BPTES inhibits anthrax lethal toxin-induced infl ammatory response
Jinling Wanga,1, Daowei Yanga,⁎,1, Xizi Shenc, Junsheng Wanga, Xiaomei Liua, Jinzhou Lina, Jiaying Zhongd, Yilin Zhaoe, Zhongquan Qib,⁎
aDepartment of Emergency, Zhongshan Hospital of Xiamen University, Xiamen 361005, China
bSchool of Medicine, Guangxi University, Nanning 530004, China
cFaculty of Medicine, The University of Queensland, Brisbane, Australia
dFaculty of Medicine, Xiamen University, Xiamen, China
eDepartment of Oncology and Vascular Interventional Radiology, Zhongshan Hospital of Xiamen University, Xiamen, China
A R T I C L E I N F O
Keywords: BPTES NLRP1b
Anthrax lethal toxin Inflammatory response
A B S T R A C T
Bacillus anthracis is a lethal agent of anthrax disease and the toxins are required in anthrax pathogenesis. The anthrax lethal toxin can trigger NLRP1b infl ammasome activation and pyroptosis. Although the underlying mechanism is well understood, the medications targeting the NLRP1b infl ammasome are not available in the clinic. Herein, we describe that BPTES, a known Glutaminase (GLS) inhibitor, is an effective NLRP1b in- fl ammasome inhibitor. BPTES could effectively and specifi cally suppress NLRP1b infl ammasome activation in macrophages but have no effects on NLRP3, NLRC4 and AIM2 infl ammasome activation. Mechanistically, BPTES alleviated the UBR2 mediated proteasomal degradation pathway of the NLRP1b N terminus, thus blocking the release of the CARD domain for subsequent caspase-1 processing. Furthermore, BPTES could prevent disease progression in mice challenged with the anthrax lethal toxin. Taken together, our studies indicate that BPTES can be a promising pharmacological inhibitor to treat anthrax lethal toxin-related inflammatory diseases.
Bacillus anthracis is the spore bearing bacillus and gram-positive pathogen, which can cause black cutaneous eschars. Cases if inhalation anthrax have taken place by respiratory route, and the inhaled anthrax can usually cause the fatal toxemia of the host . Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin, con- sisting of protective antigen (PA) and lethal factor (LF), which leads to cell death and inflammation in anthrax disease .
Anthrax lethal toxin is sufficient to reproduce most of the symptoms of the anthrax disease once it is injected into small rodent . It has been suggested that the inhibitors of anthrax lethal toxin can increase the survival of rodents challenged with Bacillus anthracis . Over the past decade, an increasing number of evidence has suggested that an- tibiotic treatment is the only eff ective therapeutic intervention for pa- tients with inhalational anthrax [5–7]. However, although most bac- teria can be eliminated by antibiotics, there are no specifi c medications to protect the patients from the lethal toxin-mediated toxemia and subsequent tissue injury.
Previous studies have suggested that macropahges death and cyto- kine release are critical events in the death of susceptible host against
anthrax lethal toxin infection [8,9]. It has been shown that the NOD- like receptor NLRP1b controls the macrophage death, caspase-1 acti- vation and IL-1β secretion in response to anthrax lethal toxin [10,11]. Considering that the NLRP1b infl ammasome is clearly involved in an- thrax lethal toxin-related disease , identifying its potential in- hibitors will be extremely benefi cial in developing new drugs for an- thrax lethal toxin-induced inflammatory disorders. Here we identify BPTES and its analog CB839 as inhibitors for NLRP1b infl ammasome activation and demonstrate the potential clinic use of BPTES in anthrax lethal toxin driven diseases.
2.1.BPTES could specifically inhibit the NLRP1b inflammasome signaling pathway in J774A.1 cells
To dissect the role of glutamine metabolism in NLRP1b inflamma- some activation induced by anthrax lethal toxin, we used the specific inhibitors of glutaminase (GLS), BPTES and its close analog termed CB839, to block the GLS activity . Since anthrax lethal toxin kills murine macrophages in a strain-specific manner by activating the
⁎ Corresponding authors.
E-mail addresses: [email protected] (D. Yang), [email protected] (Z. Qi). 1 These two authors contributed equally to this work.
Received 1 May 2020; Received in revised form 1 June 2020; Accepted 2 June 2020
Fig. 1. BPTES/CB839 could efficiently and specifi cally inhibit the NLRP1b infl ammsome signaling pathway in J774A.1 cells. (A) J774A.1 cells were treated with or without LF + PA for 3 h in the presence or absence of BPTES and CB839. The release of p20 subunit of caspase-1 into the supernatant (sup) was measured by immunoblotting using anti-Caspase-1 antibody. The cell lysate was analyzed by immunoblotting using anti-Caspase-1 and anti-GAPDH antibodies. (B) J774A.1 cells were primed with LPS for 4 h and then treated with nigericin for 2 h in the presence or absence of BPTES and CB839. The cell culture supernatant was analyzed by immunoblotting with anti-Caspase-1 antibody and the corresponding cell lysate was analyzed by immunoblotting with anti-Caspase-1 and anti-GAPDH antibodies. (C) The supernatant of the cells described in B was analyzed by IL-1β ELISA kit. (D) J774A.1 cells were treated with flagellin (500 ng/ml) for 2 h in the presence or absence of BPTES and CB839. The cell culture supernatant was analyzed by immunoblotting with anti-Caspase-1 antibody and the corresponding cell lysate was analyzed by immunoblotting with anti-Caspase-1 and anti-GAPDH antibodies. (E) J774A.1 cells were transfected with (dA:dT) for 8 h in the presence or absence of BPTES and CB839. The cell culture supernatant was analyzed by immunoblotting with anti-Caspase-1 antibody and the corresponding cell lysate was analyzed by immunoblotting with anti-Caspase-1 and anti-GAPDH antibodies. (F) The LDH released from the cells was measured by using the cytotoxicity assay kit. (G) J774A.1 cells were treated as in A, then the cells were stained with PI and representative images were captured and processed. All results except for the Fig. 6 are re- presentative of three independent experiments. Scale bar, 10 μm. ∗∗∗p < 0.001; N.S., no signifi cance, by unpaired Student’s t test. inflammatory protein NLRP1b and Caspase-1 [8,13], we used the J774A.1 cells to study the eff ect of BPTES on NLRP1b infl ammasome because of its susceptibility to anthrax lethal toxin exposure. NLRP1b inflammasome activation was monitored by immunoblotting analysis of caspase-1 p20 subunit from the medium supernatant fraction. As it was suggested that caspase-1 p20 subunit was obviously seen in the super- natant fraction of J774A.1 cells with anthrax lethal factor (LF) and protective antigen (PA) treatment (Fig. 1A). To test whether glutamine metabolism was involved in lethal toxin-induced NLRP1b infl amma- some activation, J774A.1 cells were treated with the lethal toxin in the presence or absence of BPTES or CB839. Lethal toxin administration robustly induced the caspase-1 processing, whereas, under the condi- tion where cells were treated with anthrax lethal toxin plus BPTES or CB839, the caspase-1 p20 subunit was almost eliminated (Fig. 1A). Moreover, BPTES acted more effectively than CB839 because BPTES could abolish the Caspase-1 activation at the 2 µM concentration, whereas CB839 must be used at the 5 µM to reach the same inhibitory eff ect (Fig. 1A). To assess whether the inhibitory eff ect of BPTES and CB839 was specific to the NLRP1b infl ammasome, we treated J774A.1 cells with BPTES or CB839 after treatment with LPS + nigericin, poly (dA:dT) or fl agellin, which induced the corresponding NLRP3, AIM2, and NLRC4 inflammasomes, respectively. However, our results showed that BPTES and CB839 did not aff ect the activation of NLRP3, AIM2, or NLRC4 infl ammasomes (Fig. 1B-E), which suggested that BPTES acted specifically on NLRP1b inflammasome. Given the fact that NLRP1b infl ammasome activation can result in cell death through the pore- forming protein Gasdermin D (GSDMD) , we then measure the lactate dehydrogenase (LDH) release, a marker of cell death due to the rupture of the plasma membrane, to test the eff ect of BPTES and CB839 on NLRP1b infl ammasome-induced cell death. As anticipated, BPTES and CB839 could dramatically decrease the LDH release at the 5 µM concentration (Fig. 1F). Consistently, the anthrax lethal toxin-induced cell death analyzed by propidium iodide (PI) staining was also blocked by BPTES and CB839 (Fig. 1G). Taken together, these results demon- strate that BPTES could eff ectively and specifi cally inhibit NLRP1b in- fl ammasome activation and subsequent cell death. 2.2.The reduced catabolism of glutamine is not responsible for BPTES/ CB839 to suppress the NLRP1b infl ammasome activation Glutamine plays a very important role in a variety of physiological functions, which can be converted to glutamate and ammonia by GLS. Glutamate is the main excitatory neurotransmitter in the neuron and ammonia is very important for the regulation of acid-base balance . To explore the possibility of BPTES can suppress the NLRP1b in- fl ammasome activation through alleviating the catabolism of gluta- mine. We pretreated the cells with glutamate and its downstream me- tabolite α-ketoglutarate (α-KG) to examine whether glutamate and α- Fig. 2. The reduced Glutamine metabolism is not responsible for BPTES/CB839 to suppress the NLRP1b infl ammasome activation. (A) J774A.1 cells were cultured in the medium supplemented with glutamate or α-KG, and the cells were treated with or without anthrax lethal toxin for 3 h in the presence or absence of BPTES. Subsequently the cells were stained with PI and representative images were captured and processed. Scale bar, 10 μm. (B) J774A.1 cells were treated as the same as in A except that the BPTES was replaced by CB839. Scale bar, 10 μm. (C) J774A.1 cells were treated as in A and B, then the cell culture supernatant was analyzed by immunoblotting with anti-Caspase-1 antibody and the corresponding cell lysate was analyzed by immunoblotting with anti-Caspase-1, anti-LaminB1 antibodies. KG can reverse the inhibitory effect of BPTES or CB839. However, no significant diff erence in cell death was observed between the control group and cells treated with glutamate and α-KG (Fig. 2A-B). Accord- ingly, we found that the addition of glutamate and α-KG could not increase the Caspase-1 activation following BPTES or CB839 treatment (Fig. 2C). Taken together, we conclude that the reduced catabolism of glutamine is not responsible for the role of BPTES or CB839 in the NLRP1b infl ammasome activation. 2.3.The inhibitory effect of BPTES on NLRP1b inflammasome is independent of BPTES-mediated inhibition of GLS As BPTES and CB839 are potent and specific GLS inhibitors, we then test the role of GLS and GLS2 in the NLRP1b infl ammasome signaling pathway. To verify this, we fi rstly checked the protein level of GLS and GLS2 in J774A.1 cells, and found a low expression level of GLS2 in macrophage (Fig. 3A-B). This is in line with previous studies showing that GLS2 were strongly expressed in hepatocyte, such as the hepa1-6 cells (Fig. 3A-B). We then knocked down GLS in J774A.1 cells and found that the ability of BPTES to suppress the NLRP1b infl ammasome activation was not decreased (Fig. 3C-D). Thus, we conclude that the inhibitory effect of BPTES on anthrax lethal toxin-induced NLRP1b inflammasome activation is independent of BPTES-mediated inhibition of GLS. 2.4.BPTES acts upstream of NLRP1b in the process of the NLRP1b infl ammasome activation ASC aggregates called ASC specks are formed in the cytosol of macrophages stimulated with anthrax lethal toxin . We next eval- uated the ASC specks formation in J774A.1 cells treated with BPTES or CB839. Consistent with previous results, rapid intracellular ASC specks could be observed in the J774A.1 cells treated with LF + PA (Fig. 4A). Notably, the formation of ASC specks induced by anthrax lethal toxin was abolished by BPTES or CB839 (Fig. 4A-B). In addition, the ASC oligomer after cross-linking was also abrogated by BPTES (Fig. 4C). These results suggest that BPTES acts on or upstream of ASC to suppress the anthrax lethal toxin-induced NLRP1b inflammasome. To further pinpoint the role of BPTES in the formation of ASC specks induced by activated NLRP1b, we co-overexpressed NLRP1b and ASC- GFP in HEK293T cells, and we found that the overexpression of NLRP1b was sufficient to activate ASC-GFP specks formation (Fig. 4D). How- ever, the formation of ASC-GFP specks was unperturbed in the HEK293T cells treated with BPTES or CB839 (Fig. 4D-E). Taken to- gether, our studies indicate that BPTES acts upstream of NLRP1b acti- vation. 2.5.BPTES inhibits the N-end rule mediated proteasomal degradation pathway of the NRLP1b N terminus Protective antigen can bind to the cell surface receptor, mediating the endocytosis of lethal factor into the cell . Lethal factor is a metalloprotease responsible for the proteolysis mitogen-activated pro- tein kinase kinases (MEKs) and NLRP1b . Anthrax lethal toxin can directly cleave NLRP1b at Lys44, which is essential for NLRP1b in- fl ammasome activation . Recent studies suggested NLRP1b in- fl ammasome activation was induced by the ubiquitin ligase UBR2 mediated degradation of the NLRP1b N terminus [20,21]. To dissect the mechanism underlying the inhibitory activity of BPTES on NLRP1b infl ammasome activation, we firstly detected the cleavage of a known substrate MEK2. Consistent with previous results, anthrax lethal toxin could induce notable cleavage of MEK2 (Fig. 5A). In addition, BPTES, rather than CB839, modestly reduced the level of MEK2 cleavage only at the concentration of 10 μM (Fig. 5A). We then examined whether BPTES played a role in the cleavage of NLRP1b, and we found that BPTES or CB839 could dramatically abolished the clea- vage of NLRP1b (Fig. 5B). Since it was reported that UBR2 could de- grade NLRP1bL45-L60-GFP (L45-L60 of NLRP1b N terminus fused to GFP) in HEK293T cells , we then transfected NLRP1bL45-L60-GFP in HEK293T cells and evaluated the role of BPTES or CB839 in the Fig. 3. The inhibitory eff ect of BPTES on NLRP1b inflammasome is independent of BPTES-mediated inhibition of GLS. (A) Western blot to detect GLS and GAPDH protein in indicated cell lines by using the anti-GLS, anti-GAPDH antibodies. (B) Western blot to detect GLS2 and GAPDH protein in in- dicated cell lines by using the anti-GLS2, anti- GAPDH antibodies. (C) GLS was knocked down in J774A.1 cells, and then the cells were treated with anthrax lethal toxin for 3 h in the presence or ab- sence of BPTES. The cell culture supernatant was analyzed by immunoblotting with anti-Caspase-1 antibody and the corresponding cell lysate was analyzed by immunoblotting with anti-Caspase-1, anti-GLS, anti-GAPDH antibodies. (D) J774A.1 cells were treated as in C, and then the LDH released from the cells was measured by using the cyto- toxicity assay kit. ∗∗∗p < 0.001, by unpaired Student’s t test. Fig. 4. BPTES acts upstream of NLRP1b in the process of NLRP1b inflammasome activation. (A) J774A.1 cells were treated with or without LF + PA for 3 h in the presence or absence of BPTES and CB839, then immunostained for ASC and counterstained with DAPI. The representative images were captured and presented. Scale bar, 10 μm. Arrowheads indicate the ASC specks. (B) J774A.1 cells were treated as in A, then the cells containing ASC speck were quantifi ed. (C) J774A.1 cells were treated with LF + PA for 3 h in the presence or absence of BPTES, then the cells were solubilized with triton X-100-containing buff er, and the insoluble fractions were cross-linked with disuccinimidyl suberate (DSS) to capture ASC oligomers. Western blot to detect the indicated proteins of soluble and insoluble fractions by using anti-ASC, anti-LaminB1 antibodies. (D) HEK293T cells were treated with the indicated combinations of the indicated plasmids and BPTES or CB839 for 24 h. The representative images were captured and presented. Scale bar, 10 μm. (E) HEK293T cells were treated as in D, then the cells containing ASC speck were quantifi ed. ∗∗∗p < 0.001; N.S., no signifi cance, by unpaired Student’s t test. Fig. 5. BPTES inhibits the N-end rule mediated proteasomal degradation pathway of the NRLP1b N terminus. (A) J774A.1 cells were treated with or without LF + PA for 3 h in the presence or absence of BPTES and CB839. Western blot to detect MEK2 and LaminB1 protein by using the anti-MEK2, anti-LaminB1 antibodies. (B) J774A.1 cells were treated as in A, and western blot to detect NLRP1b and LaminB1 protein by using the anti-NLRP1b, anti-LaminB1 antibodies. (C) HEK293T cells were treated with the indicated combinations of 0.1 μg NLRP1bL45-L60-GFP-FLAG plasmid and BPTES or CB839 for 24 h. Western blot to detect FLAG and LaminB1 protein by using the anti-FLAG, anti-LaminB1 antibodies. (D) HEK293T cells were transfected with 0.5 μg NLRP1bL45-L60-GFP-FLAG and 1.5 μg HA-UBR2 for 36 h, then the cells were lysed in lysis buffer and the NLRP1bL45-L60-GFP-FLAG protein was immunoprecipitated with anti-FLAG M2 magnetic beads. Western blot to detect Ub and FLAG, HA, LaminB1 proteins in immunoprecipitation fraction and cell lysate (TCL) by using the anti-Ub, anti-FLAG, anti-HA, anti-LaminB1 antibodies. stability of NLRP1bL45-L60-GFP. We found that BPTES and CB839 sig- nificantly elevated the stability of NLRP1bL45-L60-GFP (Fig. 5C). Since the N-end rule mediated ubiquitination and degradation of NLRP1b, we next examined the ubiquitination of NLRP1b. Indeed, the ubiquitina- tion of NLRP1b was decreased in HEK293T cells treated with BPTES (Fig. 5D). Thus, we conclude that BPTES blocks the N-end rule medi- ated proteasomal degradation pathway of the NRLP1b N terminus. 2.6.BPTES can prevent the anthrax lethal toxin-induced mortality in mice Our above data have suggested that BPTES efficiently inhibit an- thrax lethal toxin-induced NLRP1b inflammasome activation and cell death. We next wanted to know whether BPTES could also protect the mice from anthrax lethal toxin-driven mortality and tissues injury. To assess the effects of BPTES on anthrax lethal toxin-driven disease, we pretreated the Balb/c mice with vehicle or BPTES for 1 h through in- traperitoneal injection, then these mice were intravenously injected with lethal dose of anthrax lethal toxin. We found that all of the mice challenged with anthrax lethal toxin alone died within 70 h. However, mice challenged with anthrax lethal in the presence of 1 mg/kg of BPTES had a longer survival time, time to death and thus the number of deaths in the BPTES-treated mice group were delayed and reduced (Fig. 6A). Given the fact that the prominent effect of anthrax lethal toxin in mice is lung injury and adrenal lesions [22,23], we first ex- amined several features associated with anthrax-related disease, pleural eff usions and pulmonary edema in particular. Anthrax lethal toxin could cause severe pulmonary edema and BPTES reversed the lethal toxin-mediated increase of the lung weight (Fig. 6B). In addition, sig- nificant increase of pleural eff usions was observed in mice challenged with anthrax lethal toxin, and BPTES could get the similar rescue effect (Fig. 6C). This protection of BPTES on anthrax lethal toxin-induced lung injuries was substantiated by subsequent histological examination of lung tissues. The results showed widespread pulmonary edema in mice treated with anthrax lethal toxin (Fig. 6D). However, the lungs from the mice challenged with anthrax lethal toxin and BPTES shown normal histology (Fig. 6D). Moreover, we found that the mice chal- lenged with anthrax lethal toxin showed massive adrenal gland lesion, as evidenced by the loosen structure (Fig. 6E). The adrenal gland lesion was completely rescued by BPTES (Fig. 6E). Importantly, anthrax lethal toxin severely disrupted the crypts and villi in the intestine, which was rescued by BPTES (Fig. 6F). Taken together, these data suggest that BPTES can block the anthrax lethal toxin-induced mortality and tissue injury in mice by preventing injury to lungs, adrenal glands and in- testine. 2.7.Discussion Anthrax lethal toxin can cause severe cell death, which may be re- lated to the pathology of systemic infections. Intravenous injection of anthrax lethal toxin induces severe death in rats . NLRP1b is the primary mediator of macrophage susceptibility to anthrax lethal toxin. NLRP1b consists of a pyrin domain (PYD), a nucleotide-binding domain (NATCH), a leucine-rich repeat domain (LRR), a function to fi nd do- main (FIIND) and a caspase recruitment domain (CARD). NLRP1b re- cruits ASC and Caspase-1 through the CARD domain for the GSDMD mediated- pyroptosis and IL-1β release . After translocating into the cell, LF cleaves NLRP1b at the N-terminus and this cleavage is sufficient to activate NLRP1b inflammasome . The N-terminal Fig. 6. BPTES can prevent the anthrax lethal toxin-induced mortality in mice. (A) Four-week-old female Balb/c mice received intraperitoneal injection of vehicle or 1 mg/kg BPTES, followed by intravenous injection of 500 mg/kg LF + PA. Mice survival up to 150 h was measured. N = 9 for each group. (B) The Lungs of mice with indicated treatment were removed, and the wet-to-dry weight ratio of the lung was calculated by dividing the wet weight by the dry weight. N = 4 for each group. (C) The pleural effusions of mice with indicated treatment were drained and quantifi ed from thoracic cavities. N = 4 for each group. (D) The sections of lung were stained with H&E, and the representative images of lung of mice with indicated treatment were shown. (E) The sections of adrenal were stained with H&E, the representative images of lung of mice with indicated treatment were shown. (F) The sections of intestine were stained with H&E, and the representative images of lung of mice with indicated treatment were shown. Original magnifi cation: ×200 and × 400; scale bar, 100 µm. ∗p < 0.05, by unpaired Student’s t test. degradation of NLRP1b is essential to activate caspase-1 and pyroptosis . To our knowledge, this is the first report to demonstrate that it is possible to suppress the NLRP1b infl ammasome activation with tar- geting the ubiquitination of NLRP1b N terminus. Thus, BPTES is a po- tential therapeutic drug targeting NLRP1b inflammasome for the treatment of inflammatory diseases. Although the proteasome inhibitor NPI-0052 has been shown to prevent the lethal toxin-induced disease progression in rats and Balb/c mice . However, the rats challenged with NPI-0052 showed severe toxicity and side effects. Here we provide the evidence that a novel NLRP1b inhibitor, BPTES, exhibits no signifi cant toxicity and side ef- fects. BPTES, a known inhibitor of glutaminase and its analog CB-839 exhibits strong antitumor activity in triple-negative breast cancer [28,29]. However, such inhibitory eff ect of BPTES on GLS is not re- quired for the NLRP1b infl ammasome activation. Thus, our study broadens the substrates and medicine usages of BPTES. It remains unknown how BPTES reduces the UBR2-mediated ubi- quitination of the NLRP1b N terminus. Undoubtedly, further work is needed to elucidate the exact mechanism underlying the role of BPTES in the ubiquitination of NLRP1b N terminus. Collectively, our data demonstrates that BPTES eff ectively suppress infl ammation by reducing the UBR2-mediated ubiquination and degradation of the NLRP1b N terminus, which reveals a new anti-infl ammatory eff ect for BPTES. The information presented in this work is benefi cial for developing new therapeutic intervention targeted at anthrax lethal toxin-induced dis- eases. 3.Materials and methods 3.1.Mice Four-week-old female Balb/c mice were supplied by the Xiamen Univeristy Labortory Animal Center. These mouse experimental pro- tocols were approved by the Institutional Animal Care and Use Committee at Xiamen University. 3.2.Reagent, antibodies and plasmids L-Glutamic acid dimethyl ester hydrochloride (49560-10G) was from Sigma. BPTES (HY-12683) was from MedChem Express (MCE). Anthrax lethal factor (#172C) and Anthrax protective antigen (#171E) were from List Biological Labs, Inc. Poly(dA:dT) and Nigericin were from InvivoGen. MEK2 Antibody (11049-1-AP) was from Proteintech. NLRP1b antibody (sc-390133) and HA (sc-7392) were from Santa Cruz Biotechnology. ASC antibody (67824S) and Ubiquitin antibody (3936 s) were from Cell Signaling Technology. Caspase-1 antibody was from Adipogen. LaminB1 antibody (66095-1-Ig) and GLS1 (12855-1-AP) antibody were from Proteintech. GLS2 (ab150474) antibody was from abcam. Mouse monoclonal Flag antibody was from Sigma-Aldrich. All plasmids were constructed as previously described .
J774A.1, HEK293T cells were obtained from ATCC. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), non-essential amino acids, peni- cillin and streptomycin. All cells were grown at 37 °C in a humidified incubator containing 5% CO2.
3.4.Infl ammasome assay
The NLRP1b infl ammasome was activated by 2 μg/ml anthrax lethal toxin for 3 h. The NLRP3 inflammasome was activated by 10 μM Nigericin after 100 ng/ml LPS priming for 4 h. The NLRC4 inflamma- some was activated by flagellin (500 ng/ml) for 2 h. The AIM2 in- fl ammasome was activated by transfecting 4 μg/ml Poly (dA:dT) for 8 h.
3.5.ASC oligomerization assay
The ASC oligomerization assay was performed as previously de- scribed . Briefly, J774A.1 cells were lysed with TBS buff er
containing 0.5% triton X-100. The cell lysates were centrifuged at 6000g for 15 min, then the supernatant was used as the insoluble fractions and the pellet was cross-linked at 37 °C with 2 mM dis- uccinimidyl suberate (Pierce). The pellets were dissolved in SDS buff er.
3.6.Immunoprecipitation and immunoblotting
Cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4) supple- mented with protease inhibitor at 4 °C for 30 min. Then the cell lysates were centrifuged at 13500 rpm for 30 min. The supernatant was im- munoprecipitated with anti-FLAG M2 magnetic beads (Sigma-Aldrich) at 4 °C for overnight. After the immunoprecipitation, the beads were washed three times in lysis buffer and the immunoprecipitated proteins were subsequently eluted using the SDS sample buff er. Western blotting was performed as previously described . The luminescent signals of immunoblotting were analysed using an ImageQuant LAS 4000 Scanner (GE Healthcare).
LDH assay using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit from Promega Corporation was used to measure cell viability according to the manufacturer’s instructions and previous report .
3.8.Measurement of IL-1β
To measure IL-1β release, the culture supernatants of the cells were collected, then the release of mature IL-1β was determined by using the IL-1β ELISA kit according to the manufacturer’s instructions.
The lentivirus-based vector pLV-H1-EF1a-bsd (BiOSETTIA) was used for expression of siRNA in J774A.1 cells. The targeted sequences of siRNAs used in this study were as follows (5′ to 3′):
GLS-#1: CATCGATGAGTTATATGAAAGTGCT GLS-#2: GAGAGAAGGAGGTGATCAA
Lentiviruses were packaged in HEK293T cells using calcium phos- phate precipitation method. At 36 h post-transfection, the culture medium was collected and added to the J774A.1 cells.
J774A.1 cells were grown on coerslips (Nest) in 24 wells plate. Cells were then washed with PBS followed by fixation with 4% paraf- ormaldehyde for 15 min. After fi xation, the cells were permebilized in 0.2% trixon X-100, blocked with 5% BSA and stained with ASC anti- body and the corresponding secondary antibody. Lastly, the cells were counterstained with DAPI to visualize nucleus. Imaging was performed by using Zeiss LSM 780. All images were processed and quantifi ed by ImageJ software.
After the mice were sacrificed, tissues of diff erent organs were collected and fixed immediately in 10% neutralized formalin. The fixed tissues were dehydrated in ethanol and embedded in paraffin and cut into sections which were then stained with hematoxylin and eosin (H&
E). The representative images were captured and processed by using the Lecia DM2500.
All results are analyzed using the GraphPad 6.0 software and
expressed as means ± SEM. The analysis was performed by two-tailed unpaired Student’s test. Mice survival is presented as a Kaplan-Meyer plot and compared by log-rank (Mantel-Cox) Test. P < 0.05 was considered statistically significant. Author contributions D.Y conceived and designed the study. D.Y performed most of the biochemical and cellular experiments. J.W performed the mouse ex- periments with the help from other authors. Z.Q acquired the grant support for this project. D.Y analyzed the data and wrote the manu- script with input from other authors. All authors discussed the results and commented on the manuscript. Acknowledgement This work was supported by grants from the National Key R&D Program of China [grant number 2018YFA0108304], the National Natural Science Foundation of China [grant number 81771271] and the Natural science foundation of Fujian province [grant number 2019J01559]. Declaration of Competing Interest The authors declare that they have no competing interests. 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