Animals
A total of six—three 21-day-old (weaners; weighing 8‑10 kg) and three 0-day-old (neonates; weighing 1.10‑1.30 kg)—male cross‑bred Duroc/Landrace/Yorkshire piglets were obtained from the Jiangsu Huai'an Pig Farm (Huai'an, China). The piglets were housed indoors at the Jiangsu Huai'an Pig Farm, under constant conditions of 60 % humidity, 26 °C, and 12 h light/dark cycle as well as free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University, and the National Institutes of Health guidelines for the performance of animal experiments were followed.
Material collection and preparation
Each piglet was euthanized by an intravenous injection of pentobarbital sodium (100 mg/kg) before sample collection. To determine complete anesthetization of the piglets before opening the abdomen, their palpebral and withdrawal reflexes were checked. Duodenum, jejunum, and ileum tissue samples were collected from the piglets and fixed in 4 % paraformaldehyde for 48 h at 25 ℃. After fixation, the samples were sectioned into small pieces. Then the small pieces (1cm) dehydrated using a graded ethanol series (75, 85, 95, 100, and 100 %, each for 1 min in that order). The dehydrated blocks were embedded in paraffin wax and kept at room temperature to dry. Next, the tissue samples were sliced longitudinally into 5-μm-thick sections using a microtome. The sections were dried horizontally on a warming tray (37 °C) overnight, dewaxed in xylene, rehydrated in a graded series of ethanol (100, 90, 80, and 70 %, each for 1 min in that order), and washed in phosphate-buffered saline (PBS). These tissue sections were then used in the subsequent experimental analyses. All observations were single-blinded.
Histological analysis
The prepared tissue sections were stained with hematoxylin and eosin (H&E) and examined using light microscopy (BH-2, Olympus). The size of the ileal Peyer’s patches (PPs), villus height and crypt depth were measured using computer-assisted morphometry (Image-Pro Plus software).
Immunohistochemistry
Antigens were retrieved from the tissue sections by placing them in a citrate buffer (pH 6; 90‑95 °C) for 15 min. Then, the sections were treated with 0.3 % hydrogen peroxide at room temperature for 15 min and washed with PBS to quench endogenous peroxidase activity. To avoid the non-specific binding of antibodies, the sections were blocked using 5 % bovine serum albumin for 30 min at room temperature. After incubating with the primary antibodies overnight at 4 °C, the sections were treated with biotinylated secondary antibodies for 1 h at room temperature (Table I). To visualize the immuno-positive cells, the sections were stained with diaminobenzidine (DAB) for 60 min at room temperature and sealed with neutral balata. The respective isotypes were used as negative controls. The sections were visualized using a light microscope (Olympus CX23; Olympus Corporation, Tokyo, Japan) at a magnification of 400× or 100×. Different fields (n=10) of each tissue in each piglet were counted for the statistical analysis.
Immunofluorescence
The tissue sections were incubated with 0.4 % Triton X-100 in PBS for 5 min. After blocking with 5 % bovine serum albumin in PBS for 1 h, the sections were stained with Ulex europaeous agglutinin-1 (UEA-1) antibodies at room temperature for 2 h. PBS was used instead of the antibodies in control samples. After staining with DAPI, the sections were observed using confocal laser scanning microscopy (LSM-710; Zeiss, Oberkochen, Germany).
RNA isolation and Real-time quantitative PCR
Total RNA was extracted from the tissues using a TRIzol® Plus RNA Purification kit (Thermo Fisher Scientific, Inc.). One microgram of total purified RNA was reverse transcribed to cDNA, using the PrimeScript™ RT‑PCR kit (Takara Biotechnology Co., Ltd., Dalian, China), as follows: one cycle of 37 °C for 15 min followed by 85 °C for 5 s. RT‑qPCR analysis was performed with 2 μl of diluted cDNA (vol:vol, 1:5) was used to perform RT‑qPCR analysis, by ABI 7500 PCR system (Life Technologies; Thermo Fisher Scientific, Inc.) and SYBR‑Green qPCR Master Mix (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturers' protocols. The thermocycler reaction involved a pre-incubation period of 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 31 s. Specific primers referred from the studies by Zong et al. (2019), Temeeyasen et al. (2017), and Wang et al. (2020) are shown in Table II [14, 15] [16]. Data were normalized against GAPDH mRNA levels and are expressed as fold differences between control and treated cells, calculated using the 2-ΔΔCT method.
Flow cytometry
Isolated single mononuclear cells were obtained from the piglet peripheral blood-mononuclear cells (PBMCs) by density centrifugation using a porcine peripheral blood lymphocyte separation kit (Solarbio). PBMCs were stained with anti-CD3-APC (BD Biosciences, California, USA), anti-CD4-FITC (BD Biosciences, California, USA), and anti-CD8-PE (BD Biosciences, California, USA) (1:100 dilution) for 30 min at 4℃ in the dark. The cells were then washed twice with PBS, and the expression of surface markers was observed using flow cytometry (FACSC6, BD Biosciences). The flow cytometry data were analyzed using the FlowJo software. A total of 10 000 lymphocytes were acquired per sample.
Statistical analysis
Analysis of variance and unpaired Student’s t-tests were employed to determine statistically significant differences among multiple groups. The differences were considered significant at *P < 0.05, **P < 0.01. Results are expressed as mean ± SEM.