Reagents and antibodies
Rabbit phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, #4370) and rabbit phospho-p38 MAPK (Thr180/Tyr182, #4631) monoclonal antibodies were purchased from Cell Signaling (Danvers, MA, USA). Mouse anti-chicken GAPDH antibody was purchased from Thermo Fisher Scientific (AM4300; Waltham, MA, USA). Alexa Fluor 488 goat anti-rabbit IgG (H + L) secondary antibody was purchased from Invitrogen (A-11008, Carlsbad, CA, USA). Anti-rabbit IgG (H+L) HRP-conjugated antibody was purchased from Promega (W4011; Madison, WI, USA). Goat anti-mouse IgG HRP-conjugated antibody (A16078) was purchased from Thermo Fisher Scientific. In addition, HRP-conjugated rabbit anti-6-His antibody (A190-114P) was purchased from Bethyl Laboratories, Inc. (Montgomery, TX, USA) and 4′,6-diamidino-2-phe-nylindole (DAPI) was purchased from Invitrogen (Rockford, IL, USA). RIPA lysis and extraction buffers were purchased from Thermo Fisher Scientific.
Cloning of AvBD8
The primers were designed using DNASTAR (DNASTAR Incorporation, Madison, WI, USA) to amplify the mature sequence of AvBD8 from Gallus gallus avian beta-defensin 8 (AvBD8) mRNA sequence (NM_0 01001781.1). The AvBD8 coding sequence was amplified using total RNA derived from the intestinal mucosal layer of White Leghorn chickens, kindly provided by the Animal Biosciences and Biotechnology Laboratory (Beltsville, MD, USA) of the USDA Agricultural Research Service. The PCR product was amplified using the following specific primers: forward, 5′-CGGAATTCAACAACGAGGCACAGTGTG-3′ and reverse, 5′- CCAAGCTTGTCGTACACAGTCCG-3′ (the EcoRI and HindIII restriction enzyme sites are underlined) with DreamTaq Green PCR Master Mix (2×) (Thermo Fisher Scientific). The PCR amplification was carried out under the following conditions: a pre-denaturation step at 95°C for 5 min, a denaturation step at 95°C for 30 s, an annealing step at 55°C for 30 s, an extension step at 72°C for 30 s for 35 cycles, and a final extension at 72°C for 5 min. The PCR products were purified using the FavorPrep™ GEL/PCR purification kit (Favorgen, Ping-Tung, Taiwan), cloned into the pCR2.1-TOPO vector (Invitrogen), and transformed using Escherichia coli TOP 10 competent cells (Invitrogen) according to the manufacturer’s protocol. Through blue–white screening, positive clones were picked out and cultured overnight in Luria–Bertani (LB) broth (with 100 μg/mL ampicillin). Plasmids were extracted using the FavorPrep™ plasmid DNA extraction mini kit (Favorgen) and sequenced by Genotech (Daejeon, South Korea). The AvBD8/pCR2.1-TOPO vector was digested with the restriction enzymes EcoRI and HindIII (Thermo Fisher Scientific, USA). The protein expression vector pET32a (Novagen, Madison, WI, USA) was also digested with the same restriction enzymes. The digested fragments were purified by agarose gel electrophoresis using the FavorPrep™ GEL/PCR purification kit (Favorgen) and ligated using T4 DNA ligase (Invitrogen). The ligated vector and insert were transformed into One Shot BL21 (DE3) chemically competent E. coli (Invitrogen) and sequenced.
Production of recombinant AvBD8 protein
Recombinant AvBD8 protein was produced as previously described for chicken IL-26 . Briefly, the positive clones of AvBD8/pET32a were incubated at 37°C overnight in a shaking incubator at 225 rpm in LB broth with 100 μg/mL ampicillin. The bacterial culture was then induced for recombinant protein expression with 1 mM isopropyl-β-D-thiogalactopyranoside (USB Corporation, Cleveland, OH, USA) for 4 h at 37°C, and then centrifuged at 5000 × g for 15 min. The AvBD8 recombinant protein was extracted with B-PER bacterial protein extraction reagent (Thermo Fisher Scientific) and purified using HisPur cobalt resin (Thermo Fisher Scientific). The recombinant AvBD8 protein was eluted using 250 mM imidazole and analyzed by sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis and western blotting using HRP-conjugated rabbit anti-6-His antibody (Bethyl Laboratories). The purified recombinant protein was dialyzed using SnakeSkin™ dialysis tubing (Thermo Fisher Scientific) in phosphate-buffered saline (PBS; pH 7.4) overnight at 4°C with stirring and analyzed by SDS-PAGE and western blotting. Endotoxins in recombinant AvBD8 were evaluated using Pierce™ Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific) according to manufacturer’s protocol.
Cell culture and recombinant protein treatment
Chicken macrophage cell line HD11  was cultured in complete RPMI 1640 medium (Thermo Fisher Scientific) containing 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific) in a humidified 5% CO2 atmosphere at 41°C. The cells (1.0 × 106/well) were incubated in a 12-well plate containing 1 mL of culture medium and treated with 100 ng/mL (final concentration) recombinant AvBD8 protein and incubated for 0, 0.5, 1, 2, and 4 h in a humidified 5% CO2 atmosphere at 41°C.
Quantitative real-time polymerase chain reaction
HD11 cells were washed with ice-cold PBS, and then total RNA was extracted from the cells using TRIzol™ reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol. cDNA was synthesized from the total RNA using the RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. To analyze the cytokine gene expression, primers were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table 1) and qRT-PCR was performed using FastStart Essential DNA Green Master (Roche, Indianapolis, IN, USA), according to the manufacturer's instructions, in the LightCycler96 system (Roche). The chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the control to normalize RNA quantity. The relative quantification of gene-specific expression was calculated using the 2-ΔΔCt method after normalization with the GAPDH gene expression level . All qRT-PCRs were performed in triplicate.
HD11 cells (5.0 × 106/well) were incubated in a 6-well plate (Thermo Scientific) containing 2 mL of culture medium and stimulated with 100 ng/mL recombinant AvBD8 proteins for 0, 15, 30, 60, and 90 min in a humidified 5% CO2 atmosphere at 41°C. After incubation, the cells were washed with ice-cold PBS and the proteins were extracted from the cells using RIPA lysis and extraction buffers according to the manufacturer’s protocol. Halt™ phosphatase inhibitor cocktail (Thermo Fisher Scientific) was added to the cell lysate. The cell protein concentration was measured using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Protein samples were mixed with 4× sample buffer (200 mM Tris-Cl [pH 6.8], 20% β-mercaptoethanol, 8% SDS, 0.4% bromophenol blue, and 40% glycerol) and heated to 100°C for 5 min. The protein samples (30 µg) were electrophoresed on 12% Tris–glycine SDS polyacrylamide gels. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Rydalmere, Australia) using the Mini-PROTEAN® electrophoresis system (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk (Thermo Fisher Scientific) in PBS (pH 7.4) containing 0.05% Tween-20 (Sigma-Aldrich, MO, USA) (PBST). Antibodies were prepared with 2% skim milk in PBST (Phosphate Buffered Saline with Tween 20). The membranes were washed with PBST and treated with anti-rabbit IgG (H+L), HRP-conjugated antibody (Promega). The membranes were then developed using Western Lightning ECL Plus (Thermo Fisher Scientific) for Hyperfilm (GE Healthcare).
Immunocytochemistry was performed using the Nunc™ Lab-Tek™ Chamber Slide (Thermo Fisher Scientific) as previously described . Briefly, HD11 cells (4.0 × 104 cells/well) were cultured in a chamber slide for 30 min in a humidified 5% CO2 atmosphere incubator at 41°C in the presence or absence of the recombinant AvBD8 proteins (100 ng/mL). The cells were then fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min, and then incubated with ice-cold methanol for 10 min at 4°C. Following overnight incubation with the anti-rabbit primary antibody at 4°C, the cells were treated with Alexa Fluor®488-conjugated secondary antibody for 1 h, and then stained with DAPI for 5 min. Finally, images were captured using EVOS® FLoid® Cell Imaging Station (Life Technologies, Carlsbad, CA, USA).
HD11 cells (2 × 104 cells/well) were seeded and cultured in 24-well plates. After culturing overnight, the cells were incubated with the AvBD8 recombinant protein (50, 100, 200, 300, 400, 5000, and 1000 ng/mL) for 72 h in a humidified 5% CO2 atmosphere at 41°C to analyze cell proliferation and nitric oxide (NO) production. The NO content was measured using the Griess reagent system (Promega) and cell proliferation was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's protocols as previously described .
The purified plasmid was sequenced by Genotech (Daejeon, Republic of Korea). To compare the cloned chicken AvBD8 sequence with sequences in GenBank, the data were analyzed using a Nucleotide Basic Local Alignment Search Tool (nBLAST) search (http://www.ncbi.nlm.nih.gov/BLAST/). Protein identification was performed using the Expert Protein Analysis System (https://www.expasy.org/) to determine the molecular weight. The protein structure was predicted using RaptorX (http://raptorx.uchicago.edu/) and FirstGlance in Jmol (http://www.bioinformatics.org/firstglance/fgij/).
Data are presented as mean ± standard error of mean of three independent experiments. Statistical analyses were performed using IBM SPSS software (SPSS 23.0 for Windows; IBM, Chicago, IL, USA). The results with a p-value of < 0.05 were considered statistically significant. Differences between groups were evaluated using Duncan's multiple range test.