Extracellular vesicles (EVs) preparation from MSCs
Extracellular vesicles (EVs) had been remoted from human umbilical twine MSCs (Procell, CP-CL11) utilizing an ultracentrifugation methodology. Briefly, cells had been cultured for 3 days in DMEM/F-12 medium (Gibco, 11320033) supplemented with fetal bovine serum (FBS, Vivacell, C3801-0100) to facilitate exosome secretion. The tradition medium was collected and centrifuged at 300g after which 2000g for 15 min every at 4 °C to take away lifeless cells and cell particles. Subsequently, the supernatant was centrifuged at 20,000g for 30 min at 4 °C to take away insoluble impurities. The ensuing resolution was filtered by a 0.22 μm membrane filter (MerckMillipore, SLGP033RB). EVs had been then extracted by centrifuging at 200,000g for two h at 4 °C. After washes with PBS (200,000g, 2 h), EVs had been resuspended in a 5% trehalose PBS buffer (Beijing Bioassayon Biotechnology Co., Ltd., C1049) [20].
Preparation of modified P-D2-EVs
Modified P-D2 exosomes (P-D2-EVs) had been ready utilizing a lipid hybrid method. The P-D2 peptide (amino acid sequence: GGVTLTYQFAAGPRDK) was used for particular focusing on of DCs. In short, ldl cholesterol (Solarbio, C8280), phospholipid (AVT, PC-98T), and DSPE-PEG-P-D2 synthesized by Hangzhou Xin Qiao Biotechnology Co., Ltd. had been dissolved in 2 ml of tetrahydrofuran (Aladdin, 109-99-9) with an optimum weight ratio of 4:2:1. The combination was then subjected to ultrasonication at 50 kHz for 30 min. Subsequent, the answer was evaporated for 1 h on a rotary evaporator at 50 °C to take away the tetrahydrofuran. Subsequently, 1 ml of PBS was added, and the P-D2 liposomes had been gently resuspended. Lastly, the P-D2 liposomes had been incubated with EVs at 37 °C for 15 min and subjected to a number of gentle extrusions utilizing a mini extruder (Avanti Polar Lipids, 610020) and a 200 nm pore dimension polycarbonate membrane (Avanti Polar Lipids, 610006) to type P-D2-EVs [20, 21]. As beforehand described, EVs had been labeled with DiR covalently. EVs coupled with P-D2 peptide had been purified utilizing a Zeba spin desalting column (14K MWCO, ThermoFisher Scientific) equilibrated with PBS resolution. The conjugation effectivity of P-D2 peptide on EVs was decided utilizing 3-(4-carbpxubemzpul)quinoline-2-carboxaldehyde (CBQCA) (A6222, ThermoFisher Scientific) [22].
Characterization of EVs and P-D2-EVs
Transmission Electron Microscopy (TEM): 20 µL of EVs had been dropped onto a copper grid and left to face for 3 min. The liquid was then drawn from the aspect utilizing filter paper. Afterward, 30 μl of pH 6.8 phosphotungstic acid resolution (79690, Merck, USA) was added and allowed to relaxation for five min at room temperature. The pattern was dried utilizing an incandescent lamp and noticed below a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) with an accelerating voltage of 80 kV. Photographs had been captured utilizing the side-viewing Digicam-Megaview III system (Gentle Imaging System, Muenster, Germany).
Nanoparticle Monitoring Evaluation (NTA): EV samples had been suspended in PBS and diluted 500-fold utilizing Milli-Q water. Subsequently, the diluted EVs had been injected into the pattern chamber of a NanoSight LM10 (Malvern, UK) instrument utilizing a sterile syringe, making certain the absence of bubbles till the chamber was full. Video evaluation was carried out utilizing NanoSight Model 2.3 (Malvern, UK) software program with a achieve of 6.0 and a threshold of 11. The motion trajectories of the particles had been recorded, and dilution pattern focus and dimension distribution plots had been generated. The EV focus within the authentic resolution was calculated primarily based on the dilution issue.
Identification of EVs floor markers utilizing Western blot: EVs had been resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer (Wuhan Asben Organic Know-how Co., Ltd., China, AS1004), and floor markers CD9, CD81, CD63, and Alix, in addition to endoplasmic reticulum marker Calnexin, had been detected by Western blot evaluation. Antibody info will be discovered within the Western blot part beneath [23,24,25,26]. Every experiment was repeated thrice.
Animals
Male SPF-grade BALB/c mice (6–8 weeks outdated) had been obtained from Beijing Vitonlihua Experimental Animal Know-how Co., Ltd (Beijing, China). The mice had been housed in an SPF-grade animal laboratory with a humidity of 60–65% and a temperature of twenty-two–25 °C and had been subjected to a 12-h mild–darkish cycle. After one week of acclimation, the well being situation of the mice was assessed previous to the beginning of the experiment [27].
Moral assertion
This research adhered strictly to moral pointers and laws relating to animal experimentation. All experimental procedures had been authorised by the Institutional Animal Care and Use Committee (IACUC) with evaluation quantity No. NCULAE-20221031024. The animals had been housed and cared for in accordance with ideas of humaneness, and experiments had been carried out with the utmost care to reduce ache and misery. On the finish of the experiment, all mice had been euthanized in a humane method below ethyl ether anesthesia.
Institution and grouping of a mouse mannequin for AR
100 BALB/c mice had been obtained and randomly assigned to 2 teams: the Management group (18 mice) and the Mannequin group (81 mice). The Mannequin group was induced with ovalbumin (OVA) to develop an AR mouse mannequin. Briefly, BALB/c mice had been sensitized with 200 μl of PBS (containing 50 μg OVA from Sigma-Aldrich, 01641, and a pair of mg aluminum hydroxide from Solarbio, IA5810) through intraperitoneal injection on days 0, 7, and 14. From day 21 to day 27, mice had been intranasally challenged with 20 μl of PBS (containing 400 μg OVA) to induce allergic reactions. The variety of nostril itching (rubbing) and sneezing occasions inside 2 h after the final intranasal OVA administration had been recorded and evaluated. Particularly, we calculated the variety of sneezing episodes, period of rhinorrhea, and frequency of nostril scratching or rubbing in mice of the experimental and management teams inside 2 h after the final intranasal OVA administration. Statistical evaluation utilizing a t-test was performed to evaluate the numerous variations within the prevalence of those signs between the management and experimental teams [28, 29], aiming to verify the profitable institution of the AR mouse mannequin. Mice within the Management group solely obtained intraperitoneal injection and intranasal PBS administration [30,31,32].
Subsequently, the Mannequin group was randomly divided into the next 10 teams: Mannequin + PBS group (3 mice), Mannequin + DiR-EVs group (3 mice), Mannequin + DiR-P-D2-EVs group (3 mice), Mannequin group (6 mice), Mannequin + EVs group (6 mice), Mannequin + P-D2-EVs group (6 mice), Mannequin + NC-OE-mDCs group (6 mice), Mannequin + Fut1-OE-mDCs group (6 mice), Mannequin + P-D2-EVs + NC-OE-mDCs group (6 mice), and Mannequin + P-D2-EVs + Fut1-OE-mDCs group (6 mice).
Within the Mannequin + PBS group, on the twenty eighth day after the profitable institution of the AR mannequin, intranasal administration of 20 μl PBS was performed. Within the Mannequin + DiR-EVs group, on the twenty eighth day, intranasal administration of 20 μl PBS (containing 250 μg DiR-labeled EVs) was carried out. The DiR labeling was carried out utilizing the DiR fluorescent dye (Noninbio, NW3201) in response to the producer’s directions previous to administration. Within the Mannequin + DiR-P-D2-EVs group, intranasal administration of 20 μl PBS (containing 250 μg DiR-labeled P-D2-EVs) was performed on the twenty eighth day. In vivo, fluorescence imaging was carried out utilizing an in vivo imaging system (IVIS) at fastened time factors [20, 27].
As well as, the Mannequin group obtained no additional therapy after the AR mannequin institution and was euthanized on day 28 after anesthesia with pentobarbital sodium (80 mg/kg, Sigma-Aldrich, P3761). For the Mannequin + EVs group, aside from intranasal administration of 20 μl PBS (containing 400 μg OVA and 250 μg EVs) from days 21 to 27, the procedures had been the identical as these for the Mannequin group. The Mannequin + P-D2-EVs group intranasally obtained 20 μl PBS (containing 400 μg OVA and 250 μg P-D2-EVs) from days 21 to 27 [27].
As well as, within the Mannequin + NC-OE-mDCs group, on the sixteenth and 18th days of building the AR mannequin, 1 × 106 bone marrow-derived mature DCs, transfected with adeno-associated virus empty vector (NC-OE), had been transferred to the nasal cavity of mice. The procedures had been the identical as within the Mannequin group. Within the Mannequin + Fut1-OE-mDCs group, on the sixteenth and 18th days, 1 × 106 mature DCs transfected with adeno-associated virus vector overexpressing Fut1 (Fut1-OE) had been transferred to the nasal cavity of mice. The procedures had been the identical as within the Mannequin group. Within the Mannequin + P-D2-EVs + NC-OE-mDCs group, on the sixteenth and 18th days, 1 × 106 NC-OE-mDCs had been transferred to the nasal cavity of mice. The procedures had been the identical as within the Mannequin + P-D2-EVs group. Within the Mannequin + P-D2-EVs + Fut1-OE group, on the sixteenth and 18th days, 1 × 106 Fut1-OE-mDCs had been transferred to the nasal cavity of mice. The procedures had been the identical as within the Mannequin + P-D2-EVs group. Detailed strategies for making ready NC-OE or Fut1-OE transfected mDCs are described within the following part on cell tradition and cell grouping [33].
Furthermore, the remaining 10 untreated BALB/c mice had been used to extract bone marrow DCs and first CD4+ T cells from the spleen. Three mice from the Mannequin + PBS group, Mannequin + DiR-EVs group, and Mannequin + DiR-P-D2-EVs group had been used for in vivo fluorescence imaging. Three mice, every from the Mannequin group and Mannequin + P-D2-EVs group, had been used for single-cell sequencing, whereas an extra three mice had been used for transcriptome sequencing. Every group, together with the Management group, Mannequin group, Mannequin + EVs group, Mannequin + P-D2-EVs group, Mannequin + NC-OE-mDCs group, Mannequin + Fut1-OE-mDCs group, Mannequin + P-D2-EVs + NC-OE-mDCs group, and Mannequin + P-D2-EVs + Fut1-OE-mDCs group, consisted of six mice for detecting AR-related indicators, aside from the Mannequin + EVs group, an extra three mice had been utilized in every group for circulation cytometry to detect the proportion of goal cells and related gene expression [30,31,32].
In vivo fluorescence imaging
Following the institution of the AR mannequin, animal grouping was performed on Day 28 as follows: Mannequin + PBS group, Mannequin + DiR-EVs group, and Mannequin + DiR-P-D2-EVs group. Based on the outline of the animal teams, these teams obtained intranasal administration of PBS EVs labeled with DiR and P-D2-EVs, respectively. In vivo, fluorescence imaging of the mice was carried out at 6 and 12 h after drug administration utilizing the IVIS Lumina III in vivo imaging system (PerkinElmer, CLS136334). Previous to imaging, the mice underwent temporary anesthesia to make sure their immobility through the process [20, 34].
Detection of in vivo DC uptake of P-D2-EVs utilizing circulation cytometry
We analyzed the distribution of P-D2-EVs inside DCs in nasal tissues of AR mice utilizing in vivo fluorescence imaging. We utilized antibodies labeled with FITC in opposition to floor markers of anti-Mouse CD11c (BD, 568942), MHC-II (CST, 42594S), CD80 (BD, 553768), and CD86 (BD, 561962) to carry out DC sorting on the Cytek Aurora Movement Cytometer (Cytek). Subsequently, we decided the mobile percentages of DiR-labeled EVs and P-D2-EVs uptake [20, 35, 36].
Movement cytometric evaluation
First, single cells had been ready by enzymatic digestion of nasal tissue. The cell suspension was lysed utilizing an ACK lysis buffer (ThermoFisher, A1049201), and lifeless cells that stain constructive for 7-AAD reagent (CST, 72782S) had been excluded. Antibodies in opposition to floor markers of DCs, together with FITC-labeled anti-Mouse CD11c, MHC-II, CD80, and CD86, in addition to Alexa Fluor™ 488-labeled anti-Mouse Ki67 (ThermoFisher, 53-5698-82), PE-labeled anti-Mouse IL10 (ThermoFisher, 12-7101-82), Alexa Fluor™ 532-labeled anti-Mouse CD3 (ThermoFisher, 58-0032-82), APC-eFluor™ 780-labeled anti-Mouse CD4 (ThermoFisher, 47-0042-82), APC-labeled anti-Mouse IL4 (ThermoFisher, 17-7041-82), Alexa Fluor™ 700-labeled anti-Mouse GATA3 (ThermoFisher, 56-9966-42), PerCP-Cy™5.5-labeled Mouse Lineage Antibody Cocktail (BD, 561317), BV711-labeled Anti-Mouse CD90.2 (BD, 740647), PE-Cy™ 7-labeled anti-Mouse CD45 (BD, 552848), BUV563-labeled anti-Mouse KLRG1 (BD, 741343), and eFluor™ 660-labeled anti-Mouse IL13 (ThermoFisher, 50–7133-82) had been incubated with the cells. CD11c, MHC-II, CD80, and CD86 had been floor markers for DCs, Ki67 was an activation marker for DCs, IL10 was a cytokine for DCs, CD3 + CD4 + IL4 + represented Th2 cells, GATA3 was a marker for Th2 cell differentiation and ILC2 activation, and Lineage-CD45 + KLRG1 + CD90.2 + represented ILC2 cells, whereas IL13 was a cytokine for ILC2 cells. After including the antibodies, cells had been incubated at 4 °C in the dead of night for 30 min. The cells had been then washed twice with PBS and centrifuged at 2000 g for five min at 4 °C. Percentages of labeled markers had been analyzed utilizing the Cytek Aurora Movement cytometer (Cytek) [20, 35,36,37,38,39].
In vitro extracellular vesicle uptake
To label EVs and P-D2-EVs, the PKH67 inexperienced fluorescent cell linker mini package (Sigma-Aldrich, MINI67-1KT) was employed as per the package’s directions. Subsequent, the DCs remoted from nasal tissue by circulation cytometry had been seeded in a 24-well plate at a focus of 4 × 104 cells per nicely. The plate was incubated in a single day at 37 °C with 5% CO2 following thorough mixing. The supernatant was then discarded, and DMEM/F-12 medium containing exosome-free FBS was added to every nicely. Roughly 10 μg of PKH67-labeled EVs or P-D2-EVs was added to every nicely and totally blended. The plate was positioned in a cell tradition incubator for a complete of two or 8 h. After the incubation interval, the cells had been collected and seeded on poly-l-lysine-treated slides for 30 min for adherence. CellMembrane staining was carried out utilizing CellMask™ Cell Membrane Stain (Invitrogen, C10046) following the producer’s directions to label the cell membrane of DCs after uptake of EVs or P-D2-EVs. The cells had been then handled with PBS containing 0.1% Triton X-100 (Sigma-Aldrich, X100) for five min, adopted by three washes with PBS. Lastly, the slides had been mounted utilizing a mounting medium containing DAPI (Beyotime, P0131-5 ml) and noticed and photographed below a Zeiss LSM 880 confocal fluorescence microscope (Carl Zeiss). The fluorescence depth of three impartial fields of view was quantified utilizing ImageJ software program for every group [35, 40].
Mobile evaluation of NALF and serum immunoglobulin E (IgE)
NALF and serum had been collected from euthanized mice previous to anesthesia. After anesthetizing the mice, the higher place of the trachea was ligated, after which 1 ml of chilly PBS was gently injected into the nasopharynx utilizing a dimension 21 cannula. The collected NALF was centrifuged at 3000 g for five min at 4 °C. The mobile pellet of NALF was resuspended in PBS, and 50 μl of the cell suspension was smeared onto glass slides, adopted by staining utilizing the Diff-Fast staining package (Solarbio, G1540). The overall cell depend was decided utilizing ImageJ software program, with 3 impartial fields counted for every group. Blood was collected from the inferior vena cava, allowed to clot for two–3 h at room temperature, after which centrifuged at 3000g for five min at 4 °C to acquire serum. OVA-specific IgE ranges within the serum had been measured utilizing the ELISA package (Bioswamp, MU30065) [36].
Nasal tissue pathology
After the nasal tissues from the mice had been collected, they had been fastened in 10% impartial formalin (Biosharp, BL388A) for 48 h and embedded in paraffin. Subsequently, 4 μm thick paraffin sections had been ready (three sections per mouse), and hematoxylin and eosin (H&E) staining was carried out utilizing the staining package (Solarbio, G1120) to judge the infiltration of nasal eosinophils. Immunohistochemical staining was additionally carried out utilizing the anti-Mouse NIMP-R14 antibody (Abcam, ab2557) as a neutrophil marker, adopted by incubation with Goat anti-Rat-HRP secondary antibody (ThermoFisher, 31470) and substrate 3,3ʹ-diaminobenzidine (Sigma-Aldrich, D8001), leading to brown staining of constructive cells. A periodic acid-Schiff (PAS) staining package (Beyotime, C0142S) was utilized to find out the share of PAS-positive goblet cells. Photographs had been noticed below an inverted microscope (Olympus, BX63), and ImageJ software program was used to quantify the nasal mucosal thickness, eosinophil and neutrophil counts, and the share of goblet cells [32, 36, 41]. Three impartial fields had been quantified for every part.
RNA extraction, library development, and sequencing
Whole RNA was extracted from dendritic cells (DCs) remoted from nasal tissues of mice within the Mannequin group (n = 3) and the Mannequin + P-D2-EVs group (n = 3) utilizing Trizol reagent (15596026, Invitrogen, USA) by circulation cytometry. The focus and purity of RNA samples had been measured utilizing the Nanodrop 2000 spectrophotometer (1011U, nanodrop, USA). Whole RNA samples assembly the next standards had been used for subsequent experiments: RNA integrity quantity (RIN) ≥ 7.0 and 28S:18S ratio ≥ 1.5 [35, 42].
Sequencing libraries had been generated and sequenced by CapitalBio Know-how (Beijing, China). A complete of 5 μg RNA was used for every pattern. In short, the Ribo-Zero magnetic package (MRZE706, Epicentre Applied sciences) was used to take away ribosomal RNA (rRNA) from complete RNA. NEB Subsequent Extremely RNA library preparation package (#E7775, NEB, USA) was used to assemble libraries for Illumina sequencing. Then, the RNA was fragmented into roughly 300 base pair (bp) fragments utilizing NEB Subsequent first strand synthesis response buffer (5×). First-strand cDNA was synthesized utilizing reverse transcriptase primers and random primers, adopted by second-strand cDNA synthesis utilizing second-strand synthesis response buffer within the presence of dUTP Combine (10×). The cDNA fragments had been end-repaired, together with the addition of polyA tails and the connection of sequencing adapters. After adapter ligation, the USER enzyme (#M5508, NEB, USA) was used to digest the second strand of cDNA to assemble strand-specific libraries. Library DNA was amplified, purified, and enriched utilizing PCR. Lastly, the libraries had been assessed utilizing Agilent 2100 and quantified utilizing the KAPA library quantification package (KK4844, KAPA Biosystems). Pair-end sequencing was carried out on the NextSeqCN500 (Illumina) sequencer [35, 42].
High quality management of sequencing knowledge and alignment to the reference genome
The standard of paired-end reads within the uncooked sequencing knowledge was examined utilizing FastQC software program v0.11.8. The unique knowledge was preprocessed utilizing Cutadapt software program 1.18 to take away Illumina sequencing adapters and poly(A) tail sequences. Perl scripts had been utilized to remove reads with an N content material exceeding 5%. The FASTX Toolkit software program 0.0.13 was employed to extract reads with a base high quality of 20 or above, accounting for 70% of the bases. BBMap software program was used to restore the paired-end sequences. Lastly, the filtered high-quality learn fragments had been aligned in opposition to the mouse reference genome utilizing Hisat2 software program model 0.7.12 [43, 44].
Pattern preparation and sequencing of single cells
Three Mannequin mice and three mannequin + P-D2-EVs mice had been chosen, and nasal tissue samples had been collected. The nasal tissue samples had been ready as single-cell suspensions and loaded onto Chromium Single Cell chips. The Chromium Single Cell 3ʹ v3 package (10 × Genomics) was used to arrange the samples in response to the producer’s directions, capturing roughly 10,000 particular person cells per pattern. Throughout cDNA synthesis, the captured mRNA was barcode labeled. Subsequently, the single-cell RNA-seq libraries for Illumina sequencing had been ready utilizing the Chromium Single Cell 3ʹ Resolution (10 × Genomics) following the producer’s directions. Barcode labeling was carried out utilizing Illumina Pattern Indexes, and sequencing was performed on a single Illumina circulation cell channel utilizing the Illumina HiSeq 4000 sequencer. The sequencing parameters had been as follows: Learn 1 contained 28 bases, Learn 2 contained 98 bases, and the pattern index included 8 bases. The minimal variety of reads per cell sequenced from the RNA library was set at 25,000 [45].
TSNE clustering evaluation, cell annotation, pseudotime evaluation, and cell communication evaluation
On this research, scRNA-seq knowledge was analyzed utilizing the “Seurat” package deal in R software program. A sequence of high quality management measures had been initially carried out, together with the next filtering circumstances: nFeature_RNA > 200, nFeature_RNA < 5000 %.mt < 10. Batch results had been eradicated utilizing the canonical correlation evaluation (CCA) methodology, adopted by standardization of the info utilizing the LogNormalize operate.
To scale back the dimensionality of the scRNA-seq dataset, principal element evaluation (PCA) was utilized to the extremely variable genes primarily based on the highest 2000 genes with the very best variance. The primary 30 principal parts had been then chosen for TSNE clustering evaluation. The FindClusters operate supplied by Seurat was employed to determine the primary cell subgroups, with a default decision worth of res = 0.9. Subsequently, the UMAP algorithm was used to cut back nonlinear dimensionality of the scRNA-seq sequencing knowledge. Cell type-specific marker genes had been recognized for numerous cell subgroups utilizing the Seurat package deal. Moreover, the “SingleR” package deal was utilized to annotate the marker genes of every cell cluster, with reference knowledge units loaded utilizing the MouseRNAseqData operate. Cell annotation was carried out by contemplating identified cell lineage-specific marker genes and using the CellMarker on-line web site.
Subsequently, the “monocle” package deal in R software program was employed for pseudotime evaluation. Cell communication evaluation was carried out utilizing the “cellchat” package deal [46].
Bioinformatics evaluation of differentially expressed genes in transcriptome and single-cell sequencing
Differential expression gene (DEG) identification was carried out on the uncooked depend matrix of DCs transcriptome sequencing utilizing the “limma” package deal within the R language. A threshold of |log fold change (FC)|> 2 and P-value < 0.05 was chosen, and a volcano plot was generated utilizing the “ggplot2” package deal. A heatmap of the DEGs was created utilizing the “pheatmap” package deal. Subsequently, the R packages “clusterProfiler”, “org.Hs.eg.db”, “org.Mm.eg.db”, “enrichplot”, “ggplot2”, and “pathview” had been utilized for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses [47].
The DCs cell subpopulation was extracted from the single-cell knowledge, and the “limma” package deal within the R language was employed to determine differentially expressed genes (ScRNA-DEGs) with a threshold of |log fold change (FC)|> 0.5 and P-value < 0.05. The “EnhancedVolcano” package deal was used to generate a volcano plot. A Venn diagram was created utilizing the Jvenn web site to acquire the differentially expressed genes (B-DEGs) that intersected with DEGs and ScRNA-DEGs. KEGG and GO analyses had been then carried out [48].
RT-qPCR
RNA was extracted from ruptured cells or nasal tissue utilizing the Trizol reagent package (Invitrogen, 10296028CN). The standard and focus of RNA had been decided by UV–seen spectrophotometry utilizing the ND-1000 NanoDrop (USA). To measure mRNA expression, reverse transcription was carried out utilizing the PrimeScript™ RT-qPCR package (TaKaRa, RR037Q). Actual-time quantitative reverse transcription polymerase chain response (RT-qPCR) was carried out on a LightCycler 480 system (Roche Diagnostics, Pleasanton, CA, USA) utilizing SYBR Premix Ex Taq™ (TaKaRa, RR390A). The reference gene GAPDH was used as an inner management for mRNA expression. Primer design and synthesis had been supplied by Shanghai Common Biotech Co., Ltd. Primer sequences will be present in Desk S1. The relative expression of the goal gene within the experimental group in comparison with the management group was decided utilizing the two−ΔΔCt methodology, the place ΔΔCT = ΔCt experimental group − ΔCt management group, and ΔCt = goal gene Ct − reference gene Ct [35].
Cell tradition
To isolate DCs from the bone marrow of BALB/c mice, the mice had been euthanized by cervical dislocation, after which soaked in a 75% ethanol resolution for five min. The femur and tibia had been extracted, and the bone marrow cavity was rinsed with sterile PBS thrice to acquire a cell suspension. The suspension was filtered by a 200-mesh cell strainer (Corning, 352,350) to take away small bone fragments and muscle tissues, adopted by centrifugation at 1200g for five min. The supernatant was eliminated, and the remaining cells had been handled with pink blood cell lysis buffer (Solarbio, R1010) for 3 min, adopted by centrifugation to gather the bone marrow cells (BM cells). After counting the BM cells, they had been seeded in a 24-well plate at a focus of 1 × 106/ml in RPMI 1640 medium (Gibco, 11875093) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin, supplemented with 20 ng/ml GM-CSF (MCE, HY-P7361) and 20 ng/ml IL-4 (MCE, HY-P70644) dissolved in PBS. The cells had been incubated at 37 °C with 5% CO2. On the third and fifth days, half of the medium was changed with a recent medium containing the identical focus of cytokines. After 5 days of tradition, cells had been harvested, and the phenotype of immature DCs (iDCs) was analyzed by circulation cytometry. Moreover, on the fifth day, 100 ng/ml of LPS (MCE, HY-D1056) dissolved in PBS was added to the tradition medium for two days to gather non-adherent cells, known as mature DCs (mDCs). The collected cells had been suspended in ice-cold circulation cytometry buffer (ThermoFisher, 00-4222-26) and characterised as immature and mature DCs utilizing circulation cytometry with antibodies focusing on DC floor markers CD11c, MHC-II, CD80, and CD86 [35, 41, 49].
The spleens had been eliminated to isolate CD4+ T cells from the spleens of BALB/c mice. The Mouse Spleen Dissociation Package (Miltenyi Biotec, 130-095-926) was used in response to the producer’s directions to arrange single-cell suspensions from the spleen. The Mouse Naïve CD4+ T cell Isolation Package (Miltenyi Biotec, 8804-6824-74) was used in response to the producer’s directions to isolate naïve CD4+ T cells from mouse splenocytes. Mouse CD4+ T cells (3 × 105 cells/ml) had been co-cultured with DCs handled in numerous methods for five days. The medium used was RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin, and IL-2 (100 IU/ml, Beyotime, P5907) [38, 50].
ILC2 cells had been sorted from the nasal-associated lymphoid tissue (NALT) of OVA-sensitized mice. On day 28, OVA-sensitized mice had been euthanized, and the NALT was transferred to RPMI 1640 medium for additional processing. The cells had been handed by a 40 μm cell strainer (Fisher Scientific, FIS22-363-547). Purple blood cells had been lysed utilizing pink blood cell lysis buffer at room temperature for two–5 min. Subsequently, the cells had been washed with 10 ml of PBS, centrifuged for five min, and resuspended in 10 ml of RPMI 1640 medium. ILC2 cells had been remoted utilizing the EasySep™ Mouse ILC2 Enrichment Package (Miltenyi Biotec, 19842) in response to the producer’s directions. The sorted cells had been then cultured at 37 °C for six days in a medium containing 10% FBS, 0.1% β-mercaptoethanol (ThermoFisher, 31350010), 1% penicillin/streptomycin, and 10 ng/ml of mouse recombinant IL-33 (Abcam, ab187455), IL-7 (Abcam, ab270065), and TSLP (Abcam, ab310786) [39].
Cell grouping
Firstly, cells extracted from BALB/c mouse bone marrow had been induced into three various kinds of iDCs utilizing totally different therapies: the BM cells group, the iDCs group, and the iDCs + P-D2-EVs group. Within the BM cells group, bone marrow cells had been co-cultured with PBS for five days. Within the iDCs group, bone marrow cells had been co-cultured with GM-CSF, IL-4, and PBS for five days. Within the iDCs + P-D2-EVs group, bone marrow cells had been co-cultured with GM-CSF, IL-4, and three × 109/ml P-D2-EVs for five days [35, 51].
Subsequent, BALB/c mouse bone marrow cells had been handled or induced utilizing totally different strategies, leading to twelve various kinds of mDCs: the iDCs group, the mDCs group, the mDCs + P-D2-EVs group, NC-OE-iDCs, Fut1-OE-iDCs, NC-OE-mDCs, Fut1-OE-mDCs group, NC-OE-mDCs + P-D2-EVs group, Fut1-OE-mDCs + P-D2-EVs group, Fut1-OE-mDCs + 2DGal group, mDCs + 2DGal group, and mDCs + 2DGal + Anisomycin group.
Within the iDCs group, bone marrow cells had been co-cultured with GM-CSF and IL-4 from day 1 to 7, adopted by PBS therapy from day 5 to 7. Each the mDCs group and mDCs + P-D2-EVs group performed cell relying on day 5, adjusting the focus of P-D2-EVs primarily based on the ratio of 1000 EVs internalized per cell. The cell density of assorted mDC varieties on day 5 was roughly 2–3 × 105/ml. From day 5 to 7, LPS and PBS or LPS, together with P-D2-EVs primarily based on the 1000 EVs per cell commonplace, had been added. Subsequent cell experiments involving P-D2-EVs had been handled in accordance with this commonplace. Within the mDCs + 2DGal group, 2-deoxy-D-galactose (10 mM; MCE, HY-131892) was moreover induced from day 5 to 7. Within the mDCs + 2DGal + Anisomycin group, each 2DGal and Anisomycin (0.5 μM; MCE, HY-18982) had been induced from day 5 to 7 [52, 53].
Moreover, plasmids overexpressing the mouse Fut1 gene (Advert-Fut1, Fut1-OE) and plasmids for pulling down the mouse Fut1 gene had been synthesized by HanBio. Fut1-OE and adenoviral empty vector (NC-OE), in addition to sh-Fut1 and sh-NC, had been individually transfected into human embryonic kidney cell line AD293 (Agilent, 240085) for viral packaging utilizing LipoFiter transfection reagent (HanBio, HB-LF-1000). Supernatant containing viruses was collected 72 h later. Excessive titer adenovirus was purified utilizing Vivapur® AdenoPACK™ 20 package (Sartorius, VS-AVPQ022) and subsequently titrated by endpoint dilution methodology. Following 5 days of incubation with GM-CSF and IL-4, iDCs had been seeded into 24-well plates at a focus of 106 cells per nicely. The cells had been then contaminated with Fut1-OE or NC-OE, in addition to sh-Fut1 and sh-NC at multiplicities of an infection (MOI) of 10, 50, 100, or 1000. After 4 h of transfection, the transfection medium was changed with recent tradition medium and incubated for an extra 18 h. Subsequently, LPS was used to induce iDCs into mDCs [54, 55].
NC-OE-iDCs/sh-NC-iDCs had been transfected for 4 h earlier than being switched to a recent tradition medium for an 18-h incubation. Likewise, Fut1-OE-iDCs/sh-Fut1-iDCs underwent transfection and subsequent tradition change. The NC-OE-mDCs/sh-NC-mDCs group was subjected to continued induction with LPS and PBS for two days following the transfection of NC-OE-iDCs/sh-NC-mDCs. Equally, the Fut1-OE-mDCs/sh-Fut1-mDCs group obtained the identical induction with LPS and PBS post-transfection of Fut1-OE-iDCs/sh-Fut1-iDCs. The group NC-OE-mDCs + P-D2-EVs was maintained with LPS + P-D2-EVs induction after the NC-OE-iDCs/sh-NC-iDCs transfection. Furthermore, the group Fut1-OE-mDCs + P-D2-EVs/sh-Fut1-mDCs + P-D2-EVs underwent the identical induction with LPS + P-D2-EVs post-transfection of Fut1-OE-iDCs/sh-Fut1-iDCs. Lastly, the group Fut1-OE-mDCs + 2DGal was induced with LPS and 2DGal following the transfection of Fut1-OE-iDCs [52, 54, 55].
Within the CD4+ T group, solely CD4+ T cells had been cultured for five days with out the addition of mDCs, with an equal quantity of tradition medium. Within the CD4+ T + mDCs group, mDCs had been co-cultured with CD4+ T cells in a ratio of 1:10 for five days. The CD4+ T + P-D2-EV-mDCs group signifies the co-culturing of P-D2-EV-mDCs with CD4+ T cells. The CD4+ T + NC-OE-mDCs group signifies the co-culturing of NC-OE-mDCs with CD4+ T cells. The CD4+ T + P-D2-EV-NC-OE-mDCs group entails the co-culturing of P-D2-EV-NC-OE-mDCs with CD4+ T cells. The CD4+ T + Fut1-OE-mDCs group demonstrates the co-culturing of Fut1-OE-mDCs with CD4+ T cells. The CD4+ T + P-D2-EV-Fut1-OE-mDCs group denotes the co-culturing of P-D2-EV-Fut1-OE-mDCs with CD4+ T cells [38, 50].
Moreover, within the CD4+ T + anti-IL10Rα + P-D2-EV-mDCs group, CD4+ T cells had been pre-treated with IL10Rα blocking antibody (5 μg/ml, R&D, AF-474-NA) for 30 min earlier than co-culturing. Within the CD4+ T + anti-IL10 + P-D2-EV-mDCs group, an extra anti-mouse IL10 monoclonal antibody was added throughout co-culturing (75 ng/ml, R&D, MAB417-SP). The CD4+ T + IL10 + Fut1-OE-mDCs group concerned the extra addition of IL10 (10 ng/ml, Solarbio, P00104) throughout co-culturing [35, 38].
Lastly, the supernatant of the co-cultured mDCs and CD4+ T cells was divided into 8 teams for co-culture with ILC2, particularly ILC2 + CD4+ T, ILC2 + CD4+ T + mDCs, ILC2 + CD4+ T + P-D2-EV-mDCs, ILC2 + IL4 + CD4+ T + P-D2-EV-mDCs, ILC2 + CD4+ T + NC-OE-mDCs, ILC2 + CD4+ T + Fut1-OE-mDCs, ILC2 + anti-IL4 + CD4+ T + Fut1-OE-mDCs, and ILC2 + anti-IL4Rα + CD4+ T + Fut1-OE-mDCs.
Within the ILC2 + CD4+ T group, the supernatant from the CD4+ T group was co-cultured with ILC2 for 3 days. Within the ILC2 + CD4+ T + mDCs group, the supernatant from the CD4+ T + mDCs group was co-cultured with ILC2. Within the ILC2 + CD4+ T + P-D2-EV-mDCs group, the supernatant from the CD4+ T + P-D2-EV-mDCs group was co-cultured with ILC2. Within the ILC2 + IL4 + CD4+ T + P-D2-EV-mDCs group, an extra 20 ng/ml IL-4 was added throughout co-culturing. Within the ILC2 + CD4+ T + NC-OE-mDCs group, the supernatant from the CD4+ T + NC-OE-mDCs group was co-cultured with ILC2. Within the ILC2 + CD4+ T + Fut1-OE-mDCs group, the supernatant from the CD4+ T + Fut1-OE-mDCs group was co-cultured with ILC2. Within the ILC2 + anti-IL4 + CD4+ T + Fut1-OE-mDCs group, an extra 100 ng/ml IL-4 and anti-IL4 antibody (ThermoFisher, MA5-23,722) had been added throughout co-culturing. Within the ILC2 + anti-IL4Rα + CD4+ T + Fut1-OE-mDCs group, an IL-4Rα blocking antibody (1 µg/ml, Santa Cruz Biotechnology, sc-28361) was added throughout co-culturing [35, 38, 56].
Intracellular cytokine staining of extracellular cells utilizing circulation cytometry
For intracellular cytokine staining, cells had been first fastened with 4% paraformaldehyde (Biosharp, BL539A) for 20 min, adopted by permeabilization utilizing eBioscience™ Intracellular Fixation & Permeabilization Buffer (ThermoFisher, 88-8824-00). The fastened and permeabilized cells had been then resuspended in eBioscience™ Movement Cytometry Staining Buffer (ThermoFisher, 00-4222-57) and co-incubated with the next antibody markers in the dead of night for 30 min: PE-conjugated IL10 (ThermoFisher, 12-7101-82), APC-conjugated IL4 (ThermoFisher, 17-7041-82), Alexa Fluor™ 700-conjugated GATA3, eFluor™ 660-conjugated IL13 (ThermoFisher, 50-7133-82), and eFluor™ 450-conjugated IL5 (ThermoFisher, 48-7052-82). After co-incubation, cells had been washed twice with FACS washing buffer and subjected to differential expression evaluation utilizing an LSRII Fortessa circulation cytometer (BD Biosciences). Knowledge obtained had been analyzed utilizing FlowJo-X software program [57].
ELISA
Mouse serum or cell tradition supernatant was collected and the degrees of IL-10, IL-4, IL-13, and IL-5 had been quantified utilizing Mouse ELISA kits bought from ThermoFisher (IL-10: BMS614, IL-4: BMS613, IL-13: BMS6015, IL-5: M551). The assays had been carried out in response to the producer’s directions to measure the concentrations of IL-10, IL-4, and IL-13 within the serum or cell tradition supernatant [35].
Western blot
RIPA complete protein lysis buffer (AS1004, Wuhan Aspen Biotechnology Co., Ltd.) was used to lyse EVs, P-D2-EVs, and cells. The protein concentrations had been measured utilizing the BCA protein quantitation assay package (Thermo Fisher, 23227). The proteins had been separated by SDS-PAGE and transferred to a PVDF membrane, which was blocked with 5% BSA at room temperature for 1 h. Subsequently, the next main antibodies, diluted as indicated, had been added: CD9 (1:1000, Abcam, ab307085), CD81 (1:1000, Abcam, ab109201), CD63 (1:1000, Abcam, ab134045), Alix (1:1000, Abcam, ab275377), Calnexin (1:20,000, Abcam, ab92573), Fut1 (1:500, Proteintech, 17956-1-AP), ICAM1 (1:250, Thermo Fisher, MA5407), UEA1 antibody (1:500, BIOSS, bs-10025R), p-P38 MAPK (1:1000, CST, #9212), P38 MAPK (1:1000, CST, #9211), and GAPDH (1:10,000, Thermo Fisher, AM4300). The first antibodies had been incubated in a single day at 4 °C.
CD9, CD81, CD63, and Alix are markers of EVs, whereas Calnexin is a marker of the endoplasmic reticulum membrane. FUT1 and ICAM1 function key regulatory proteins, and UEA1 particularly acknowledges α-(1,2)-fucose glycans. p-P38 MAPK and P38 MAPK are markers of the P38 MAPK signaling pathway.
The membrane was washed thrice with TBST (5 min every wash) after which incubated at room temperature for two h with the suitable secondary antibodies: Anti-Mouse-HRP (1:10,000, Thermo Fisher, 31430) or Goat anti-Rabbit-HRP (1:10,000, Thermo Fisher, 31460). Afterwards, the membrane was washed thrice with TBST (5 min every wash). TBST was eliminated, and an applicable quantity of ECL working resolution (Millipore, WBKLS0500) was added, adopted by incubation at room temperature for 1 min. Extra ECL reagent was eliminated, and the membrane was sealed with plastic wrap, positioned in a darkish field, and uncovered to X-ray movie for five–10 min. The movie was then developed and stuck. Picture J software program was used for densitometric evaluation of the Western blot bands, with GAPDH used as an inner reference [38].
Enrichment experiment of Ulex europaeus agglutinin 1 (UEA1)
To gather totally different handled cell traces, we lysed the cells utilizing NP40 cell lysis buffer (ThermoFisher, FNN0021) supplemented with a protease inhibitor (ThermoFisher, A32953). The cell lysate (1000 µg) was then blended with 100 μl of UEA1 (0.1 µg/ml; Xian QiYue Biotechnology Co., Ltd., Q-0003008) labeled with biotin. The combination was incubated in a single day at 4℃ with mild rotation. Streptavidin Agarose (ThermoFisher, SA10004) beads had been added, containing an affinity for biotin, and the incubation was continued for an additional 4 h. The samples had been extracted and separated by SDS-PAGE after which transferred onto a PVDF membrane. The membrane was incubated with antibodies in opposition to FUT1, ICAM1, and GAPDH, with GAPDH serving as a management. The antibody info is in line with the Western blot part. FUT1 and ICAM1 had been used to detect the entire protein in numerous enter samples, whereas ICAM1 was additionally used to evaluate the extent of ICAM1 fucosylation after UEA1 enrichment [52, 58].
Immunoprecipitation (IP)
Immunoprecipitation was carried out in response to the producer’s directions utilizing the Protein A Immunoprecipitation Package (Beyotime, 2006). mDCs cells subjected to totally different therapies had been lysed utilizing NP40 cell lysis buffer supplemented with protease inhibitors and incubated on ice for 1 h. The lysates containing proteins had been incubated in a single day at 4 °C with Sepharose beads coupled with main antibodies in opposition to ICAM1 and UEA1. The proteins had been eluted by the lysing buffer, resuspended in SDS pattern buffer, and subsequently analyzed by Western blot. The antibody info was in line with the Western blot outcomes [58].
Immunofluorescence staining
Cells had been cultured on glass slides and stuck with 4% paraformaldehyde. Cell membranes had been permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, X100), and non-specific binding was blocked with BSA (Solarbio, SW3015). Slides had been incubated in a single day with main antibodies in opposition to ICAM1 (Thermofisher, MA5-43106) at a dilution of 1:60. After washing with PBST, cells had been incubated with Alexa Fluor™ 594 goat anti-rabbit IgG secondary antibody (Thermofisher, A-11012) at a focus of two μg/ml. Following a PBST wash, FITC-labeled UEA1 (Thermofisher, L32476) was used for UEA1 staining at a focus of 5 μg/ml for 1 h at 37 °C. Nuclei had been counterstained with DAPI (Invitrogen, D3571) for five min at room temperature, after which the slides had been sealed. ICAM1 and UEA1 co-localization fluorescence intensities had been quantitatively analyzed utilizing ImageJ software program with three impartial fields of view for every group below an Olympus BX40 fluorescence microscope [59, 60].
Statistical evaluation
All experiments had been performed independently a minimum of thrice, and the info are offered because the imply ± SD (commonplace deviation). Impartial samples t-test or one-way evaluation of variance (ANOVA) had been employed to match variations between teams. If the ANOVA outcomes indicated vital variations, additional Tukey’s actually vital distinction (HSD) post-hoc exams had been performed to match variations amongst particular person teams. For non-normal distribution or unequal variances, the Mann–Whitney U check or Kruskal–Wallis H check had been employed. All statistical analyses had been carried out utilizing GraphPad Prism 8.0 [61]. Statistical significance was set at P < 0.05.