C. butyricum strain MIYAIRI 588 (CBM588) was obtained from Miyarisan Pharmaceutical Co. Ltd, Tokyo, Japan and was cultured in reinforced clostridial medium (Solaribio, China) or in tryptone–peptone–glucose–yeast extract (TPGY) broth (pH = 7.0) under anaerobic conditions (10% H2, 80% N2, and 10% CO2) at 37 °C.
Isolation, purification, characterization of EVs
After pelleting of bacterial cultures (10,000×g for 20 min), the obtained supernatants were filtred (0.22 μm) to remove parental bacterial debris and other impurities. To get the crude EVs, the supernatant was further concentrated to 1/8 of its initial volume using 100 kDa ultrafiltration membranes (Millipore, Germany) and ultracentrifuged at 4 °C and 60,000×g (Hitachi C21G, HITACHI, Maru, Chiyoda, Tokyo, Japan) for 30 min, and washed with PBS twice. The purified EVs were obtained with filtering (0.22 μm) again before ultracentrifugation in a 45 Ti rotor at 150,000×g at 4 °C for 2 h using a sucrose density gradient, followed by using Detoxi-gel endotoxin removing columns (Thermo Scientific, USA) to remove endotoxin . The final pellets were resuspended in PBS and stored at − 80 °C.
To confirm the morphological characteristics of the isolated EVs, transmission electron microscope (TEM) was conducted. Briefly, 15 μl of EV solution was applied on a copper grid for 1 min, and the excess solution was blotted with filter paper. For negative staining of EVs, 2% uranyl acetate (15 μl) was applied to the grid for 1 min, and then blotted with filter paper. Grids were thoroughly dry before imaging under an FEI Tecnai transmission electron microscope (FEI, USA).
To detect the diameter and particle number of the EVs, nanoparticle tracking analysis (NTA) was performed using a ZetaView PMX 110 instrument (Particle Metrix, Germany). For each measurement, the sample was diluted in 1xPBS, and 11 different cell positions were recorded for three cycles under the following settings: shutter: 70; scattering intensity: 4.0; temperature: 25 °C. Polystyrene particles (110 nm) were used to calibrate the instrument before the measurement. Collected data for each sample was analyzed with the corresponding software, ZetaView 8.04.02.
Wild-type male C57BL/6 mice (20–24 g; 40–60 days old) purchased from the Laboratory Animal Centre of Southern Medical University (Guangzhou, China) were kept at 21 ± 2 °C, with a relative humidity of 45 ± 10% on a 12 h dark/light cycle with free access to water/food under specific pathogen-free (SPF) barrier conditions. Euthanasia was performed by CO2 gas in a closed chamber, followed by cervical dislocation. All animal experiments were performed at the SPF Animal Room, Southern Medical University. The protocols of experiments were confirmed and approved by the Institutional Animal Care and Use Committee of Southern Medical University, China.
Treatment of animals
Three treatment groups (Control, DSS, and DSS+ EVs) were randomly assigned to six mice each. UC was induced by administering 2% (w/v) DSS (36–50 kDa, Millipore Corporation, Billerica, MA, USA) in the drinkable water for 5 days, succeeded by ordinary water for 5 days. Mice in the DSS+ EVs group received intragastrically 0.2 ml of PBS containing 15 μg of EVs and in the control and DSS groups received 0.2 ml PBS once every day. The treatment dosage of 15 µg EVs was chosen based on previously available studies and a dose–effect relationship experiment on DSS induced colitis model [20,21,22].
For gut microbiome depletion
Mice were administered a blend of the following antibiotics: metronidazole (1.0 g/l); ciprofloxacin (0.2 g/l), dissolved in drinking water for 14 days. Gut microbiota depletion was confirmed by collecting faecal samples and assessing the total DNA. Three groups (Control, antibiotics+ DSS, and antibiotics+ DSS+ EVs) were randomly assigned to six mice each. The control group did not receive any treatment; the groups with antibiotics+ DSS received an antibiotics blend, followed by DSS for UC induction. The antibiotics+ DSS+ EVs group received 0.2 ml of PBS containing 15 μg of EVs intragastrically once every day, whereas mice in the groups with antibiotics+ DSS received 0.2 ml of normal PBS orally.
For faecal transplantation assays
Four groups (DSS+ EVs+ , DSS+ EVs−, DSS−EVs+ , and DSS−EVs−) were randomly assigned six mice each. The groups with DSS+ were given DSS for UC induction; the DSS−EVs− group received regular feed; the groups with EVs+ were given oral EVs once every day, and the groups with EVs− were given 0.2 ml PBS treatment. Ten days later, stool samples were collected every day for the next 10 days under sterile conditions. The stool samples from each group were combined to obtain a 100 mg sample, which was resuspended in 1.0 ml of sterile saline, stirred potently for 10 s, followed by centrifugation for 3 min at 800×g. The supernatant was used for transplantation. Changes in bacterial composition were prevented by preparing each transplantation sample 10 min before intragastrical gavage. Each recipient group was randomly assigned six mice who were given DSS for UC induction, followed by intragastrical administration of 100 µl of fresh transplant material every day for 10 days.
Disease activity index (DAI)
The monitoring of disease activity index (DAI) was carried out daily. It comprised of stool consistency, body weight, and stool bleeding. Scoring was done as follows: stool consistency (3–4 = diarrhea; 1–2 = loose stool; and 0 = normal); Weight loss (0 = none; 1 = 1–5%; 2 = 5–10%; 3 = 10–15%; and 4 = > 15%); Stool bleeding (0 = negative; 1 = + ; 2 = ++ ; 3 = +++ ; and 4 = ++++) .
After removal, the spleen, liver, small intestine, kidney, and colon of the mice were fixed using paraformaldehyde, inserted in paraffin, and cut into segments (5 µM). The extent of inflammation in the H&E stained sections was determined based on a histological colitis score: 0 to 14 (maximum score).
Ex vivo and in vivo imaging
EVs were incubated with 10 µM DiI (Beyotime Biotechnology, Shanghai, China), a lipophilic fluorescent dye, for 30 min at room temperature. DiI-labeled EVs were isolated using ultracentrifugation and then injected orally into C57BL/6 mice. After 8 h, the mice were killed, and gastrointestinal tissues were acquired. DiI fluorescence and EV distribution were detected by the IVIS spectrum.
Intestinal permeability measurement was performed upon oral gavage of FITC-dextran solution (4 kDa, 600 mg/kg; Sigma-Aldrich). After 4 h absorption and excretion, mice were anaesthetized and exposed to the IVIS Spectrum CT system, and the fluorescent retentions of FITC-dextran were measured.
Isolation of lamina propria mononuclear cells (LPMCs)
The large intestines were opened longitudinally, rinsed with PBS (Gibco, USA) to eliminate fecal matter, cut into 1 cm long strips, followed by stirring in Hanks’ Balanced Salt Solution (HBSS, Gibco, USA) containing 1 mM DTT (Fudebio, Hangzhou, China) and 2 mM EDTA at 37 °C for 30 min. The isolation of the cells was performed from the intestinal lamina propria (LP) following a previously described method. In brief, the intestinal tissue was digested using 1 mg/ml of collagenase (type IV), and 5% FBS (Gibco, Grand Island, NY, USA) in RPMI 1640 medium (Gibco, USA) at 37 °C for 30 min with stirring. Every 30 min, we centrifuged the released cells and stored them in the culture medium. Fresh collagenase was added to replace the mucosal pieces at least 2 times. Finally, the purification of the LP cells was carried out utilizing a discontinuous Percoll (Yeasen, Shanghai, China) gradient collecting at the 40–70% interface.
The cellular suspensions of LP were kept at 4 °C for 30 min with anti-CD16/32, anti-F4/80, and anti-CD206 fluorochrome-conjugated antibodies (BD Pharmingen, USA) to evaluate the M1/M2 macrophage equilibrium. Data were collected on a FACSCalibur flow cytometry instrument and analyzed using FlowJo software 7.6 (FlowJo, Ashland, OR, United States).
Isolation of fecal DNA and 16S rRNA analysis
Total DNA from fresh stool samples was extracted using the E.Z.N.A.® Stool DNA Kit (Omega Bio-tek, GA, USA) and quantified following a previously described technique . Briefly, the V3–V4 regions of the 16S rRNA genes were amplified utilizing the extracted and diluted DNA (1 ng/µl) from each sample as a template. The following primers were used: (806R: 5′-GGACTACHVGGGTWTCTAAT-3′; 338F: 5′-ACT CCTACGGGAGGCAGCAG-3′) in a TransStart® Fastpfu DNA polymerase (TransGen, Beijing, China). A 2% agarose gel electrophoresis was used to detect the products of PCR and purified utilizing the Kit of AxyPrep DNA gel extraction (Axygen Biosciences, CA, USA). Next, sequencing libraries were created utilizing the TruSeq DNA PCR-Free Library Preparation Kit (Illumina, USA) on the Illumina Miseq platform according to the manufacturer’s recommendations. The raw sequencing data were quality-filtered using fastp version 0.20.0  and merged using FLASH version 1.2.7 . Raw data were further analyzed by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).
The expression of the protein related to MUC2, ZO-1, IL-10, TNF-α, arginase 1(Arg1), inducible nitric oxide synthase (NOS2) (1:1000; Affinity, USA) was evaluated using western blot following a previously described method . Quantification of band intensities expressed the modifications in the relative levels of the specific proteins. ImageJ computer program (National Institutes of Health, Bethesda, MD, USA) was utilized for densitometric studies.
After deparaffinization and rehydration of the paraffin-embedded sections, antigen retrieval was performed following the standard protocol. Next, treatment with 3% H2O2 was followed by blocking with goat serum for 1 h at 37 °C, succeeded by incubation at 4 °C during the night, with the specific primary antibody. After washing the sections with cold PBS three times, they were incubated with a second antibody (ZSGB-BIO), developed with the DAB reagent and counterstained with haematoxylin. Negative controls were incubated without antibodies.
After deparaffinization using xylene and dehydration using gradient ethanol, the slides were permeabilized. Next, the slides were kept in nightly incubation with the specific primary antibody in 5% normal goat serum at 4 °C. After washing the sections with cold PBS, they were incubated with the secondary antibody of Alexa Fluor 488/594-labelled (Yeasen, Shanghai, China) for 1 h at 25 °C. DAPI (Boster, Wuhan, China). was used to stain the cell nuclei. The sections were observed under the DM4B fluorescence microscope system (Leica, Wetzlar, Germany), and the mean fluorescence intensity of images was analyzed at identical microscopic settings.
A one-way analysis of variance (ANOVA) was utilized for data analysis, succeeded by comparison multiple tests of Newman–Keuls. Data was studied utilizing SPSS version 21.0 (Chicago, IL, USA) and expressed as mean ± standard deviation (mean ± SD). GraphPad Prism 7 (San Diego, CA, USA) was used for curve-fitting. P-values were considered statistically significant < 0.05.
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