Animals

All animal experimental procedures were approved by the Dong-A University Medical School Institutional Animal Care and Use Committee (Approval No. DIACUC-21-11) at 22.03.2021. Female BALB/c mice that were 78weeks old were purchased from Orient Bio Inc. (Gyeonggi-do, korea). The classification of experimental groups involves the random assignment of mice of the same age and within a 1g difference in body weight after the acclimatization process. The mice are then housed in a pathogen-free facility at Dong-A University (Busan, Korea). Mice were maintained at Dong-A University facility at 22C1C room temperature, 4060% humidity, on a 12h lightdark cycle (7a.m. to 7p.m.), and given food and water freely, according to institutional guidelines. All experiments were performed under inhalation anesthesia with isoflurane, and mice were euthanized by CO2 inhalation at the end of the experiment. This study adhered to the guidelines set forth by the laboratory animal ethics committee of Dong-A University and the ARRIVE guidelines. To ensure statistical significance, 5 or more mice per group were used, and all experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Dong-A University. Inhalational anesthesia using isoflurane was used to induce anesthesia when sacrificing all experimental animals.

Contact urticaria mouse model was induced according to a previously reported method31,32. Mice were initially sensitized by applying 100l of a TMA (trimellitic anhydride; 500mg/ml, Alfa Aesar, Ward Hill, MA, USA) in acetone/olive oil (4:1, v/v) on the shaved hind flank. This sensitization process is essential for inducing an immune response to TMA. Secondary sensitizations were performed on the hind flank to reinforce the immune response. On the 7th and 10th days after the first sensitization, mice sensitized 50l of a TMA solution (250mg/ml) in acetone/olive oil (4:1, v/v). On day 13 after the initial sensitization, contact urticaria (CU) was induced by challenging the ears with 25l of a TMA solution (100mg/ml) dissolved in acetone/olive oil (4:1, v/v). The disease symptoms were assessed by measuring ear thickness, itching, and lesions on the skin. Particularly, skin lesions were evaluated by determining the ratio of the affected area, indicating erythema and edema on a 3 cm2 area of the dorsal skin. The symptom evaluation of experimental animals was evaluated based on all animals without exclusion criteria. For histological analysis, H&E and mast cell staining were conducted. Cellular and molecular analysis involved the use of flow cytometry to assess immune cell activity in mouse lymphoid organs, along with genetic analysis of the lesions. Experimental animals of 10 mice per group were performed by blindly selecting 56 mice for efficient handling of animal-derived flow cytometric and genetic analysis. The corresponding author and one of the first authors (S.Y. Hyun) were aware of the group assignment, and the symptoms, flow cytometry, and other molecular analysis results were evaluated together by multiple blinded co-authors.

For the purposes of in vitro experiments, we used the nave CD3+ T cell isolation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) to enrich nave CD3+ cells from the spleens of BALB/c mice (6weeks old). All steps were conducted strictly following the manufacturers protocol.

M-MSCs used in this study was provided Mirae Cell Bio (Seoul, Korea). M-MSCs differentiated from H9 hESCs30 were maintained in EGM2-MV medium (Lonza, San Diego, CA, USA) containing supplement Mix (promocell, Heidelberg, Germany) and 50 ug/ml Gentamicin (Gibco, NewYork, USA) in a humidified atmosphere containing 5% CO2 at 37, as previously described33. M-MSCs at less than ten passages were used for in vitro cell culture and in vivo animal experiments. Bone marrow-derived MSCs (BM-MSCs) were maintained in MSCBM medium (Lonza, Basel, Switzerland) containing supplement kit (Lonza) in a humidified incubator at 5% CO2 at 37. Bone marrow-derived mast cells (BMMCs) derived from BALB/c mice were cultured in RPMI 1640 medium containing 2mM L-glutamine, 0.1mM nonessential amino acids, antibiotics, 10% fetal bovine serum (FBS), and IL-3 (10ng/ml; PeproTech Inc., Rocky hill, NJ, USA). After 4weeks,>98% of the cells were verified as BMMCs, as previously described34. Mouse splenic T cells were presorted by CD3 mAb-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by the manufacturer's method. For T cell polarization, splenic nave CD3+ T cells were cultured onto a 24-well plate coated with 1g/ml of anti-CD3 (eBioscience, San Diego, CA, USA) in complete RPMI 1640 medium and supplemented with TH1 reagents [IL-2 (20ng/ml, PeproTech Inc.), IL-12 (20ng/ml, PeproTech Inc.), and anti-IL-4 (10g/ml, Bio X cell, West Lebanon, NH, USA)] or TH2 reagents [IL-2 (20ng/ml, PeproTech Inc.), IL-4 (100ng/ml, PeproTech Inc.), and anti-IFN- (10g/ml, Bio X cell)]. After 48h, the cells were co-cultured with M-MSCs for 24h under polarized conditions. To confirm the regulation of immune cells by M-MSCs in vitro, BMMCs (5.0105 cells/well), splenic T cells (5.0105 cells/well), or polarizing splenic T cells (5.0105 cells/well) were co-cultured with the indicated ratio of M-MSCs for 24h. Co-cultured splenic T cells were identified by flow cytometry analysis. The analysis of polarized T cells was assessed by distinguishing them using the gating strategy as depicted in Supplementary Fig.S1. The ratio of degranulation of BMMCs was analyzed by -hexosaminidase secretion.

After the primary sensitization of the contact urticaria model, M-MSCs were injected subcutaneously into the ear as a single administration on the 10th days or twice on the 10th and 12th days. A primary disease improvement evaluation was performed through single administration or two administrations, and an appropriate cell administration group for disease efficacy was selected. The administration of an equal amount of BM-MSCs and oral administration of cetirizine (50mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. To deplete TGF-, BALB/c mice were intraperitoneally injected with 300g of anti-TGF- mAb (1D11.16.8, Bio X cell) or an isotype-matched control mAb (Bio X cell) twice on days 0 and 3 of M-MSCs administration.

Single-cell suspensions were isolated from the spleen, cervical lymph node (cLN), and ear. Ear tissues were isolated into single cells using the gentleMACS dissociator (Miltenyi Biotec) followed by the manufacturers method. For the detection of intracellular cytokines, the isolated cells were stimulated with PMA (50ng/ml; Sigma-Aldrich), ionomycin (500ng/ml; Sigma-Aldrich), and brefeldin A (3g/ml; eBioscience) for 4h before analysis and a fixation/permeabilization kit were from eBioscience. Before cell surface markers were stained, Fc receptors were blocked with anti-CD16 and anti-CD32 mAbs (2.4G2, BD Biosciences), and conjugated and dead cells were excluded by analysis on the basis of forward and side light scatter parameters and staining with a Zombie NIR Fixable Viability Kit (Biolegend, San Diego, CA, USA). The antibodies against proteins were as follows: Antibodies against CD3 (17A2) and CD8a (53-6.7) were obtained from BioLegend. Antibodies for CD4 (RM4-5), IFN- ((XMG1.2), IL-4 (11B11) were obtained from eBioscience. Antibodies for CD3 (17A2), CD45 (30-F11), and CD127 (A7R34) were obtained from BioLegend. The cells were then analyzed with a NovoCyte flow cytometer (Agilent) and FlowJo version 10 software (Tree Star, Ashland, OR, USA).

BMMCs (5.0105 cells/well) co-cultured with M-MSCs (0.5 to 2106 cells/well) for 24h were sensitized for 4h with Monoclonal dinitrophenol (DNP)-specific IgE (100ng/ml; Sigma). The IgE-primed BMMCs were then stimulated with 50ng/ml of DNP-human serum albumin (DNP-HSA, Sigma-Aldrich) in Tyrode-BSA buffer (20mM Hepes (pH 7.4), 135mM NaCl, 5mM KCl, 1.8mM CaCl2, 1mM MgCl2, 5.6mM glucose, and 0.1% BSA) for 15min in the presence or absence of the M-MSCs.

Degranulation was determined by measuring the release of the granule marker -hexosaminidase as previously described35. The degree of degranulation of BMMCs was expressed as the % of the activity of -hexosaminidase secreted out of the cells compared to the total activity of -hexosaminidase.

M-MSCs were co-cultured with splenic T cells or BMMCs for 24h and then effector cells were removed. M-MSCs were rinsed with PBS and left on ice for 5min to stop the reaction. Total RNA was extracted using AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, Korea), and cDNA was synthesized using AccuPower CycleScript RT PreMix (Bioneer) according to the manufacturers instructions. The PCR reaction was amplified using AccuPower PCR PreMix (Bioneer) and PCR was performed at 95 for 2min, 95 for 20s, 58 for 40s, 72 for 30s, 72 for 5min for 30 cycles. Primers used as follow: human Hgf (forward 5-TCCATGATACCACACGAACACAGC-3, reverse 5-TGCACAGTACTCCCAGCGGGTGTG-3); human Ido1 (forward 5-TTTGCTAAAGGCGCTGTTGG-3, reverse 5-CCTTCATACACCAGACCGTCTGA-3); human Pdl1 (forward 5-TATGGTGGTGCCGACTACAA-3, reverse 5-TGCTTGTCCAGATGACTTCG-3); human Il10 (forward 5-AGACATCAGGGTGGCGACTCTAT-3, reverse 5-GGCTCCCTGGTTTCTCTTCCTAAG-3); human Pge2 (forward 5-ACCATCTACCCCTTCCTTT-3, reverse 5-CCGCTTCCCAGAGGATCT-3); human Tgfb (forward 5-GGGACTATCCACCTGCAAGA -3, reverse 5-CCTCTTGGCGTAGTAGTCG-3); human Gapdh (forward 5-ACCACAGTCCATGCCATCAC-3, reverse 5-TCCACCACCCTGTTGCTGTA-3). Snap-frozen disease-inducing mouse ear tissues were ground to powder. Total RNA isolation and PCR reaction were performed in the same manner as above. Real-time PCR was performed Thermal Cycler Dice Real Time System III TP950 (Takara, Shiga-ken, Japan). Primers used as follow: mouse Il4 (forward 5-ACAGGAGAAGGGACGCCAT-3, reverse 5-GAAGCCCTACAGACGAGCTCA-3); mouse Il6 (forward 5-GAGGATACCACTCCCAACAGACC-3, reverse 5-AAGTGCATCATCGTTGTTCATACA-3); mouse Ifng (forward 5-CAGCAACAGCAAGGCGAAAAAGG-3, reverse 5-TTTCCGCTTCCTGAGGCTGGAT-3); mouse Tnfa (forward 5-AGTGACAAGCCTGTAGCCCACGT -3, reverse 5-CCATCGGCTGGCACCACTAGTT-3); mouse Gapdh (forward 5-CATCACTGCCACCCAGAAGACTG-3, reverse 5-ATGCCAGTGAGCTTCCCGTTCAG-3);

After the induction of contact urticaria in mice, their ear tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline for 24h and then embedded in paraffin. The tissues were dehydrated in a graded ethanol series (70 to 100%), rinsed three times with xylene for 3min each, and then embedded in paraffin. Sections of paraffin-embedded tissues, with a thickness of 6m, were prepared and stained with hematoxylin (Sigma-Aldrich) and eosin (Sigma-Aldrich) to compare and analyze the degree of cell invasion and epidermal thickness in the tissue. Additionally, sections of tissues with a thickness of 6m were stained with a 1% toluidine blue (Sigma-Aldrich) solution to assess the number of infiltrating mast cells and the degree of degranulation.

The in vitro experiment was repeated three independent times, and the animal experiment was based on five or more animals per group, and if the results of the first experiment were insufficient, the significance was evaluated within a total of 10 animals in the group. The data are presented as the meanstandard error (SEM) from three or more independent experiments for in vitro experiments. Statistical analysis was done by unpaired Student's t-test. One-way analysis of variance (ANOVA) with Tukey's post hoc test was performed for multiple comparisons. Statistical significance (*P<0.05 and **P<0.01) was determined with Prism version 7.0 (GraphPad, San Diego, CA).

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Dong-A University(DIACUC-21-11). All animal experiments were performed in accordance with the guidelines and regulationsof the institutional guidelines.

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