L. reuteri pressure tradition
L. reuteri (BNCC192190) was bought from the BeNa Tradition Assortment (BNCC), and cultured in MRS broth (Huankai) at 37 °C below anaerobic situations. For oral gavage, L. reuteri was harvested on the logarithmic section by centrifugation, washed, and resuspended in PBS to succeed in a density of 1*109 colony forming models (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 twenty-four h was collected and inoculated into MRS broth (Huankai) at 37 ◦C for 12 h. The tradition was then refreshed by a 1:100 dilution in contemporary MRS broth and incubated at 37 °C for about 16 h till the OD600 reached 1. After centrifugation at 15,000g for 30 min at 4 °C, the supernatant was filtered via a 0.45 μm polyvinylidene fluoride filter (Millipore) and concentrated roughly 20-fold utilizing 50 kDa extremely centrifugal filters (Millipore). The supernatant was once more filtered with a 0.22 μm filter (Millipore), after which CMVs have been collected by ultracentrifugation at 150,000 g for 3 h at 4 °C (Ultracentrifuge Optima™ XE-100 with a Sort 70 Ti rotor, Beckmann Coulter, USA). The pellet was lastly resuspended in PBS and filtered aseptically via a 0.22 μm filter to take away intact micro organism or cell particles. The filtrate was collected and saved at -80 °C for additional use. The protein focus of CMVs was quantified utilizing the BCA protein assay package (EpiZyme). The morphology of CMVs was visualized by transmission electron microscopy (TEM) (HT7700 electron microscope), and the scale of CMVs was analyzed utilizing dynamic gentle scattering (DLS, Zetasizer Nano AS90) in keeping with our earlier research [20].
Lipidomic evaluation of L. reuteri-CMVs and proteomic profiles of L. reuteri-CMVs and L. reuteri
Lipidomic composition evaluation of L. reuteri-CMVs was investigated based mostly on our earlier research [20]. The proteins in L. reuteri-CMVs and L. reuteri have been extracted and separated utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein lysates have been ready and subjected to nano-LC-MS/MS evaluation to determine and quantify their proteins utilizing Orbitrap mass spectrometry (Thermo Fisher Scientific, Bremen, Germany).
Cell tradition
HT-29 and Caco2 human intestinal epithelial cell strains have been obtained from the American Sort Tradition Assortment (ATCC). HT-29 cells have been 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 have been cultivated in Minimal Important Medium (MEM) (Gibco™) supplemented with 20% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). All cells have been grown at 37 °C in an incubator with 5% CO2.
Stimulation of IECs in vitro
Upon reaching 70% confluence in full media, HT-29 or Caco2 cells have been stimulated with 2% DSS (MP Biomedicals) and hrTNF (PeproTech, 10 ng/ml) for twenty-four h. Cells have been harvested for additional experiments as described under.
L. reuteri-CMVs labeling
To allow flurescent monitoring, L. reuteri-CMVs have been labeled utilizing two distinct dyes: IRDye 800CW near-infrared fluorescent dye (IRDye® 800CW NHS Ester) and Dil dye (Thermo Fisher Scientific). The IRDye 800CW labeling was carried out as described beforehand [20]. Briefly, a 0.21-mM answer of fluorescent IRDye 800CW was added to 1 mg of L. reuteri-CMVs in 1 ml of PBS. Then, the combination was incubated with a 0.2-M sodium bicarbonate buffer (pH 8.3) for two h at room temperature. To take away unbound dye, the labeled CMVs have been purified utilizing a 100 kDa extremely centrifugal filter (Millipore). For Dil labeling, 5 µl of Dil (1 mM) was added to 1 mL of CMVs, blended completely, and incubated for two h at room temperature. The Dil-labeled CMVs have been equally purified utilizing a 100 kDa extremely centrifugal filter (Millipore) to take away any unbound dye. Each IRDye 800CW-labeled and Dil-labeled CMVs have been resuspended in PBS and saved for additional experiments.
L. reuteri-CMVs stability
The soundness of IRDye® 800CW-labeled L. reuteri-CMVs was investigated based mostly on our earlier research [20]. IRDye® 800CW-labeled L. reuteri-CMVs have been incubated in stomach-like answer or intestine-like answer at 37 °C for 30 min. Subsequently, the IRDye® 800CW-labeled L. reuteri-CMVs have been collected via exosome spin columns with a molecular weight cutoff of 4,000 (MW4000).
Depletion of L. reuteri in mice
So as to deplete the intestine commensal L. reuteri, C57BL/6 mice have been handled with a cocktail containing vancomycin (Sigma-Aldrich, 0.5 g/L) and ampicillin (Sigma-Aldrich, 1 g/L) in ingesting water for 7 days. The abundance of L. reuteri was recurrently detected in contemporary stool samples by qRT-PCR.
Animals and housing
C57BL/6 and IL-10-deficient (IL-10−/−) mice have been bought from Biotechnology Co., Ltd, Beijing, China and Gempharmatech CO., Ltd, Jiangsu, China, respectively. All mice have been housed in a germ-free setting with a 12-hour gentle/darkish cycle. At 8 weeks of age, they have been used for experiments in keeping with the rules and laws of the Animal Care Committee of Shenzhen Individuals’s Hospital, Shenzhen, China (No. 2024 − 118). All institutional and nationwide tips for the care and use of animals have been adopted.
Efficacy of L. reuteri-CMVs in treating DSS-induced colitis
All mice got 3% DSS in ingesting water constantly for six–7 days to induce colitis as described beforehand [20, 21]. The intervention was carried out for six–7 consecutive days, beginning on day 1 of the DSS therapy. C57BL/6 mice have been divided into 4 teams: wholesome management group, PBS group, L. reuteri group, and CMVs group. IL-10−/− mice have been divided into three teams: wholesome management group, PBS group, and CMVs group. Mice depleted of L. reuteri have been divided into three teams: PBS group, L. reuteri group, and CMVs group. The colitis mice have been fed day by day with both 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 management.
The physique weight, fecal traits, and bodily exercise of the mice have been assessed day by day all through the experiment. The illness exercise index (DAI) was calculated utilizing a beforehand established scoring system [20, 21]. On days 6 or 7, the mice have been euthanized utilizing CO2 inhalation. Blood samples have been obtained from the orbits of the mice for additional use. The colon size was measured from the cecum to the rectum. Colon samples, feces, and main organs (coronary heart, liver, spleen, lung, and kidney) have been harvested for additional experiments.
Biodistribution and uptake of L. reuteri-CMVs within the gastrointestinal tract
To evaluate the biodistribution of L. reuteri-CMVs within the gastrointestinal tract, colitis mice and untreated mice have been gavaged with 200 µl of PBS containing IRDye® 800CW-labeled L. reuteri-CMVs (2.5 mg/ml). At numerous time factors after oral administration (4, 12, and 24 h), the mice have been euthanized utilizing CO2 inhalation. Gut tissues and main organs (mind, coronary heart, liver, spleen, lung, and kidney) have been then obtained for fluorescence imaging via an IVIS spectrum imaging system (Hopkinton, USA), which permits for visualization and quantification of the fluorescence emitted by the IRDye® 800CW-labeled L. reuteri-CMVs in these tissues.
So as to examine the uptake of L. reuteri-CMVs within the gastrointestinal tract, colitis mice have been administered 200 µl of Dil-labeled L. reuteri-CMVs (2.5 mg/ml) by oral gavage. The abdomen, small gut, and colon have been harvested 8 h post-administration. The tissues have been mounted in 4% paraformaldehyde for twenty-four h at 4 °C. Subsequently, the tissues have been immersed in PBS containing 30% sucrose for 48 h at 4 °C till they bottomed out. The tissues have been then buried in optimum slicing temperature compound (OCT), frozen, and sectioned into 5 μm slices. The frozen slices have been cleaned with PBS, blocked with 3% bovine serum albumin (BSA), and subsequently incubated with main antibodies at 4 °C in a single day. Following 3 washes with PBS, the slices have been incubated with fluorescent secondary antibodies for 1 h at room temperature. Lastly, the nuclei have been stained utilizing 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime). Photographs have been acquired utilizing a fluorescence scanner (Pannoramic MIDI, 3DHISTECH, Hungary).
Cell uptake of L. reuteri-CMVs
A quantity 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 have been washed thrice 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 evaluate the cytotoxicity of L. reuteri-CMVs on Caco-2 and HT-29 cells. Cells have been seeded in 96-well plates at a density of 1*104 cells/nicely and incubated for twenty-four h. Cells have been then incubated with 100 µl of full medium with L. reuteri-CMVs for twenty-four and 48 h. After the medium containing CMVs was eliminated, the cells have been completely rinsed as soon as with PBS and incubated with 100 µl of MTT (5 mg/ml) for 3 h at 37 °C. Lastly, the media have been discarded and 100 µl of dimethylsulfoxide was added to every nicely previous to spectrophotometric studying at 490 nm. Untreated cells have been outlined as detrimental controls.
In vivo, blood samples have been 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 have been handled with L. reuteri-CMVs for 7 days. The wholesome mice and untreated colitis mice have been controls. As well as, coronary heart, liver, spleen, lung, and kidney samples have been harvested and carried out to guage the toxicity of the L. reuteri-CMVs utilizing hematoxylin-eosin (H&E) staining.
H&E and alcian blue (AB) staining
Briefly, the colon tissues have been mounted in a 4% polyformaldehyde answer. Then, they have been dehydrated in an ethanol gradient, cleared in xylene, and embedded in paraffin. Subsequently, they have been lower into 5 μm-thick slices for H&E and AB staining. Histological scores have been estimated as beforehand described [22]. Goblet cell counts have been carried out by calculating the variety of AB-stain constructive cells in every crypt (≥ 6 crypts per mouse have been examined) [23].
FITC-dextran permeability assay
FITC-dextran (Sigma-Aldrich, 0.6 mg/g) was administered orally to mice for 12 h with out consuming or ingesting. Serum samples have been collected 4 h after the administration of the FITC-dextran. The FITC fluorescence depth of the serum was measured utilizing a multimode reader (Thermo Fisher Scientific) at an excitation wavelength of 490 nm and an emission wavelength of 530 nm.
Intestine microbiota 16 S rRNA sequencing
Briefly, the HiPure Stool DNA Package from Magen (Guangzhou, China) was used to extract whole fecal DNA based mostly on the producer’s directions. The DNA high quality and focus have been scrutinized utilizing agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer, respectively. The v3-v4 areas of the 16 S rRNA gene have been amplified utilizing primers: 341 F: CCTACGGGNGGCWGCAG and 806R: GGACTACHVGGGTATCTAAT. Sequencing was carried out on the Illumina NovaSeq PE250 platform (Illumina, San Diego, USA). Sequencing information processing and bioinformatics evaluation have been carried out utilizing the Illumina HiSeq 2500 platform (Guangzhou Genedenovo Bio-Know-how Firm Restricted, Guangzhou, China).
RNA extraction and qRT-PCR
Whole RNA was remoted from cells and colon tissues utilizing the TRIzol Reagent (Thermo Fisher Scientific). The focus of RNA was detected utilizing the NanoDrop 2000 (Thermo Fisher Scientific). Gene expression was carried out utilizing the PrimeScript RT Grasp Combine (Takara). Primers have been designed and obtained in Complement 1.
Enzymelinked immunosorbent assay (ELISA)
The concentrations of TNF-α, IL-1β, IL-6, IL-12, interferon-γ (IFN-γ), and IL-10 have been decided in serum samples utilizing ELISA kits (Elabscience) in keeping with the producer’s directions.
Immunohistochemistry (IHC)
For IHC evaluation, colon sections have been put in 0.01 M sodium citrate buffer (pH 6.0) and high-pressure handled for 3 min for antigen restore. The sections have been then blocked by 5% BSA for 30 min and underwent subsequent incubation with a myeloperoxidase (MPO) main antibody (Abcam, ab208070, 1:200) in a single day at 4 °C. The sections have been then incubated with an acceptable HRP-conjugated secondary antibody for 1 h, and the immunoreactivity was detected utilizing a DAB substrate package.
Immunofluorescence (IF)
Cells and colon sections have been mounted in a 4% paraformaldehyde answer, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. They have been then incubated with main 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 acceptable dilutions in a single day at 4 °C. Following the removing of the first antibodies, samples have been then washed with PBS and incubated with Alexa Fluor 488 (Abcam) and/or Alexa Fluor 555 (Abcam) at room temperature for 1 h. Lastly, the nuclei have been stained with DAPI. The pictures have been acquired by a fluorescence scanner and CLSM.
Western blot evaluation
Cells or colon tissue have been lysed on ice for 30 min utilizing RIPA lysis buffer (EpiZyme) containing a phosphatase inhibitor (EpiZyme). Protein focus was measured utilizing the BCA protein assay package (EpiZyme). Proteins have been separated by SDS-PAGE (EpiZyme) and transferred to PVDF membranes (Sigma-Aldrich). Membranes have been blocked with 5% skim milk for 1 h at room temperature after which incubated in a single day at 4 °C with numerous main antibodies at acceptable 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 management. Membranes have been then handled with secondary antibodies (mouse secondary antibody (Affinity, S0002), rabbit secondary antibody (Affinity, S0001)) for 1 h at room temperature. Lastly, the outcomes have been imaged by a Bio-Rad Imaging System.
Progress curve experiment of micro organism and bacterial uptake of L. reuteri-CMVs
Akkermansia muciniphila (AKK) (BNCC341917) was bought from the BNCC and cultured in mind coronary heart infusion (BHI) broth supplemented with Threonineat (6 g/L) and N-acetylglucosamine (GlcNAc) (4.4 g/L) below anaerobic situations at 37 °C. Subsequently, AKK cultures have been supplemented with 10 µl of PBS containing L. reuteri-CMVs (2.5 mg/ml), and untreated AKK was outlined as a management. Optical density at 600 nm (OD600) was measured each 4 h at 37 °C utilizing a NanoDrop 2000 to watch bacterial progress and calculate the entire variety of bacterial cells.
To evaluate bacterial uptake, 1 ml of logarithmic section AKK answer was co-cultured with 10 µl of PBS containing Dil-labeled L. reuteri-CMVs (2.5 mg/ml). The combination was centrifuged at 7,000 g for five min after 24 h. The collected samples have been washed thrice with PBS to take away unincorporated Dil-labeled L. reuteri-CMVs. The uptake of L. reuteri-CMVs by AKK was visualized utilizing CLSM.
Bacterial RNA-sequencing evaluation
Below anaerobic situations at 37 °C, AKK was co-incubated with PBS or CMVs for 72 h. After incubation, bacterial pellets have been harvested, and whole RNA was extracted utilizing a bacterial RNA extraction package (TIANGEN). RNA sequencing libraries was ready utilizing the Quick RNA-seq Lib Prep Package V2 (ABclonal, RK20306). Transcriptome sequencing was carried out on the Illumina platform at Novogene Co.,Ltd. (Beijing, China). The clear reads have been mapped to the reference AKK genome (https://www.ncbi.nlm.nih.gov) utilizing HISAT2 (v2.0.5). HTSeq (v0.9.1) was used to depend the reads numbers mapped to every gene. Differential expression evaluation of two teams was carried out utilizing the DESeq2 R bundle (v1.20.0). Genes with P adj < 0.05 and |log2(foldchange)| > 0 have been recognized as differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) have been used for enrichment evaluation of DEGs.
Mobile RNA-sequencing evaluation
Whole RNA was extracted from cells utilizing TRIzol Reagent (Thermo Fisher Scientific) in keeping with the producer’s directions. Then, the quantity and amount of mRNA have been evaluated utilizing a Nanodrop 2000. RNA-seq libraries have been ready, sequenced, and analyzed on the Illumina sequencing platform by Guangzhou Genedenovo Bio-Know-how Firm Restricted (Guangzhou, China). Reads obtained from the sequencing machines have been additional filtered by fastp (v0.18.0). Quick reads alignment instrument Bowtie2 (v2.2.8) was used for mapping reads to ribosome RNA (rRNA) database. An index of the reference genome was constructed, and paired-end clear reads have been mapped to the reference genome utilizing HISAT2 (v2.1.0). Principal part evaluation (PCA) was carried out with R bundle gmodels (http://www.r-project.org/) on this expertise. Correlation evaluation was carried out by R. RNAs differential expression evaluation was carried out by DESeq2 software program between two completely different teams. The genes with the parameter of false discovery fee (FDR) under 0.05 and absolute fold change > 1.5 have been thought of DEGs. Consequence, gene expression warmth map, volcano plot, GO evaluation, and KEGG pathway evaluation of those DEGs have been carried out. Gene set enrichment evaluation (GSEA) was carried out to determine the numerous enrichment of gene units in related pathways.
Availability and evaluation of transcriptomic datasets
Single-cell datasets have been accessible at http://scibd.cn24. The relative expression of HMOX1 was evaluated in epithelial cell subsets.
Statistical evaluation
A comparability of a number of experimental teams was carried out by one-way or two-way evaluation of variance (ANOVA). A t-test was calculated to match the technique of the 2 teams. Knowledge are introduced as means ± SEM. The p < 0.05*, p < 0.01**, p < 0.001***, and p < 0.0001**** signify statistically significance, and ns represents non-significance.
