L. reuteri strain culture
L. reuteri (BNCC192190) was purchased from the BeNa Culture Collection (BNCC), and cultured in MRS broth (Huankai) at 37 °C under anaerobic conditions. For oral gavage, L. reuteri was harvested at the logarithmic phase by centrifugation, washed, and resuspended in PBS to reach a density of 1*109 colony forming units (CFUs)/mL.
L. reuteri-CMVs isolation, purification, and physicochemical characterization
In an anaerobic chamber, a single colony of L. reuteri after grown on MRS Agar Plate for 24 h was collected and inoculated into MRS broth (Huankai) at 37 ◦C for 12 h. The culture was then refreshed by a 1:100 dilution in fresh MRS broth and incubated at 37 °C for about 16 h until the OD600 reached 1. After centrifugation at 15,000g for 30 min at 4 °C, the supernatant was filtered through a 0.45 μm polyvinylidene fluoride filter (Millipore) and concentrated approximately 20-fold using 50 kDa ultra centrifugal filters (Millipore). The supernatant was again filtered with a 0.22 μm filter (Millipore), and then CMVs were collected by ultracentrifugation at 150,000 g for 3 h at 4 °C (Ultracentrifuge Optima™ XE-100 with a Type 70 Ti rotor, Beckmann Coulter, USA). The pellet was finally resuspended in PBS and filtered aseptically through a 0.22 μm filter to remove intact bacteria or cell debris. The filtrate was collected and stored at -80 °C for further use. The protein concentration of CMVs was quantified using the BCA protein assay kit (EpiZyme). The morphology of CMVs was visualized by transmission electron microscopy (TEM) (HT7700 electron microscope), and the size of CMVs was analyzed using dynamic light scattering (DLS, Zetasizer Nano AS90) according to our previous study [20].
Lipidomic analysis of L. reuteri-CMVs and proteomic profiles of L. reuteri-CMVs and L. reuteri
Lipidomic composition analysis of L. reuteri-CMVs was investigated based on our previous study [20]. The proteins in L. reuteri-CMVs and L. reuteri were extracted and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein lysates were prepared and subjected to nano-LC-MS/MS analysis to identify and quantify their proteins using Orbitrap mass spectrometry (Thermo Fisher Scientific, Bremen, Germany).
Cell culture
HT-29 and Caco2 human intestinal epithelial cell lines were obtained from the American Type Culture Collection (ATCC). HT-29 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco™) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml). Caco-2 cells were cultivated in Minimum Essential Medium (MEM) (Gibco™) supplemented with 20% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). All cells were grown at 37 °C in an incubator with 5% CO2.
Stimulation of IECs in vitro
Upon reaching 70% confluence in complete media, HT-29 or Caco2 cells were stimulated with 2% DSS (MP Biomedicals) and hrTNF (PeproTech, 10 ng/ml) for 24 h. Cells were harvested for further experiments as described below.
L. reuteri-CMVs labeling
To enable flurescent tracking, L. reuteri-CMVs were labeled using two distinct dyes: IRDye 800CW near-infrared fluorescent dye (IRDye® 800CW NHS Ester) and Dil dye (Thermo Fisher Scientific). The IRDye 800CW labeling was performed as described previously [20]. Briefly, a 0.21-mM solution of fluorescent IRDye 800CW was added to 1 mg of L. reuteri-CMVs in 1 ml of PBS. Then, the mixture was incubated with a 0.2-M sodium bicarbonate buffer (pH 8.3) for 2 h at room temperature. To remove unbound dye, the labeled CMVs were purified using a 100 kDa ultra centrifugal filter (Millipore). For Dil labeling, 5 µl of Dil (1 mM) was added to 1 mL of CMVs, mixed thoroughly, and incubated for 2 h at room temperature. The Dil-labeled CMVs were similarly purified using a 100 kDa ultra centrifugal filter (Millipore) to remove any unbound dye. Both IRDye 800CW-labeled and Dil-labeled CMVs were resuspended in PBS and stored for further experiments.
L. reuteri-CMVs stability
The stability of IRDye® 800CW-labeled L. reuteri-CMVs was investigated based on our previous study [20]. IRDye® 800CW-labeled L. reuteri-CMVs were incubated in stomach-like solution or intestine-like solution at 37 °C for 30 min. Subsequently, the IRDye® 800CW-labeled L. reuteri-CMVs were collected through exosome spin columns with a molecular weight cutoff of 4,000 (MW4000).
Depletion of L. reuteri in mice
In order to deplete the gut commensal L. reuteri, C57BL/6 mice were treated with a cocktail containing vancomycin (Sigma-Aldrich, 0.5 g/L) and ampicillin (Sigma-Aldrich, 1 g/L) in drinking water for 7 days. The abundance of L. reuteri was regularly detected in fresh stool samples by qRT-PCR.
Animals and housing
C57BL/6 and IL-10-deficient (IL-10−/−) mice were purchased from Biotechnology Co., Ltd, Beijing, China and Gempharmatech CO., Ltd, Jiangsu, China, respectively. All mice were housed in a germ-free environment with a 12-hour light/dark cycle. At 8 weeks of age, they were used for experiments according to the guidelines and regulations of the Animal Care Committee of Shenzhen People’s Hospital, Shenzhen, China (No. 2024 − 118). All institutional and national guidelines for the care and use of animals were followed.
Efficacy of L. reuteri-CMVs in treating DSS-induced colitis
All mice were given 3% DSS in drinking water continuously for 6–7 days to induce colitis as described previously [20, 21]. The intervention was performed for 6–7 consecutive days, starting on day 1 of the DSS treatment. C57BL/6 mice were divided into four groups: healthy control group, PBS group, L. reuteri group, and CMVs group. IL-10−/− mice were divided into three groups: healthy control group, PBS group, and CMVs group. Mice depleted of L. reuteri were divided into three groups: PBS group, L. reuteri group, and CMVs group. The colitis mice were fed daily with either 1*109 CFUs/ml of L. reuteri (200 µl of bacterial suspension), 200 µl of PBS containing CMVs (2.5 mg/ml), or 200 µl of PBS as a control.
The body weight, fecal characteristics, and physical activity of the mice were assessed daily throughout the experiment. The disease activity index (DAI) was calculated using a previously established scoring system [20, 21]. On days 6 or 7, the mice were euthanized using CO2 inhalation. Blood samples were obtained from the orbits of the mice for further use. The colon length was measured from the cecum to the rectum. Colon samples, feces, and major organs (heart, liver, spleen, lung, and kidney) were harvested for further experiments.
Biodistribution and uptake of L. reuteri-CMVs in the gastrointestinal tract
To assess the biodistribution of L. reuteri-CMVs in the gastrointestinal tract, colitis mice and untreated mice were gavaged with 200 µl of PBS containing IRDye® 800CW-labeled L. reuteri-CMVs (2.5 mg/ml). At various time points after oral administration (4, 12, and 24 h), the mice were euthanized using CO2 inhalation. Intestine tissues and major organs (brain, heart, liver, spleen, lung, and kidney) were then obtained for fluorescence imaging through an IVIS spectrum imaging system (Hopkinton, USA), which allows for visualization and quantification of the fluorescence emitted by the IRDye® 800CW-labeled L. reuteri-CMVs in these tissues.
In order to investigate the uptake of L. reuteri-CMVs in the gastrointestinal tract, colitis mice were administered 200 µl of Dil-labeled L. reuteri-CMVs (2.5 mg/ml) by oral gavage. The stomach, small intestine, and colon were harvested 8 h post-administration. The tissues were fixed in 4% paraformaldehyde for 24 h at 4 °C. Subsequently, the tissues were immersed in PBS containing 30% sucrose for 48 h at 4 °C until they bottomed out. The tissues were then buried in optimal cutting temperature compound (OCT), frozen, and sectioned into 5 μm slices. The frozen slices were cleaned with PBS, blocked with 3% bovine serum albumin (BSA), and subsequently incubated with primary antibodies at 4 °C overnight. Following 3 washes with PBS, the slices were incubated with fluorescent secondary antibodies for 1 h at room temperature. Finally, the nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime). Images were acquired using a fluorescence scanner (Pannoramic MIDI, 3DHISTECH, Hungary).
Cell uptake of L. reuteri-CMVs
A volume of 10 µl Dil-labeled L. reuteri-CMVs (2.5 mg/ml) was incubated with Caco-2 and HT-29 cells for 8 h at 37 °C, respectively. After incubation, the cells were washed three times with PBS and stained with DAPI. The uptake of L. reuteri-CMVs was then visualized by a confocal laser scanning microscope (CLSM) (ZEISS LSM 900, Germany).
Biosafety of L. reuteri-CMVs in vitro and in vivo
In vitro, the MTT assay was used to assess the cytotoxicity of L. reuteri-CMVs on Caco-2 and HT-29 cells. Cells were seeded in 96-well plates at a density of 1*104 cells/well and incubated for 24 h. Cells were then incubated with 100 µl of complete medium with L. reuteri-CMVs for 24 and 48 h. After the medium containing CMVs was removed, the cells were thoroughly rinsed once with PBS and incubated with 100 µl of MTT (5 mg/ml) for 3 h at 37 °C. Finally, the media were discarded and 100 µl of dimethylsulfoxide was added to each well prior to spectrophotometric reading at 490 nm. Untreated cells were defined as negative controls.
In vivo, blood samples were collected for the detection of creatine kinase isoenzymes (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (CREA), and serum urea (UREA) after colitis mice were treated with L. reuteri-CMVs for 7 days. The healthy mice and untreated colitis mice were controls. In addition, heart, liver, spleen, lung, and kidney samples were harvested and performed to evaluate the toxicity of the L. reuteri-CMVs using hematoxylin-eosin (H&E) staining.
H&E and alcian blue (AB) staining
Briefly, the colon tissues were fixed in a 4% polyformaldehyde solution. Then, they were dehydrated in an ethanol gradient, cleared in xylene, and embedded in paraffin. Subsequently, they were cut into 5 μm-thick slices for H&E and AB staining. Histological scores were estimated as previously described [22]. Goblet cell counts were performed by calculating the number of AB-stain positive cells in each crypt (≥ 6 crypts per mouse were examined) [23].
FITC-dextran permeability assay
FITC-dextran (Sigma-Aldrich, 0.6 mg/g) was administered orally to mice for 12 h without eating or drinking. Serum samples were collected 4 h after the administration of the FITC-dextran. The FITC fluorescence intensity of the serum was measured using a multimode reader (Thermo Fisher Scientific) at an excitation wavelength of 490 nm and an emission wavelength of 530 nm.
Gut microbiota 16 S rRNA sequencing
Briefly, the HiPure Stool DNA Kit from Magen (Guangzhou, China) was used to extract total fecal DNA based on the manufacturer’s instructions. The DNA quality and concentration were scrutinized using agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer, respectively. The v3-v4 regions of the 16 S rRNA gene were amplified using primers: 341 F: CCTACGGGNGGCWGCAG and 806R: GGACTACHVGGGTATCTAAT. Sequencing was performed on the Illumina NovaSeq PE250 platform (Illumina, San Diego, USA). Sequencing data processing and bioinformatics analysis were performed using the Illumina HiSeq 2500 platform (Guangzhou Genedenovo Bio-Technology Company Limited, Guangzhou, China).
RNA extraction and qRT-PCR
Total RNA was isolated from cells and colon tissues using the TRIzol Reagent (Thermo Fisher Scientific). The concentration of RNA was detected using the NanoDrop 2000 (Thermo Fisher Scientific). Gene expression was performed using the PrimeScript RT Master Mix (Takara). Primers were designed and obtained in Supplement 1.
Enzymelinked immunosorbent assay (ELISA)
The concentrations of TNF-α, IL-1β, IL-6, IL-12, interferon-γ (IFN-γ), and IL-10 were determined in serum samples using ELISA kits (Elabscience) according to the manufacturer’s instructions.
Immunohistochemistry (IHC)
For IHC analysis, colon sections were put in 0.01 M sodium citrate buffer (pH 6.0) and high-pressure treated for 3 min for antigen repair. The sections were then blocked by 5% BSA for 30 min and underwent subsequent incubation with a myeloperoxidase (MPO) primary antibody (Abcam, ab208070, 1:200) overnight at 4 °C. The sections were then incubated with an appropriate HRP-conjugated secondary antibody for 1 h, and the immunoreactivity was detected using a DAB substrate kit.
Immunofluorescence (IF)
Cells and colon sections were fixed in a 4% paraformaldehyde solution, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. They were then incubated with primary antibodies (anti-ZO-1 (Invitrogen, 33-9100, 1:200), anti-Occludin (Ocln) (Abcam, ab216327, 1: 100), anti-Mucin2 (Muc2) (Servicebio, GB11344, 1:1000), anti-E-cadherin (Cdh1) (Servicebio, GB12083, 1:1000), anti-CD4 (Servicebio, GB15064-100, 1:200), anti-CD8 (Servicebio, GB15068-100, 1:400), and anti-HMOX1 (Proteintech, 66743-1-Ig, 1:200)) at appropriate dilutions overnight at 4 °C. Following the removal of the primary antibodies, samples were then washed with PBS and incubated with Alexa Fluor 488 (Abcam) and/or Alexa Fluor 555 (Abcam) at room temperature for 1 h. Finally, the nuclei were stained with DAPI. The images were acquired by a fluorescence scanner and CLSM.
Western blot analysis
Cells or colon tissue were lysed on ice for 30 min using RIPA lysis buffer (EpiZyme) containing a phosphatase inhibitor (EpiZyme). Protein concentration was measured using the BCA protein assay kit (EpiZyme). Proteins were separated by SDS-PAGE (EpiZyme) and transferred to PVDF membranes (Sigma-Aldrich). Membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with various primary antibodies at appropriate concentrations: ZO-1 (Invitrogen, 33-9100, 1:400), Ocln (Abcam, ab216327, 1:1000), Cdh1 (CST, 3195T, 1:000), and HMOX1 (Proteintech, 66743-1-Ig, 1:2000) with β-actin (Proteintech, 66009-1-Ig, 1:50000) serving as a control. Membranes were then treated with secondary antibodies (mouse secondary antibody (Affinity, S0002), rabbit secondary antibody (Affinity, S0001)) for 1 h at room temperature. Finally, the results were imaged by a Bio-Rad Imaging System.
Growth curve experiment of bacteria and bacterial uptake of L. reuteri-CMVs
Akkermansia muciniphila (AKK) (BNCC341917) was purchased from the BNCC and cultured in brain heart infusion (BHI) broth supplemented with Threonineat (6 g/L) and N-acetylglucosamine (GlcNAc) (4.4 g/L) under anaerobic conditions at 37 °C. Subsequently, AKK cultures were supplemented with 10 µl of PBS containing L. reuteri-CMVs (2.5 mg/ml), and untreated AKK was defined as a control. Optical density at 600 nm (OD600) was measured every 4 h at 37 °C using a NanoDrop 2000 to monitor bacterial growth and calculate the total number of bacterial cells.
To assess bacterial uptake, 1 ml of logarithmic phase AKK solution was co-cultured with 10 µl of PBS containing Dil-labeled L. reuteri-CMVs (2.5 mg/ml). The mixture was centrifuged at 7,000 g for 5 min after 24 h. The collected samples were washed three times with PBS to remove unincorporated Dil-labeled L. reuteri-CMVs. The uptake of L. reuteri-CMVs by AKK was visualized using CLSM.
Bacterial RNA-sequencing analysis
Under anaerobic conditions at 37 °C, AKK was co-incubated with PBS or CMVs for 72 h. After incubation, bacterial pellets were harvested, and total RNA was extracted using a bacterial RNA extraction kit (TIANGEN). RNA sequencing libraries was prepared using the Fast RNA-seq Lib Prep Kit V2 (ABclonal, RK20306). Transcriptome sequencing was performed on the Illumina platform at Novogene Co.,Ltd. (Beijing, China). The clean reads were mapped to the reference AKK genome (https://www.ncbi.nlm.nih.gov) using HISAT2 (v2.0.5). HTSeq (v0.9.1) was used to count the reads numbers mapped to each gene. Differential expression analysis of two groups was performed using the DESeq2 R package (v1.20.0). Genes with P adj < 0.05 and |log2(foldchange)| > 0 were identified as differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for enrichment analysis of DEGs.
Cellular RNA-sequencing analysis
Total RNA was extracted from cells using TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Then, the amount and quantity of mRNA were evaluated using a Nanodrop 2000. RNA-seq libraries were prepared, sequenced, and analyzed on the Illumina sequencing platform by Guangzhou Genedenovo Bio-Technology Company Limited (Guangzhou, China). Reads obtained from the sequencing machines were further filtered by fastp (v0.18.0). Short reads alignment tool Bowtie2 (v2.2.8) was used for mapping reads to ribosome RNA (rRNA) database. An index of the reference genome was built, and paired-end clean reads were mapped to the reference genome using HISAT2 (v2.1.0). Principal component analysis (PCA) was performed with R package gmodels (http://www.r-project.org/) in this experience. Correlation analysis was performed by R. RNAs differential expression analysis was performed by DESeq2 software between two different groups. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change > 1.5 were considered DEGs. Consequence, gene expression heat map, volcano plot, GO analysis, and KEGG pathway analysis of these DEGs were performed. Gene set enrichment analysis (GSEA) was performed to identify the significant enrichment of gene sets in relevant pathways.
Availability and analysis of transcriptomic datasets
Single-cell datasets were available at http://scibd.cn24. The relative expression of HMOX1 was evaluated in epithelial cell subsets.
Statistical analysis
A comparison of multiple experimental groups was carried out by one-way or two-way analysis of variance (ANOVA). A t-test was calculated to compare the means of the two groups. Data are presented as means ± SEM. The p < 0.05*, p < 0.01**, p < 0.001***, and p < 0.0001**** represent statistically significance, and ns represents non-significance.