Wild-type specific pathogen free animals
Wild-type (WT) C56BL/6J mice purchased at 8 weeks of age (Vital River Laboratory Animal Technology. Co., Ltd., China) were randomly paired (1:1) after adapting for one week and fed a standard diet (#12450B, Research Diets, Inc.). After cohousing for 4 days (one estrous cycle for house mouse), females were separated from the males and fed solely. During and after cohousing, pregnancies and due dates were monitored and calculated according to the formation of vaginal plugs and body weight changes, respectively.
Pups (and their mothers) were randomly assigned to four groups: (1) low-dose penicillin (LDP)-free control group (Ctr) that received no antibiotics, (2) LDP-treated control group (LDP) that received antibiotics only, (3) FMT-treated group (F) that received LDP and fecal microbiota transplantation treatments, and (4) probiotics-treated group (P) that received LDP and probiotic cocktail treatments. Pups were separated from their mothers at 21 days. Pups in every group were from at least three dams and born within 24 hours. Pups in the Ctr and F groups were born within 24 hours. For the LDP, F and P groups, dams received antibiotics at a dose of 10 mg/L to deliver approximately 1.5 mg per kg body weight about one week prior to birth and were continuously maintained on penicillin. Pups in the three groups were exposed to penicillin until 30 days of age either through their mother or through drinking water. After the LDP treatment, pups were divided by sexes and kept 2~3/cages. Pups in the F and P groups were gavaged with fecal microbiota from Ctr and probiotics, respectively at 12, 16, 20, 24 and 28 days of age. For the FMT group, feces were collected from pups in the Ctr group and immediately placed in prereduced anaerobically sterilized PBS, pooled under anaerobic conditions, settled with gravity for 2 minutes, and then the supernatant was transferred to pups in the F group. For the P group, pups were gavaged with prereduced anaerobically sterilized PBS containing Lactobacillus bulgaricus and Lactobacillus rhamnosus GG at ~108/mL for each bacterium. For the Ctr and LDP groups, pups were gavaged with prereduced anaerobically sterilized PBS on the same days. For 12 day and 16 day mice, 50 μL and 100 μL liquid were gavaged, respectively; for the rest, 150 μL was gavaged. All pups were fed on normal diet (#D12450B, Research Diets, Inc.) and allowed ad libitum access to food and water.
For the microbiota transfer to germ-free (GF) animals experiment, feces were collected from 30-day-old donors from each group and immediately placed in prereduced anaerobically sterilized PBS, homogenized under anaerobic conditions, settled with gravity for 2 minutes, and then the supernatant was transferred to even-aged GF C57BL/6 mice (n = 8 for each group, n (females) = n (males) = 4). After transfer, the conventionalized GF mice were housed in standard SPF conditions, and food and water were provided ad libitum.
It is known that SIgA-deficient mice have a different gut microbiota (GM) compared to WT mice. Therefore to ensure a comparable GM was obtained by SIgA-deficient pups to WT during birth and nursing, Pigr+/- males and females, which can produce normal SIgA and therefore have similar GM to WT mice, were used to generate SIgA-deficient pups. Specifically, 8-week-old Pigr+/- females and males were mated as above specified, genotypes of pups were identified at 10 days of age and only Pigr-/- female pups were chosen for the following experiment as specified above.
B cell-deficient (μMT) mice
Separation and transfer of pan B cells to B cell-deficient (μMT) mice was conducted as previously described with some modification . Pan B cells (includes plasma cells) from the spleens, Peyer’s patches, mesenteric lymph nodes and ileal and colonic lamina propria of 30 day LDP-treated or -free WT SPF mice were purified using negative selection (>90% purity, EasySep; StemCell Technologies) and injected intraperitoneally (~5.5 × 106 cells) in even-aged μMT mice.
Animal management and sampling
Mice were housed in standard specific pathogen-free (SPF) conditions (12/12-hour light-dark cycle, humidity at 50 ± 15%, temperature of 22 ± 2°C), and food and water were provided ad libitum. The food used in this study was sterilized using radiation (25.0 kGy). Food intake was recorded every week. Body weight was recorded weekly. At the end of the experimental period, the mice were fasted for 12 hours, and plasma was collected by eyeball extirpation. The lumen contents of the distal ileum and proximal colon were collected by washing the lumen with sterilized PBS, and the mucosal samples were collected by scraping the intestinal wall with sterilized glass slides. Then, the samples were stored at -80°C for microbial analysis. The contents of the cecum were collected and stored at -80°C for the analysis of secreted IgA (SIgA). The weights of the liver, inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT) and mesenteric white adipose tissue (mWAT) were measured. Tissues were preserved at -80°C for gene expression analysis (for ileum, all fat and mesentery were removed, and Peyer’s patches were excised from the ileum), and the liver, iWAT, colon and ileum were fixed using 4% paraformaldehyde and used for hematoxylin-eosin (H&E) staining, immunofluorescence and immunohistochemistry analysis.
Derivation of “pure or none Fimicutes” animals
Wild-type C57BL/6 mice purchased at 4 weeks of age (Vital River Laboratory Animal Technology. Co., Ltd., China) were randomly assigned to 3 groups (n = 3 for each group) after adapting for 2 weeks: (1) “pure Firmicutes” group 1 that fed a HFD (60% energy from fat, #D12492, Research Diets, Inc.), (2) “pure Firmicutes” group 2 that fed a standard diet with 0.5 g/L vancomycin and streptomycin in their drinking water, and (3) “none Firmicutes” group that fed a standard diet with 1g/L norfloxacin and 0.5g/L cefotaxime in their drinking water. All mice were maintained in a standard SPF environment as specified above, and food and water (with 4g/L sucralose) were provided ad libitum. After 4 weeks experiment, mice were killed and cecal contents were collected and stored at -80°C for further analysis.
The guidelines of the institute regarding the care and use of laboratory animals were followed. This study was approved by the Animal Experiment Committee of the College of Food Science and Nutritional Engineering at China Agricultural University.
Ileum tissue culture
Mouse ileum was obtained and cultured as previously described with some modification . Distal ileum samples were washed and cultured using RPMI 1640 supplemented with 10% FCS and penicillin/streptomycin 50 mg/ml at 37°C and 5% CO2 in 24-well plates. Two ileum samples per well (~ 3 mm) were cultured for each group (n = 4). Two days after co-culture with antigens, culture medium was collected and supernatants were centrifuged and stored at -80°C for the analysis of IgA and cells were collected for the analysis gene expression.
Quantitative Real-time PCR (qPCR) Analysis
Total RNA was extracted using TRIzolTM reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription of the total RNA (2.5 μg) was performed with a high-capacity cDNA reverse transcription kit (Promega Biotech Co., Ltd). qPCR was run in triplicate for each sample and analyzed in a LightCycler 480 real-time PCR system (Roche). Data were normalized to the internal control β-actin and analyzed using the △△CT method. The expression of genes in iWAT, the liver, ileum and colon, as well as the bacterial and fungal load, were determined through qPCR (the related genes and primers used are shown in Table S1).
Quantification of the bacterial and fungal loads through qPCR was conducted as previously described. Briefly, the total bacterial DNA was isolated from the samples with a QIAamp DNA Stool Mini Kit (Qiagen) following the manufacturer’s instructions. For the isolation of fungal DNA, samples were suspended in 50 mM Tris buffer (pH 7.5) supplemented with 1 mM EDTA, 0.2% b-mercaptoethanol and 1000 units/ml of lyticase (Sigma), incubated at 37°C for 30 min to disrupt fungal cells as described, prior to processing through the QIAamp DNA Stool Mini Kit (Qiagen). The DNA was then subjected to qPCR using a QuantiFast SYBR Green PCR kit (Bio-Rad) with specific primers (Table S1).
Determination of Body Composition through MRI
MRI experiments were performed on 30-day-old and 25-week-old mice. The body composition was determined using MesoQMR instrument (Testniumag, Shanghai, China) with a 60 mm receiver and 0.5 ± 0.08 T magnetic field strength. To obtain high resolution scanned MRI images, MRI measurements were performed on a 7.0 T Varian MRI instrument (Varian Medical Systems, Palo Alto, CA, USA) using a 40 mm volume and receiver coil at the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences. Prior to the experiments, the mice were initially anesthetized with 2% isoflurane in a dedicated chamber. During the course of MRI, anesthesia levels were reduced to 1.5–1% in a combination of medical air and medical oxygen. The mice were positioned in the prone position, and respiratory-gated image acquisition was performed. MRI images of the mice were analyzed by Argus software.
Plasma Biochemical Parameters
The plasma biochemical parameters, including, glucose, cholesterol and triglyceride (TG) levels, were determined by a 3100 Clinical Analyzer (Hitachi High-Technologies Corporation, Japan).
Quantification of Serum Hormones and SIgA
Serum hormones including leptin, insulin, PYY, GLP-1, IGF-1 and GIP, as well as total caecal SIgA levels were determined using ELISA kits (Sangon Biotech, Shanghai, China) according to the manufacturer’s recommendations.
Separation of antigens and determination of IgA to the specific antigens were conducted as described with some modification[8, 11]. Briefly, to prepare antigens from cecal contents of mice carrying GM composed of “pure or none Firmicutes”, cecal contents of mice from each group were normalized by bacterial loads (determined by qPCR as previously described), pooled (n = 3) in PBS (sterilized with a 0.22 μm filter), vortexed for 5 min, and centrifuged for 5 min at 13000 RPM, 4°C. Samples were then sonicated for 15 min and centrifuged for 15 min at 13000 RPM, 4°C. Supernatant was taken and protein was measured using BCA assay (Thermo Fisher). For the determination of IgA to specific antigens, 96-well microtiter plates (Costar, Corning, New York) were coated with 1 mg/ml of antigen in 9.6 pH bicarbonate buffer overnight at 4 °C, then IgA from the cecal contents of 30 day or 25 week mice were loaded at 1:20 dilution for 1 h at 37°C. Anti-IgA Biotin and HRP Conjugated Streptavidin were used to detect binding of IgA.
Immunofluorescence for immunoglobulin A was conducted using FITC-conjugated secondary antibody at a 1:500 dilution and applied to the section for 2 h. Observations and analyses were performed with a Zeiss LSM 700 confocal microscope.
Tissues fixed in 4% paraformaldehyde were cut into 5 μm sections after being embedded in paraffin. Multiple sections were prepared and stained with hematoxylin and eosin (H&E) for general morphological observation.
The microbial community of fecal, mucosal and lumen samples of colon and ileum were analyzed through the sequence of 16S rRNA gene V4 region. Briefly, Total genome DNA from samples was extracted using CTAB/SDS method. DNA concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/μL using sterile water. 16S rRNA gene V4 region were amplified used specific primer for V4 region (515F-806R) with the barcode. All PCR reactions were carried out in 30 μL reactions with 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs); 0.2 μM of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s. Finally 72°C for 5 min. Mix same volume of 1×loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. PCR products were mixed in equidensity ratios. Then, mixture PCR products were purified with GeneJETTM Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) following manufacturer's recommendations. The library quality was assessed on the [email protected] Fluorometer (Thermo Scientific). At last, the library was sequenced on an Ion S5TM XL platform and 400 bp/600 bp single-end reads were generated.
Single-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Quality filtering on the raw reads was performed under specific filtering conditions to obtain the high-quality clean reads according to the Cutadapt (V1.9.1, http://cutadapt.readthedocs.io/en/stable/) quality controlled process. The reads were compared with the reference database (Silva database, https://www.arb-silva.de/) using UCHIME algorithm (UCHIME Algorithm, http://www.drive5.com/usearch/manual/uchime_algo.html) to detect chimera sequences, and then the chimera sequences were removed. Then the Clean Reads finally obtained.
Alpha diversity is applied in analyzing complexity of species diversity for a sample through 6 indices, including Observed-species, Chao1, Shannon, Simpson, ACE, Good-coverage. All this indices in our samples were calculated with QIIME (V1.7.0) and displayed with R software (V2.15.3). Beta diversity analysis was used to evaluate differences of samples in species complexity, Beta diversity on both weighted and unweighted unifrac was calculated by QIIME software (V1.7.0). Principal Coordinate Analysis (PCoA) was performed to get principal coordinates and visualize from complex, multidimensional data. A distance matrix of weighted or unweighted unifrac among samples obtained before was transformed to a new set of orthogonal axes, by which the maximum variation factor is demonstrated by first principal coordinate, and the second maximum one by the second principal coordinate, and so on. PCoA analysis was displayed by WGCNA package, stat packages and ggplot2 package in R software (V2.15.3).
Total RNA of the liver and ileum were extracted using TRIzolTM reagent (Invitrogen) according to the manufacturer’s instructions. RNA degradation and contamination was monitored on 1% agarose gels; RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA); RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA); RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Then a total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 (V2.0.5) and paired-end clean reads were aligned to the reference genome using Hisat2. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools. featureCounts (V1.5.0-p3) was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (V1.16.1). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed. Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (V3.18.1). The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.05 and |log2foldchange| > 1.2 was set as the threshold for significantly differential expression.
All data reported in this paper are expressed as the means ± SEMs. The data were evaluated by one-way ANOVA, Wilcox, or Mann-Whitney U tests. All statistics were analyzed by SPSS software, and all analyses were performed with GraphPad Prism 7.