Animals
Wild type male C57BL/6J mice, aged 6–8 weeks, were purchased from Academy of Military Medical Science (Beijing, China). CD11c-GFP-DTR mice were a gift from Professor Xuetao Cao of Nankai University. Foxp3-GFP-DTR mice were a gift from Professor Xiaoming Feng of Institute of Hematology and Hospital of Blood Disease, Chinese Academy of Medical Sciences. All mice were bred and maintained under specific pathogen free condition and a standard chow diet ad libitum in the animal facility at Tianjin Medical University. Animal procedures and protocols were performed with 6 to 8 weeks male mice and were approved by Animal Care and Use Committee, Tianjin Medical University.
Antibiotic treatment
Mice were provided with the following antibiotics (SangonBiotech, Shanghai, China) in drinking water individually or in combination: ampicillin (A610028-0025, 1 g/L), chloramphenicol (A100230-0010, 500 mg/L), ciprofloxacin (A600310-0025, 500 mg/L), clindamycin (A600312-0025, 500 mg/L), metronidazole (A600633-0025, 1 g/L), neomycin (A610366-0025, 1 g/L), polymycin (A610318-0001, 1 g/L), tetracycline (A600504-0025, 500 mg/L), vancomycin (A600983-0001, 500 mg/L). Antibiotics were pretreated for 7 days and then given with DSS until the end of the experiment. In addition, we used a combination of ampicillin, vancomycin, neomycin and metronidazole (ABX) to clear most of intestinal bacteria [45]. Antibiotic-containing water was renewed every 3 to 4 days to maintain efficacy.
DSS-induced colitis mouse model
To study antibiotic effect on mice susceptibility to DSS-induced colitis, male C57BL/6J mice were pretreated with antibiotics for seven days, and exposed with the same antibiotics in addition with 3% DSS (0216011090, M.W. = 36,000–50,000 Da; MP Biomedicals, Solon, OH, USA) in drinking water for another seven days [46]. Feces were collected on the zero and fifth day, and tissues were collected on the seventh day when mice were euthanized.
To test the effect of individual bacterial strain on DSS-induced colitis, the strain was inoculated into 10 ml of sterile BHI broth (E. gallinarum, K. huaxiensis, E. ludwigii, E. lactis and K. michiganensis) or MRS broth (L. johnsonii) containing 2.5% L-cysteine, grown in anaerobic penicillin bottles at 37 °C for 24 h. Cultured bacteria were pelleted by centrifugation (5000 × g for 5 min at 4 °C) and resuspended in PBS to obtain a density of 5 × 109 CFU/ml. Each male C57BL/6J mouse was gavaged with 200 μl liquid bacterial strains (109 CFU) once per day for fourteen days, and 3% DSS was added to the drinking water of the mice from the seventh day to the fourteenth day during the bacteria administration.
To check the effect of bacterial strain mixture on DSS-induced acute colitis, individual strain was cultured separately in anaerobic penicillin bottles containing BHI broth at 37 °C for 24h and mixed together in the same proportion before gavage. Each male C57BL/6J mice were pretreated with 109 CFU strain mixture for seven days, and exposed with the same strain mixture plus 3% DSS in drinking water for another seven days.
To evaluate the therapeutic effect of E. ludwigii on experimental colitis without pretreatment, male C57BL/6J mice were treated with 3% DSS in drinking water for 7 days and gavaged with single bacterial strain once a day during the same period.
To study the major effect of individual bacterial strain on DSS-induced acute colitis, male C57BL/6J mice were pretreated with ABX for 5 days and water for 1 day, then mice were gavaged with 109 CFU bacterial strains once per day for twelve days. 3% DSS was added to the drinking water of mice, and mice were gavaged with LPS (8 mg/kg body weight/day, L2880, Sigma-Aldrich, St. Louis, MO, USA) simultaneously from the fifth day to the twelfth day during the bacteria administration.
Body weight loss was calculated relative to starting weight before giving DSS of the same mouse. DAI is the sum of stool consistence score, stool bleeding score and body weight loss score. Stool consistency was scored as follows: 0, normal; 1, mild soft stools; 2, very soft stools; 3, watery stools. Stool bleeding was scored as follows: 0, normal; 1, brown color; 2, reddish color; 3, bloody stool. Body weight loss was scored as follows: 0 (<2%), 1 (2-5%), 2 (5-10%), 3 (10-15%), 4 (≥15%) [47]. Body weight loss and DAI were scored every day during the DSS treatment. A distal part of colon was fixed in 4% paraformaldehyde and embedded into paraffin. Slides (5µm) were stained with hematoxylin and eosin, then images acquired with a microscope (BX46, Olympus, Tokyo, Japan) were used to assess histopathological scores. Seven parameters were used to score histopathology: (A) extent of inflammation (score of 0 to 4), (B) extent of crypt damage (score of 0 to 4), (C) infiltration of neutrophils and lymphocytes (score of 0 to 3), (D) submucosal edema (score of 0 to 3), (E) loss of goblet cells (score of 0 to 3), (F) reactive epithelial hyperplasia (score of 0 to 3), (G) crypt abscesses (score of 0 to 2) [46, 47].
DNA extraction and 16S rRNA gene sequencing
Microbial genomic DNA was extracted from each sample using the Stool DNA Kit (D4015-02, Omega Bio-tek, Norcross, GA, USA) according to manufacturer’s protocols. Total DNA was eluted in 50 μl of Elution buffer. The amount of DNA was determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The DNA was evaluated using 1.5% (wt/vol) agarose gel electrophoresis. All DNA samples were stored at -80 °C until sequencing.
16S rRNA gene sequencing was performed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) were used to amplify the V3-V4 hypervariable region of the 16S rRNA gene by thermocycler PCR system (GeneAmp 9700, ABI, USA).The process of PCR program was as follows: 95 °C for 3 min, followed by 27 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s, and a final extension at 72 °C for 10 min. The 20μl PCR reaction system contained 4 μL 5 × FastPfu Buffer, 2 μL dNTPs (2.5 mM), 0.8 μL V3 primer (5 μM), 0.8 μL V4 primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA and PCR reactions were performed in triplicate. The PCR products were separately extracted from a 2% agarose gel and further purified through the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Then using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s protocol quantified the purified products. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA).
Processing of sequencing data
Illumina sequencing generated raw fastq files, and the sequence reads were then merged, trimmed, filtered, aligned, and clustered by operational taxonomic unit (OTU) using UPARSE (version 7.1 http://drive5.com/uparse/). The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database using confidence threshold of 70%. OTU sequences were clustered, and those with > 97% similarity were binned into the same OTU. Alpha diversity metrics at the OTU level based on Shannon’s index [48] were mainly calculated by MOTHUR program (https://www.mothur.org/wiki/Main_Page). Beta diversity at the OTU level based on Bray-Curtis [49], were qualitatively examined by MOTHUR program and visualized in Principal Coordinate Analysis (PCoA) using the vegan R packages [50]. Bacterial taxonomic distributions of each group communities at the genus level were visualized using the vegan R packages. A heatmap with a base-10 logarithmic transformation of the absolute abundance at the genus level was generated through R package vegan. Analyses of alpha diversity, beta diversity, bacterial taxonomic distributions, and generation of the heatmap were performed at Majorbio I-Sanger Cloud platform (https://cloud.majorbio.com). 16S rRNA gene sequencing data have been submitted to NCBI Sequence Read Archive (SRA) with the accession number of PRJNA657382.
Isolation of bacterial strains
Fresh feces pellets collected from mice with metronidazole treatment were homogenized in 1 ml sterile phosphate buffered saline containing 2.5% L-cysteine and serially diluted with PBS, seeded onto multiple agar plates (hopebiol, Qingdao, China), including Man Rogosa Sharpe agar (MRS) and Brain Heart Infusion agar (BHI). After incubated under aerobic conditions or anaerobic conditions at 37 °C for 48 h to 72 h, individual colonies with distinct morphologies were picked up and the selected colonies were stream plated in the same media for another 2 or 3 days for further purification and identification.
Bacterial genomic DNA was extracted using Bacterial Genomic DNA Extraction Kit (DP302-02, TIANGEN, Beijing, China). To identify the isolated strains, 16S rRNA gene sequence was amplified by PCR using 16S rRNA gene-specific primer pairs: 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’), and sequenced in Sangon Biotech (Shanghai, China). The resulting sequences were compared to sequences in EzBioCloud 16S rRNA databases (www.ezbiocloud.net).
Preparation of lymphocytes
Collected colons were opened longitudinally after excising fat tissues and washed with PBS to remove luminal feces, followed by shaking in PBS containing 2 mM dithiothreitol (DTT, D1070, Solarbio, Beijing, China) for 10 min once and PBS containing 5 mM EDTA for 10 min thrice at 37 °C to remove epithelial cells. Lamina propria tissues were sliced into small pieces and digested with PBS containing 2% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, 1 mg/ml collagenase IV (C5138,Sigma-Aldrich) and 100 ng/ml DNaseI for 30 min at 37 °C in a shaking water bath. The digested cell tissues were then filtered through a 70 μm nylon cell strainer (352350, BD Biosciences, San Jose, CA, USA) to obtain single cell suspensions and resuspended in 40% percoll to perform percoll gradient separation for obtaining lamina propria lymphocytes. Spleens and mesenteric lymph nodes were ground directly into a single cell suspension mechanically, in which spleen cells were subjected to lyse erythrocytes on ice for 15 min.
Flow cytometry
For cell surface staining, single cell suspensions were incubated on
ice at 37 ℃ for 30 min with following antibodies: anti-CD45 conjugated to PE-Cy7 (25-0451-82, eBioscience, San Diego, CA, USA), anti-CD11c conjugated to APC (117309, Biolegend, San Diego, CA, USA), anti-MHCII conjugated to PE (107607, Biolegend), anti-CD103 conjugated to PerCP/Cy5.5 (46-1031-82, eBioscience), anti-CD4 conjugated to APC-Cy7 (100414, Biolegend), anti-CD3 conjugated to FITC (100204, Biolegend), anti-CD4 conjugated to PE-Cy7 (25-0041-81, eBioscience), anti-CD3 conjugated to APC-Cy7 (05112-87-100, eBioscience), anti-CD25 conjugated to PE (102008, Biolegend), anti-CD44 conjugated to PE-Cy7 (103029, Biolegend), anti-CD62L conjugated to APC (104411, Biolegend).
For Treg cell analysis, after staining of cell surface with CD3, CD4 and CD25 antibodies, lymphocyte suspensions were fixed, permeabilized using Foxp3/transcription factor staining buffer sets (00-5523-00, eBioscience) according to the manufacturer’s instructions and stained with anti-Foxp3-APC (320014, Biolegend). For analysis of Th1 and Th17 cells, isolated tissue lymphocytes were stimulated for 5 h with cell activation cocktail with Brefeldin A (423303, Biolegend). After incubation for 5 h, cells were washed in PBS and stained with dead cells using Zombie NIRTMFixable Viability Kit (423105, Biolegend), anti-CD3 conjugated to PE (100205, Biolegend) and anti-CD4 conjugated to FITC (100405, Biolegend). Stained cells were fixed in fixation buffer (420801, Biolegend), permeabilized with intracellular staining permeabilization wash buffer (421002, Biolegend), and stained with anti-IL-17A conjugated to PE-Cy7 (25-7177-80, eBioscience) and anti-IFN-γ conjugated to PerCP/Cy5.5 (505821, Biolegend).
Stained cells were then analyzed on a FACS Canto II Flow Cytometer (BD Biosciences) using the FlowJo software (FlowJo, Ashland, OR, USA) or performed flow sorting to obtain purified cells on FACSAriaTMⅡ Cell Sorter (BD Biosciences).
Identification of knockout mice
Genomic DNA was extracted from mouse tail, and the mouse genotype was identified by PCR with specific primers (Table S1, additional file 11). To investigate the role of DC and Treg deletion in E. ludwigii-mediated protection, CD11c-GFP-DTR transgenic mice were injected intraperitoneally (i.p.) with 200 µl PBS containing 100 ng of Diphtheria toxin (4 ng DT/g body weight, D0564-1MG, DTx, Sigma-Aldrich) to rapidly deplete CD11c+DC, and transgenic Foxp3-DTR-GFP mice were given intraperitoneal injections of 1 µg DTx (50 ng DT/g body weight) to deplete Treg. DTx was injected on one day prior to bacteria gavage, and subsequent doses were given every other day to maintain DC and Treg depletion throughout the whole phase of bacteria gavage. When the mice were sacrificed, the spleens, MLNs, and colons were removed and analyzed using flow cytometry to verify the elimination efficiency of DC and Treg cells (FACS Canto II Flow Cytometer, BD Biosciences; the FlowJo software, FlowJo).
Tissue immunofluorescence analysis
Small segment colons were embedded in OCT compound with liquid nitrogen and cut into 5-µm sections followed by fixing with cold acetone for 10 min. After blocked with 5% BSA for 1 h, sections were incubated with CD103 antibody (1:200, ab224202, Abcam, Cambridge, MA, USA) in blocking buffer overnight at 4 ℃. After that, slides were incubated with Alexa Fluor 594 - labeled second antibody (1:200, SA00006-8, Proteintech, Chicago, IL, USA) for 1 h and counterstained with DAPI to stain nuclei. Images were acquired using a confocal fluorescence microscope (Leica TCS-SP8, Leica Microsystems, Germany).
RNA extraction and qRT-PCR
Sorted DCs (CD45+MHCII+CD11c+) from MLNs of C57BL/6J mice were seeded into a 96 well plate, then incubated with indicated live bacteria (MOI=10, 2h), heat-killed bacteria (MOI = 10, 24h) or bacterial culture supernatant (bacterial culture supernatant: cell culture medium = 1:1, 24h). RNA of stimulated DCs, flow sorted CD103+DCs (CD45+MHCII+CD11c+CD103+ from C57BL/6J mice), or flow sorted Foxp3+Tregs (CD3+CD4+CD25+Foxp3+ from Foxp3-GFP-DTR mice) was extracted using Micro RNA extraction kit (74104,QIAGAN, Duesseldorf, Germany) and converted to cDNA using HiFiScript cDNA Synthesis Kit (CW2569M, CWBio, Beijing, China) according to the manufacturer’s protocol. The PCR reactions were performed with Ultra SYBR Mixture (CW0957M, CWBio) on a 7900 Fast Real-Time PCR System (Roche, Basel, Switzerland). Transcript levels of the indicated genes were normalized to endogenous control β-actin for each individual sample using the primers (Table S1, additional file 11) and quantified using the comparative critical threshold cycle 2–ΔΔCt method.
T cell and DC co-cultures
Sorted DCs were incubated with heat-killed Escherichia coli K12 (MOI = 10) and indicated bacterial culture supernatant for 24 h followed by washing. 1×105 flow purified Naive T cells (CD4+CD62L+CD25-CD44-, from spleens of Foxp3-GFP-DTR mice) were cultured with 1×104 stimulated DCs or 2×104 sorted CD103+ DCs (CD45+MHCII+CD11c+CD103+) from MLNs of C57BL/6J mice, in RPMI 1640 containing 1 μg/ml of immobilized anti-CD3 (100339, Biolegend), 100 U/ml penicillin, 100 U/ml streptomycin, 5 ng/ml recombinant mouse IL-2 (575402, Biolegend) and 5 ng/ml recombinant mouse TGF-β1 (7666-MB-005/CF, R & D Systems, Minneapolis, MN, USA) in 96-well plates. After 72 hours at 37 °C in 5% CO2, cells were stained with anti-CD4 conjugated to PE-Cy7 and anti-CD25 conjugated to PE, and Foxp3 expression were detected by GFP through flow cytometry on a FACS Canto II Flow Cytometer (BD Biosciences) using the FlowJo software (FlowJo).
Stimulated DC and Naive T cell were co-cultured on a Lab-Tek chambered cover glass for 72 h, then fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 10 min and incubated with DAPI for 10 min. Cells were imaged using a confocal fluorescence microscope (Leica TCS-SP8, Leica Microsystems).
Statistical analysis
Data were presented as the mean ± SEM. The statistical significance of the differences between three or more groups was tested using one-way ANOVA or two-way ANOVA. All operations are performed in commercially available software (Graphpad Prism 6, San Diego, CA) and values of P < 0.05 were considered statistically significant.