The LPS from L. interrogans serovar Hebdomadis was extracted by the method of Isogai et al. (1989) as follows. The leptospires were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH; Difco Laboratories, Detroit, MI, USA) medium with 10% normal rabbit serum (Gibco, Carlsbad, CA, USA) at 30°C for 7 days. The LPS was prepared by the hot phenol-water method and was further purified using a procedure to remove LPS-associated proteins. Escherichia coli serotype O111:B4 LPS (E-LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA), which was used as a positive control.

The pig embryonic fibroblast cell line was immortalized by simian vacuolating virus 40 large T fragment (PEFs_SV40) as established previously (Fukuda et al., 2012). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai-tesque Inc., Kyoto, Japan) containing 10% heat-inactivated fetal bovine serum (FBS; Biowest SAS, Rue de la Caille, Nuaillé, France), 1% antibiotic and antimycotic solution (Nacalai-tesque). The cells were seeded at 1×10⁶ cells/well in 6 well tissue culture plates and cultured at 37°C in an incubator with 5% CO₂. Before LPS stimulation, the cells were incubated in an antibiotic-free medium for 2 h. In all experiments, the cell viability was more than 96%, which was evaluated by Trypan blue staining (Invitrogen, Carlsbad, CA, USA). An unstimulated well was prepared as a control in the same cell culture plate. The cell culture supernatants were collected from each well, and adherence cells were harvested using the lysing buffer contained in NucleoSpin RNA II kit (Takara Bio Inc., Shiga, Japan).

Total RNA was isolated from the cells using the NucleoSpin RNA II kit (Takara Bio, Japan) according to manufacturer’s protocol, and reverse transcription was performed using PrimeScript RT reagent kit (Takara Bio, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) reactions were performed on a Thermal Cycler Dice Real-time PCR System II (Takara Bio, Japan) as same procedure as previous experiment (Guo et al., 2014), using SYBR green I mixture (Takara Bio, Japan) and the following primers: swine TLR2 (forward: 5′-GCATGAAGATGATGTGGGCC-3′, reverse: 5′-TAGGAGTCCTGCTCACTGTA-3′), TLR4 (forward: 5′-TGGAACAGGTATCCCAGAGG-3′, reverse: 5′-CAGAATCCTGAGGGAGTGGA-3′), interleukin (IL)-6 (forward: 5′-TGGATAAGCTGCAGTCACAG-3′, reverse: 5′-ATTATCCGAATGGCCCTCAG-3′), IL-8 (forward: 5′-GCTCTC TGTGAGGCTGCAGTT-3′, reverse: 5′-TTTATGCACTGG CATCGAAGTT-3′) and swine β-actin (forward: 5′-CATCACCATCGGCAACGA -3′, reverse: 5′-GCGTAGAGGTCCTTGCGGATGT-3′) serves as an internal control. Each sample was prepared in triplicate and the average value was used as the expression value. Final results were reported as the relative expression level compared with that of non-treatment group after normalization using β-actin. For visualization, 10 μL of the qRT-PCR reaction were subjected to electrophoresis in 10% Del-gel (Kitasato Medical Service Co., Ltd, Tokyo, Japan) stained with GelRed (Biotium Inc., Hayward, CA, USA). A 100 bp ladder (TrackIt; Invitrogen, Carlsbad, CA, USA) was used as a marker. Pig peripheral blood mononuclear cells (PBMCs) and pig fibroblasts (PEFs_NCC1) which can express of TLR4 mRNA in the previous studies (Uddin et al., 2012; Guo et al., 2014) were used as positive controls.

The protein level of cytokines (IL-6 and IL-8) were measured from PEFs_SV40 cell culture supernatant using pig enzyme-linked immunosorbent assay (ELISA) Kits (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. The value of the optical density was detected at 450 nm wavelengths using an ELISA plate reader (BioTek, Winooski, VT, USA) and the concentrations were determined (pg/mL) with known standards by using Microsoft Excel. To ensure the reproducibility of the experiment, each sample was measured twice and the average concentration was used as protein level (pg/mL) in cell culture supernatants.

PEFs_SV40 cells were cultured in 10% FBS DMEM and stimulated with or without 0.1 μg/mL L-LPS for 3 h and 72 h. To obtain total protein extract, cells were lysed in solution which containing 50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 2.5 mg/mL sodium deoxycholate (Wako Pure Chemical Industries, Osaka, Japan), and a protease inhibitor cock-tail (1:200 dilution, Nacalai-tesque). After calibrating the total protein concentration of cell lysate by a DC Protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA), 10 μg proteins was subjected to SDS-PAGE (ATTO Corporation, Tokyo, Japan) and blotted onto polyvinylidene fluoride membrane (Immobilon-P; Millipore Corporation, MA, USA). The membrane was blotted using the polymer immunocomplexes method as described previously (Fukuda et al., 2000). TLR2 protein expression level was detected with the ECL chemiluminescence Western blot system for immunostaining (GE healthcare, Buckinghamshire, UK). Anti-α-tubulin antibody (dilution 1:1,000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used as a loading control.

Anti-L-LPS antiserum was prepared from hyper-immunized rabbit as described previously (Isogai et al., 1998). Briefly, one mg of Hebdomadis LPS was emulsified in 1 mL of complete freund’s complete adjuvant. The mixture was injected twice intracutaneously into 10 sites on the shaved back and into the foot of rabbit. Then the rabbit was bled on the 4th post-inoculation week and the serum was used for the experiments. The negative control serum is the preimmune serum (PIS).

All experiments were done in triplicates. Means and standard deviations were calculated from the multiple data. The data were evaluated using Steel’s test in Excel software. The values of p<0.05 (*) and p<0.01 (**) were considered as statistically significant.

The TLR2 and TLR4 mRNA expressions were analyzed in cultured PEFs_SV40 cells. The TLR4 mRNA with the size of 125 bp expressed in PBMCs and pig fibroblasts (PEFs_NCC1), but not in PEFs_SV40 cells (Figure 1). Thus, the PEFs_SV40 cells which do not express TLR4 mRNA have a potential to be used as a good model in researches on immune response in which TLR2 is involved.

TLR2 mRNA expression in PEFs_SV40 cells was examined while various concentrations of LPSs were applied. Under the stimulation by L-LPS, correspondingly, the expression level of TLR2 mRNA increased gradually while the dose concentration was increasing (p<0.05), the relative values of the expression level in stimulated cells were more than 2.1 folds to that of unstimulated cells (Figure 2A). The E-LPS was used as a positive control, showing drastic and significant increase of expression of TLR2 mRNA from 4 to 25 folds when the dose-concentration range from 0.1 to 10 μg/mL (p<0.05; Figure 2A). We then tested the time-dependent change of TLR2 mRNA expression in PEFs_SV40 cells under 0.1 μg/mL LPSs stimulation. When the cells were incubated with 0.1 μg/mL L-LPS, the expression of TLR2 mRNA was markedly up-regulated to 2.3 folds at 3 h and then rapidly declined at 12 h. A significant difference of the expression level between L-LPS stimulated cells and unstimulated cells was maintained until 24 h (p<0.01; Figure 2B). When incubated with E-LPS, the maximum expression of TLR2 mRNA was shown at 3 h. After 12 h, the expression level was rapidly decreased up to half of the expression level of 3 h but the expression level was still higher than that of unstimulated cells (p<0.05; Figure 2B). These results indicate that TLR2 of pig cells can be activated strongly and quickly after being stimulated by L-LPS.

To examine the protein expression level of TLR2 by the stimulation of L-LPS in the PEFs_SV40 cells, we performed the Western blotting. In unstimulated PEFs_SV40 cells, TLR2 protein with a molecular mass of 74 kDa was stably expressed at low level, and the expression level of TLR2 protein was constant until 72 h after stimulation (Figure 3). Measurement of brightness of protein band by using ImageJ software showed that, at 72 h, the expression level of TLR2 protein in L-LPS stimulated cells was about 3.3 folds of that of unstimulated cells. These indicate that TLR2 plays a vital role during L-LPS stimulation, while the protein expression of TLR2 is considerably later than the mRNA transcription (Figure 2B).

To understand the inflammatory response triggered by L-LPS, we examined the expression level of cytokines mRNA in the PEFs_SV40 cells by qRT-PCR. The cells were treated with various concentrations of L-LPS, showing significant increase of the mRNA expression of IL-6 (p<0.05) and IL-8 (p<0.05) up to more than 30 folds and 40 folds of unstimulated cells, respectively (Figure 4A and B). Also marked enhancement of IL-6 and IL-8 mRNA expression were caused at 3 h after L-LPS stimulation and then rapidly decreased but the expression level was still higher than that of unstimulated cells (p<0.05; Figure 4C and D). As a positive control, the cells were treated with E-LPS, shows significantly high expression of IL-6 and IL-8 as compared with unstimulated cells (p<0.05; Figure 4A and B). These expressions were gradually decreased over time (Figure 4C and D).

The amount of IL-6 and IL-8 secreted in culture supernatant were quantitatively determined, showing considerable up-regulation of these cytokines by E-LPS and L-LPS stimulations (p<0.05; Figure 5). In L-LPS stimulations, the secretion level of IL-6 protein was increased until dose of 1.0 μg/mL L-LPS and was slightly decreased at 10 μg/mL L-LPS stimulation (Figure 5A). In contrast, IL-8 secretion was monotonically enhanced with dose concentration of L-LPS (Figure 5B). The secretion levels of IL-6 and IL-8 reached the highest level at 3 h after L-LPS or E-LPS stimulation and retained the level throughout the experiment. Thus, we conclude that L-LPS can rapidly induce production of a large amount of cytokines.

When the cells were incubated with both L-LPS (10 μg/mL) and anti-L-LPS antibody for 6 h, the enhancement of TLR2 mRNA was completely inhibited by anti-L-LPS antibody comparing with that of L-LPS stimulated cells (Figure 6). While replacing anti-L-LPS antibody by PIS, there was no effect on the TLR2 mRNA expression induced by L-LPS (Figure 6). Altogether, these results indicate that anti-L-LPS can interrupt the interaction between L-LPS and TLR2 in PEFs_SV40 cells, thereafter might reduce the occurrence of antibiotics-triggered endotoxin shock.