Materials
Dulbecco’s modified Eagle’s medium nutrient mix F12 (DMEM/F12), fetal bovine serum (FBS), double-antibiotics (containing 100 U/mL penicillin and 100 U/mL streptomycin), and epidermal growth factor (EGF) were from Invitrogen (Fisher Scientific, Ottawa, ON, Canada).
Cell culture
IPEC-J2 cells (ACC 701, RRID: CVCL_2246, DSMZ-German Collection of Microorganisms) were cultured in DMEM/F12 culture medium supplemented with 5% FBS, 1% double-antibiotics and 3 ng/mL EGF in the humidified incubator (Thermo Electron Corporation) with atmosphere of 5% CO2 at 37℃. The culture medium was replaced every two days. After growing to 80-90% confluence, the cells were digested with 0.25% trypsin and inoculated into cell culture plates for further experiments.
Bacterial strains
The strain of ETEC F4 was isolated from feces of piglets infected with post-weaning diarrhea in the Veterinary Diagnostic Services Laboratory, Government of Manitoba, Canada. The ETEC F4 strain was preserved in the 2 mL cryovials including cryopreservative-added broth. ETEC F4 was first incubated in Tryptic soy broth (TSB, Millipore Sigma) at 37℃ for 16-18 h. After dilution with fresh medium, the cultures were incubated under the same condition until the appearance of logarithmic phase. The optical density (OD) value at 600 nm was measured by spectrophotometer (Biochrom™, Fisher scientific). The OD values around 0.3-0.5 were used for the following experiments.
The strain of B. licheniformis PF9 was obtained from the Guelph Food Research Centre, Agriculture and Agri-Food Canada and preserved in sterile glycerol (10-20%). The B. licheniformis PF9 strain was isolated from feces of piglets and incubated in TSB at 37 ℃ overnight (about 16 h). The OD values around 0.8-1.0 were used for the following experiments.
Experimental design
IPEC-J2 cells were detached with 0.25% trypsin (GibcoTM, Thermo fisher scientific) and diluted to 4×105 cells/mL. IPEC-J2 cells were then inoculated into different well plates and cultured in DMEM/F12 medium for about 2-3 days until growing to 80-90% confluence. For different treatments, the control group was cultured in DMEM/F12 medium without treatment, the ETEC group was treated with ETEC F4 (106 CFU/mL) for 3 h, the ETEC + BL group was pretreated the selected concentrations of B. licheniformis PF9 for 2 h prior to both ETEC F4 and B. licheniformis PF9 treatment together (1:5, 1:10, 1: 25, 1:50, or 1:100) for 3 h, and the BL group was pretreated the selected concentrations of B. licheniformis PF9 for 2 h prior to its alone treatment (3 h).
Lactate dehydrogenase (LDH) assay
LDH, a cytosolic enzyme present in many different cell types, will be released into the surrounding cell culture media when there is a damage to cells. IPEC-J2 cells were detached with 0.25% trypsin and diluted to 4×105 cells/mL. IPEC-J2 cells were then inoculated into 24 well plate at 2×105 cells/well and cultured in DMEM/F12 medium for 3 days. After the previously described treatments, the supernatants containing released LDH of cultured IPEC-J2 cells were collected to determine the integrity of cells. After centrifugation at 12,000 r/min for 5 min at 4℃, supernatants were carefully collected and transferred into new tubes. The supernatants of each sample (50 μL) were added into a 96-well plate and the reaction mix for the assay was prepared by using LDH assay kit (CyQUANTTM, Thermo fisher scientific) according to the manufacturer’s instructions. The absorbance at 490 nm and 680 nm was immediately measured by using a Synergy H4 hybrid multi-mode microplate reader (BioTek, Winooski, VT, USA). The activity of LDH was presented as a percentage of the LDH activity by the control group.
Cytokine measurement by ELISA
The IL-8 concentrations of culture supernatants were measured by ELISA kit (Invitrogen, Fisher Scientific) following the manufacturer’s instructions. Briefly, 100 μL of culture supernatant was used for IL-8 assay. At the end of the reaction process, the plates were read at 450 nm, using a Synergy H4 hybrid multi-mode microplate reader (BioTek, Winooski, VT, USA).
Quantitative PCR (qPCR)
IPEC-J2 cells were seeded with a density of 5×105 cells per well in 12-well culture plates. After different treatments, total RNA was extracted by using Trizol (Invitrogen, Carlsbad, CA, U.S.) according to the manufacturer’s instructions. The total RNA was dissolved in 20 µL RNase-free water and stored at −80℃. The RNA concentration was determined using a NanoDrop 2000 Spectrophotometer (Nano-Drop Technologies, Wilmington, DE, U.S.) with purity ascertained (A260/A280) between 1.8 and 2.0. About 1 µg total RNA from each sample was converted into cDNA by using iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instruction. The primers were designed with Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and synthesized by Integrated DNA Technologies, Inc. The qPCR was performed to quantify the target genes, such as cytokines genes, barrier function genes, toll-like receptors genes, NF-κB signaling pathway genes, and virulence-related genes (Supplemental Tables S1 and S2). The cyclophilin-A (CycA) gene was used as the housekeeping gene in IPEC-J2 cells. The glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene was used as the housekeeping gene in ETEC F4. The relative changes in gene expression levels in IPEC-J2 cells or ETEC F4 normalized against CycA or gapA were determined by using the 2−∆∆CT method [24].
Immunofluorescent Staining
Cells cultured onto cover-slips (Fisher Scientific) were fixed with 4% paraformaldehyde for 20 mins and permeabilized with 0.3% Triton X-100 in PBS for 20 min. The cells were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h and then incubated with anti-rabbit polyclonal antibodies of zona occludens 1 (ZO-1, 1:100 dilution in PBS) and occludin (OCLN, 1:100 dilution, Thermal Scientific) at 4°C for 16 h, respectively. The cells were then washed 3 times with PBS and incubated with an Alexa Fluor 488 goat anti-rabbit (green) or Alexa Fluor 584 goat anti-rabbit (red) antibody (Thermal Scientific, catalogue no. A-11034 or A-11037) for 1 h at 22°C. For F-actin staining, the cells were incubated with phalloidin, CF488A (1:40 dilution in PBS, Biotium, Inc., Fremont, CA 94538, USA) at 22°C for 1 h. Rinsed cells were mounted with Vectashield mounting medium with 4,6-Diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc. Burlingame, CA, USA). Images were taken by a Zeiss fluorescence microscope (Car-Zeiss Ltd., Toronto ON, Canada).
Transepithelial electric resistance (TEER) and cell permeability measurement
The IPEC-J2 cells with a density of 5×105 cells per well were seeded into Millicell membrane cell inserts (12 wells, Corning Costar). The TEER of cell monolayers was measured using a Millicell Electrical Resistance System (ESR-2, Millipore-Sigma). The TEER was monitored every two days until a monolayer of cells was completely differentiated. The TEER of four treatment groups (control, ETEC F4 alone, ETEC F4 plus B. licheniformis PF9, and B. licheniformis PF9 alone) was measured before (0 h) and after ETEC F4 treatments (1, 2, 3, 4, and 5 h), respectively. The TEER of monolayers with/without adding ETEC F4 represented the ETEC F4 group and control group, respectively. The ETEC F4 plus B. licheniformis PF9 group were pre-treated with B. licheniformis PF9 for 2 h and then incubated with ETEC F4 plus B. licheniformis PF9 for 3 h. The value of TEER was corrected for background resistance by subtracting the contribution of cell free filter and the medium (200 Ω).
To quantify the paracellular permeability of cell monolayers, 0.5 mg/mL of 4 kDa FITC-dextran (FD4, Sigma-Aldrich) was added to the apical side of the inserts. The basolateral medium aliquots were taken after 48 h of incubation. The diffused fluorescent tracer was then measured by fluorometry (excitation, 485 nm, emission, 528 nm) using a microplate reader (BioTek, Winooski, VT, USA). Measurements were performed in quadruplicate.
Western blot
Proteins from IPEC-J2 were extracted by using RIPA Lysate Buffer (Sigma-Aldrich) and their concentrations were determined by the BCA Protein Assay Kit according to the manufacturer’s protocol (Fisher Scientific). Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane using a Trans-Blot® TurboTM transfer system (Bio-Rad, Hercules, CA, USA). The membrane was then blocked for 2 h with 5% skim milk in Tris-buffered saline (TBS) at 22°C, and was subsequently immunolabeled in primary antibodies diluted in TBS at 4°C for 16 h. Primary antibodies to rabbit polyclonal anti-ZO-1 (1:1000 dilution), anti-OCLN (1:1000 dilution), anti-claudin-3 (1:1000 dilution) and anti-NF-κB p65 (1:1000 dilution), rabbit monoclonal anti-NF-κB phospho-p65 (1:1000 dilution) and mouse monoclonal anti-β-actin (1:5000 dilution) from Invitrogen by Thermo Fisher scientific were used, respectively. After washing for 3×10 min with TBST, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse (1:350 dilution) or goat anti-rabbit IgG (1:4000 dilution) as a secondary antibody for 1 hour at 22°C, respectively. Visualization of the antigen-antibody complex was conducted with a Clarity Max ECL Western Blotting Substrate (Bio-Rad) and immunoreactive proteins were visualized using the ChemiDocTM MP imaging system (2.4.0.03, Bio-Rad). The protein bands were analyzed by Image Lab 6.0 software (Bio-Rad). The β-actin was used as the internal control. Values of target proteins were represented as the ratio of the optical density of the protein bands to the density of the respective β-actin band. The value of the normalized phosphorylation of p65 protein was represented as the ratio of the density of phosphor-p65 to that of p65.
Statistical analysis
Data were presented as means ± standard deviations (SD). Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Differences between means were evaluated by one-way ANOVA. Tukey’s multiple comparisons test was used. Difference of the mRNA abundance of virulence-related factors in ETEC F4 between ETEC and ETEC+BL groups were evaluated by unpaired t test. Level of significance was set at P < 0.05.