Glaucocalyxin A alleviates LPS-mediated septic shock and inflammation via inhibiting NLRP3 inflammasome activation

International Immunopharmacology 81 (2020) 106271

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Glaucocalyxin A alleviates LPS-mediated septic shock and inflammation via inhibiting NLRP3 inflammasome activation

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Xiaorong Houa,b,1, Guang Xub,1, Zhilei Wangb,d,1, Xiaoyan Zhanb,c, Huifang Lia, Ruisheng Lie, ⁎ Wei Shib,f, Chunyu Wangb,g, Yuanyuan Chenb,h, Yongqiang Aib,f, Xiaohe Xiaoa,b, , ⁎ Zhaofang Baib,c, a

Institute of Pharmaceutical & Food Engineering, Shanxi University of Traditional Chinese Medicine, Jinzhong 030619, China China Military Institute of Chinese Materia, The Fifth Medical Centre, Chinese PLA General Hospital, Beijing 100039, China c Integrative Medical Center, The Fifth Medical Centre, Chinese PLA General Hospital, Beijing 100039, China d School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China e Research Center for Clinical and Translational Medicine, The Fifth Medical Centre, Chinese PLA General Hospital, Beijing 100500, China f School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China g College of Pharmacy, Jinzhou Medical University, Jinzhou 121000, China h School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Glaucocalyxin A NLRP3 inflammasome Caspase-1 IL-1β Septic shock

Glaucocalyxin A (GLA) is a bioactive ent-kauranoid diterpenoid derived from the herbal medicine, Rabdosia japonica var. glaucocalyx, and it has been reported to possess marked anti-inflammatory properties. However, the underlying mechanisms are not fully understood. Here, we reported that GLA dramatically inhibited canonical and non-canonical NLRP3 inflammasome activation induced by multiple agonists. In addition, GLA also blocked NLRC4 inflammasome activation but had no effect on AIM2 inflammasome. Furthermore, we found that GLA inhibited NLRP3 or NLRC4 agonists-induced ASC oligomerization, which is an upstream event of the inflammasomes assembly. Most importantly, administration of GLA significantly reduced lipopolysaccharide (LPS)-induced mortality in septic-shock mouse model. Additionally, GLA dose-dependently inhibited the production of interleukin (IL)-1β, but had no effect on NLRP3-independent TNF-α production induced by LPS in vivo. In conclusion, our study suggests that GLA alleviates LPS-induced septic shock and inflammation via inhibiting NLRP3 inflammasome activation and provides a promising candidate drug for the treatment of NLRP3driven diseases.

1. Introduction Inflammasomes are a group of cytosolic protein complexes that lead to innate immune responses to microbial infection and endogenous damage. Inflammasome assembly requires the nucleotide-binding-andoligomerization domain (NOD) and leucine-rich-repeat–containing (NLR) family or pyrin domain (PYD) and HIN domain–containing (PYHIN) family in response to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) that

control the production of interleukin-1β (IL-1β) and IL-18 [1,2]. NLR family members—including NLRP1, NLRP3, NLR family CARD domain containing 4 (NLRC4), and NLRP6—and the PYHIN family member, absent in melanoma 2 (AIM2), have all been demonstrated to form inflammasomes [3,4]. The NLRP3 inflammasome is the most comprehensively and clearly investigated member among these inflammasomes. The NLRP3 inflammasome consists of the sensor protein, NLRP3, the adapter protein, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), and pro-

Abbreviations: NOD, nucleotide-binding oligomerization domain; NLR, NOD like receptor; NLRP3, NOD–like receptor family pyrin domain–containing 3; PYHIN, pyrin domain (PYD) and HIN domain–containing (PYHIN) family; PAMP, pathogen-associated molecular pattern; DAMP, damage-associated molecular pattern; NLRC4, NLR family CARD domain containing 4; AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain; NF-κB, nuclear factor-kappa B; LPS, lipopolysaccharide; IL, interleukin; DSS, dextran sulfate sodium; CAPS, cryopyrin-associated periodic syndrome; IκB, inhibitor κB ⁎ Corresponding authors at: The Fifth Medical Centre, Chinese PLA General Hospital, No. 100 West 4th Ring Middle Road, Fengtai District, Beijing 100039, China. E-mail addresses: [email protected] (X. Xiao), [email protected] (Z. Bai). 1 These authors made equal contributions to this work. https://doi.org/10.1016/j.intimp.2020.106271 Received 11 October 2019; Received in revised form 20 January 2020; Accepted 29 January 2020 1567-5769/ © 2020 Elsevier B.V. All rights reserved.

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2.2. Mice

caspase-1 [5]. The activation of the NLRP3 inflammasome requires two signals. Signal 1, also known as the priming signal, is mediated by microbial ligands recognized by toll-like receptors (TLRs) such as LPS [4,6]. Signal 1 activates the NF-κB pathway, which in turn upregulates the transcription of inflammasome-related components, including inactive NLRP3 and pro-IL-1β [7,8]. Basal level of pro-IL-1β is very low in unstimulated cells. Signal 2 (the activation signal) is mediated by the stimulation of numerous PAMPs or DAMPs, such as extracellular ATP, MSU crystal, and the pore-forming toxin, nigericin [9–11]. Once activated, NLRP3 recruits ASC and pro-caspase-1 and promotes the autocatalytic activation of caspase-1. Caspase-1 leads to pyroptosis and the cleavage of biologically inactive pro-IL-1β and pro-IL-18 into mature and functional IL-1β and IL-18, respectively [8,12,13]. Many studies have demonstrated that the aberrant activation of the NLRP3 inflammasome is involved in the pathogenesis of several metabolic and inflammatory diseases, such as type-2 diabetes [14], colitis [15], idiosyncratic liver injury [16], and septic shock [17,18]. Some small molecules play an important role in treating various NLRP3driven diseases, including MCC950 [19] and β-hydroxybutyrate [20]. It has been reported that curcumin derived from the herb, Curcuma longa, suppresses dextran sulfate sodium (DSS)-induced NLRP3 inflammasome activation and alleviates DSS-induced colitis in mice [21]. Tranilast targeting the NLRP3 inflammasome has preventive effects in mouse models of gouty arthritis and cryopyrin-associated periodic syndrome (CAPS) [22]. Cardamonin as a specific inhibitor of the NLRP3 inflammasome and protects against LPS-induced septic shock [23]. Thus, the NLPR3 inflammasome has been considered as a potential drug target for the treatment of many diseases. Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid isolated from Rabdosia (R.) japonica var. glaucocalyx, which is an herbal medicine distributed widely in East Asia [24]. It has been reported that GLA has wide range of biological activities, such as antibacterial, anti-oxidative, anti-coagulative, immune, and anti-neuroinflammatory activities [25]. It has been documented that GLA attenuates lipopolysaccharide (LPS)-mediated neuroinflammation by inhibiting NF-κB and p38 MAPK signaling pathways [26]. Furthermore, GLA also suppresses nuclear-factor-κB activation through blocking degradation of inhibitor κB (IκB)-α in LPS-stimulated BV-2 cells. Until now, the antiinflammatory mechanism of GLA has been mainly attributed to the inhibition of NF-κB signaling pathways, but whether GLA blocks NLRP3 inflammasome activation and protects against NLRP3-driven diseases remains unclear. In the present study, we found that GLA dramatically inhibited activation of NLRP3 and NLRC4 inflammasomes but had no effect on AIM2 inflammasome activation. More importantly, we found that GLA had preventative and beneficial effects on NLRP3-driven septic shock and inflammation in vivo.

Pathogen-free female C57BL/6 mice (6–8 weeks old; 18–20 g) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). Mice were housed in a specific pathogen-free (SPF) standard room with a 12-h light-dark cycle at a temperature of 21–25 °C and were given free access to a standard laboratory diet and water for the duration of the experiment, except during fasting tests. Protocols for the animal experiments were permitted by the guidelines for the care and use of laboratory animals. All of the experiments were performed in accordance with the approved guidelines of the animal ethics committee of the Fifth Medical Centre, Chinese PLA General Hospital. 2.3. Cell culture Mouse bone marrow-derived macrophages (BMDMs) were prepared from the femurs of C57BL/6 mice and cultured in Dulbecco’s modified eagle’s medium (DMEM; Macgene, Beijing, China) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, CA, USA), 1% penicillin and streptomycin (100 µg/mL; Sigma-Aldrich), and 50 ng/mL murine macrophage colony-stimulating factor (M-CSF; R&D systems, MN, USA). Cultures were stimulated at 5–7 days after differentiation. Human THP-1 cells were obtained from ATCC (Shanghai, China) and maintained in RPMI 1640 complete medium (Macgene, Beijing, China) containing 10% FBS supplemented with 100 nM of PMA overnight to differentiate into macrophages. All of the cell lines were incubated in a humidified 5% CO2 atmosphere at 37 °C. 2.4. Salmonella typhimurium infection in vitro Salmonella was kindly provided by Dr. Tao Li from the National Center of Biomedical Analysis. The protocol for Salmonella typhimurium infection has been detailed previously [27]. 2.5. Inflammasome activation Specifically, 9 × 105 cells/mL of BMDMs and 1.5 × 106 cells/mL of THP-1 cells were plated in 24-well plates for 12–18 h. The overnight medium was replaced with fresh medium, and cells were treated with 50 ng/mL of LPS or 1 μg/mL of Pam3CSK4 (for non-canonical inflammasome activation) for 4 h. After that, the medium was changed with Opti-MEM (Gibco, New York, USA) containing GLA (0.625, 1.25, or 2.5 μM) for 1 h. To induce NLRP3 inflammasome activation, cells were triggered with the following inflammasome activators: 10 μM of nigericin (1 h), 5 mM of ATP (1 h), 200 μg/mL of MSU (6 h), or 2 μg/mL of poly (I:C) transfected with Lipofectamine 2000 for 6 h according to the product manual. For AIM2 and noncanonical inflammasome activation, cells were transfected with poly (dA:dT) (2 μg/mL) and ultrapure LPS (1 μg/mL) for 6 h by using Lipofectamine 2000 (Invitrogen, CA, USA). For NLRC4 inflammasome activation, cells were stimulated with Salmonella typhimurium (MOI 50–100) for 6 h.

2. Materials and methods 2.1. Drug, reagents and antibodies

2.6. Western blotting

GLA was purchased from TargetMol (Boston, MA, USA). Nigericin, monosodium urate crystals (MSU), adenosine triphosphate (ATP), polyinosinic:polycytidylic acid (poly (I:C)), poly (deoxyadenylic-deoxythymidylic; poly [dA:dT]), phorbol 12-myristate 13- acetate (PMA), dimethyl sulfoxide (DMSO), and ultrapure lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (Munich, Germany). Pam3CSK4 was purchased from InvivoGen (Toulouse, France). Anti-mouse CASPASE-1 (1:1000, AG-20B-0042), anti-ASC (1:1000, sc-22514-R) and antiGAPDH (1:2000, 60004-1-1g) were purchased from Adipogen (San Diego, CA, USA), Santa Cruz Biotechnology (CA, USA) and Proteintech Group (Chicago, IL, USA), respectively. Anti-NLRP3 (1:2000, 15101S), anti-mouse IL-1β (1:1000, 12507), anti-human CASPASE-1 (1:2000, 4199S), and anti-human cleaved IL-1β (1:2000, 12242) were obtained from Cell Signaling Technology (CST, MA, USA).

Cell extracts and precipitated supernatants were detected by immunoblotting. Processing of protein samples from cell culture supernatants have been described previously [28], and the protein samples were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After cells were washed twice with phosphate buffered saline (PBS; Macgene, Beijing, China), the whole cells were lysed by using cold RIPA Lysis buffer (Thermo Scientific; IL, USA), and were then collected and boiled in SDS sample buffer (Dingguo, Beijing, China) and resolved on 10% SDS–PAGE. Subsequently, samples were then transferred onto 0.45-μm polyvinylidene difluoride membranes (PVDF, Millipore, MA, USA) in a wet system. Blots were blocked for 1 h in TBST (GenStar, CA, USA) containing 5% non-fat dried milk at 2

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room temperature and were hybridized with specific primary antibodies overnight at 4 °C. The membranes were washed three times with TBST and were then incubated with peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Reactive signals were visualized with enhanced chemiluminescent reagents (ECL; Promega, Beijing, China) detection system. All of the blots were normalized with GAPDH to ascertain equal loading of proteins.

Differences between the experimental groups were considered statistically significant as follows P < 0.05(*), P < 0.01(**), and P < 0.001(***).

2.7. Caspase-1 activity assay

To evaluate the effect of GLA on NLRP3 inflammasome activation, we first tested whether GLA could inhibit the production of IL-1β and caspase-1 triggered by NLRP3 inflammasome agonists. The CaspaseGlo® 1 Inflammasome Assay is a homogeneous, bioluminescent method to selectively measure the activity of caspase-1, and it is often used to evaluate the activation of inflammasomes [17,32]. When LPS-primed BMDMs were stimulated with nigericin, caspase-1 activity in the supernatant increased noticeably. Consistent with the results of caspase-1 activity, the protein expression of caspase-1 are significantly increased, as shown by the western blotting assay under the same conditions. Caspase-1 activation can also directly induce a distinct form of programmed cell death (pyroptosis), accompanied by the release of lactate dehydrogenase (LDH) [33,34], which is a widely used marker in cytotoxicity studies. When LPS-primed BMDMs were pretreated with GLA before nigericin stimulation, we observed that GLA dose-dependently suppressed caspase-1 activation, IL-1β secretion, and LDH release (Fig. 1B–E and Suppl. Fig. 2) but had no effect on NLRP3-independent cytokine TNF-α production (Fig. 1F). In addition, NLRP3, pro-caspase-1 (p45), pro-IL-1β, and ASC expression in whole cell lysates were not impaired by GLA (Fig. 1B). We further tested the effect of GLA in human THP-1 cells. The results showed that nigericin-induced caspase1 activation and IL-1β production in PMA-differentiated THP-1 cells were also inhibited by GLA in a dose-dependent manner (Fig. 1G–J). Similarly, GLA did not alter TNF-α production in PMA-differentiated THP-1 cells (Fig. 1K). These data illuminate that GLA had a robust inhibitory effect on NLRP3 inflammasome in BMDMs and human THP-1 cells. To further clarify the influence of GLA on NLRP3 inflammasome activation, we stimulated LPS-primed BMDMs with other NLRP3 inflammasome agonists, including ATP, MSU crystals, and poly (I:C) in the presence or absence of GLA. Consistent with the effect of GLA on nigericin-induced NLRP3 inflammasome activation, GLA strongly inhibited caspase-1 activation and IL-1β secretion triggered by all the tested agonists (Fig. 2A, C, D). Similarly, GLA did not exhibit an obvious inhibitory effect on TNF-α production (Fig. 2E). These results suggest that GLA is a broad-spectrum inhibitor of the NLRP3 inflammasome.

3. Results 3.1. GLA suppresses canonical NLRP3 inflammasome activation in BMDMs and human THP-1 cells

Caspase-1 activity detected in the medium can be tested through assays that transfer supernatant via the Caspase-Glo® 1 Inflammasome Assay (Promega, Beijing, China). This monitoring system provides a rapid and convenient method to specifically and quantitatively detect caspase-1 activation in cells in a plate-based format [29]. 2.8. Lactate dehydrogenase (LDH) assay Cells treated with inflammasome stimulants cause cell death. Quantification of lactate dehydrogenase (LDH) is a well-established assay for cell viability [30]. LDH released into the culture medium was measured by the LDH cytotoxicity assay kit (Promega, Beijing, China) according to the manufacturer’s indications. 2.9. Cytokine analysis via enzyme-linked immunosorbent assay (ELISA) The secretion levels of mouse and human TNF-α and IL-1β in culture medium or in serum and peritoneal lavage fluid were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Dakewe, Beijing, China and R&D systems, MN, USA), according to the manufacturer’s protocol. Levels of cytokines are reported in pg/mL. All of the assays were performed in triplicates. 2.10. ASC oligomerization assay The assay for ASC oligomerization has been described previously [31]. 2.11. Immunofluorescence The protocols for immunofluorescence have been described previously [22]. 2.12. LPS-induced mouse septic-shock model in vivo GLA was dissolved in 10% DMSO and was then diluted in sterile saline. Mice were allowed to adapt to a laboratory environment for at least three days before the start of experiments. Experimental animals were divided into three groups (n = 10/group). Mice were injected intraperitoneally with 20 mg/kg and 40 mg/kg of GLA or vehicle control for 1 h before injection of 20 mg/kg LPS (L2880, SigmaAldrich), and their health status and living conditions were observed at regular intervals. In the next experiment, the route of administration to mice (n = 6/group) followed the same operation as that above. After 2 h of injection of LPS, serum was collected, and 10 mL of ice-cold PBS was utilized to wash the peritoneal cavities [10]. The levels of IL-1β and TNF-α in sera and peritoneal lavage fluid were detected by ELISAs.

3.2. GLA inhibits activation of non-canonical NLRP3 and NLRC4 inflammasomes but has no effect on AIM2 inflammasome activation Intracellular LPS derived from Gram-negative bacteria is sensed by the non-canonical NLRP3 inflammasome, the activation of which results in caspase-11-dependent production of IL-1β and IL-18 [35,36]. Additionally, NLRP3, NLRC4, and the cytosolic receptor, AIM2, are crucial components of inflammasomes and link microbial infections and endogenous danger signals to the activation of caspase-1 [1]. Next, we examined whether GLA could inhibit the non-canonical NLRP3 inflammasome, as well as inhibit the activation of NLRC4 and AIM2 inflammasomes. LPS-primed BMDMs were stimulated with Salmonella typhimurium infection and poly (dA:dT) transfection to activate the NLRC4 and AIM2 inflammasomes, respectively. Pam3CSK4-primed BMDMs were transfected with LPS to active the non-canonical NLRP3 inflammasome. GLA significantly prevented caspase-11-dependent caspase-1 cleavage and IL-1β secretion (Fig. 2B, F-G), suggesting that GLA restrained activation of the non-canonical NLRP3 inflammasome. Results also showed that GLA blocked maturation of caspase-1 (p20) and IL-1β (p17) triggered by Salmonella typhimurium infection,

2.13. Statistical analyses GraphPad Prism software, version 6, was used for statistical analysis. All of the data are expressed as the mean ± standard error of the mean (s. e. m.) of a representative experiment performed in triplicate. Statistical analyses were carried out using unpaired two-tailed Student’s t-tests to analyze the significant differences between two groups. 3

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Fig. 1. GLA suppresses canonical NLRP3 inflammasome activation in BMDMs and human THP-1 cells. (A) GLA structure (B) LPS-primed BMDMs treated with different concentrations (0.625, 1.25, 2.5 μM) of GLA for 1 h and then stimulated with nigericin for 1 h. Western blot analysis of caspase-1 (p20), IL-1β (p17) in culture supernatants (Sup.) and caspase-1 (p45), pro-IL-1β, NLRP3, ASC in cell lysates (Lys.). (C-E) Activity of caspase-1 (C), ELISA of IL-1β (D), LDH release assay (E) and TNF-α (F). (G) Western blot analysis of caspase-1 (p20), IL-1β (p17) in culture supernatants (Sup.) and caspase-1 (p45), pro-IL-1β, NLRP3, ASC in cell lysates (Lys.) in culture supernatants (Sup.) from PMA-differentiated human THP-1 cells stimulated with nigericin and treated with GLA (2.5, 5, 10 μM). (H-K) Activity of caspase-1, production of IL-1β, LDH and TNF-α in culture supernatants (Sup.) described in (G) were measured by Caspase-1 activity assay(H), ELISA (I, K) and LDH assay (J). Coomassie blue staining is provided as the loading control for the Sup. (B, G). GAPDH serves as a loading control in Lys. (B, G). Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate (C-F, H-K). P < 0.05(*), P < 0.01(**), and P < 0.001(***) vs. the control by unpaired two-tailed Student’s t-tests. NS: not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 4

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Fig. 2. GLA inhibits activation of non-canonical NLRP3 and NLRC4 inflammasomes but has no effect on AIM2 inflammasome activation. (A) Western blot analysis of caspase-1 (p20), IL-1β (p17) in culture supernatants (Sup.) and caspase-1 (p45), pro-IL-1β, NLRP3, ASC in cell lysates (Lys.) from LPS-primed BMDMs treated with GLA (2.5 μM) and stimulated with ATP, nigericin, MSU, poly(I:C). (B) Western blot analysis of caspase-1 (p20), IL-1β (p17) in culture supernatants (Sup.) and caspase-1 (p45), pro-IL-1β, NLRP3, ASC in cell lysates (Lys.) from LPS-primed BMDMs treated with GLA (2.5 μM) and then stimulated with nigericin, poly (dA:dT) and Salmonella, or Pam3CSK4-primed BMDMs treated with GLA (2.5 μM) and then stimulated with intracellular LPS. (C-E) Activity of caspase-1 (C), ELISA of IL-1β (D), TNF-α (E) in Sup. from BMDMs described in (A). (F-H) Activity of caspase-1 (F), ELISA of IL-1β (G), TNF-α (H) in Sup. from BMDMs described in (B). Coomassie blue staining is provided as the loading control for the Sup. (A, B). GAPDH serves as a loading control in Lys. (A, B). Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate (C-H). P < 0.05(*), P < 0.01(**), and P < 0.001(***) vs. the control by unpaired two-tailed Student’s t-tests. NS: not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

findings suggest that GLA inhibited activation of NLRP3 and NLRC4 inflammasomes but had no effect on activation of the AIM2 inflammasome in vitro.

indicating that GLA also suppressed activation of the NLRC4 inflammasome. However, we found that GLA did not inhibit poly (dA:dT)-mediated caspase-1 (p20) production or IL-1β secretion (p17), suggesting that GLA had no effect on the activation of the AIM2 inflammasome. The expression of NLRP3, pro-caspase-1 (p45), pro-IL-1β, and ASC in cell lysates and the production of TNF-α in cell supernatants were not influenced by GLA treatment (Fig. 2B, H). Collectively, these 5

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Fig. 3. GLA has no effect on upstream signaling of NLRP3 inflammasome activation. (A) Western blot analysis of the indicated proteins in Lys. from BMDMs stimulated with LPS for 4 h and remove the supernatant, then treated with different doses of GLA (0.625, 1.25, 2.5 μM) for 2 h (GLA after LPS), or BMDMs treated with different doses of GLA (0.625, 1.25, 2.5 μM) for 2 h and then stimulated with LPS for 4 h (GLA before LPS). GAPDH was used as an internal control. (B) Production of TNF-α (as measured by ELISA) in Sup. from BMDMs described in (A). (C) Immunofluorescence was performed to analyze mitochondrial damage from LPS-primed BMDMs treated with GLA (2.5 μM) and next stimulated with nigericin. Data shown represent the mean ± SEM of three independent experiments carried out in triplicate (B). P < 0.05(*), P < 0.01(**), and P < 0.001(***) vs. the control by unpaired two-tailed Student’s t-tests. NS: not significant.

LPS treatment in subsequent experiments. In addition, mtDNA and reactive oxygen species (mtROS) acting as upstream events also drive the activation of the NLRP3 inflammasome [38]. It has been demonstrated that mitochondrial DNA released by mitochondrial damage is linked with NLRP3 activation [39]. Thus, we examined whether GLA inhibited NLRP3 inflammasome activation by regulating mitochondrial damage. The degree of mitochondrial damage was assayed by immunofluorescence. As shown in Fig. 3C, nigericin treatment led to mitochondrial damage, but GLA had no effect on nigericin-induced mitochondrial damage. These results suggest that GLA did not affect mitochondrial damage, which is one of the important upstream events of NLRP3 activation.

3.3. GLA has no effect on upstream signaling of NLRP3 inflammasome activation The expression of NLRs and pro-IL-1β are induced by priming with TLR ligands, such as LPS and Pam3CSK4. Earlier studies have shown that GLA inhibits TLR-dependent NF-κB signaling pathways [26]. We next tested whether GLA affected LPS-mediated expression of NLRP3 and pro-IL-1β. When BMDMs were treated with GLA for 2 h before LPS stimulation for 4 h, the protein expression levels of NLRP3, pro-IL-1β, and TNF-α were detected. Pretreatment with GLA dose-dependently inhibited the expression levels of NLRP3, pro-IL-1β, and the production of TNF-α in LPS-stimulated BMDMs (Fig. 3A, B and Suppl. Fig. 1A). However, when BMDMs were triggered with LPS for 4 h and then treated with GLA for 2 h, the expression levels of NLRP3, pro-IL-1β, and the production of TNF-α were not affected by GLA. To further elucidate the influence of GLA on NLRP3 inflammasome and TLR4 signaling pathway, BMDMs were treated with GLA, Resatorvid (a small-moleculespecific inhibitor of TLR4 signaling) [37] or MCC950 (a specific inhibitor of NLRP3 inflammasome) [17] for 2 h, and then the cells were stimulated with LPS for 4 h. Consistent with previous studies [17,26,37], MCC950 has no impact on the expression of pro-IL-1β and NLRP3, and GLA and Resatorvid inhibited the expression of pro-IL-1β and NLRP3 significantly, suggesting that GLA and Resatorvid indeed suppressed NF-κB signaling (Suppl. Fig. 1A). However, MCC950, GLA and Resatorvid had no effect on LPS-induced NLRP3 and pro-IL-1β expression, when BMDMs were primed with LPS for 4 h and treated with these drugs. In addition, unlike Resatorvid, GLA and MCC950 suppressed IL-1β secretion and caspase-1 activation when LPS-primed BMDMs were treated these drugs and then stimulated with nigericin (Suppl. Fig. 1C). It can be seen that the inhibitory effect of GLA on NLRP3 inflammasome activation was not caused by the downregulation of NLRP3 or pro-IL-1β via the TLR4 signaling pathway under these conditions. Taken together, the results suggested that, although GLA influences TLR4 signaling pathway, GLA-induced suppression of the NLRP3 inflammasome activation does not occur via the TLR4 signaling pathway. In order to clarify the mechanisms underlying GLA-induced inflammasome inhibition, we stimulated BMDMs with GLA after 4 h of

3.4. GLA inhibits ASC oligomerization ASC oligomerization is an essential step for NLRP3 and AIM2 inflammasome activation [40,41]. Although ASC oligomerization is not necessary for NLRC4 inflammasome activation, it can enhance NLRC4 inflammasome activation. To further elucidate how GLA inhibits inflammasome activation [42], we assessed the effect of GLA on ASC oligomerization. LPS-primed BMDMs or PMA-primed THP-1 cells were treated with GLA or vehicle and were then stimulated with nigericin. Cytosolic fractions from cell lysates were cross-linked and ASC monomers and higher-order complexes in the lysate were detected via immunoblotting. We observed that GLA treatment dose-dependently inhibited ASC oligomerization triggered by nigericin in LPS-primed BMDMs or PMA-primed THP-1 cells (Fig. 4A-B). We next investigated the effect of GLA on ATP, MSU, and poly (I:C)-induced ASC oligomerization in LPS-primed BMDMs (Fig. 4C), and the results showed that GLA also inhibited all examined agonist-induced ASC oligomerizations. Taken together, these results suggest that GLA inhibited ASC oligomerization during activation of the canonical NLRP3 inflammasome. Additionally, we also found that GLA strongly prevented ASC oligomerization induced by Salmonella infection and intracellular LPS but had no effect on poly (dA:dT)-induced ASC oligomerization (Fig. 4D). These data indicate that GLA suppressed inflammasome activation by blocking ASC oligomerization. 6

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Fig. 4. GLA inhibits ASC oligomerization. (A) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellets and cell lysates of LPS-primed BMDMs stimulated with nigericin in the presence or absence of GLA (0.625, 1.25, 2.5 μM). (B) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellets and cell lysates of PMA-differentiated human THP-1 cells treated with GLA (2.5, 5, 10 μM) and then stimulated with nigericin. (C) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellets and cell lysates of LPS-primed BMDMs treated with GLA (2.5 μM) or vehicle and next stimulated with ATP, MSU, and poly (I:C). (D) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellets and cell lysates of LPS-primed BMDMs treated with GLA (2.5 μM) and then stimulated with poly (dA:dT) transfection and Salmonella infection, or Pam3CSK4-primed BMDMs treated with GLA (2.5 μM) and then stimulated with intracellular LPS. GAPDH serves as a loading control in Lys.

3.5. GLA attenuates LPS-induced septic shock and inflammation in vivo

4. Discussion

Since GLA abrogated NLRP3 inflammasome activation in vitro, we next examined the activity of GLA in vivo. The sepsis and production of IL-1β induced by intraperitoneal injection of LPS is shown to be NLRP3 inflammasome-dependent [17,43–45]. Evidences also show that genetic deficiency of NLRP3 inhibited inflammatory responses and enhanced survival of septic mice [46]. To investigate the role of GLA in a mouse model of LPS-induced septic shock, we first assessed the survival rate of LPS (20 mg/kg)-induced septic shock mice in the presence or absence of GLA. GLA clearly attenuated the mortality of mice in a dosedependent manner (Fig. 5A). Additionally, we further found that GLA inhibited the production of IL-1β in the peritoneal lavage fluid and sera of mice (Fig. 5B, D) but did not considerably diminish the amount of TNF-α (Fig. 5C, E). These results suggest that NLRP3 inflammasome activation, which causes IL-1β secretion, occurs during LPS-mediated septic shock and also demonstrate that GLA attenuated LPS-induced septic shock and inflammation in vivo via inhibiting NLRP3 inflammasome.

Rabdosia (R.) japonica var. glaucocalyx (Lan’e’xiangchacai in Chinese) has been common in traditional folk medicine for centuries; this herb remains widely used to treat various inflammation [47]. Glaucocalyxin A, a major active compound in R. japonica, had noticeable immune and anti-neuroinflammatory effects [25]. However, the mechanisms underlying these effects are not well understood. Thus, the clarification of the anti-inflammatory mechanisms of this compound may support clinical application. It has been shown that inappropriate activation of NLRP3 inflammasome can cause severe inflammation during the pathogenesis of various human inflammatory diseases [15,48,49]. Thus, NLRP3 inflammasome may be a promising target of anti-inflammatory therapies. In this study, we aimed to explore whether GLA exerts an immunosuppressive effect via NLRP3 inflammasome signaling pathway. Here, GLA blocked canonical and non-canonical NLRP3 inflammasome activation in LPS-primed BMDMs and PMA-primed THP-1 cells. Additionally, GLA had a strong inhibitory effect on NLRC4 inflammasome but no inhibitory effect on AIM2 inflammasome activation. These results suggested that GLA is the inhibitor of NLRP3 and NLRC4 inflammasomes activation, and could be used to treat diseases caused by 7

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Fig. 5. GLA attenuates LPS-induced septic shock and inflammation in vivo. (A) Survival of 6–8 weeks old female C57BL/6 mice injected intraperitoneally with LPS (20 mg/kg of body weight) in the presence of GLA (20 and 40 mg/kg of body weight). Lethality was recorded for 72 h. (B-C) levels of IL-1β (B) and TNF-α (C) in the peritoneal lavage fluid from C57BL/6 mice pretreated with GLA or vehicle control (1 h) and intraperitoneal LPS (20 mg/kg) injection (2 h)were measured in control, LPS-treated and LPS plus GLA-treated mice by ELISAs. (D-E) levels of IL-1β (D) and TNF-α (E) in the sera described in (B) as measured by ELISAs. Data are expressed as the mean ± SEM. n = 6 (B-E). P < 0.05(*), P < 0.01(**), and P < 0.001(***) vs. the control by unpaired two-tailed Student’s t-tests. NS: not significant.

efficacy of drugs targeting the NLRP3 inflammasome. In the present study, in vivo experiments demonstrated that administration of GLA dramatically improved the survival of LPS-produced septic shock in mice, and also inhibited NLRP3-dependent IL-1β production in the sera and peritoneal lavage fluid of mice. Therefore, our present study suggests that GLA blocks NLRP3 inflammasome activation in vivo and provides a potential candidate drug for the treatment of NLRP3-driven diseases. In conclusion, our findings reveal an anti-inflammatory effect of GLA via suppressing the activation of NLRP3 and NLRC4 inflammasomes through the regulation of ASC oligomerization. Moreover, our findings demonstrate that GLA is helpful in improving the survival of mice suffering from lethal endotoxic shock, which is NLRP3-dependent. Hence, our findings offer a mechanistic basis to support the therapeutic potential of GLA for the prevention or treatment of septic shock, as well as other NLRP3- and NLRC4-inflammasome-related diseases.

these inflammasomes. We also studied GLA-caused inhibitory mechanisms of NLRP3 inflammasome activation. We found that GLA inhibited the NF-κB signaling pathway, which is consistent with previous studies [26]. Although GLA influences TLR4 signaling pathway, GLA-induced suppression of the NLRP3 inflammasome activation does not occur via the TLR4 signaling pathway. During inflammasome activation, various inflammasome-activating stimuli lead to upstream events of inflammasome activation, which is vital for inflammasome activation. Stimuli-mediated mtROS and oxidized mitochondrial DNA promote the activation of NLRP3 inflammasome [50–52]. As such, mitochondrial perturbations contribute to NLRP3 inflammasome activation. Our data indicate that GLA has no effect on upstream signaling of NLRP3 inflammasome activation triggered by inflammasome-activating stimuli. Thus, we speculate that GLA may directly target assembly proteins of the NLRP3 inflammasome. ASC oligomerization is a common step for NLRP3 inflammasome activation [40,41]. Consistent with the effect of GLA on inflammasomes activation, our results showed that GLA strongly inhibited NLRP3- and NLRC4-dependent ASC oligomerization but had no effect on AIM2-dependent ASC oligomerization. These results demonstrate that GLA inhibited activation of NLRP3 and NLRC4 inflammasomes by blocking ASC oligomerization. In the present study, we found that GLA inhibited NLRP3 inflammasome activation in vitro, but whether it also would inhibit the activity of the NLRP3 inflammasome and attenuate NLRP3 inflammasome-driven diseases in vivo has remained unclear. Sepsis is a complex disease that is considered to be one of the most important causes of mortality in intensive care units (ICU) and results in multiple organdysfunction syndrome [53,54]. Intraperitoneal injection of LPS has been an acknowledged approach to produce animal models of septic shock [55,56]. Several studies have confirmed that intraperitoneal injection of LPS results in the activation of the NLRP3 inflammasome [57,58], so LPS-mediated septic shock is often used to evaluate the

CRediT authorship contribution statement Xiaorong Hou: Investigation, Validation, Visualization, Formal analysis, Software, Writing - original draft. Guang Xu: Investigation, Validation, Visualization, Formal analysis, Writing - original draft. Zhilei Wang: Investigation, Validation, Visualization, Formal analysis, Writing - original draft. Xiaoyan Zhan: Investigation, Visualization, Formal analysis. Huifang Li: Visualization, Formal analysis. Ruisheng Li: Visualization, Formal analysis. Wei Shi: Investigation, Visualization. Chunyu Wang: Visualization. Yuanyuan Chen: Investigation, Visualization. Yongqiang Ai: Visualization. Xiaohe Xiao: Investigation, Validation, Resources, Formal analysis, Data curation, Visualization, Supervision, Project administration, Writing review & editing, Funding acquisition. Zhaofang Bai: Conceptualization, Methodology, Investigation, Validation, Resources, Formal analysis, Data curation, Visualization, Supervision, Project 8

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administration, Writing - review & editing, Funding acquisition.

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Acknowledgments This work was supported by grants from the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (No. 2017ZX09301022, 2018ZX09101002-001-002), the National Natural Science Foundation of China (No. 81874368, 81630100, 81903891), Beijing Nova Program (No. Z181100006218001), the Innovation Groups of the National Natural Science Foundation of China (No. 81721002) and Youth Foundation of Chinese PLA General Hospital of China (No. QNF19040). Declaration of Competing Interest The author declared that there is no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2020.106271. References [1] P. Broz, V.M. Dixit, Inflammasomes: mechanism of assembly, regulation and signalling, Nat. Rev. Immunol. 16 (2016) 407–420. [2] F. Martinon, A. Mayor, J. Tschopp, The inflammasomes: guardians of the body, Annu. Rev. Immunol. 27 (2009) 229–265. [3] L. Franchi, R. Munoz-Planillo, G. Nunez, Sensing and reacting to microbes through the inflammasomes, Nat. Immunol. 13 (2012) 325–332. [4] Y. He, H. Hara, G. Nunez, Mechanism and regulation of NLRP3 inflammasome activation, Trends Biochem. Sci. 41 (2016) 1012–1021. [5] E. Latz, T.S. Xiao, A. Stutz, Activation and regulation of the inflammasomes, Nat. Rev. Immunol. 13 (2013) 397–411. [6] W. Zhang, X. Xie, D. Wu, X. Jin, R. Liu, X. Hu, et al., Doxycycline attenuates leptospira-induced IL-1beta by suppressing NLRP3 inflammasome priming, Front. Immunol. 8 (2017) 857. [7] B.Z. Shao, Z.Q. Xu, B.Z. Han, D.F. Su, C. Liu, NLRP3 inflammasome and its inhibitors: a review, Front. Pharmacol. 6 (2015) 262. [8] T. Gong, Y. Yang, T. Jin, W. Jiang, R. Zhou, Orchestration of NLRP3 inflammasome activation by ion fluxes, Trends Immunol. 39 (2018) 393–406. [9] H. Shi, Y. Wang, X. Li, X. Zhan, M. Tang, M. Fina, et al., NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component, Nat. Immunol. 17 (2016) 250–258. [10] H. He, H. Jiang, Y. Chen, J. Ye, A. Wang, C. Wang, Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity, Nat. Commun. 9 (2018) 2550. [11] P. Duewell, H. Kono, K.J. Rayner, C.M. Sirois, G. Vladimer, F.G. Bauernfeind, et al., NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals, Nature 464 (2010) 1357–1361. [12] M.A. Katsnelson, K.M. Lozada-Soto, H.M. Russo, B.A. Miller, G.R. Dubyak, NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx, Am. J. Physiol. Cell Physiol. 311 (2016) C83–C100. [13] O. Gross, C.J. Thomas, G. Guarda, J. Tschopp, The inflammasome: an integrated view, Immunol. Rev. 243 (2011) 136–151. [14] S. Ding, S. Xu, Y. Ma, G. Liu, Modulatory mechanisms of the NLRP3 inflammasomes in diabetes, Biomolecules 9 (2019). [15] E. Tourkochristou, I. Aggeletopoulou, C. Konstantakis, C. Triantos, Role of NLRP3 inflammasome in inflammatory bowel diseases, World J. Gastroenterol. 25 (2019) 4796–4804. [16] Z. Wang, G. Xu, X. Zhan, Y. Liu, Y. Gao, N. Chen, et al., Carbamazepine promotes specific stimuli-induced NLRP3 inflammasome activation and causes idiosyncratic liver injury in mice, Arch. Toxicol. 93 (2019) 3585–3599. [17] R.C. Coll, A.A. Robertson, J.J. Chae, S.C. Higgins, R. Munoz-Planillo, M.C. Inserra, et al., A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases, Nat. Med. 21 (2015) 248–255. [18] E.K. Jo, J.K. Kim, D.M. Shin, C. Sasakawa, Molecular mechanisms regulating NLRP3 inflammasome activation, Cell. Mol. Immunol. 13 (2016) 148–159. [19] A.P. Perera, R. Fernando, T. Shinde, R. Gundamaraju, B. Southam, S.S. Sohal, et al., MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice, Sci. Rep. 8 (2018) 8618. [20] E.L. Goldberg, J.L. Asher, R.D. Molony, A.C. Shaw, C.J. Zeiss, C. Wang, et al., Betahydroxybutyrate deactivates neutrophil NLRP3 inflammasome to relieve gout flares, Cell Reports 18 (2017) 2077–2087. [21] Z. Gong, S. Zhao, J. Zhou, J. Yan, L. Wang, X. Du, et al., Curcumin alleviates DSSinduced colitis via inhibiting NLRP3 inflammsome activation and IL-1beta production, Mol. Immunol. 104 (2018) 11–19. [22] Y. Huang, H. Jiang, Y. Chen, X. Wang, Y. Yang, J. Tao, et al., Tranilast directly

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