Quantitative expression analysis by real-time polymerase chain reactions (PCR) in swine tissues
Tissues (spleen, lung, duodenum, jejunum, ileum, ileal PP, MLN and colon) were obtained from adult (age 6 months) LWD swine (n = 9; genotype 1/4 Landrace, 1/4 Large White, 1/2 Duroc; Hiruzu Co., Ltd., Miyagi, Japan). The swine were clinically healthy and free of infectious diseases. The present study was conducted in accordance with the Guidelines for Animal Experimentation of Tohoku University, Japan.
Total RNA was isolated from the various adult swine tissues as described previously [11, 12]. Briefly, cDNA were prepared by reverse transcription of 1 μg total RNA using Oligo d(T)18 primer (Invitrogen, Carlsbad, CA, USA). An equivalent volume of cDNA solution (5 μL) from each sample was used for quantification of porcine TLR3-specific cDNA. The real-time quantitative PCR reactions were performed with the 7300 Real-time PCR System (Applied Biosystems, Warrington, UK) using Platinum SYBR Green qPCR SuperMix UDG with ROX (Invitrogen, Tokyo, Japan) and the following primers: sense TAGAGACATGGATTGCTCCC, antisense AACTTCTGGAATGCAGGTCC. PCR was carried out with an initial denaturation for 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 10 s at 60 °C, and 5 s at 72 °C.
To quantify cytokine mRNA using real-time RT-PCR, cDNA standards were produced for TLR3 and β-actin as previously described . Briefly, the cDNA were prepared by reverse transcription from 5 μg of total RNA using oligo (dT)18 primers (Invitrogen, Tokyo, Japan) and ThermoScript RNase H− reverse transcriptase (Invitrogen, Tokyo, Japan). The PCR products were inserted into the vector pGEM-T easy DNA (Promega, Madison, WI, USA). We confirmed the homology of each insert with dideoxy chain termination methods using a DNA sequencer (4000 L; Li-Cor, Lincoln, NE, USA) and the SequiTherm EXCELTM II DNA Sequencing Kit-LC (Epicentre Biotechnologies, USA). The cDNA standards were amplified by PCR from the vector pGEM-T easy DNA-TLR3 or -β-actin and standards were purified and quantified spectrophotometrically. The copy number for each mRNA was determined using the following formula: Copy number (copies) = 6.02 × 1023 (copies/mol) × measurement (g)/MW (g/mol). MW = size in bp × 660 (g/mol/bp). Aliquots of standard cDNA containing 107-102 fg/μL were created for β-actin and TLR3 for use as assay standards. TLR3 mRNA levels in different tissues were calibrated by the swine β-actin level, and normalized by common logarithmic transformation in comparison to the TLR3 mRNA level in the spleen (as 1.00). Values represent means and error bars indicate the standard deviations. The results are means of 3 measures repeated by 9 times of independent experiments using various tissues from at least 9 different adult swine. Amplification products of contaminants such as primer dimers were not detected by SYBR green chemistry using serial dilutions of cDNA.
Analysis of TLR3 expression in porcine ileal PP cells
Expression levels of TLR3 protein in porcine ileal PP immunocompetent cells were determined by flow cytometry. Porcine PP immune single-cell suspensions were prepared from the ileum of adult swine as previously described [11, 13–15]. Briefly, after cutting the specimens into small fragments, they were gently pressed through a nylon mesh and washed three times in complete RPMI 1640 medium (Sigma) supplemented with 10% FCS (Sigma). Residual erythrocytes were lysed by resuspension in hypotonic salt solution (0.2% NaCl), followed by hypertonic rescue in an equal volume of 1.5% NaCl. Finally, immune cells were fractionated with Lympholyte-Mammal (Cedarlane, Hornby, Ontario, Canada) density gradient centrifugation and the cells obtained were suspended in complete DMEM (Invitrogen, Tokyo, Japan) supplemented with 10% FCS (Sigma), 50 μg/mL penicillin/streptomycin and 50 μg/mL gentamycine (Nakalai tesque, Kyoto, Japan). This mononuclear cell suspension contains a mixed population of T (CD4+ and CD8+), B (CD21+) and antigen presenting cells (CD4- CD8- MHCII+) .
For the detection of cell surface and intracellular TLR3 in the different populations of immune cells: leukocytes (CD45+), antigen-presenting cells (CD172a+CD11R1-, CD172a-CD11R1low and CD172a+CD11R1high), B cells (CD21+) and T cells (CD4+ and CD8+), the following primary antibodies were used: anti-mouse TLR3(unlabeled) rabbit-IgG (M-300:sc-28999, Santa Cruz Biotechnology, CA, USA), anti-porcine CD3(R-PE) mouse IgG1k (SouthernBiotech, 4510-09), anti-porcine CD4(unlabeled) mouse IgG2a (PT90A VMRD Inc., WA, USA), anti-porcine CD4(FITC) mouse IgG2b (4515-02, SouthernBiotech, Alabama, USA), anti-porcine CD8a(Biotin) mouse IgG2b (732844, Beckman Coulter, Tokyo, Japan) anti-porcine CD11R1(unlabeled) mouse IgG1 (MCA1220, AbD serotec, Kidlington, UK), anti-porcine CD172a(R-PE) SWC3 mouse IgG1 (4525-09, SouthernBiotech), anti-porcine CD172a(Biotin) SWC3 mouse IgG1 (4525-08, SouthernBiotech), and anti-porcine CD45RA(unlabeled) mouse IgG1 (PG96A, VMRD). In addition the following secondary antibodies were used: goat anti-rabbit IgG (H+L) Alexa Fluor488 (A-11008, Invitrogen, Tokyo, Japan), goat anti-mouse IgG2a-PE (sc-3765, Santa Cruz Biotechnology), goat anti-mouse IgG1-PerCP/Cy5.5 (sc-45103, Santa Cruz Biotechnology) and streptavidin(PE) (12-4317-87, eBioscience, CA, USA). Cells stained with irrelevant mouse IgG2b-FITC (11-4732, eBioscience), IgG1-PerCP/Cy5.5 (45-4714, eBioscience), IgG2b-PE (12-4732, eBioscience), IgG2a-PE (12-4724, eBioscience), IgG1-PE (12-4714, eBioscience) and rabbit IgG-Alexa Fluor488 isotype control (4340S, Cell Signaling Technology Japan KK, Tokyo, Japan) antibodies were included as isotype controls.
The cells were collected, washed twice with washing buffer (2% FCS, 0.01% NaN3/PBS) and the live cell counts were adjusted to 1 × 106 cells/tube. Immune cells were resuspended and labeled with primary and secondary antibodies for detection of CD3, CD4, CD8, CD21, CD45, CD172a and CD11R1. Finally, the cells were permeabilized with the BD cytofix-cytoperm kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions and then labeled with primary anti-mouse TLR3(unlabeled) rabbit-IgG and secondary goat anti-rabbit IgG (H+L) Alexa Fluor488 antibodies for detection of intracellular TLR3. In addition, non-permeabilized immune cells were used to evaluate cell surface expression of TLR3.
Analysis of the stained cells was performed using FACS-Calibur™ (BD, Franklin Lakes, NJ, USA) equipped with Cell-Quest software. Data analysis was performed by using FlowJo software (Tree star, Ashland, Oregon, USA). For the analysis, fluorescence of permeabilized and non-permeabilized cells stained with anti-TLR3 antibody were compared simultaneously with isotype control and presented in the same histogram. In addition, intracellular and cell surface expression of TLR3 is presented as a log of MFI.
Analysis of TLR3 expression in PIE cells
PIE cells, which are non-transformed intestinal cell lines originally derived from intestinal epithelia isolated from an unsuckled neonatal swine, were isolated and cloned previously . When PIE cells are cultured, they assume a monolayer, cobblestone and epithelial-like morphology, with close contact between the cells. In addition, PIE cells are strongly positive for cytokeratin K8.13, a marker for porcine intestinal epithelial cells . For the passage, PIE cells were treated with a sucrose/EDTA buffer for 4 min, detached using 0.04% trypsin in PBS, and then plated at 1.5 × 104 cells/cm2 in culture flasks (Nalge Nunc International, Rochester, New York, USA) at 37 °C in an atmosphere of 5% CO2. PIE cells were cultured in 10% FCS DMEM and passaged every 3 or 4 days. In the present study, we used PIE generations from 20 to 30.
Immunohistochemical analysis of PIE cells was conducted according to our previous reports . Briefly, PIE cells were grown on COL-coated cover glass (12 mm; Iwaki, Japan) in 24-well plates and cultured over-night. CellLight™ Early Endosomes-RFP BacMam 2.0 (Invitrogen) or Null control were added after culture according to the manufacturer's instruction, and cultured for 1 day to visualize endosomes. These cover glasses were then fixed in 4% paraformaldehyde solution in PBS for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. PIE cells were then blocked with 2% normal goat-serum (Sigma) in PBS for 30 min. After removal of the blocking solution, PIE cells were incubated for 16 h at 4 °C with anti-mouse TLR3 (unlabeled) rabbit-IgG (Santa CruzBiotechnology, M-300:sc-28999) or rabbit-IgG isotype control (20304E, IMGENEX, CA, USA) in Can Get Signal® immunostain solution (Toyobo, Tokyo, Japan), and then stained with secondary anti-rabbit IgG (H+L) Alexa Fluor488 (Invitrogen A-11008) antibody. After labeling, the cells were washed with PBS, rinsed in distilled water and mounted in Fluoroshield with DAPI (Gentaur, Kobe, Japan). The samples were analyzed using a confocal laser microscope (FluoView FV1000, OLYMPUS, Tokyo, Japan).
Flow cytometric analysis of PIE cells was conducted according to our previous reports . PIE cells were collected and washed twice with washing buffer (2% FCS, 0.01% NaN3/PBS). The live cell counts were adjusted to 1 × 106 cells/tube and the cells were fixed and permeabilized with the BD cytofix-cytoperm kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Then, fixed and permeabilized cells were resuspended and labeled with primary anti-mouse TLR3 (unlabeled) rabbit-IgG and secondary goat anti-rabbit IgG (H+L) Alexa Fluor488 antibodies for detection of intracellular TLR3. In addition, non-permeabilized PIE cells were used to evaluate cell surface expression of TLR3. Intracellular and cell surface expression of TLR3 is presented as log of MFI.
Lactobacilli immunomodulatory activity in PIE cells
The following lactobacilli strains were used in this study: Lactobacillus reuteri MEP221101 and MEP221102, Lactobacillus casei MEP221103, TL2768, MEP221104, MEP221105, MEP221106, MEP221107, MEP221108, MEP221109, MEP221114 and MEP221115, Lactobacillus rhamnosus MEP221110, MEP221111, MEP221112 and GG, Lactobacillus salivarius MEP221113, Lactobacillus jensenii TL2937 and Lactobacillus gasseri MEP221117. The lactobacilli strains were grown in MRS medium (Difco, Detroit, USA) for 16 h at 37 °C and washed with PBS. Lactobacilli were re-suspended in DMEM, enumerated in a microscope using a Petroff-Hausser counting chamber, and stored at -80 °C until use.
PIE cells were plated at 1.5 × 104 cells/24 well plate on type I collagen-coated plates (Iwaki, Tokyo, Japan), and cultured for three days. After changing medium, lactobacilli (5 × 107 cells/mL) were added and 48 h later, each well was washed vigorously with medium at least 3 times to eliminate all the stimulants, and then stimulated with poly(I:C) for the time indicated.
We performed two-step real-time quantitative PCR to characterize the expression of mRNA in PIE cells [11, 12]. Total RNA from each sample was isolated from the PIE cells using TRIzol reagent (Invitrogen). All cDNA were synthesized by the same method mentioned above. Real-time quantitative PCR was carried out with a 7300 Real-time PCR System (Applied Biosystems) using Platinum SYBR Green qPCR SuperMix UDG with ROX (Invitrogen). The primers used in this study were described previously . The PCR cycling conditions were 2 min at 50 °C; followed by 2 min at 95 °C; then 40 cycles of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. The reaction mixture contained 5 mL of the sample cDNA and 15 mL of the master mix including the sense and antisense primers. Cytokine mRNA levels were calibrated by the swine β-actin level and normalized by common logarithmic transformation. Values represent means and error bars indicate the standard deviations. The results are means of 3 measures repeated 4 times with independent experiments.
Lactobacilli immunomodulatory activity in PIE-immune cell co-culture system
Porcine PP immune single-cell suspensions were prepared from the ileum of adult swine as described above [11, 13, 15]. In the Transwell culture system, PIE cells were seeded in the apical surface at a concentration of 1.5 × 105 cells/well in 12-well tissue culture plates (Transwell-Col. (PTFE), pore size 0.2 mm) while porcine PP immunocompetent cells were seeded in the basolateral compartment at a concentration of 2 × 107 cells/well. For the evaluation of the immunomodulatory activity of lactobacilli in the PIE-immune cell co-culture system, the apical surface containing PIE cells was stimulated with lactobacilli strains for 48 h and then washed twice with PBS. Finally, PIE cells were stimulated with poly(I:C) for 12 h. Studies of mRNA expression in PIE cells were performed as described above. In addition, levels of IL-1β, IL-2, IL-6, IL-10, IL-12p40, TNF-α, IFN-α, IFN-β, IFN-γ and TGF-β mRNA were studied in immune cells by using previously described primers .
The statistical analysis for all data was performed using the GLM of SAS computer program (SAS, 1994). All of the means and standard deviations were calculated 3 times with repeated measurements and experiments. Normalized fold expression was calculated as the ratio of mRNA expression of cytokines to one of β-actin normalized by common logarithmic transformation of expression. To examine the significance of the fixed effect of repeated experiments, the REG procedure of SAS programs was used while considering the linear regression of expression on the repeated experiments. Comparisons among mean normalized fold expressions to evaluate relative mRNA expressions of cytokines in cells were carried out using one-way ANOVA for an effect of time or bacteria strains as a fixed effect, and then examined by the Fisher least significant difference (LSD) test. For these analyses, the p < 0.05 level was used to define significance.