The experimental protocol involving animals was approved by the Ethics Committee on the Use and Care of Animals at Northeast Agricultural University (Approval No. NEAUEC20240383), in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 2000). All experiments were conducted in compliance with the relevant guidelines and regulations.

T. pyogenes (strain 0912) was isolated and stored in our laboratory and was cultured in Martin broth medium with 10% fetal bovine serum (FBS) under aerobic conditions. The culture was harvested during the late-log phase. The density of T. pyogenes cells was determined by colony counting method. T. pyogenes was inactivated by adding formaldehyde into the culture to a final concentration of 0.5% and incubating at 37°C for 48 h. The inactivation of the T. pyogenes cells was determined by bacterial culture in liquid cultural medium. Then, the inactivated T. pyogenes cells (iTP) were washed twice with ice-cold sterile phosphate buffer saline (PBS) and were finally suspended in sterile PBS. The T. pyogenes suspension was adjusted to a concentration of 4 × 10¹⁰ colony-forming units per milliliter (CFU/mL) and stored at -80°C until use.

Recombinant plasmid pET-30a (+)-plo, which encodes recombinant mature PLO (rPLO) with an N-terminal extension of a 52-aa fusion peptide, was laboratory constructed (19). Recombinant plasmid pET-30a (+)-plo W497F was constructed according to another previous study (20). pET-30a (+)-plo W497F was constructed using a PCR-mediated DNA mutation system fast mutagenesis system (Transgen Biotech, Beijing). The recombinant plasmid pET-30a-plo was used as a template, and the sequences of the primers are 5′-GCAACTGGCCTAGCGTTCGATCCGTGGTGG-3′ and 3′-TGACCGGATCGCAAGCTAGGCACCACCTGG-5′. The resulting pET-30a (+)-plo W497F encodes rPLO W497F with the tryptophan (W) at the position of 497 of rPLO, which was replaced with a phenylalanine (F). The cell membrane binding capacity of rPLO W497F [named His-PLO. F₄₉₇ in Billington, et al. (20)] was reduced compared with that of rPLO.

The recombinant plasmids were transformed into Escherichia coli (E. coli) Rosetta (DE3)™ competent cells. rPLO and rPLO W497F were expressed by inducing with isopropyl-β-D-thiogalactoside (IPTG) and then purified with nickel-charged resin. The purified proteins were dialyzed against PBS with 5% glycerol at 4°C for 48 h. The proteins were quantified by Bradford method and stored at -80°C until use.

The assays for detecting the membrane binding ability of rPLO and rPLO W497F were performed as previously described (21). Briefly, sheep red blood cell membranes (RBCM) were prepared. rPLO and rPLO W497F, or PBS (as a negative control), were incubated with the RBCM suspension for 30 minutes at 37°C and then washed twice with PBS. The proteins in the samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (NC) membranes. The binding of rPLO and rPLO W497F to RBCM was determined by Western blot analysis using an anti-His tag monoclonal antibody as the primary antibody.

For the hemolysis assay, the concentrations of rPLO and rPLO W497F were first adjusted to 200 μg/mL. The proteins were then subjected to two-fold serial dilutions with PBS in V-bottomed 96-well microtiter plates. An equal volume of a 2% suspension of sheep red blood cells (sRBCs) was added to each well. The mixtures were incubated at 37°C for 30 minutes. Complete hemolysis was defined as the absence of visible sRBC precipitate at the bottom of the well.

To observe the pores formed by the recombinant proteins, the concentrations of rPLO and rPLO W497F were adjusted to 400 μg/mL. Then, 100 μL of each protein (rPLO and rPLO W497F) was mixed separately with 900 μL of a 2% sRBC suspension. The mixtures were incubated at 37°C for 30 minutes, then centrifuged at 5000 r/min for 7 minutes at 4°C. The supernatant was discarded, and the precipitates were resuspended in 200 μL of fresh PBS and negatively stained. The specimens were observed under a Hitachi H-7650 electron microscope at an acceleration voltage of 100 kV.

Bone marrow-derived macrophages (BMDMs) were obtained from 3- to 6-week-old C57BL/6 mice, with slight modifications to a previously described method. After sacrificing the mice, the bone marrow was flushed from the femurs. The cells were then washed and resuspended in Roswell Park Memorial Institute-1640 (RPMI-1640) medium, supplemented with 10% FBS and 15% (v/v) L929 cell-conditioned medium, which served as a biological source of macrophage colony stimulating factor (M-CSF). The medium was replaced on days 3 and 5, and non-adherent cells were removed. The cells were used for experiments starting on day 7.

The sublytic concentration of rPLO against BMDMs was determined using a lactate dehydrogenase (LDH) release assay. Briefly, BMDMs were seeded into a 48-well cell plate at a density of 1×10⁵ cells per well. The cells were then incubated with various concentrations of rPLO (8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL) in RPMI-1640 medium without FBS for 30 minutes at 37°C. For the control group, cells were treated with RPMI-1640 medium supplemented with PBS. The cells were observed and photographed using an inverted microscope. The amount of released LDH in the medium was measured using an LDH cytotoxicity assay kit (C0016, Beyotime Biotechnology) according to the manufacturer’s instructions. The sublytic concentration of rPLO was defined as the lowest concentration that did not induce significant morphological changes or cell detachment but resulted in a significant increase in LDH release compared to the control group. LDH release was also measured in cells treated with various concentrations of rPLO W497F.

For the in vivo experiment, nine 6-week-old female BALB/c mice were randomly divided into three groups. On days 0, 7, and 14, mice in the two experimental groups received subcutaneous injections of rPLO (25 μg in a final volume of 300 μL) and iTP (1 × 10¹⁰ CFU in a final volume of 300 μL), respectively. Mice in the control group received subcutaneous injections of PBS containing 5% glycerol (300 μL). On day 21, the mice were humanely sacrificed, and tissues near the injection sites were collected. Part of the tissues was weighed and homogenized in 5 mL sterile PBS on ice. The homogenates were centrifuged at 12,000 r/min at 4°C for 5 min, and the supernatants were harvested. The expression levels of cytokines in the supernatants were detected by enzyme-linked immunosorbent assay (ELISA). The amount of cytokines in the homogenate supernatants was converted to the amount per gram of muscle tissue for further statistical analysis. The remaining tissues were frozen in liquid nitrogen and ground into powder. Total RNA was extracted using TRIzol™ reagent (Invitrogen, USA) to measure cytokine expression at the mRNA level by quantitative real-time PCR (qRT-PCR) assays.

In the in vivo IL-1β blocking assay, each mouse received a mixture containing 30 μg of IL-1β-specific polyclonal antibody (Proteintech, China) and 50 μg of rPLO subcutaneously. Control mice received 50 μg of rPLO only. Two days later, the mRNA level of interleukin-6 (IL-6) in the tissues surrounding the injection sites was determined by qRT-PCR.

For the in vitro experiment, BMDMs were seeded in a 6-well cell plate at a density of 1×10⁶ cells per well. The cells were incubated with rPLO (0.03 μg/mL) or iTP (4 × 10⁹ CFU) in RPMI-1640 medium supplemented with 10% FBS for 16 h at 37°C. Supernatants were harvested after the incubation and stored at -80°C. The amount of cytokines in the supernatants was determined by ELISA. Total RNA was extracted from the cells using TRIzol™ reagent, and cytokine expression at the mRNA level was determined by qRT-PCR assays.

For patch-clamp experiments, RAW 264.7 cells were plated overnight in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS on sterile glass coverslips in six-well plates at a density of 1×10⁴ cells per well. On the day of the experiment, the cells were incubated with or without rPLO at the sublytic concentration for 30 minutes. Whole-cell recordings of potassium ion (K⁺) currents were performed using standard patch-clamp techniques as previously described with slight modifications (22). The cells were superfused at room temperature with an extracellular solution containing (in mM): 150 NaCl, 6 KCl, 10 HEPES, 1.5 CaCl₂, 1 MgCl₂, and 1 ethylene glycol tetraacetic acid (EGTA) (pH 7.3). The pipette solution contained (in mM): 145 KCl, 10 HEPES, 1 MgCl₂, and 1 EGTA (pH 7.3). Voltage steps ranging from -40 mV to +120 mV were applied from a holding potential of -60 mV to determine the current-voltage (I-V) relationship. Each pulse lasted for 500 ms with an interval of 5 s. All recordings were performed at least 5 minutes after establishing the whole-cell configuration. Currents were recorded using an Axon 700B Amplifier (Axon Instruments, Foster City, CA). Data were filtered at 2 kHz and digitized at 10 kHz. A CED Power 1401 Analogue/Digital Interface (Cambridge Electronic Design Limited, Cambridge, United Kingdom), controlled by Signal 3.06 software (Cambridge Electronic Design Limited), was used for generating voltage commands, recording the current, and collecting data. Data were analyzed offline using Origin v8.0 (Northampton, MA). Currents were normalized to cell capacitance (pA/pF). Five to six cells were recorded for each experimental condition. All recordings were performed within a 1-hour window after exposing the cells to the recombinant proteins.

BMDMs were seeded in a six-well cell culture plate at a density of 1×10⁶ cells per well and were subsequently treated with rPLO at the sublytic concentration, rPLO W497F (at a concentration equal to that of rPLO), nigericin (5 mM), and rPLO plus 120 mM KCl at 37°C for 30 minutes. The intracellular K⁺ concentrations were then determined using a cellular potassium concentration quantitative test kit (chemical colorimetric) (HL70032.2, Shanghai Haling Biological Technology Co., Ltd.) according to the manufacturer’s instructions.

J774A.1 cells were transduced with pooled Nlrp3 siRNA/shRNA/RNAi lentivirus (mouse) (iV043412, Abm Inc.) or scrambled siRNA GFP lentivirus (LVP015-G, Abm Inc.). The cells were transduced with Nlrp3 siRNA or control siRNA and were selected using puromycin. The efficiency of NLRP3 silencing was confirmed by Western blot analysis.

Cells were treated with iTP or lipopolysaccharides (LPS) (1 μg/mL) for various durations to examine the expression of pro-IL-1β. In some experiments, cells were co-treated with iTP plus TPCA-1 (a NF-κB pathway inhibitor, 1 μM) or BAY11-7082 (a NF-κB pathway inhibitor, 10 μM).

The culture medium was discarded, and a fresh medium without FBS but containing rPLO, rPLO W497F, or nigericin was added. Cells were then incubated at 37°C for 30 min. In some experiments, cells treated with rPLO were also exposed to KCl (5, 10, 25, 50, 100, or 120 mM), NaCl (125 mM), Z-VAD-FMK (a pan-caspase inhibitor, 20 μM), Z-YVAD-FMK (a caspase-1 inhibitor, 20 μM), or Z-DEVD-FMK (a caspase-3 inhibitor, 20 μM).

After treatment, cells were lysed with a cell lysis buffer for Western blotting and IP (Beyotime, China) supplemented with PMSF. The culture supernatant components were precipitated by adding trichloroacetic acid. The precipitates were then re-dissolved in NaOH. The samples were denatured in 5× reducing sample buffer for SDS-PAGE and boiled for 10 min. Proteins separated by SDS-PAGE were transferred to NC or polyvinylidene difluoride membranes and immunoblotted with primary antibodies against caspase-1 p20 (B200721, Biolegend), NLRP3 (AG-20B-0014, Adipogen), IL-1β (12507S, Cell Signaling Technology), and GAPDH (TA-08, ZSGB-BIO). The membranes were then treated with HRP-linked secondary antibodies: anti-rabbit (ZDR-5306, ZSGB-BIO), anti-rat (ZB-2307, ZSGB-BIO), or anti-mouse (ZDR-5307, ZSGB-BIO).

BMDMs were seeded in 24-well cell cultural plate at a density of 2 × 10⁵ cells per well. BMDMs was primed with or without LPS and then treated with rPLO. Active caspase-1, caspase-3, and caspase-8 was quantified by using caspase activity assay Kit (caspase-1 C1102; caspase-3 C1116; caspase-8 C1152, Beyotime Biotechnology) according to the manufacturer’s instructions.

Total cellular RNA was reverse-transcribed into cDNA using a reverse transcriptase kit. qRT-PCR was performed using the SYBR Green method. The specific primer sequences for mouse IL-1β, interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis-α factor (TNF-α), and β-actin are listed in Table 1 . Expression levels were normalized to that of β-actin. All measurements were performed in triplicate, and expression changes were normalized to untreated controls.

The amount of TNF-α, IL-1β, IL-6 and IL-10 in samples were detected by commercial ELISA kits from Uscn Life Science Inc., China (TNF-α, SEA133Mu; IL-1β, SEA563Mu; IL-6, SEA079Mu; IL-10, SEA056Mu) according to the manufacturers’ instructions.

All statistical analyses were performed based on data acquired from three or five biological replications of the experiment. One-way or two-way AVONA test was used to analyze the data using Graphpad Prism 10.4.0 software (GraphPad Software), the normality of the data distribution was determined by Shapiro-Wilk test. The results are presented as mean ± SD. A p value < 0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

The purified rPLO and rPLO W497F were serially diluted and then incubated with RBCs to determine the differences in their hemolytic activities. The results showed that a concentration of 3.125 µg/mL of rPLO was sufficient to cause complete hemolysis, whereas rPLO W497F only caused partial hemolysis even at a concentration as high as 200 µg/mL ( Figure 1A ). These results suggested that rPLO W497F had almost completely lost hemolytic activity. TEM observations revealed that rPLO could form pores in the membranes of RBCs, whereas rPLO W497F could not ( Figure 1B ). RBCM binding assays further showed that rPLO could bind to RBCM, while rPLO W497F could not ( Figure 1C ).

When incubated with BMDMs, rPLO at concentrations higher than 0.25 µg/mL caused significant LDH release (p < 0.01) from BMDMs within a 30-min incubation period ( Figure 1D , upper panel). In contrast, rPLO W497F did not cause significant LDH release even at a concentration as high as 8 µg/mL ( Figure 1D , lower panel). Microscopic observations indicated that rPLO at concentrations up to 0.5 µg/mL did not induce obvious morphological changes or exfoliation in BMDMs ( Figure 1E ). These results suggested that 0.5 µg/mL of rPLO could disrupt the cell membrane integrity of BMDMs without causing cell death within 30 min. Therefore, 0.5 µg/mL was considered a sub-cytotoxic concentration of rPLO for BMDMs and was used in subsequent experiments.

iTP and purified rPLO were used to treat mice and BMDMs, respectively. qPCR and ELISA results showed that rPLO treatment significantly upregulated the levels of IL-1β, TNF-α, and IL-6 at both the mRNA and protein levels in the muscles of mice and in BMDMs. While not as potent as rPLO treatment, iTP treatment also increased the expression levels of IL-1β, TNF-α, IL-6, and IL-10 in both mice and cells ( Figure 2A ). Injection of anti-IL-1β-specific antibody with rPLO significantly inhibited the synthesis of IL-6 mRNA in the tissues of rPLO-challenged mice ( Figure 2B ). Since IL-1β can induce the expression of IL-6, this result indicates that IL-1β plays an important role in the occurrence of PLO-induced inflammation.

Western blot assays showed that iTP could induce the synthesis of pro-IL-1β in BMDMs ( Figure 3A ). LPS, a classical TLR4 agonist, was used as a positive control ( Figure 3A ). A 2-h treatment of BMDMs with iTP was sufficient to induce pro-IL-1β synthesis ( Figure 3B ). However, iTP was not efficient in promoting the maturation of IL-1β, as the mature IL-1β (the 17 kDa band) could only be detected in BMDMs incubated with iTP for 10 h, and the 17 kDa band on the NC membrane was very weak ( Figure 3B ). TPCA-1 and BAY11-7082, two inhibitors of the NF-κB pathway, significantly inhibited the iTP-induced synthesis of pro-IL-1β at both the protein ( Figure 3C ) and mRNA levels ( Figure 3D ). These results indicate that iTP induces the synthesis of pro-IL-1β in a NF-κB–dependent manner.

BMDMs were primed with LPS for 4 h and then incubated with rPLO or rPLO W497F. Western blot assays showed that pretreatment with LPS promoted the synthesis of pro-IL-1β but did not induce the maturation of IL-1β in BMDMs ( Figure 4A ). Subsequent incubation with rPLO led to the production of mature IL-1β (as shown by the 17 kDa band on the NC membrane) within 30 min ( Figure 4A ). In contrast, rPLO W497F failed to elicit the maturation of IL-1β in LPS-primed BMDMs ( Figure 4A ). ELISA results revealed that the amount of IL-1β in the culture supernatants of cells treated with rPLO was significantly higher (p < 0.01) than that in the untreated controls, regardless of whether the cells were primed with LPS ( Figure 4B ). The IL-1β production in LPS-primed cells was significantly higher than that in unprimed cells after treatment with rPLO (p < 0.01) ( Figure 4B ). By contrast, the IL-1β production in cells treated with LPS + rPLO W497F did not significantly differ from that in untreated cells ( Figure 4B ). These findings suggested that a 30-min incubation with rPLO could induce rapid maturation of IL-1β and the cell membrane binding and pore-forming activities of rPLO are essential for promoting pro-IL-1β maturation in BMDMs.

Enzymatic activity measurements showed that rPLO treatment activated caspase-1 and caspase-3, but not caspase-8, within 30 min ( Figure 5A ). To determine which caspase is responsible for the maturation of IL-1β, cells were treated with rPLO in the presence of caspase inhibitors. The results showed that Z-VAD-FMK (a pan-caspase inhibitor) and Z-YVAD-FMK (a caspase-1 inhibitor) completely abolished the maturation of IL-1β, whereas Z-DEVD-FMK (a caspase-3 inhibitor) did not ( Figure 5B ). To confirm that caspase-1 was activated in rPLO-treated cells, LPS-primed BMDMs were treated with rPLO or nigericin. The results showed that rPLO treatment indeed activated caspase-1 within 30 min ( Figure 5C ). Therefore, caspase-1 is responsible for PLO-induced IL-1β maturation.

Since K⁺ plays an essential role in caspase-1 activation (23), we first evaluated the K⁺ flux in rPLO-treated macrophages using electrophysiological techniques. As shown in Figure 6A , exposure of RAW 264.7 cells to rPLO significantly increased the whole-cell outward K⁺ current density at depolarizing potentials. Next, we assessed the intracellular K⁺ concentrations in BMDMs treated with recombinant proteins. The results showed that BMDMs treated with rPLO (p < 0.05) or nigericin (p < 0.01), a well-characterized K⁺ ionophore, exhibited significant reductions in intracellular K⁺ concentration compared to untreated cells, whereas BMDMs treated with rPLO W497F had intracellular K⁺ concentrations similar to those of untreated BMDMs ( Figure 6B ). BMDMs treated with rPLO plus 120 mM KCl showed increased intracellular K⁺ concentrations compared to untreated cells ( Figure 6B ), indicating a reversal of intracellular K⁺ depletion in rPLO-treated cells in the presence of high extracellular K⁺ concentrations.

Western blot assays revealed that 120 mM KCl, but not 120 mM NaCl, inhibited the activation of caspase-1 and the processing of pro-IL-1β induced by rPLO in LPS-primed BMDMs ( Figure 6C ). Even at a low extracellular KCl concentration of 5 mM, the activation of caspase-1 and processing of pro-IL-1β induced by rPLO in LPS-primed BMDMs were partially inhibited ( Figure 6D ).

The above results indicate that PLO can efficiently induce K⁺ efflux in macrophages, leading to caspase-1 activation and pro-IL-1β processing.

We first constructed J774A.1 cell lines expressing either nlrp3 siRNA or control siRNA via lentivirus-mediated transduction. The cells were then treated as described. Figure 7 shows that rPLO induced caspase-1 activation and pro-IL-1β processing in J774A.1 cells expressing control siRNA, but not in those expressing nlrp3 siRNA. These results indicate that rPLO-induced caspase-1 activation and pro-IL-1β processing are dependent on the NLRP3 inflammasome.