inhibition of NLRP3 is an explicit method for the therapies addressing of inflammatory diseases [31]. In the past decades, a few NLRP3 inhibitors have shown benefical effects on curing diseases in both models in vitro and in vivo, many of which have reached the clinical trials [32]. Based on the ATPase activity of NACHT domain, some drugs have been shown as competitive inhibitors of ATP. CRID3 (also named MCC950), which is the best inhibitor with nanomolar inhibitory activity, binds to the NACHT domain of NLRP3, inhibiting ATP-ADP exchange and ATP hydrolysis [33, 34]. However, drugs with a wide range of indications were stopped in phase II trials because of potential drug-induced liver injury [1]. OLT1177, another NLRP3 inhibitor in phase II/III trial, has excellent therapeutic effects on mouse LPS-induced systemic inflammation by inhibiting the ATPase activity of NLRP3 [32, 35], underscoring the druggability of NLRP3 as a target for treating a range of diseases. In our study, Britannin was recognized as an ATPase-independent inhibitor of NLRP3, and its inhibitory effects depend on blocking the NLRP3-NEK7 interaction. We will further explore the development and application of inhibitors based on the NLRP3-NEK7 interaction to treat NLRP3-related diseases.

In our study, we showed for the first time that Britannin exerted anti-inflammasome activity in vitro and in vivo. The pharmacological effects of Britannin on previously published studies had focused on inhibiting cancers. A previous study reported that Britannin suppressed the invasion and metastasis of triple-negative breast carcinomas cells by degrading ZEB1, which might be used to prevent tumor metastasis and recurrence [13]. Moeinifard et al. reported that Britannin induced mitochondrial apoptosis through ROS production and modulation of the AKT-FOXO1 signaling axis in human pancreatic cancer cells [14]. Inula japonica Thunb, from which Britannin is isolated, is commonly used as a traditional Chinese medicine. The use of this medicine to treat respiratory diseases can be traced back to the Treatise on Febrile Diseases, one of the earliest complete clinical texts in the world. Although Britannin was reported to exert anti-inflammatory effects via NF-κB and MAPK inactivation in RAW 264.7 cells [18], the mechanism and the targets of Britannin in vivo remain unclear. Our study showed that Britannin suppressed NLRP3 inflammasome activation and assembly by blocking the interaction between NLRP3 and NEK7, elucidating the anti-inflammatory mechanism of Britannin. We also showed for the first time that Britannin alleviated inflammatory diseases, such as MSU-induced gouty arthritis and LPS-induced ALI in mouse models. Furthermore, the multiple anti-inflammatory activities and potential multitarget effects of Britannin, particularly its synergistic inhibition of inflammation-related signals, may enhance its efficacy as a clinical treatment. Therefore, the study of drug development based on Britannin, including derivative construction, the discovery of new inflammatory indications, and more discoveries about the anti-inflammatory mechanisms of natural products, will continue.

In this study, we identified Britannin, which is a natural product derived from traditional Chinese medicine, that is a novel NLRP3 NACHT targeting inhibitor. Britannin efficiently suppressed IL-1β secretion and Caspase-1 cleavage and inhibited assembly of the NLRP3 inflammasome by blocking the interaction between NLRP3 and NEK7 in an ATPase-independent manner. Arg335 and Gly271 in the NACHT domain might be the specific binding sites that mediate Britannin’s inhibitory activity. Britannin showed effective therapeutic effects on mouse models of human diseases, such as gouty arthritis and ALI. Britannin is a potential lead compound for the design and development of novel NLRP3 inhibitors.

Author contributions

GL and HWX contributed to the literature search and study design. HWX and JJS participated in the drafting of the article. JJS, WFL, JFS, ZSZ, JLAM, and XHL carried out the experiments. GL and HWX revised the manuscript. JFS and GJW contributed to data collection and analysis.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-023-01212-5.