The wild-type (WT) C57BL/6 mice were purchased from Chongqing Academy of Chinese Material Medical (Chongqing, China). Nlrp3^–/–, Asc^–/– and Casp1/11^–/– mice were kindly gifted by Dr. Feng Shao from the NIBS (National Institute of Biological Sciences, Beijing, China). All gene knockout mice were on a C57BL/6 background and maintained in Specific Pathogen Free (SPF) conditions for being used at 8-10 weeks old. All of animal experiments were approved by the Southwest University Ethics Committee, Chongqing, China (IACUC-2019-0112-02).

The highly virulent bovine P. multocida capsular type A PmCQ2 (GenBank accession number: LIUN00000000) was isolated from the lungs of calves with pneumonia in Chongqing, China (Du et al., 2016). The bacteria were stored at −80 °C and incubated on Martin’s agar plates at 37°C for 18–24 h. Then, the single colony was incubated in 5 mL Martin Broth (Solarbio, China) at 37°C for 12 h. Bacterial concentration was determined by colony counting, and the bacteria was diluted as indicated concentration for assays described below.

Peritoneal exudate cells (PECs) were collected as previously reported (Fang et al., 2019). Briefly, mice were injected intraperitoneally with 2 mL of 4% thioglycolate (Eiken, Japan) and PECs were collected 3 days later. PECs were suspended with RPMI 1640 medium containing 10% FCS. Cells were seeded into each well at 2 × 10⁵ cells/well for 48-well plates or 1.0 × 10⁶ cells/well for 12-well plates and incubated at 37°C with 5% CO₂. After 2 h incubation, the non-adherent cells were removed and adherent cells were infected with PmCQ2 at a multiplicity of infection (MOI) of 1 for 9 h. Then 100 μg/mL of ciprofloxacin (Solarbio, China) was added for an additional 15 h. After 24 h incubation, supernatants and cell lysates were collected for assays described below. To inhibit the outflow of potassium ions, cells were pretreated with KCl, Quinine (Sigma, United States) or Glibenclamide (dilute with DMSO, TCI, China) for 30 min before bacterial infection.

Cells were prepared in 48-well plates and infected with PmCQ2 as described above. After infection, supernatants were collected and Enzyme Linked Immunosorbent Assay (ELISA) was used to measure the levels of cytokines according to the manufacturers’ instructions. The kits of IL-1β, TNF-α, and IL-6 were purchased from Invitrogen (CA, United States).

Cells were prepared in 12-well plates and infected with PmCQ2 as described above. After infection, supernatants were collected and concentrated using 20% (w/v) trichloroacetate, and the cells were lysed with 1 × SDS Loading buffer (Beyotime, China). Cell lysates were subjected to 12% SDS-PAGE and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane by electroblotting. The membranes were blocked with 5% non-fat dry milk and then immunoblotted with indicated antibodies (Abs) including anti-IL-1β Ab (R&D, United States), anti-Caspase1-p20 Ab (AdipoGen, United States), anti-ASC Ab (Cell signaling technology, Danvers, MA, United States), anti-Nek7 Ab (Abcam, Cambridge, UK), anti-NLRP3 Ab (Wanlei Life Sciences, Shenyang, China) and anti-GAPDH Ab (Beyotime, Beijing, China). Finally, the distinct protein bands were detected by ECL detection reagent (Biosharp, China).

Cells were prepared in 12-well plates and infected with PmCQ2 as described above. After infection, cells were lysed with cold PBS containing 0.5% Triton X-100 and cell lysates were centrifuged at 13,000 rpm for 15 min at 4°C to obtain cell pellets. Then, the pellets were washed twice with cold PBS and suspended in 200 μL PBS. Subsequently, the resuspended pellets were cross-linked with 2 mM fresh disuccinimidyl suberate (DSS) at 37°C for 30 min and the pellets were centrifuged at 13,000 rpm for 15 min at 4°C. Finally, the cross-linked pellets were redissolved in 30 μL 1 × SDS-PAGE sample loading buffer and samples were boiled for 5 min before the western blot analysis.

PECs were infected with PmCQ2 as described above. After infection, total RNA was extracted using the TRIzol Reagent (Life Technologies Carlsbad, CA, United States) according to the manufacturers’ instructions, and then cDNA was synthesized using PrimeScript^® RT reagent Kit (Perfect Real Time) (Takara, Japan). Subsequently, quantitative real time-PCR (RT-PCR) was performed using the CFX96 (Bio-Rad, United States). Primers were used as follows: β-actin forward 5′-TGGAATCCTGTGGCATCCATGAAAC and reverse 5′-TAAAACGCAGCTCAGTAACAGTCCG, NLRP3 forward 5′-CTTTCTGGACTCTGACCGGG and reverse 5′-CTCCCATTCTGGCTCTTCCC. The relative mRNA expression was analyzed against the expression level of β-actin.

After infection, cells were lysed in ice-cold cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, and sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, leupeptin) (Beyotime, Beijing, China) for western blot and Immunoprecipitation (IP). Cell lysates were clarified by centrifugation (12,000 rpm) at 4°C for 10 min, and were incubated with anti-NLRP3 (1:100) or Rabbit IgG (Beyotime, Beijing, China) as negative control at 4°C overnight. The proteins bound to antibody were pulled down by protein A + G beads (Beyotime, Beijing, China) and subjected to immunoblotting analysis.

PECs were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, United States) with 60 nM of Nek7 siRNA (Sangon Biotech, 5′-GAUAGACUGUGUUUAUAGATT-3′) or 60 nM of control siRNA (Sangon Biotech, 5′-UUCUCCG AACGUGUCACGUTT-3′) for 48 h before infection and then were infected with PmCQ2 as described before. Finally, cell lysates were collected for RT-PCR and western blot. The relative Nek7 gene expression levels were normalized against β-actin expression.

Cells were prepared as described and infected with PmCQ2 for 3 or 4 h. After infection, cells were fixed with 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 30 min at room temperature (RT) and then blocked with 5% Bovine Serum Albumin (BSA) for 1 h at RT. After washing steps, primary antibodies (anti-ASC Ab, Santa Cruz, CA; anti-NLRP3 Ab, Wanlei Life Sciences, Shenyang, China) were added and incubated overnight at 4°C. Next, Goat anti-rabbit IgG (H&L) Alexa fluor 594 (Abcam, United Kingdom) was added after washing with PBS at RT for 1 h. Subsequently, DAPI (Beyotime Biotechnology, Shanghai, China) was added and incubated in the dark for 5 min. Finally, anti-fluorescence attenuation mounting tablets (Solarbio, Beijing, China) were used and the results were observed an inverted fluorescence microscope (Olympus, Tokyo, Japan).

To show the extent of speck formation more intuitively, the percentage of cells that contained a speck was determined. Cells from five different fields (average of 100 cells/field) were counted based on DAPI-stained nuclei for each of different experiments. Images were analyzed using ImageJ. The data is expressed as the percentage of cells with specks per number of cells per field.

All data were presented as mean ± SD of three independent experiments and analyzed by Student’s t-test for two-group comparison. Statistical significance was shown as *p < 0.05, ^**p < 0.01, and n.s means no significance. All the graphs were made by GraphPad Prism software.

Our previous study showed that PmCQ2-induced IL-1β secretion was NLRP3-dependent (Fang et al., 2019), but the exact mechanism of PmCQ2-induced NLRP3 inflammasome activation remains unknown. Therefore, to explore the role of K⁺ efflux in PmCQ2-induced NLRP3 inflammasome activation, different K⁺ channel inhibitors including KCl, Quinine and Glibenclamide were used to pretreat macrophages prior to infection to inhibit potassium efflux. Our results showed that these K⁺ channel inhibitors significantly reduced PmCQ2-induced IL-1β secretion in macrophages in a concentration-dependent manner (Figures 1A–C) while the secretion of TNF-α and IL-6 were not affected (Figures 1D–G). These results suggest that potassium efflux is essential for IL-1β secretion in PmCQ2-infected macrophages.

Our previous study has shown that PmCQ2-induced IL-1β secretion in macrophages was caused by NLRP3 inflammasome assembly to activate caspase-1 activity. To identify the role of K⁺ efflux in PmCQ2-induced NLRP3 inflammasome assembly, caspase-1 activation and the formation of ASC oligomerization were investigated. Our results showed that the inhibition of K⁺ efflux significantly reduced PmCQ2-induced IL-1β secretion and caspase-1 (p20) maturation (Figure 2A). Notably, PmCQ2-induced NLRP3 transcription was not affected by inhibited K⁺ channel in macrophages (Figure 2B). Similarly, the expression of adaptor protein ASC as the important component of NLRP3 inflammasome was not regulated by K⁺ efflux (Figure 2C). However, the formation of ASC dimer and oligomer, which is critical for NLRP3 activation, were significantly reduced via inhibition of K⁺ efflux in PmCQ2-infected macrophages (Figure 2C). Interestingly, Quinine seems to be different with KCl, Quinine exerts an inhibitory effect on ASC monomer after ASC cross-linking but has no effect on ASC in lysate. Similarly, the results of immunofluorescence also showed that blocking K⁺ efflux significantly decreased the formation of ASC specks (Figure 2D, white arrows). In addition, we analyzed the percentage of cells (compared to the total number of cells) with ASC specks, the treatment of PmCQ2-infected cells with inhibitors showed a significant reduction in ASC specks (Figure 2E). These results suggest that K⁺ efflux is a necessary upstream signaling of PmCQ2-induced NLRP3 inflammasome activation, which induces ASC oligomerization and caspase-1 activation, leading to IL-1β.

It has been discovered that Nek7 acts on the downstream of K⁺ outflow, directly binds to NLRP3 and regulates NLRP3 activation. To investigate whether Nek7 mediates PmCQ2-induced NLRP3 inflammasome activation, resulting in IL-1β secretion in macrophages, Nek7 expression was genetically modified in primary macrophages using siRNA knock-out. The results showed that the transcription level of Nek7 in macrophages after Nek7 siRNA transfection was significantly lower than that of negative control siRNA transfection (Figure 3A) and these Nek7 knock-down cells also showed decreased Nek7 protein expression by western blot analysis (Figures 3F,G), indicating the silence of nek7 gene. Furthermore, knockdown of Nek7 significantly inhibited the secretion of IL-1β (Figure 3B), but the secretion of IL-6, TNF-α, and IL-1α were not affected during PmCQ2 infection (Figures 3C–E). Similarly, PmCQ2-induced protein expression of caspase-1 and IL-1β were also attenuated in si-Nek7 cells while the NLRP3 protein expression was not affected (Figures 3F,G). In addition, the results of immunofluorescence also showed the formation of ASC specks and the combination of ASC and NLRP3 was significantly reduced after Nek7 knockdown (Figure 3H). Similarly, the percentage of cells with ASC specks after transfection is also consistent with this result (Figure 3I). These results demonstrate the involvement of Nek7 in NLRP3 inflammasome activation in response to PmCQ2 infection.

Nek7 plays an important role in the regulation of NLRP3 inflammasome activation as shown in Figure 3. Next, Nek7-NLRP3 interaction induced by PmCQ2 infection and the role of K⁺ efflux in this process were further studied. The results of western blot clearly showed Nek7 protein expression in infected and non-infected macrophages including Nlrp3^–/–, Asc^–/–, Casp1/11^–/– cells (Figure 4A), suggesting that these components of inflammasome are not involved in Nek7 expression. Interestingly, PmCQ2 promoted Nek7-NLRP3 interaction by the detection of Co-IP not only in WT macrophages but also in Asc^–/– and Casp1/11^–/– cells (Figure 4B), demonstrating that inflammasome components are not associated with Nek7-NLRP3 interaction. Finally, to understand the mechanism of PmCQ2-induced Nek7-NLRP3 interaction, inhibitors of potassium ions were used to disrupt K⁺ efflux to investigate its role in Nek7-NLRP3 interaction. Our results showed that PmCQ2-induced combination of Nek7 and NLRP3 was abrogated in KCl-treated macrophages while Nek7 protein expression was not affected (Figures 4C,D). These results indicate that Nek7 just binds to NLRP3 protein to regulate inflammasome in response to PmCQ2 infection and this process is mediated by K⁺ efflux.