Female C57BL/6 mice (6–8 weeks old) were purchased from the Changsheng Experimental Animal Center (Changchun, China), and TLR2-deficient (TLR2^-/-) mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). The mice were supplied with sterile food and water and maintained under specific pathogen-free conditions in the National Experimental Teaching Demonstration Center of Jilin University (Changchun, China).

Neospora caninum tachyzoites (Nc-1 strain) were maintained by serial passages in VERO cells in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2% fetal bovine serum (FBS) (Biological Industries, Ltd., Beit HaEmek, Israel), 2 mM L-glutamine, 100 U of penicillin, and 100 μg of streptomycin (P/S) (all from Life Technologies, Carlsbad, CA, United States) in a 5% CO₂ atmosphere at 37°C. Free N. caninum tachyzoites were obtained and harvested from VERO cells [centrifuged at 500 ×g/min for 10 min at room temperature (RT)] and then passed through a 1-ml syringe and centrifuged at 1,500 ×g for 30 min to remove host cell debris by gradient density centrifugation with a 40% Percoll (GE Healthcare, Uppsala, Sweden) solution (v/v). The parasite pellet was collected and washed twice with RPMI 1640, and the N. caninum concentration was determined in a hemocytometer.

The wild-type (WT) and TLR2^-/- mice were inoculated intraperitoneally with thioglycollate medium (BD Biosciences, New Zealand, United States) for 4 days, and PMs were harvested in cold phosphate-buffered saline (PBS) as previously described (Li et al., 2017; Wang et al., 2017). In addition, PMs were cultured in complete RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 2 M L-glutamine.

The total DNA of N. caninum was extracted from purified tachyzoites according to the manufacturer’s protocol. The 14-3-3 gene [GenBank: U31542.1] was amplified by polymerase chain reaction (PCR) using the primers 14-3-3-F (5′-CGCGGATCCATGGCGGAGGAAATCAAAAATCT-3′) and 14-3-3-R (5′-CCGGAATTCCTGATCAGCCTGCTCAGCGG-3′), which contained Bam HI and EcoR I sites (underlined), respectively. The obtained 801-bp PCR product that corresponded to 14-3-3 was digested with Bam HI/EcoR I and cloned into the corresponding sites of the pGEX-4T-1 vector. The positive recombinant plasmid pGEX-Nc14-3-3 was transformed into the E. coli expression strain Rosetta DE3a (TIANGEN, Beijing, China), and glutathione S-transferase (GST) fusion protein expression was induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 4 h at 37°C. The protein was then solubilized and purified with Proteinlso GST Resin (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. Briefly, after sonication and centrifugation, the supernatant was filtered through a 0.45-μm membrane and loaded slowly into a 2.5 cm × 10 cm column in which the GST-Bind resin had been equilibrated with 10 column volumes of binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄, pH 7.3). The column was then washed with the wash buffer. Finally, the fusion protein GST-14-3-3 was eluted with elution buffer (50 mM Tris-HCl, pH 8.0, and 10 mM reduced-glutathione). The purified protein was dialyzed in PBS for at least 5 h at 4°C with two buffer changes. To remove possible contaminants from the process, purification was performed as previously described, with a slight modification (Liu et al., 1997; Macedo et al., 2013). Briefly, Triton X-114 was added to the purified protein fraction to a final concentration of 1%. The mixture was incubated at 4°C for 10 min, with constant stirring to ensure a homogenous solution, and then transferred to a 37°C water bath and incubated for 10 min until oily matter appeared; the sample was then centrifuged at 13,000 ×g for 10 min (RT). The upper aqueous phase was removed and subjected to additional Triton X-114 phase separation two times. Finally, the upper water phase containing the protein was collected carefully and stored at -80°C until use. In addition, N. caninum lysate antigen (NLA), excretory secretory antigens (ESAs) and extracellular vesicles (EVs) were prepared as previously described (Li et al., 2018). The collected proteins were either stored at -80°C or directly used in additional experiments. The pellets were resuspended in protein loading buffer and stored at -20°C or immediately analyzed. All protein concentrations were measured using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, United States).

To assess the level of endotoxin in rNc14-3-3 proteins, the ToxinSensor^TM chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, United States) was used according to the manufacturer’s instructions.

Mouse polyclonal antibodies against Nc14-3-3 were prepared according to previously described protocols (Dong et al., 2017). Briefly, six 8-week-old BALB/C mice were subcutaneously immunized with rNc14-3-3 protein (100 μg/mouse), which was emulsified with Freund’s complete adjuvant (Sigma, St. Louis, MO, United States). Boosters were administered at 2, 4, and 6 weeks with rNc14-3-3 protein (50 μg/booster), which was emulsified with Freund’s incomplete adjuvant (Sigma, St. Louis, MO, United States). One week after the last injection, mouse blood was collected via orbital bleeding, and the sera were obtained. The levels of antibodies in mouse sera were detected by enzyme-linked immunosorbent assay (ELISA) as follows. Briefly, 96-well microtiter plates were coated with 1 μg of SNAg in 100 μl of carbonate buffer (150 mM Na₂CO₃, 349 mM NaHCO₃, pH 9.6) and incubated at 4°C overnight. After washing three times, the plates were blocked with 3% bovine serum albumin (BSA) for 2 h at 37°C and subsequently incubated with mouse sera diluted in PBS for 2 h at 37°C. HRP-conjugated goat anti-mouse IgG antibody (Proteintech, Wuhan, China) was used as a secondary antibody at a 1:2000 dilution, and the reaction were revealed by TMB (Beyotime, Shanghai, China) for 15 min at 37°C and stopped by 2 M H₂SO₄. The reaction was measured at 490 nm with an ELISA reader. Finally, the generated mouse polyclonal antibodies were purified using HisTrap for protein G (GE Healthcare Bio-Sciences, Ltd., Marlborough, MA, United States) according to the instructions.

Indirect immunofluorescence assays (IFAs) were performed in both extracellular and intracellular N. caninum tachyzoites. Free N. caninum tachyzoites (Nc-1) were washed three times with PBS, added to poly-L-lysine (0.1 mg/ml, Sigma Aldrich) precoated cover glass slides, dried at RT and fixed in 4% paraformaldehyde for 10 min. The cover slides were then washed three times with PBS, permeabilized with 0.25% Triton X-100 in PBS for 10 min, washed and blocked in 3% BSA/PBS for 2 h at RT. After blocking, the samples were incubated with a 1:100 dilution of the 14-3-3 antibody overnight at 4°C, then washed and incubated with the secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ig-G (Proteintech, Wuhan, China) for 1 h at RT. The coverslips were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Scientific, Waltham, MA, United States) for 10 min before analysis on an Olympus FV1000 Laser Scanning Confocal microscope (Japan).

Neospora caninum tachyzoite-infected VERO cells were stained with the 14-3-3 antibody as follows. The intracellular tachyzoites were washed with cold PBS three times and fixed on glass coverslips with 4% formaldehyde for 20 min at 4°C. The infected cells were permeabilized with 0.25% Triton X-100 in PBS for 8 min, washed and blocked in 3% BSA/PBS for 2 h at RT. All procedures were the same as those described above.

Free N. caninum tachyzoites or VERO cells challenged with N. caninum tachyzoites for 12 h (MOI = 1:1, parasite:cell) were collected and fixed with 4% paraformaldehyde (pH 7.2) for 2 h. Then, the samples were dehydrated with 50, 70, 90, and 100% N,N-Dimethylformamide (DMF) for 15 min at 4°C in sequence, incubated with mixtures DMF: LR White Resin = 2:1 and DMF: LR White Resin = 1:2 for 30 min at 4°C in sequence. Then, they were incubated with LR White Resin overnight at 4°C, replaced fresh LR White Resin, embed and UV irradiation polymerization for 10 days at -20°C. The samples were washed, cut into 80-nm sections and applied to a carbon-coated copper grids and stored at 4°C. For immunolabeling, the grids were blocked in 3% BSA/PBS for 30 min at RT; then, the 14-3-3 antibody (1:100) was diluted in 1% BSA/PBS and added to the grid for 40 min. Following washes with PBS, the secondary antibody conjugated to 10-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA, United States) was used at a 1:100 dilution and incubated for 40 min. The grids were examined used a Hitachi 7650 transmission electron microscope operating at an accelerating voltage of 80 kV. Images were obtained using an Olympus SIS Megaview II digital camera and iTEM software.

We examined the effect of rNc14-3-3 on PMs viability using Cell Counting Kit (CCK-8) (Dojindo Laboratories, Japan) according to manufacturer’s directions. The mouse PMs were plated in 96-well plates at the density of 1 × 10⁴ and incubated for 24 h. Then cells were stimulated with rNc14-3-3 at concentration of 12.5, 25, 50, 100, 200 ug/ml in 100 μl sterile PBS or PBS alone, respectively. After incubation for 24 h, 10 μl of CCK-8 reagent was added to each well and incubation continued for another 3 h. Finally, the absorbance was measured at 450 nm.

The cytoplasmic and nuclear proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (KeyGen Biotech, Nanjing, China) according to the instructions. Briefly, 6 × 10⁶ cells were collected in 1.5 ml tubes and washed twice with ice-cold PBS and resuspended in 450 μl of prechilled Buffer A with 50 μl of Buffer B, then incubated on ice for 30 min. The cell lysates were centrifuged at 800 ×g for 10 min, and the resulting supernatants were the cytoplasmic fraction. The pellets were lysed in 100 μl of Buffer C (17 μl of 100 mM PMSF and 1 μl of protease inhibitor/1 ml of Buffer C), vortexed strongly for 15 s and then incubated on ice for 60 min. The lysates were then centrifuged at 13,000 ×g for 30 min, and the resulting supernatants were the nuclear fraction.

The PMs were seeded in 6-well culture plates (Corning Incorporated, Corning, NY, United States) at a density of 3 × 10⁶ cells/well and treated with rNc14-3-3 for the indicated times. Then, the cell lysates were extracted from PMs using a total protein extraction kit (KeyGen Biotech, Nanjing, China). Protein concentrations were measured using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, United States). Thirty micrograms of each protein sample was separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, United States). After 2 h of blocking in TBS-0.05% Tween 20 containing 5% skim milk, the membranes were incubated at 4°C overnight with the following antibodies: mouse anti-14-3-3 and primary rabbit antibodies against p-P38 (1:1,000), total P38 (1:1,000), p-ERK1/2 (1:1,000), total ERK1/2 (1:1,000), p-JNK (1:1,000), total JNK (1:1,000), p-AKT (1:1,000), total AKT (1:1,000), p-IκB (1:1,000), total IκB (1:1,000) (all from Cell Signaling Technology, United States); p-p65 (1:1,000), total p65 (1:1,000) (from Abcam, United Kingdom); and GAPDH (1:1,000). For all secondary antibody incubations, horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit antibodies (Proteintech, Wuhan, China) were used at a 1:2000 dilution. The membranes were visualized by an enhanced chemiluminescence (ECL) Western Blot Detection System (Clinx Science Instruments, Co., Ltd., Shanghai, China).

To further confirm the role of the AKT signaling pathway in mouse PMs after treatment with rNc14-3-3, a small-interfering RNA (siRNA) specific for AKT was designed and synthesized by Ruibo Biology Company (Guangzhou, China). The mouse PMs were seeded in 6-well culture plates at a density of 3 × 10⁶ cells/well and transfected with siRNA-control and siRNA-AKT for 24 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s protocol. To detect interference effects, the cells were collected for mRNA and protein analyses. In the formal experiment, after transfection, the cells were further stimulated with 50 μg/ml rNc14-3-3 for 24 h. The supernatants were harvested for cytokine measurement.

The PMs were seeded in 24-well culture plates (Corning Incorporated, Corning, NY, United States) at a density of 5 × 10⁵ cells/well and treated with rNc14-3-3. In some experiments, the PMs were pretreatment for 2 h with a p38 inhibitor (SB203580; 30 μM) (Sigma, St. Louis, MO, United States), for 1 h with an ERK inhibitor (PD98059; 40 μM) (Sigma), for 1 h with a JNK inhibitor (SP600125; 10 μM) (Sigma), or for 1 h with an AKT inhibitor (IV; 10 μM) (Santa Cruz Biotechnology, United States); each well was incubated in the presence or absence of a p38, ERK, or JNK inhibitor or PBS before rNc14-3-3 treatment. The concentration of each inhibitor was chosen based on previously published manuscripts (Jin et al., 2017b; Li et al., 2017). After 24 h of treatment with rNc14-3-3, the supernatants were harvested for cytokine measurement, and Cytokine ELISA Ready-SET-Go kits (eBioscience, San Diego, CA, United States) were used to detect IL-12p40, TNF-α, and IL-6 levels following the manufacturer’s instructions. The assays were read at 450 nm, and the optical density (OD) values obtained were converted to pg/ml by extrapolation using a standard curve.

To determine the localization of NF-κB/p65, 5 × 10⁵ PMs were seeded in 24-well culture plates (Corning Incorporated, Corning, NY, United States) with sterile glass slides, and the cells were treated with rNc14-3-3 (50 μg/ml) or Pam3CSK4 (TLR2/TLR1 Agonist, InvivoGen, United States, 10 ug/ml) at 37°C. After 30 min, the cells were washed twice with sterile PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min, and washed. Then, the cells were incubated with a rabbit anti-phospho-NF-κB/p65 antibody (Santa Cruz, CA, United States) at a 1:100 dilution at 4°C overnight. The cells were washed and incubated with FITC-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China) for 1 h at RT. The cells were washed, and the coverslips were stained with DAPI before analysis on a Zeiss LSM 710 confocal microscope equipped with a 633, 1.4-NA, oil-immersion objective (Carl Zeiss).

Data were expressed as the standard error of the mean ± standard deviation (SD), and the means were compared by one-way analysis of variance using SPSS 19.0 software (SPSS, Inc., Chicago, IL, United States). All graphs were generated in GraphPad Prism 7.00, and independent experiments were performed with at least three technical replicates.

An 801-bp PCR product was generated that corresponded to Nc14-3-3 (Figure 1A, left); the product was digested with Bam HI/EcoR I and cloned into the corresponding sites of the pGEX-4T-1 vector. To confirm the process, we detected the product by PCR and restriction enzyme analysis. The results of the restriction enzyme digestion of plasmids are shown that we have successfully constructed expression plasmid (Figure 1A, right). To further assess the localization of Nc14-3-3, a recombinant fusion protein of Nc14-3-3 (rNc14-3-3) was successfully expressed and purified using a GST Resin (Figure 1B). Western blotting analysis showed good immunoreactivity of rNc14-3-3 to serum (1:200) from a mouse infected with N. caninum (Figure 1C).

Next, we detected the localization of 14-3-3 in N. caninum tachyzoites. Free N. caninum tachyzoites and VERO cells infected with N. caninum tachyzoites were subjected to immunofluorescence analysis using the polyclonal anti-N. caninum 14-3-3 antibody, and the results showed that the 14-3-3 protein was mainly found in the cytosol and cell membrane (Figure 2A). To obtain additional information, we used electron microscopy, which allows ultrastructural observations of the cells, and immunogold labeling of 14-3-3 protein. 14-3-3 was detected in the cytosol and cell membrane, in agreement with the immunofluorescence results. In addition, VERO cells infected with N. caninum tachyzoites showed that 14-3-3 was also seemingly to present in the parasitophorous vacuole (PV) (Figure 2B), certainly, this conclusion still required further evidence.

To determine the appropriate concentration of rNc14-3-3 used in our experiments, we investigated the influence of it on the viability of Mouse PMs using CCK-8 assay. As shown in Figure 3, rNc14-3-3 did not affect the cell viability at concentrations lower than 50 μg/mL. However, the cell viability was significantly reduced at the concentration of 100 μg/mL (P < 0.05 vs. untreated cells). Thus, the appropriate concentration of rNc14-3-3 in vitro for Mouse PMs was 50 μg/ml.

LAL assays showed that the concentration of endotoxin in rNc14-3-3 was 0.03 EU/ml, which could not induce TLR2/TLR4 activation or the production of IL-8 or TNF-α (Nozoe et al., 2017; Teodorowicz et al., 2017). Our previous work demonstrated that mouse bone marrow-derived macrophages (BMDMs) exposed to N. caninum tachyzoites, NLA, ESA, and EVs can induce the phosphorylation of MAP kinases 30 min after treatment (Li et al., 2018). These data suggested that N. caninum activates predominantly the MAPK signaling pathway, probably through a component of its ESA or EV proteins. We hypothesized that rNc14-3-3 participates in the activation of the MAPK signaling pathway. First, Western blotting analysis using the anti-14-3-3 antibody detected the 14-3-3 protein in N. caninum-derived polypeptides, including NLA, ESA, and EVs (Figure 4A). We hypothesized that rNc14-3-3 participates in the activation of the MAPK signaling pathway. Specifically, we examined the phosphorylation of MAP kinases in mouse PMs after treatment with rNc14-3-3. WT mouse PMs were treated with 50 μg/ml rNc14-3-3 for the indicated times, and the results revealed that rNc14-3-3 induced strong phosphorylation of p38, ERK, and JNK at 1, 1, 0.5 h, respectively; the phosphorylation of these proteins subsequently weakened over time (Figure 4B). To estimate whether rNc14-3-3 induced the phosphorylation of the p38, ERK, and JNK MAP kinases through TLR2, TLR2^-/- and WT mouse PMs were treated with rNc14-3-3 for 1 or 0.5 h. The phosphorylation of p38, ERK, and JNK was observed in both WT and TLR2^-/- mouse PMs (Figure 4C). These data suggested that rNc14-3-3 induced phosphorylation of p38, ERK, and JNK MAP kinases not by TLR2. In addition, we treatment mouse PMs with N. caninum lysate antigen (NLA) as positive control, the results showed that NLA could induce the phosphorylation of p38, ERK, and JNK after treatment for 0.5 h, while the phosphorylation levels of p38, ERK and JNK were significantly reduced in the TLR2^-/- cells, which demonstrated that NLA induced phosphorylation of P38, ERK and JNK via TLR2. Considering that the rNc14-3-3 protein was a GST fusion protein, we further proved that the GST-labeled protein had no effect on our results (Figure 4C).

To obtain more information about the effects of rNc14-3-3 on the N. caninum-induced phosphorylation of MAP kinases, we used the MAPK inhibitors SB203580 (p38), PD98059 (ERK), and SP600125 (JNK). WT mouse PMs were pretreated with or without inhibitors for the indicated time at 37°C and then incubated with 50 μg/ml rNc14-3-3 or NLA for 24 h. ELISA demonstrated that PMs treated with the inhibitors could significantly decrease the production of IL-12p40 and TNF-α, whether rNc14-3-3 or NLA. In contrast, significantly less production of IL-6 was observed in rNc14-3-3 cells treated with p38, ERK and JNK inhibitors, while no significant difference in NLA cells treated with p38 and ERK inhibitor (Figure 5). We also detected the production of IL-1β, but the expression of this factor was almost undetectable (data not shown).

To investigate whether rNc14-3-3 was important for inducing the activation of AKT in N. caninum infection, we employed Western blotting and siRNA interference technology. The results showed that rNc14-3-3 induced a remarkable phosphorylation of AKT after stimulation for 1 h, and there was minimal phosphorylation in negative control cells (Figure 6A, down). N. caninum lysate antigen (NLA) could induced the phosphorylation of AKT, the phosphorylated AKT peaked at 2 h and then returned to weaken (Figure 6A, up). To estimate whether rNc14-3-3 induced the phosphorylation of AKT through TLR2, WT and TLR2^-/- mouse PMs were treated with rNc14-3-3 or PBS as a negative control. AKT phosphorylation was detected in both WT and TLR2^-/- mouse PMs (Figure 6B), suggesting that rNc14-3-3 induced the activation of AKT but had little relationship with TLR2.

To confirm this result, WT PMs were transfected with siRNA-control or siRNA-AKT for 24 h using the Lipofectamine 2000 transfection reagent. Following transfection, the cells were further stimulated with 50 μg/ml rNc14-3-3 for 24 h. AKT expression levels were detected by Western blotting and reverse transcription (RT)-PCR, and the results showed that siRNA-AKT significantly reduced AKT expression (Figures 6C,D). Next, we attempted to identify the role of the AKT signaling pathway in the regulation of inflammatory cytokines when mouse PMs were exposed to rNc14-3-3. The results showed that AKT inhibitor IV significantly blocked the production of IL-6, IL-12p40 and TNF-α, similar results were found in NLA treatment group (Figure 7A). In addition, siRNA-AKT also significantly reduced the production of IL-6, IL-12p70 and TNF-α, which was consistent with the results obtained with the AKT inhibitor (Figure 7B).

When NF-κB/p65 is activated, it translocates from the cytoplasm to the nucleus (Inta et al., 2006), and its transcriptional activity is also regulated by phosphorylation by serine/threonine kinases (Ryu et al., 2011). Thus, we isolated the total protein, the cytoplasmic fraction and the nuclear fraction. As shown in Figure 8A, phosphorylated NF-κB/p65 was observed in WT PMs treated with rNc14-3-3, and the protein level of NF-κB/p65 was reduced in the cytoplasm but increased correspondingly in the nucleus at 2 h. As the degradation of IκBα is required for NF-κB activation, we also examined the changes in phosphorylated IκBα. The level of IκBa in WT PMs was significantly reduced after exposed to rNc14-3-3 for the indicated times. To further clarify whether NF-κB/p65 translocates from the cytoplasm to the nucleus, we used laser scanning confocal microscopy, as shown in Figure 8B, NF-κB/p65 was located in the cytoplasm in the control group and distributed mainly in the nucleus after rNc14-3-3 treatment for 2 h. In addition, the expression levels of NF-κB/p65 and IκBα in TLR2^-/- mouse PMs were similar to those in WT mouse PMs (Figure 8C). Taken together, our results indicated that rNc14-3-3 could induce the translocation of NF-κB from the cytosol to the nucleus during N. caninum infection.