Animals and management
The experimental protocols involving the management and care of pigs and mice were approved by the Animal Care and Use Committee of Guangdong Ocean University, Zhanjiang, China (Permit No. 206-1108).
Twenty-four pigs (Luchuan sows × Duroc boars; 12 males and 12 females) each weighing 15 ± 2 kg were housed in two animal rooms at the Animal Hospital of Guangdong Ocean University, Zhanjiang, China. The pigs were randomly divided into two animal room groups. Each group had six males and six females of similar body weight. There were four replicates (collection dates) of three per group. The animals were maintained for 2 wks at 20 ± 2 °C and RH = 75–85% to acclimatize them to the environment. The photoperiod was maintained under 12-h light / 12-h darkness over the adaptation and trial periods. Throughout the study, the pigs were fed a complete formula in the morning, afternoon, and evening with ~6-h intervals between feedings. Drinking water was freely available. To minimize acute heat stress, the animal facility was gradually warmed over a 7-d period. The trial lasted 21 d. The control animals were subjected to 25 ± 3 °C and the HS animals were exposed to 34 ± 1 °C at 75–85% RH.
Pseudo-germ-free animals (SPF grade) were induced by feeding mice (n = 130) with a mixture of vancomycin (200 mg kg-1), metronidazole (200 mg kg-1), and neomycin (200 mg kg-1) for 5 d consecutively. Throughout the study period, all mice were maintained under a 12-h light / 12-h dark photoperiod and an air circulation cycle under the Exhaust Ventilated Closed-System Cage Rack. They had ad libitum access to autoclave-sterilized chow and water.
Ten mice were controls (BC group) and received only chow and ad libitum water. The other mice (n = 120) were divided into four groups of 30 each. They were administered FMT after pig feces collection on days 1, 7, 14, and 21, respectively. Each group was further subdivided into three treatment groups of 10 mice each. They were administered intragastric infusions of either phosphate-buffered saline (PBS group), a 0.5-mL mixture of control pig feces homogenized in PBS (CF group), or a 0.5-mL mixture of HS pig feces homogenized in PBS (HF group). All mice were sacrificed by break the neck after ether anesthesia 7 d after the intragastric infusion.
All pigs were observed for diarrhea and weight gain. Fecal shape and color were recorded daily and scored  as indicated in Table 5. The Diarrhea Index was calculated according to the scores for six pigs sacrificed at each sampling time.
Diarrhea Index = sum of scores / 6 (1)
Forehead and rectal temperatures were measured daily with a far-infrared thermometer. At each sampling time (days 1, 7, 14, and 21), pigs from the control (n = 3) and treatment groups (n = 3) were electric shock to sacrificed. Colonic feces and colon tissue samples were collected and immediately stored at -80 °C until the subsequent cell culture studies, microbiome and transcriptomic analyses, and mouse FMT studies. After FMT administration, mice (n = 30 per collection day) were sacrificed on day 7. Blood, colonic feces, and colon tissue samples were collected and immediately stored at -80 °C until the subsequent analyses.
The data were subjected to t-tests using SPSS v. 21.0 (IBM Corp., Armonk, NY, USA). Data are expressed as means ± standard deviation. P ≤ 0.05 indicates significant difference. P ≤ 0.01 indicates highly significant difference.
Colonic tissue was fixed in buffered formalin (10% v/v) and stained with H&E for histopathological examination. Image-Pro Plus v. 6.0 (Media Cybernetics Inc., Silver Spring, USA) was used to measure villus height, crypt depth, and width. Hydrated colonic tissue sections were treated with amylase at 37 °C for 1 h, rinsed under running water for 10 min, and stained with periodic acid solution at room temperature (25 °C) for 7 min according to the instructions for the Glycogen D-PAS Staining Kit ( Leagene Biotechnology, Beijing, China). The tissue sections were rinsed with tap water, immersed in Schiff’s reagent in the dark for 15 min, and rinsed with tap water for 10 min to remove the stain. The sections were dehydrated with an alcohol concentration gradient (75%, 85%, 95%, and then 100%), cleared of alcohol with xylene, and sealed with neutral gum. Image-Pro Plus v. 6.0 (Media Cybernetics Inc., USA) was used to evaluate the goblet cells per unit area in the colonic mucosa .
Western blot analysis
To measure the responses of the critical TLR4/NF-κB signaling pathway proteins in the colonic tissues, total protein was extracted with RIPA (radioimmunoprecipitation assay) lysis buffer (Beyotime, China) and the nuclear and cytoplasmic protein fractions were extracted with NE-PERTM nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, USA), respectively. The total protein concentrations were determined using a BCA (bicinchoninic acid) protein assay kit (CWBIO, China). Equal amounts of protein lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Merck Millipore, Germany). The membranes were blocked for 1 h with 5% skimmed milk powder and incubated with primary antibody of Anti- TLR4, TRAF6, MyD88, p65 (Abcam, USA) and β-actin (Beyotime，China）overnight at 4 °C. The blots were incubated under the temperature of 4°C for 2h with a corresponding secondary antibody conjugated to horseradish peroxidase (HRP). Positive bands were visualized by enhanced chemiluminescence (ECL; Tanon, China). The band intensities were semi-quantitatively analyzed by densitometry with Gel-Pro Analyzer v. 4.0 (Meyer Instruments, Houston, TX, USA). The relative protein expression levels were normalized to β-actin. Antibodies against TLR4, TRAF6, MyD88, p65, and β-actin were obtained from Abcam (Cambridge, UK). HRP-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Cell Signaling Technology (Danvers, USA).
TLR4, MyD88, and TRAF6
Paraffin sections of the intestinal tissues were prepared. TLR4-, MyD88-, and TRAF6-positive cells and p65 (NF-κB) activity were detected by immunohistochemistry (IHC). Data are expressed as means ± standard deviation. One-way ANOVA and Tukey's multiple comparisons test were performed on the data in SPSS v. 21.0 (IBM Corp., Armonk, NY, USA). P ≤ 0.05 indicates significant difference and P ≤ 0.01 indicates highly significant difference.
Detection of p65 protein entry into the nucleus
Frozen intestinal tissue sections (8 μm) stored at -80 °C were fixed in 4% (v/v) paraformaldehyde for 10 min, rinsed thrice with PBS for 2 min each time, and blocked with 1% (v/v) bovine serum albumin (BSA) for 1 h. The p65 primary antibody was diluted 1:100 and 100 μL of it was added to each tissue section. These were stored in the dark overnight at 4 °C. The tissue sections were then rinsed thrice with PBS (pH 7.4) for 10 min each time. Alexa Fluor 647-labeled goat anti-rabbit IgG (H+L) (Beyotime China). To measure the responses of the critical TLR4/NF-κB signaling pathway proteins in the colonic tissues, total protein was extracted with RIPA (radioimmunoprecipitation assay) lysis buffer (Beyotime , China) and the nuclear and cytoplasmic protein fractions were extracted with NE-PERTM nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Waltham, USA), respectively. The total protein concentrations were determined using a BCA (bicinchoninic acid) protein assay kit (CWBIO, China). Equal amounts of protein lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Merck Millipore, Germany). The membranes were blocked for 1 h with 5% skimmed milk powder and incubated with primary antibody of Anti- TLR4, TRAF6, MyD88, p65 obtained from Abcam (Cambridge, UK) and β-actin (Beyotime, China) for 12 h at 4 °C. The blots were incubated under the temperature of 4°C for 2h with a corresponding secondary antibody of HRP-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Cell Signaling Technology (Danvers, MA, USA) conjugated to horseradish peroxidase (HRP). Positive bands were visualized by enhanced chemiluminescence (ECL; Tanon, China). The band intensities were semi-quantitatively analyzed by densitometry with Gel-Pro Analyzer v. 4.0 (Meyer Instruments, Houston, TX, USA). Alexa Fluor 647-labeled goat anti-rabbit IgG (H+L) (Beyotime, China) was diluted 1:1,000 and added dropwise to the tissue sections which were then incubated in an opaque wet box for 1.5 h. The tissue section slides were then rinsed thrice with PBS (pH 7.4) for 10 min each time, immersed in 4’,6-diamidino-2-phenylindole (DAPI) fluorescent stain for 10 min, and rinsed thrice in PBS for 10 min each time. The slides were then mounted with anti-quenching sealer and photographed under a fluorescence microscope (Olympus BX51, Japan).
Microbial genomic sequencing and analysis
Total genomic DNA was extracted from the samples with a QIAamp DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA concentration and purity were evaluated on 1% agarose gels. The quantity of DNA was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA) after zeroed with sample solvents and the DNA was diluted to 1 ng µL-l with sterile water. The V3-V4 distinct regions of the 16S rRNA genes were amplified with specific barcoded primers[66, 67]. The PCR reactions were performed in triplicate in a total volume of 25 µL consisting of 1μL of each the primers (5 µM ), 10 μL of 10 ng DNA template, 4 µL of 1×FastPfu buffer, 1μL of 2.5 mM dNTPs, 0.4 µL FastPfu polymerase, and 7.6 μL nuclease-free water.
The PCR program was as follows: initial denaturation at 94 °C for 5 min, 30 cycles at 94 °C for 50 s, 55 °C for 30 s, 72 °C for 50 s, and a final extension at 72 °C for 6 min. The PCR products were purified with an AxyPrep DNA Gel Extraction Kit (Axygen Scientific, USA). Amplicons from all samples were sent to a commercial company (Biomarker, China) for sequencing on an Illumina HiSeq 2500 platform (Illumina, USA). Species classification information corresponding to each OTU was obtained by comparing the representative OTU sequence with the microbial reference database. Sample community compositions were calculated at the phylum, class, order, family, genus, and species levels and generated in QIIME (Version 1.8.0). GraphPad Prism v. 6.0c (GraphPad Software, USA), R v. 3.0.3, Metastats, and STAMP (Statistical Analysis of Metagenomic Profiles) were used for the statistical analyses. The weighted UniFrac distances among the groups were statistically compared by analysis of similarities in the ‘vegan’ package of R v. 3.0.3. In the univariate analysis of gut microbiota and predicted KEGG biochemical pathways for each group, one-way ANOVA with Bonferroni’s multiple comparison test was performed to compare the alpha diversities among the groups. Metastats identified differentially abundant phyla, genera, and species in the groups. Significant differences between groups were identified by the LEfSe (line discriminant analysis effect size) method.
Transcriptome sequencing and analysis
An enzyme-free cryopreservation tube was pre-cooled in liquid nitrogen and colonic epithelium was quickly excised and cut into pieces similar in size to soybean granules. RNase-free water was used to prepare 1 × PBS or saline, the tissue surface stains were quickly removed, and the surface liquid was absorbed and collected in the cryopreservation tube which was rapidly transferred to liquid nitrogen and sent to Majorbio Bio-farm Technology Co. (Shanghai, China) for sequencing. The eukaryotic mRNA sequencing was based on the HiSeq platform used to sequence all mRNAs transcribed at specific times from specific eukaryote tissues. Total RNA was extracted from the tissue samples and its concentration and purity were detected with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) after zeroed with sample solvents. RNA integrity was assessed by sodium dodecyl sulfate polyacrylamide agarose gel electrophoresis (SDS-PAGE) and the RNA integrity number was determined with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The eukaryotic mRNA 3’-end had a polyA tail structure. Magnetic beads treated with Oligo (dT) were used for A-T base pairing with flo A and mRNA isolated from the total RNA for transcriptome analysis. Fragmentation buffer was added to break the mRNA into small ~300-bp fragments. Six-base random hexamers were added by reverse transcription and mRNA was used as a template to reverse the synthesis of one-strand cDNA and for two-strand synthesis to form a stable double-stranded structure. The short fragments were connected to the adaptor sequence and then sequenced on the Illumina HiSeq platform (Illumina, USA). A single Illumina sequencing run generated billions of reads. Statistical methods were used to measure the sequences visually reflecting library construction and sample sequencing quality. Quality of the data after the quality control (reads) was compared against the reference genome to obtain mapped data (reads) for the subsequent analysis. Quality of the sequencing comparison was also evaluated. Based on existing reference genomes, the mapped reads were assembled, spliced with Cufflinks, and compared against known transcripts, transcripts without annotation information, and functional annotations of potential new transcripts. Read counts for each sample gene/transcript were obtained using featureCounts alignment to genome data and annotation files. Fragments per kilobases per million reads (FPKM; number of reads on one million bases per gene alignment) were calculated. The FPKM method eliminates the effects of differences in gene length and sequencing quantity on gene expression calculation. The calculated gene expression may then be used to compare gene expression differences among samples. After securing the number of read counts for the gene/transcript, analysis of differential gene/transcript expression between samples was performed in the multi-sample (≥ 2) project and the differentially expressed genes/transcripts were identified.
RNA extraction and cDNA synthesis
The synthetic primer sequences (BBI Life Sciences, Shanghai, China) are listed in Table 6. A ~0.5 g tissue block was pulverized in liquid nitrogen and transferred to a 1.5-mL centrifuge tube. Then 1 mL TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added and the tube was placed on ice for 20 min. The tube was inverted to ensure complete lysis and prevent sedimentation. Then 200 µL NH4Cl was added and the tube was vigorously shaken to emulsify the contents. The tube was then left to stand at 25 °C for 5 min and centrifuged with speed in 12000 rpm at 4 °C for 10 mins. The supernatant (200 µL) was transferred to a new centrifuge tube and an equal volume of isopropanol was added. The contents were mixed by inversion, left to stand at 25 °C for 10 min, and centrifuged with speed in 12000 rpm at 4 °C for 10 min. The supernatant was discarded and 1 mL pre-cooled 75% (v/v) ethanol (4 °C) was added. The tube was gently inverted, left to stand for 2 min, and centrifuged at 12,000 × g and 4 °C for 5 min. The supernatant was removed and dried at 25 °C for 5 min. Twenty microliters RNase-free water was added to dissolve the RNA and the solution was stored at -80 °C. Then 2 μL RNA mixture was extracted and 1% agarose gel electrophoresis was performed to check its integrity. The rest of the mixture was diluted 100×. Diethyl pyrocarbonate water was used as a blank control and OD260/OD280 were measured by OD-1000+ Spectrophotometer (ONE Drop, USA) after zeroed with sample solvents.
After removing genomic DNA, a reverse transcription reaction system was prepared on ice according to the instructions for the HiScript® Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech, Nanjing, China), The PCR program was 25 °C for 10 min, 50 °C for 30 min, and 85 °C for 5 min. After the reaction was completed, the cDNA fractionation apparatus was stored at -80 °C until later use.
Relative quantitative real-time RT-PCR
The RT-qPCR system was configured according to the instructions for the ChamQTM SYBR® qPCR Master Mix Kit (Vazyme Biotech, Nanjing, China) for fluorescence quantitative PCR. The relative expression level was calculated by the 2-ΔΔCt method.
The intestinal porcine epithelial cell (IPEC-J2) model was a gift from Dr. Bruce Schultz of Kansas State University. The cells were cultured in Dulbecco’s modiﬁed Eagle’s medium: nutrient mixture F-12 (DMEM/F12; Sigma Aldrich Corp., St. Louis, MO, USA) containing 10% (v/v) fetal bovine serum (FBS), penicillin (100 U mL-l), and streptomycin (100 U mL-l) in a 25-cm, two-cell culture ﬂask (Corning Inc., USA) stored in a humidiﬁed incubator at 37 °C and 5% CO2. When the cells grow to 80% confluence, discard the medium, wash with PBS 2-3 times, add 1ml Trypsin-EDTA (0.25%, Thermo Fisher, USA) for 3 minutes, and then add medium to terminate the treatment. After centrifugation at 1000 rpm for 5 min at room temperature, the supernatant was discarded, diluted with the medium in proportion, and transferred to a 24-well plate. After the cells grew to sub-conﬂuence in 24-well plates, the culture medium was removed and the cells were washed twice with PBS.
In vitro LPS validation experiment
The IPEC-J2 cells were subjected to 10 μg mL-l LPS for 3 h. Western blot was run to detect TLR4 expression and p65 entry in the nucleus. One gram of each fecal sample was weighed out and washed thrice with PBS. The final fecal mass: PBS volume was 1:9. The fecal suspensions were atomized for 15 min with an ultrasonic cell pulverizer (Φ6 horn ultrasonic treatment, 1.5 s; interval, 2 s; power, 25%). After centrifugation at 5,000 × g for 10 min under
4 °C, the supernatant was collected for enzyme-linked immunosorbent assay (ELISA).