V. alginolyticus ATCC 17749 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Caspase-1 (p20, AG-20B-0042) and NLRP3 (AG-20B-0014) were purchased from AdipoGen Life Sciences (Liestal, Switzerland). IL-1β (p17) was purchased from R&D (AF-401-SP, Minnesota, USA). Ac-YVAD-CHO was purchased from Enzo Life Sciences (ALX-260-027, Lausen, Switzerland). Z-VAD-FMK and CA-074 methyl ester were obtained from Selleck (S7023, S7042, Shanghai, China). Glyburide was obtained from MedChemExpress (HY-15206, St. New Jersey, USA). RIPA was purchased from Solarbio (R0020, Beijing, China). RPMI 1640 (01-100-1ACS), fetal bovine serum (FBS, 04-001-1ACS), and gentamicin (03-035-1C) were all obtained from Biological Industries (Israel). The TRIzol reagent was purchased from Monad (MI20101S, Wuhan, China). The LDH Cytotoxicity Assay Kit was obtained from Beyotime (C0016, Shanghai, China).

All the animal experiments were conducted in accordance with the requirements of the Animal Welfare and Research Ethics Committee, Jiangsu Ocean University, certificate number 2019220341. Six- to 8-week-old female C57BL/6 mice were purchased from Pizhou Oriental Breeding Company (Pizhou, China). NLRP3 ^-/- mice were purchased from The Jackson Laboratory (ME, USA), and Caspase-1/11^-/- mice were provided by Dr. Feng Shao (Beijing, China). Each mouse was intraperitoneally injected with 2.5 ml of the 2.98% Difco Fluid Thioglycolate Medium (BD, USA) 3–4 days in advance. Then, they were humanely euthanized by cervical dislocation and soaked in 75% alcohol for 5 min. Phosphate-buffered solution (PBS) was used to lavage mice’s abdominal cavity to isolate macrophages. Cells were washed three times with PBS and centrifuged at 1,000 × g for 10 min. The cell precipitation was resuspended in 1 ml of RPMI 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin. Ten microliters of cell suspension was diluted for cell counting using the counting formula: cell count/mL=cell count/80 × 400 × 10⁴ × dilution multiple. According to the counting results, 4.5 × 10⁶ cells/well were added into six-well plates and cultured in an incubator containing 5% CO₂ at 37°C for 4–6 h. After sedimentation, unattached cells were discarded and the culture medium was replaced with RPMI 1640 medium supplemented with 1% FBS and 1% penicillin–streptomycin, and cells were continued to culture for 12 h and then used for subsequent experiments. The cell purity was verified with APC anti-mouse/human CD11b (1:200, BioLegend, USA) via flow cytometry.

V. alginolyticus was enriched in alkaline peptone water (APW) under conditions of 30°C/200 rpm. V. alginolyticus at a logarithmic growth period (OD_{600 nm} = 0.6) was counted, washed three times in sterile PBS, and then resuspended in RPMI 1640 medium supplemented with 1% FBS. Peritoneal macrophages were infected with V. alginolyticus at different multiples of infection (MOI). After incubating for 3 h, V. alginolyticus were removed followed by washing three times with PBS. Then, the cells were incubated in RPMI 1640 medium supplemented with 1% FBS and 3 µg/ml gentamicin at 37°C for 3, 6, or 12 h.

Total RNA was extracted from cell pellets using the TRIzol reagent according to the manufacturer’s recommendations. One microliter of total RNA was diluted 10 times using diethyl pyrocarbonate (DEPC) water, and the concentration and purity were measured with the NanoDrop ND-2000 apparatus (Thermo Scientific, USA). Two to three micrograms of total RNA was reverse transcribed to cDNA and then amplified using MonAmp SYBR Green qPCR Mix (None ROX, China). The quantitative real-time PCR (qPCR) protocol was set as follows: the initial cDNA template was denatured at 95°C for 30 s, followed by amplification of the cDNA template for 40 cycles, in which the amplified cycles include denaturation at 95°C for 10 s, primer annealing, and extension at 60°C for 30 s. qPCR assays were performed on a LightCycler 480 II machine (Roche, Germany). Relative mRNA fold changes were normalized to an internal control of β-actin. The data were calculated by the comparative Ct pattern (2^-ΔΔCt formula). Primers were synthesized from Sangon Biotech (Shanghai, China), and the primer sequences are listed in Table 1 .

The culture supernatants were collected and concentrated as previously described (Zhao et al., 2021). Cell pellets were washed in ice-cold PBS and lysed with RIPA containing the protease inhibitor of phenylmethanesulfonyl fluoride (PMSF, 1 mM). The protein samples were quantified with a BCA Protein Assay Kit (Thermo Scientific, USA) according to the manufacturer’s protocol. Each sample (30 μg) of cell lysates or cell supernatants (10 μl) were separated by 12% SDS-PAGE, and the electrophoresis protocol was set as follows: 80 V 1 h 05 min and 120 V 30 min. Then, the protein was transferred onto 0.22 or 0.45 μm PVDF membrane (Millipore, USA) under conditions of 200 mA/1 h or 1 h 30 min at 4°C. The membranes were blocked in 5% bovine serum albumin (BSA) in PBST for 2 h at room temperature and incubated at 4°C overnight with different primary antibodies (Abs), including IL-1β (1:1,000), caspase-1 (1:1,000), NLRP3 (1:1,000), and β-actin (1:5,000). The HRP-conjugated secondary antibodies were rabbit anti-goat IgG (H+L) (1:5,000) or goat anti-mouse IgG (H+L) (1:5,000, EarthOx, USA). The blots were developed using the ChemiScope Western Blot Imaging System (Clinx Science Instruments, China).

The culturing supernatants were collected from infected cells, centrifuged to remove cell precipitation, and stored at -80°C for subsequent experiments. ELISA Kits (Invitrogen, USA) were used to analyze the secretion levels of IL-1β, IL-6, IL-12, and TNF-α in the supernatants according to the instructions provided by the manufacturer. The absorbance at 450 nm was read using a microplate reader (BioTek, USA). The OD values were converted to pg/mL according to the standard curve.

To explore whether V. alginolyticus triggered cell death in mouse macrophages, the LDH levels were tested in infected cell culture supernatant at 12 h (MOI = 0.1, 0.5, 1, or 10). The absorbances were read under 490- and 600-nm wavelength according to the manufacturer’s recommendations.

Macrophages previously prepared on coverslips in 24-well plates were inoculated with V. alginolyticus (MOI = 1) for 3 h, and then the culture medium was replaced by RPMI 1640 medium supplemented with 1% FBS and 3 µg/ml gentamicin after washing three times with PBS. After 12 h, the infected cells were fixed in 4% paraformaldehyde (Biosharp, Beijing), and then the cell membrane was penetrated in 0.5% Triton X-100. Cells were blocked in 5% BSA at room temperature for 2 h and incubated with NLRP3 Rabbit pAb (1:100) at 4°C overnight. Then, cells were incubated with an FITC-labeled secondary antibody of goat anti-mouse (H+L) (1:200) at 37°C for 1 h; the nucleus was then stained with 1 μg/ml 4′,6-diamidino-2-phenylindole, dihydrochloride (62247, DAPI, Thermo Scientific, USA) as recommended by the manufacturer. The coverslips were fixed to Adhesion Microscope Slides (Servicebio, China), and the fluorescence images were observed on the fluorescence microscope (Olympus, Japan).

Inhibitors of Ac-YVAD-CHO, Z-VAD-FMK, CA-074 methyl ester, and glyburide were prepared in the appropriate storage solutions according to the product instructions and stored at -80°C. The inhibitors were cocultured with peritoneal macrophages for 1 h prior to stimulation. The corresponding final concentrations are as follows: Ac-YVAD-CHO (100 μM), Z-VAD-FMK (10 μM), CA-074 methyl ester (25 μM), and glyburide (50 μM). Then, the inhibitor was discarded and washed three times with sterile PBS and replaced with fresh medium. Subsequently, the cells were infected with V. alginolyticus for 3 h and the culture medium was replaced by RPMI 1640 medium supplemented with 1% FBS and 3 µg/ml gentamicin after washing three times with PBS. After 12 h, cells were collected for qPCR analysis and Western blotting. The supernatants were used for ELISA assays.

The results of the bar chart were presented through GraphPad Prism 8 (Inc., La Jolla, USA) with mean ± standard deviation (SD). The gray value comparison results were analyzed by ImageJ (National Institutes of Health, USA). The dataset comparisons were performed with IBM SPSS Statistics 20 (Chicago, IL), and there were two cases: the independent sample t-test was used to compare between two groups of data; the data of three groups and above were analyzed by one-way ANOVA, and the uniformity of variances was taken as Bonferroni or the inhomogeneity of variances was taken as Tamhane’s T2. All experiments were conducted at least three times with representative results shown in the figures. Data were regarded as statistical significance when ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and not significant (ns), p > 0.05.

Exposure of RAW264.7 macrophages to V. parahaemolyticus triggered inflammatory response by secretion of inflammation-related cytokines, such as IL-1β, IL-6, and TNF-α (Gulati et al., 2019). Therefore, the secretion of inflammatory cytokines in V. alginolyticus-infected peritoneal macrophages was first measured. The qPCR data showed that the transcriptional levels of various inflammatory cytokines in the lysates of macrophages were obviously upregulated (^** p < 0.01 or ^*** p < 0.001) at 2, 4, and 6 h except for IL-12, and IL-1β occupied the highest expression level ( Figure 1A ). To further determine the secretion of IL-1β, the protein expression levels of IL-1β/p17 were detected in cell supernatants collected from infected cells at MOI = 0.1, 0.5, and 1. Western blotting combined relative gray values to β-actin data, suggesting that the IL-1β p17 protein expression levels in different infection doses were significantly higher than those in the untreated control (C) group (^*** p < 0.001). Moreover, the relative gray value data showed that the protein levels of IL-1β p17 continued to increase with the increase in infectious dose ( Figure 1B ). In addition, the release of LDH, a marker of lytic cell death, occurred in response to V. alginolyticus stimulation with no obvious change under different MOI treatments ( Figure 1C ). These data together indicated that V. alginolyticus infection triggered inflammation and induced the release of IL-1β.

Previous studies confirmed that V. parahaemolyticus infection induced IL-1β release by activation of caspase-1 in mouse bone marrow-derived macrophages (Higa et al., 2013). To test whether IL-1β secretion depends on the classical caspase-1 cleavage pathway or the non-classical caspase-11 cleavage pathway, the secretion of IL-1β or activation of caspase-1 in the culturing supernatants and the expression of pro-caspase-1 in cell lysates were detected by Western blotting. Results showed that after treatment with the caspase-1 inhibitor of Ac-YVAD-CHO or pan-caspase inhibitor of Z-VAD-FMK, caspase-1/p20 activation was significantly inhibited (^*** p < 0.001). Interestingly, no obvious changes were found in the expression of pro-caspase-1 in cells (p > 0.05). Inhibition of caspase-1 activation significantly led to decrease in IL-1β/p17 secretion (^*** p < 0.001) in comparison to the V. alginolyticus infection group. This was consistent with the mRNA expression levels of IL-1β measured by qPCR (^*** p < 0.001) and IL-1β secretion levels detected by ELISA (^* p < 0.05, Figure 2 and Figure S1 ). Overall, these data manifested that the IL-1β release was dependent on the activation of caspase-1 in V. alginolyticus infected mice macrophages.

PRRs were the key molecules in recognizing PAMPs or DAMPs; the transcription levels of central molecules of different inflammasome families were detected in macrophages at 2, 4, 6, and 12 h after V. alginolyticus infection. The qPCR data showed that the expression levels of NOD1, NOD2, NLRP3, NLRC5, and NLRP6 in peritoneal macrophages significantly elevated at 2, 4, 6, and 12 h (^** p < 0.01 or ^*** p < 0.001). The gene levels of NLRP1, NLRP2, NLRC4, and AIM2 markedly increased at 12 h (^** p < 0.01), and the transcription levels of NLRP12 remained unchanged at 12 h (p > 0.05). On the basis of these data, the NLRP3 receptor upregulated to the highest levels when compared with other NLR family members including the AIM2 receptor ( Figure 3A ). To further explore whether the NLRP3 inflammasome was assembled during V. alginolyticus infection, the subcellular localization of the NLRP3 protein was observed by immunofluorescence staining. Images captured by fluorescence microscopy showed that NLRP3 aggregated in the perinuclear region after infection with V. alginolyticus, whereas no signals were observed in untreated cells ( Figure 3B ). In order to confirm the NLRP3 protein expression levels, Western blotting assays were conducted to detect the expression of NLRP3 protein at different infection doses (MOI = 0.1, 0.5, and 1). Relative gray values showed that the NLRP3 protein was significantly upregulated at various infection doses compared to the control group (***p < 0.001) and no protein was detected in the C group ( Figure 3C ). To further explore the activating ways of NLRP3 inflammasome and its roles in the regulation of IL-1β during V. alginolyticus infection, cells were pretreated with glyburide or CA-074 methyl ester prior to stimulation and the protein expression levels of NLRP3 and IL-1β were detected using Western blotting and ELISA assays. The protein expression levels of IL-1β p17 significantly decreased in both inhibition groups when compared to the V. alginolyticus infection group (^*** p < 0.001); in contrast, the protein expression levels of pro-IL-1β and NLRP3 in cells displayed no significant differences between the experiment group and the inhibitor-treated groups (p > 0.05). ELISA results also indicated that both glyburide and CA-074 methyl ester downregulated the IL-1β protein secretion levels (^* p < 0.05) and CA-074 methyl ester reduced IL-1β to relatively lower levels, suggesting that V. alginolyticus infection mainly activates the NLRP3 inflammasome through the release of cathepsin B ( Figure 3D ).

Similarly, the results of qPCR are indistinguishable from those of western blotting and ELISA (^*** p < 0.001, Figure S2 ). Taken together, these data revealed that V. alginolyticus induced the release of the proinflammatory cytokine of IL-1β in mouse peritoneal macrophage, which was highly dependent on the activation of NLRP3 inflammasome.

To better make sense of the relationship between activation of NLRP3 inflammasome and infection by V. alginolyticus, an experiment with V. alginolyticus on different infection doses was conducted to detect the secretion of IL-1β in peritoneal macrophages using qPCR, Western blotting, and ELISA methods. The grayscale analysis results showed that the protein expression levels of NLRP3 were significantly upregulated in a dose-dependent manner along with the increased infection dose of V. alginolyticus (^** p < 0.01 or ^*** p < 0.001). The qPCR data showed that V. alginolyticus could induce the upregulation of IL-1β mRNA transcription at different infection doses (^*** p < 0.001) and the levels showed a trend of increasing and then decreasing ( Figure S3A ). Similar results were found in the protein expression levels of IL-1β p17 in the supernatants (^* p < 0.05, ^** p < 0.01, or ^*** p < 0.001), although pro-IL-1β levels remained unchanged at MOI = 1 vs. MOI = 0.1 and MOI = 1 vs. MOI = 10 (p > 0.05). In addition, caspase-1 p20 in the infected supernatants was significantly upregulated at MOIs of 1 and 10 compared to MOI of 0.1 (^** p < 0.01 or ^*** p < 0.001), and the levels remained the same when MOIs of 1 and 10 were used; on the contrary, there were no noticeable changes in pro-caspase-1 levels in the cell precipitation (p > 0.05, Figure 4A ).

The infection dose of MOI = 1, which secreted the highest levels of IL-1β p17, was selected for the different inoculation time assays. Western blotting combined with relative gray value analysis data showed that the protein expression levels of NLRP3 were significantly upregulated in a time-dependent manner during the monitored infection time of V. alginolyticus (^* p < 0.05 or ^** p < 0.01). Meanwhile, the secretion of mature IL-1β was in a time-dependent manner, with the maximum amount at 12 h, as indicated by Western blotting and ELISA (^** p < 0.01 or ^*** p < 0.001). However, the protein levels of pro-IL-1β in the cell lysates did not vary significantly (p > 0.05) and qPCR data showed that IL-1β mRNA expression markedly spiked at 3 h during exposure to V. alginolyticus and then remained in a continuous downward trend at 6 and 12 h (^*** p < 0.001, Figure S3B ). The activation of caspase-1 p20 was positively correlated with the secretion amounts of IL-1β p17, which significantly increased during the monitored time period of 3 to 12 h, and there are significant differences at different times (^** p < 0.01 or ^*** p < 0.001), but pro-caspase-1 expression levels in cells remained steady (p > 0.05, Figure 4B ). Generally, these results confirm that the activation of the NLRP3 inflammasome was accumulated when infection time and dose were increased within a certain range.

V. alginolyticus-mediated macrophages inflammation upregulated the expression of various pro-inflammatory cytokines. Therefore, inhibition combined with ELISA assays was performed to detect the protein release levels of IL-6, IL-12, and TNF-α. It appeared that the IL-6, IL-12, and TNF-α protein release levels were significantly decreased in inhibitor groups (^** p < 0.01 or ^*** p < 0.001) except for IL-6 levels in CA-074 methyl ester and Ac-YVAD-CHO pretreatment groups (p > 0.05, Figures 5A–C ). To further determine the roles of the NLRP3 inflammasome, the protein production levels of IL-1β, IL-6, IL-12, and TNF-α were measured in the wild-type, NLRP3^-/-, and caspase-1/11^-/- macrophages after V. alginolyticus infection. The results confirmed that the protein levels of IL-1β, IL-6, IL-12, and TNF-α remarkably reduced in the supernatant of NLRP3^-/- and caspase-1/11^-/- macrophages compared with the supernatant of WT macrophages infected with V. alginolyticus at 12 h (^*** p < 0.001, Figure 5D ). The validation of gene deletion efficiency in macrophages from mice that lacked NLRP3 and caspase-1/11 was performed in Figures S4A, B . Overall, these results illustrated that blocking the NLRP3 inflammasome downregulated V. alginolyticus-triggered inflammatory response in mouse macrophages.