Belnacasan (VX-765, S2228, Purity: 99.99%) was obtained from Selleck Chemical (Houston, TX, USA), Belnacasan powder was dissolved in dimethyl sulfoxide (DMSO) at 20 mM and stored at −20°C. Propidium iodide (PI, P4170), Hoechst 33342 (B2261), CF568-conjugated goat-anti-rabbit IgG (SAB4600084), CF488-conjugated goat-anti-mouse IgG (SAB4600237), lipopolysaccharide (LPS, Escherichia coli O111:B4, L4391), adenosine triphosphate (ATP) (A6419), Tween-20 (P1379), dimethyl sulfoxide (DMSO) (D8418) and DL-dithiothreitol (DTT) (D0632) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nigericin (tlrl-nig, Purity: ≥98.0%) powder was bought from InvivoGen (San Diego, CA, USA), which was dissolved in ethanol. Dulbecco’s Modiffed Eagle’s Medium (DMEM) with high glucose (C11995500BT), RPMI Medium 1640 basic (C11875500BT), β-mercaptoethanol (21985-023), fetal bovine serum (FBS, 10099141C), 100 × streptomycin/penicillin (15140122) were bought from Invitrogen/Gibco (Carlsbad, CA, USA). Phorbol 12-myristate 13-acetate (PMA) (S1819), Co-IP lysis buffer (P0013), JC-1(C2005), Cell Mitochondria Isolation Kit(C3601M), Quick Genotyping Assay Kit for Mouse Tail (D7283S), Reactive oxygen species (ROS) assay kit (S0033S) and phenylmethanesulfonyl fluoride (PMSF) (ST505) were obtained from Beyotime (Shanghai, China). Specific antibodies against ASC (#67824), ASC-AlexaFluor647 conjugated (#23640), β-actin (#3700), cleaved caspase-3 (#9664), cleaved caspase-8 (#8592), caspase-9 (#9508), IL-1β (#12242), PARP (#9532), NLRP3 (#15101), TOMM20 (#42406), α-Tubulin (#3873), horseradish peroxidase (HRP)-conjugated horse-anti mouse IgG (#7076), HRP-conjugated goat-anti-rabbit IgG (#7074) mouse were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against pro-caspase1 + p10 + p12 (ab179515), GSDMD (ab209845), GSDME (ab215191) and pro-caspase-8 (ab108333) were bought from Abcam (Cambridge, UK). Antibodies against NLRP3 (AG-20B-0014) was purchased from Adipogen AG (Liestal, Switzerland). Antibodies against ASC (B-3) (sc-514414) was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

C57BL/6J mice (WT) aged 6–8 weeks were obtained from GemPharmatech (Nanjing, China). Heterozygous caspase-1^+/− mice on a C57BL/6J background were purchased from Cyagen Biosciences (Guangzhou, China). The caspase-1 ^−/−, and WT littermates used in this study were generated in-house by breeding heterozygous parents (5th to 9th generations). Mice with caspase-1 deletion exhibited no apparent physiological defects, and their size, body weight, and appearance were comparable to those of WT littermates. Genotyping was conducted using PCR-based methods with the Quick Genotyping Assay Kit for Mouse Ears (D7283, Beyotime; Shanghai, China), following the manufacturer’s protocol. All animals were housed at 25 °C under a 12-h light/dark cycle with ad libitum access to food and water, and were acclimatized for at least one week prior to experiments. All experimental procedures were approved by the Ethical Institutional Animal Care and Use Committee of Jinan University (EIACUC-JNU), Guangzhou, China.

Human THP-1 WT cells (kindly provided by Dr. Yao Wang of Sun Yat-sen University, Guangzhou, China) and THP-1 caspase-1-deficient cells (kindly provided by Professor Jun Cui in Sun-yat Sen University) were cultured in RPMI 1640 medium supplemented with 10% FBS and antibiotics. THP-1 cells were differentiated into macrophages incubation with 100 nM of PMA for 3 h (17).

RAW 264.7 cells were bought from Kunming cell bank, CAS (Kunming, China). The cells were maintained in complete DMEM medium (containing 10% FBS, 1% penicillin and streptomycin) and cultured at 37 °C in a humidified incubator with 5% CO₂.

BMDMs were differentiated from mouse bone marrow cells as described previously (18). Briefly, WT and caspase-1^−/− C57BL/6J mice were sacrificed by cervical dislocation and sterilized with 75% ethanol. The bone marrow cells in hind femora and tibias were flushed out by 10 mL sterile 1×PBS and separated from the mixture by centrifugation at 1500 rpm for 5 min at 4 °C. Then the bone marrow cells were re-suspended in BM-Mac medium which is composed of 80% complete DMEM medium and 20% M-CSF-conditioned medium from L-929 cells, and cultured in the 10-cm petri dish with 10 mL BM-Mac medium at 37 °C in a humidified incubator of 5% CO₂. On the third day of cell culture, half volume of fresh medium (5 mL/dish) was added and let them differentiate into BMDMs after 6 d. The cells were then collected by using cell-scraper and cultured in 6-well plates at 1.6 × 10⁶ cells/well (1.7 mL) or 24-well plates at 2.5 × 10⁵ cells/well (0.5 mL) with complete DMEM medium. The cells were ready for experiments after overnight incubation.

WT BMDMs were primed with LPS (500 ng/mL) for 4 h and stimulated with ATP (3 mM) or nigericin (5 μM) for 0.5 h or 1 h to induce NLRP3 inflammasome activation and pyroptosis, respectively. For delayed NLRP3 activator stimulation assays, WT BMDMs were treated with LPS (200 ng/mL) for 4 h, washed, and cultured in fresh medium (without LPS) for 12 h (the resting step), and then were treated with or without the same dose of LPS for 4 h again. After that, the cells were stimulated with ATP (3 mM) for 0.5 h; or alternatively, cells were primed with LPS (500 ng/mL) for 4 h, washed and cultured in fresh medium. Nigericin (5 μM) was added either immediately or 12 h later for 0.5 h. For caspase-1 inhibitor assays, BMDMs, PMA-differentiated THP-1 cells (WT or caspase-1-deficient) or RAW264.7 cells (lacking ASC expression) were incubated with LPS (500 ng/mL) for 4 h, pre-treated with VX-765 (at indicated concentrations) or vehicle for 1 h, and then stimulation with nigericin (5 μM for BMDMs and 10 μM for THP-1 cells) for specified durations.

To prepare a positive control (PC) sample for Western blotting, BMDMs were treated with 5Z-7-oxozeaenol (TAK1 inhibitor) plus LPS to induce PANoptosis (concurrent Pyroptosis, Apoptosis, and Necroptosis) as previously described (19).

Cell death was measured by PI incorporation assay or lactate dehydrogenase (LDH) release assay. Briefly, after indicated treatment, PI (2 µg/mL) and Hoechst 33342 (5 µg/mL) solutions were added into medium to stain the dying cells and the nuclei, respectively. Apoptotic cells are detected by using Annexin V-EGFP Apoptosis Detection Kit (C1062M, Beyotime). Among them, Annexin-V-EGFP⁺PI^— indicates early apoptotic cells, while Annexin-V-EGFP⁺PI⁺ indicates late apoptotic or necrotic cells. The stained cells were observed by live imaging using Zeiss Axio Observer D1 microscope equipped with a Zeiss LD Plan-Neofluar 20×0.4 Korr M27 objective lens (Carl Zeiss MicroImaging GmbH, Go¨ttingen, Germany). Fluorescence images were captured with the Zeiss AxioCam MR R3 cooled CCD camera controlled with ZEN software (Carl Zeiss). For LDH release assay, the lytic cells were evaluated by using the CytoTox 96 non-radioactive cytotoxicity assay kit (G1780, Promega) according to instructions of the manufacturer.

Western blotting was performed as previously described (18). Briefly, whole cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to PVDF membranes (03010040001; Roche Diagnostics GmbH, Mannheim, Germany), which were blocked by blocking buffer for 1 h once the electro-transfer had been completed. Then, the membrane was incubated with primary antibody at 4 °C overnight, followed by incubation with appropriate HRP-conjugated secondary antibody. The target bands were visualized with an enhanced chemiluminescence kit (BeyoECL Plus; Beyotime, Shanghai, China) and captured on X-Ray films. The results were recorded by FluorChem 8000 imaging system (AlphaInnotech, San Leandro, CA, USA) and analyzed by AlphaEaseFC 4.0 software (AlphaInnotech).

After the cells were treated, the cell culture supernatant was collected in a 1.5 ml Eppendorf tube, centrifuged at 300 ×g for 5 min, and the supernatant was transferred to a new 1.5 mL Eppendorf tube. The protein in the cell culture supernatant was precipitated with 10% sodium deoxycholate and trichloroacetic acid. Overnight precipitation at 4 °C and centrifugation at 14,000 rpm for 30 min; The supernatant was discarded, 800 μL acetone pre-cooled at -20 °C was added, ultrasonic dissolution and precipitation were allowed to stand for 10 min, centrifuge at 14,000 rpm for 5 min, and acetone was repeatedly washed and precipitated for 3 times. After the acetone was completely volatilized, 2×SDS-PAGE loading buffer was added, the protein precipitate was completely dissolved by full shock, and the solution was boiled in boiling water for 5 min and stored at -80 °C.

Immunofluorescence analysis as described previously (20), in short, BMDMs were performed in a glass-bottomed confocal dish (#801002; NEST, Wuxi, China), 1.5×10⁵ cells/well, 37 °C overnight. After the indicated treatment, the cells were fixed with 4% paraformaldehyde for 15 min and permeated with 2 mL cold methanol at -20 ˚C for 10 min. After penetration, the cells are blocked with a blocking buffer. After overnight incubation with the specified antibody, incubated cells were incubated with CF568 anti-Rabbit IgG (1:500) and CF488 anti-mouse IgG (1:500) at room temperature for 1 h, then coupled with anti-ASC-AlexaFluor647. The nucleus was then displayed with Hoechst 33342 solution (5 μg/mL).

Reactive oxygen species (ROS) assay kit (S0033, Beyotime) was adopted to detect the total intracellular ROS produced in living cells according to the supplier’s instructions. The fluorescence of intracellular DCFH-DA was observed with Axio Observer D1 microscope (Carl Zeiss) and images were captured. The average fluorescence intensity was quantified by ZEN software (Carl Zeiss).

After the cells were treated, JC-1 working solution (5 μg/mL) was added for evaluating mitochondrial membrane potential (MMP), and the cells were incubated at 37 °C without light for 30 min, and then washed twice with warm serum-free DMEM or staining buffer. Stained cells were observed by live imaging using Zeiss Axio Observer D1 microscope. Once entering cells, JC-1 becomes aggregates (red fluorescence) in intact mitochondria, but becomes monomers (green fluorescence) if the mitochondrial membrane is damaged (loss of MMP). Data were acquired and analyzed by using the software of ZEN (Carl Zeiss). Image analysis of fluorescence intensity was performed using ImageJ, while statistical analysis was conducted with GraphPad Prism 6.0. Loss of MMP was presented as the ratio of cells with mitochondrial monomer.

Isolation of mitochondria was performed using Beyotime’s Cell Mitochondria Isolation Kit (C3601), according to the instruction of the manufacturer. The purity of isolated mitochondria is monitored by Western blotting, with TOMM20 serving as a mitochondrion marker as it is distributed in the outer membrane of mitochondria, and α-Tubulin, a cytoskeleton protein, as a cytosol marker. The distributions (cytosol or mitochondrion) and levels of GSDMD and Caspase-1 were evaluated by Western blotting.

Each experiment was performed three times independently. Data were expressed as mean ± standard deviation (SD) and analyzed for statistical significance using GraphPad Prism 6.0 (GraphPad Software Inc, San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey post hoc test was used to analyze the statistical significance among multiple groups. Significance was defined as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

In this study, we first explored the response of LPS-tolerant macrophages to a triggering signal of NLRP3 inflammasome (nigericin or ATP). To address this, we established an LPS tolerant cellular model by stimulating BMDMs with double 4-h LPS treatments (with a 12-h interval between them). The results showed that LPS tolerant macrophages were less sensitive to ATP stimulation in view of the NLRP3 inflammasome activation (as evidenced by GSDMD-NT and caspase-1p10) ( Figure 1A ), showing a reduced response of LPS tolerant macrophages, although the expression of pro-IL-1β (due to NF-κB activation) could still be increased in response to the second LPS treatment. Similarly, a 12-h interval between the LPS (only once) and nigericin treatments, compared to immediate stimulation with nigericin after LPS priming, induced a reduced NLRP3 inflammasome and caspase-1 activation, but an increased caspase-8 activation ( Figure 1B ), suggesting activation of an alternative pathway. Immunofluorescence microscopy was used to observe the distribution of NLRP3, caspase-1, and caspase-8 in LPS-primed WT BMDMs that were stimulated with nigericin (immediately or delayed). In the cells immediately stimulated with nigericin, NLRP3, ASC, and caspase-1 were co-localized and formed a speck (indicating the assembly of NLRP3 inflammasome) ( Figure 1C ). But in those stimulated with nigericin 12-h later, no NLRP3 inflammasome containing caspase-1 were observed; instead, puncta of NLRP3, ASC, and caspase-8 were co-localized ( Figure 1D ), suggesting that a delayed triggering signal leads to caspase-8 recruitment to the NLRP3/ASC platform, which could trigger apoptotic caspase activation.

Although the above-mentioned results showed that nigericin induced caspase-8 activation in LPS-tolerant macrophages, it is unclear whether caspase-1 is required for the NLRP3/ASC platform to recruit caspase-8 and other apoptotic caspases. Thus, we evaluated whether nigericin could induce apoptotic caspase activation in caspase-1-deficient macrophages. Western blotting corroborated that apoptotic caspases, including caspase-8, -9, and -3 (indicative of apoptosis), were all activated in caspase-1-deficient BMDMs but not in WT cells ( Figure 2A ). Similar results of caspase-8 activation were achieved using caspase-1-deficient THP-1 macrophages ( Figure 2B ). These results suggest that NLRP3 activators prefer activating caspase-1 to caspase-8 and other apoptotic caspases, but when caspase-1 is deficient, the NLRP3 activators activate apoptotic caspases in macrophages.

Unexpectedly, caspase-1 inhibitor VX-765 could also inhibit the activation of apoptotic caspases (caspase-8, -9, -3, etc.) in both BMDMs and THP-1 macrophages ( Figures 2C, G ). Lower concentrations of VX-765 (1-25 μM), which inhibits caspase-1 activation, still inhibited the activation of caspase-8, -9, and -3 upon nigericin stimulation in a dose-dependent manner ( Figures 2D-F, H-J ). Consequently, nigericin induced the cleavage of GSDME by activative caspase-3 in caspase-1-deficient BMDMs and THP-1 macrophages. This effect could be mitigated dose-dependently by VX-765 ( Figure 2 ). Thus, VX-765 shows a property of pan-caspase inhibitor, at least in circumstances of NLRP3 activator stimulation.

Previous studies have reported that the AIM2/ASC complex can become a platform for caspase-8 activation when caspase-1 is deficient (21). It is proposed that ASC serves as an adaptor between NLRP3 and caspase-8. Indeed, our results showed that nigericin could not induce the activation of caspase-8 in ASC-deficient RAW 264.7 cells, although it activated caspase-9 weakly. Consequently, cleaved caspase-3 and GSDME fragments upon nigericin treatment were quite weak in RAW 264.7 cells as compared to their respective full-length proteins ( Figure 3A ).

Next, we explored whether NLRP3, ASC, and apoptotic caspases could form a complex upon nigericin stimulation in WT and caspase-1-deficient macrophages by immunofluorescence microscopy. The images showed that NLRP3, ASC, and caspase-1, rather than caspase-8, were co-localized with each other in WT BMDMs ( Figures 3B, C ); but in their caspase-1-deficient counterparts, NLRP3, ASC, and instead caspase-8 were co-localized to form a speck together ( Figures 3D, E ). Similarly, in caspase-1-deficient THP-1 cells, caspase-8 was observed to form a speck with ASC and NLRP3, respectively ( Figure 3F ). The co-localization of NLRP3, ASC, and caspase-3 or caspase-9 (using anti-C. CASP3 and anti-C. CASP9 antibodies) were also observed in caspase-1-deficient cells upon the stimulation with nigericin ( Figure 4 ), suggesting that the NLRP3/ASC complex can serve as a platform for apoptotic caspases activation when caspase-1 is deficient.

Unlike their WT counterparts, caspase-1-deficient BMDMs did not exhibit typical morphological features of pyroptosis, such as cell membrane bubbling and eventual membrane rupture, upon nigericin stimulation. Instead, most cells appeared normal or became rounded and shrunken while maintaining intact plasma membranes, consistent with an apoptotic morphology ( Figures 5A, C ). PI incorporation ( Figures 5A, B ) and LDH release ( Figure 5E ) assays indicated that nigericin induced lytic cell death in WT macrophages, which could be inhibited by VX-765 in a dose-dependent manner. In contrast, caspase-1-deficient cells showed less PI incorporation ( Figures 5C, D ) and LDH release ( Figure 5F ) compared to their WT counterparts upon the same dose of nigericin stimulation. Consistent with the activation of apoptotic caspases ( Figure 2 ), nigericin induced a higher proportion of Annexin-V⁺ (an early apoptosis marker) cells in caspase-1-deficient BMDMs compared to WT cells ( Figures 5G-J ), suggesting differential cell death signaling pathways in these two types of cells.

Characterizing the lytic cell death in the above-mentioned cells, Western blotting corroborated that LPS plus nigericin induced the activation of caspase-1 (as indicated by caspase-1p10 fragment), leading to the release of IL-1β and cleavage of GSDMD (indicative of pyroptosis), in both WT BMDMs and THP-1 macrophages, but not in their caspase-1-deficient counterparts, respectively. As expected, VX-765 inhibited the release of IL-1β and the cleavage of GSDMD in these WT macrophages ( Figures 6A, B ). As indicated above, nigericin could induce weak GSDME cleavage by caspase-3 in caspase-1-deficient macrophages instead of WT ones ( Figure 2 , Figure 6 ), leading to secondary necrosis ( Figures 5C, H ). These results indicate that nigericin induces GSDMD-mediated pyroptosis in WT macrophages while inducing apoptosis and GSDME-mediated secondary necrosis in caspase-1-deficient cells.

Consistent with previous studies that NLRP3 activators can induce mitochondrial damage (2, 22), an upstream event that triggers NLRP3 activation (23–25), our Western blot data showed that nigericin- and ATP-induced GSDMD-NT fragments were present in the mitochondrial parts, while VX-765 dose-dependently reduced their levels ( Figures 7A, B ). Consistently, nigericin increased ROS in WT macrophages, which could be inhibited by VX-765 ( Figures 7C, D ). Interestingly, nigericin also increased ROS in caspase-1-dificient BMDMs ( Figures 7E, F ). It induced the loss of MMP in both caspase-1-deficient and WT macrophages, but could be rescued by VX-765 treatment ( Figures 7G-J ). These results indicate that NLRP3 activators induce mitochondrial damage in both WT and caspase-1-deficient macrophages, suggesting mitochondrial injury being an upstream event in this process.