L. reuteri pressure tradition
L. reuteri (BNCC192190) was bought from the BeNa Tradition Assortment (BNCC), and cultured in MRS broth (Huankai) at 37 °C underneath anaerobic circumstances. For oral gavage, L. reuteri was harvested on the logarithmic part by centrifugation, washed, and resuspended in PBS to achieve a density of 1*109 colony forming items (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 recent 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 by means of 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 had 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 by means of 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 dimensions of CMVs was analyzed utilizing dynamic mild scattering (DLS, Zetasizer Nano AS90) in keeping with our earlier examine [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 examine [20]. The proteins in L. reuteri-CMVs and L. reuteri had been extracted and separated utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein lysates had been ready and subjected to nano-LC-MS/MS evaluation to establish and quantify their proteins utilizing Orbitrap mass spectrometry (Thermo Fisher Scientific, Bremen, Germany).
Cell tradition
HT-29 and Caco2 human intestinal epithelial cell traces had been obtained from the American Sort Tradition Assortment (ATCC). HT-29 cells had 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 had been cultivated in Minimal Important Medium (MEM) (Gibco™) supplemented with 20% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). All cells had 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 had been stimulated with 2% DSS (MP Biomedicals) and hrTNF (PeproTech, 10 ng/ml) for twenty-four h. Cells had been harvested for additional experiments as described beneath.
L. reuteri-CMVs labeling
To allow flurescent monitoring, L. reuteri-CMVs had 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 had 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 had 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 had been resuspended in PBS and saved for additional experiments.
L. reuteri-CMVs stability
The steadiness of IRDye® 800CW-labeled L. reuteri-CMVs was investigated based mostly on our earlier examine [20]. IRDye® 800CW-labeled L. reuteri-CMVs had been incubated in stomach-like answer or intestine-like answer at 37 °C for 30 min. Subsequently, the IRDye® 800CW-labeled L. reuteri-CMVs had been collected by means of exosome spin columns with a molecular weight cutoff of 4,000 (MW4000).
Depletion of L. reuteri in mice
With a view to deplete the intestine commensal L. reuteri, C57BL/6 mice had been handled with a cocktail containing vancomycin (Sigma-Aldrich, 0.5 g/L) and ampicillin (Sigma-Aldrich, 1 g/L) in consuming water for 7 days. The abundance of L. reuteri was frequently detected in recent stool samples by qRT-PCR.
Animals and housing
C57BL/6 and IL-10-deficient (IL-10−/−) mice had been bought from Biotechnology Co., Ltd, Beijing, China and Gempharmatech CO., Ltd, Jiangsu, China, respectively. All mice had been housed in a germ-free setting with a 12-hour mild/darkish cycle. At 8 weeks of age, they had 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 had been adopted.
Efficacy of L. reuteri-CMVs in treating DSS-induced colitis
All mice got 3% DSS in consuming water repeatedly 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 had been divided into 4 teams: wholesome management group, PBS group, L. reuteri group, and CMVs group. IL-10−/− mice had been divided into three teams: wholesome management group, PBS group, and CMVs group. Mice depleted of L. reuteri had been divided into three teams: PBS group, L. reuteri group, and CMVs group. The colitis mice had been fed every 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 had been assessed every 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 had been euthanized utilizing CO2 inhalation. Blood samples had 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) had 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 had 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 had been euthanized utilizing CO2 inhalation. Gut tissues and main organs (mind, coronary heart, liver, spleen, lung, and kidney) had been then obtained for fluorescence imaging by means of 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.
With a view to examine the uptake of L. reuteri-CMVs within the gastrointestinal tract, colitis mice had been administered 200 µl of Dil-labeled L. reuteri-CMVs (2.5 mg/ml) by oral gavage. The abdomen, small gut, and colon had been harvested 8 h post-administration. The tissues had been fastened in 4% paraformaldehyde for twenty-four h at 4 °C. Subsequently, the tissues had been immersed in PBS containing 30% sucrose for 48 h at 4 °C till they bottomed out. The tissues had been then buried in optimum slicing temperature compound (OCT), frozen, and sectioned into 5 μm slices. The frozen slices had been cleaned with PBS, blocked with 3% bovine serum albumin (BSA), and subsequently incubated with major antibodies at 4 °C in a single day. Following 3 washes with PBS, the slices had been incubated with fluorescent secondary antibodies for 1 h at room temperature. Lastly, the nuclei had been stained utilizing 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime). Pictures had 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 had 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 had been seeded in 96-well plates at a density of 1*104 cells/effectively and incubated for twenty-four h. Cells had 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 had 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 had been discarded and 100 µl of dimethylsulfoxide was added to every effectively previous to spectrophotometric studying at 490 nm. Untreated cells had been outlined as detrimental controls.
In vivo, blood samples had 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 had been handled with L. reuteri-CMVs for 7 days. The wholesome mice and untreated colitis mice had been controls. As well as, coronary heart, liver, spleen, lung, and kidney samples had 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 had been fastened in a 4% polyformaldehyde answer. Then, they had been dehydrated in an ethanol gradient, cleared in xylene, and embedded in paraffin. Subsequently, they had been reduce into 5 μm-thick slices for H&E and AB staining. Histological scores had been estimated as beforehand described [22]. Goblet cell counts had been carried out by calculating the variety of AB-stain constructive cells in every crypt (≥ 6 crypts per mouse had 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 consuming. Serum samples had 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 had been scrutinized utilizing agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer, respectively. The v3-v4 areas of the 16 S rRNA gene had 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 knowledge processing and bioinformatics evaluation had been carried out utilizing the Illumina HiSeq 2500 platform (Guangzhou Genedenovo Bio-Expertise Firm Restricted, Guangzhou, China).
RNA extraction and qRT-PCR
Complete 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 had 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 had been decided in serum samples utilizing ELISA kits (Elabscience) in keeping with the producer’s directions.
Immunohistochemistry (IHC)
For IHC evaluation, colon sections had been put in 0.01 M sodium citrate buffer (pH 6.0) and high-pressure handled for 3 min for antigen restore. The sections had been then blocked by 5% BSA for 30 min and underwent subsequent incubation with a myeloperoxidase (MPO) major antibody (Abcam, ab208070, 1:200) in a single day at 4 °C. The sections had 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 had been fastened in a 4% paraformaldehyde answer, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. They had been then incubated with major 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 had 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 had been stained with DAPI. The pictures had been acquired by a fluorescence scanner and CLSM.
Western blot evaluation
Cells or colon tissue had 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 had been separated by SDS-PAGE (EpiZyme) and transferred to PVDF membranes (Sigma-Aldrich). Membranes had been blocked with 5% skim milk for 1 h at room temperature after which incubated in a single day at 4 °C with numerous major 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 had 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 had 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) underneath anaerobic circumstances at 37 °C. Subsequently, AKK cultures had 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 observe bacterial progress and calculate the whole variety of bacterial cells.
To evaluate bacterial uptake, 1 ml of logarithmic part 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 had 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
Underneath anaerobic circumstances at 37 °C, AKK was co-incubated with PBS or CMVs for 72 h. After incubation, bacterial pellets had 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 had 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 had been recognized as differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) had been used for enrichment evaluation of DEGs.
Mobile RNA-sequencing evaluation
Complete 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 had been evaluated utilizing a Nanodrop 2000. RNA-seq libraries had been ready, sequenced, and analyzed on the Illumina sequencing platform by Guangzhou Genedenovo Bio-Expertise Firm Restricted (Guangzhou, China). Reads obtained from the sequencing machines had 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 had been mapped to the reference genome utilizing HISAT2 (v2.1.0). Principal element 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 price (FDR) beneath 0.05 and absolute fold change > 1.5 had been thought of DEGs. Consequence, gene expression warmth map, volcano plot, GO evaluation, and KEGG pathway evaluation of those DEGs had been carried out. Gene set enrichment evaluation (GSEA) was carried out to establish the numerous enrichment of gene units in related pathways.
Availability and evaluation of transcriptomic datasets
Single-cell datasets had 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 check the technique of the 2 teams. Information are introduced as means ± SEM. The p < 0.05*, p < 0.01**, p < 0.001***, and p < 0.0001**** characterize statistically significance, and ns represents non-significance.
