The specimens of skin (5 psoriasis patients and 5 trauma patients with healthy skin) and umbilical cord were obtained from The First Affiliated Hospital of Soochow University following institutional guideline and the ethical approval (20220238). The procedures were applied in accordance with the ethical standards of the Ethics Committee of The First Affiliated Hospital of Suzhou. Informed consent was achieved from patients or their families/legal guardians with psoriasis, who visited dermatology department without any concurrent systemic diseases and therapeutic interventions.

The procedures were conducted in accordance with the guidelines of the Ethics Committee of The First Affiliated Hospital of Suzhou. To isolate MSCs, we removed all blood vessels from the umbilical cord, which was stored in the Phosphate buffered saline (PBS). And the umbilical cord was then minced into small segments, which were subsequently transferred to culture plates containing the complete medium. Next, the cells were collected upon reaching 80% confluence. The MSCs were identified based on their cell surface markers, including cluster of differentiation 73 (CD73), CD29, CD90, CD105, CD45, CD31, CD34, CD11b, human leukocyte antigen (HLA) and HLA- D related isotype (HLA-DR). Antibodies (IgG) were diluted at 1:1000 and procured from BioLegend (San Diego, CA, USA). All samples were collected using a Cytoflex Flow Cytometer (Beckman Coulter, CA, USA), and the data were analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA).

C57BL/6 male mice, aged 10–12 weeks, were procured from Vital River Laboratory (Beijing, China). They were kept under specific pathogen-free conditions. The experiments received approval from the Institutional Animal Care and Use Committee of Soochow University (SUDA20210916A02). The mice received the daily application of 62.5 mg Imiquimod (IMQ) cream (Aldara, 3M Pharmaceuticals, St. Paul, MN, USA) on their shaved backs for a continuous seven-day period. Mice were euthanized by cervical dislocation on day 8. The skin was taken for histological staining and flow cytometry analysis. We evaluated the symptoms of the psoriatic mice by psoriasis area and severity index (PASI), which utilizes a scoring system ranging from 0 to 4 for scaling, erythema, and thickness: minimal (0), mild (1), moderate (2), severe (3), and very severe (4).

Cell suspensions of skin were prepared using collagenase I (1.5 mg/mL, 17100017, Gibco, Waltham, MA, USA) and incubated for 1.5 h at 37 °C, followed by immunolabeling with targeted antibodies, such as CD45 (10 µg/mL, 982322, Biolegend, San Diego, CA, USA) and CD3 (10 µg/mL, 981002, Biolegend). Isotype control antibodies (10 µg/mL, Biolegend) were also used. All samples were then examined by using a Cytoflex Flow Cytometer (Brea, CA, USA).

10⁶ IMSCs and IDO-Knockdown-IMSCs (IDO-KD-IMSCs) were dissolved in 100 µL PBS, and subcutaneously administered on days 1 and 4 post-IMQ application. The control mice received an equivalent volume of PBS and scramble-KD-IMSCs (Scr-KD-IMSCs).

For siRNA transfection, cells were seeded in a 6-well plate and transfected with IDO-siRNA (sense sequence 5^′-CCCUGACUUAUGAGAACAUTT-3^′, antisense sequence 5^′- AUGUUCUCAUAAGUCAGGGTT-3^′) and Scramble-siRNA (sense sequence 5^′-UUCUCCGAACGUGUCACGUTT-3^′, antisense sequence 5^′- ACGUGACACGUUCGGAGAATT-3^′) complexes formed by mixing siRNA with a transfection reagent in optimized-minimum essential medium (Opti-MEM). After incubating for 20 minutes to allow complex formation, the mixture was added to the cells and incubated for 6–8 h. Following this, the transfection medium was replaced with fresh complete medium, and cells were cultured for 24–72 h before assessing knockdown efficiency.

Protein was extracted and quantified from the cells. Then, it was separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, which was incubated overnight with IDO (13268-1-AP, Proteintech, Rosemont, Illinois, USA) and $\beta$-actin antibody (66009-1-Ig, Proteintech). The antibodies were diluted at 1:5000 for Western blot. After washing, secondary antibodies (Proteintech) conjugated with horseradish peroxidase (HRP) were applied for 1 h. The final signals were visualized by the chemiluminescent substrate.

The samples were fixed with 4% paraformaldehyde for 48 h. They were then dehydrated sequentially in 75%, 85%, 90%, 95%, and 100% ethanol solutions. The dehydrated samples were cleared with xylene and then embedded in paraffin. Finally. All samples were sectioned into 3–4 µm slices, and stained with hematoxylin (Beyotime, Nantong, Jiangsu, China) for 6 min and eosin (Beyotime) for 1 min.

Skin sections were then stained with CD3 antibody (10 µg/mL, ab16669, Abcam, Cambridge, UK), and incubated with secondary antibody (10 µg/mL, Abcam) for 1 h at room temperature. Subsequently, they were next stained with hematoxylin, and the images were captured using a microscope (Leica, Wetzlar, Hesse, Germany). Additionally, the results are quantified by enumerating the positive cells in each field, and the scorers were blinded to the experiment.

The experiment consisted of three groups: a positive control group stimulated by CD3 and CD28 antibody, a scramble-KD-IMSC group with the stimulation, and an IDO-KD-IMSC group also stimulated in the same manner. Peripheral Blood Mononuclear Cells (PBMCs) are isolated from whole blood using density gradient centrifugation. Both MSCs and PBMCs were seeded in 96-cell plates, and their proliferative capacity was evaluated using the water-soluble tetrazolium salt-1 (WST-1) cell proliferation and cytotoxicity assay kit (05015944001, Roche, Basel, Switzerland).

Statistical analysis was conducted using Graphpad prism 5 (San Diego, CA, USA). The data were presented as means $\pm$ SEM. The statistical significance for comparing two groups was evaluated using Student’s t-test. For multiple group comparisons, one-way or two-way ANOVA was employed if the F-test showed no significance. The significance levels are indicated as follows: * for p 0.05, ** for p 0.01, *** for p 0.001, and **** for p 0.0001.

We first isolated MSCs from umbilical cord tissues obtained from the First Affiliated Hospital of Soochow University. After removing the arteries and veins, we dissected the umbilical cords and cultured them onto plates (Fig. 1A). Through adherent purification, we obtained a population of homogeneous fibroblast-like spindle cells with robust proliferative capacity. When MSCs were expanded to the second generation, the cells were identified based on their common characteristic, and their surface markers were examined by flow cytometry. We observed positive expression of CD73, CD29, CD90 and CD105 in almost all cells, while CD45, CD31, CD34, CD11b, HLA and HLA-DR were negatively expressed (Fig. 1B). Thus, MSCs were successfully isolated.

Fig. 1.

Mesenchymal stem cells (MSCs) derived from umbilical cord were successfully isolated and identified. (A) The way to isolate MSCs. (B) The surface markers of MSCs were identified by flow cytometry: cluster of differentiation 105⁺ (CD105⁺), CD29⁺, CD73⁺, CD90⁺, CD11b^–, CD45^–, CD31^–, CD3^–, human leukocyte antigen^– (HLA^–) and HLA- D related isotype^– (HLA-DR^–).

The previous study has demonstrated that MSCs primed by IFN-$\gamma$ possess immunomodulatory capabilities to alleviate myocardial infarction [11]. To investigate the therapeutic efficacy of IMSCs on psoriasis, we administered human umbilical cord-derived MSCs to treat psoriasis model. The psoriatic mice were injected with PBS and IMSCs with low immunogenicity subcutaneously on days 1 and 4 after the construction of psoriasis model. Then they were euthanized on day 8 (Fig. 2A). Histological examination was performed to evaluate the efficacy of the treatment. Our results showed that epidermal thickness was decreased in the group treated with IMSCs compared to the group treated with PBS (Fig. 2B). PASI score was also assessed. Likewise, PASI score was decreased following IMSCs treatment (Fig. 2C). Thus, IMSCs can alleviate the inflammation of psoriasis, which play a crucial role in psoriasis treatment.

Fig. 2.

IFN-$\gamma$ primed mesenchymal stem cells (IMSCs) alleviated the inflammation of psoriatic mice. (A) The establishment of IMSCs treatment. (B) Hematoxylin and eosin (H&E) staining of the lesion in the indicated groups. The images were captured at 20 $\times$ magnification (Scale bars: 50 µm); Measurement of epidermal thickness. (C) Psoriasis area and severity index (PASI) scores in the indicated groups, and the scorers were blind to the experiment. Two-way ANOVA was employed (n = 5 mice for each group), * for p 0.05, ** for p 0.01and *** for p 0.001. IMQ, Imiquimod; PBS, Phosphate buffered saline.

As we known, T cells are the main pathogenic factor contributing to psoriasis. To investigate the accumulation of T cells within psoriatic lesions, we conducted an IHC experiment to examine T cell infiltration in samples from both psoriatic patients and mice. As predicted, the lesions were heavily infiltrated by T cells (Fig. 3A,B). To elucidate the underlying mechanisms through which IMSCs exert their therapeutic efficacy, we detected T cell accumulation by flow cytometry after IMSCs treatment (Fig. 3C). Our results indicated that T cells were significantly reduced after IMSCs treatment. Thus, IMSCs can treat psoriasis by inhibiting T cell infiltration.

Fig. 3.

IMSCs inhibited the proliferation of T cell. (A,B) Immunohistochemistry (IHC) staining of the lesion in the psoriatic patients and mice compared with healthy skin. The images were captured at 20$\times$ magnification (Scale bars: 50 µm). (C) T cells of the skin were identified by flow cytometry in the indicated groups (n = 5 mice for each group), *** for p 0.001, and **** for p 0.0001.

It is suggested that the immunomodulatory properties of MSCs depend on the key mediators they produce [10]. Additionally, IDO is the key effector regulating T cell proliferation [12]. However, it still needs to be determined whether IDO is implicated in T cell inhibition in IMSCs-treated psoriasis. To investigate this issue, we examined the expression of IDO by western blot analysis. As predicted, IDO was overexpressed in IMSCs (Fig. 4A). To confirm the immunomodulatory effect of IDO on T cells, we knocked down IDO in IMSCs (Fig. 4B). Moreover, we cocultured PBMCs with Scr-KD-IMSCs and IDO-KD-IMSCs. We observed that the proliferation was inhibited when PBMCs were cocultured with Scr-KD-IMSCs, but this inhibitory effect was eliminated upon IDO knockdown (Fig. 4C). Therefore, it can be concluded that IDO is the key factor in regulating T cell infiltration.

Fig. 4.

IMSCs inhibited the proliferation of T cells by indoleamine-2,3-dioxygenase (IDO). (A) IDO expression in MSCs and IMSCs were examined by western blot. (B) IDO expression in MSCs knocked down by scramble small Interfering RNA (Scr-KD-MSCs) and IDO small Interfering RNA (IDO-KD-MSCs) were examined by western blot. (C) IMSCs inhibited the proliferation of T cells through IDO. Vehicle group was the positive group, which was stimulated by CD3 and CD28 antibody. For three group comparisons, one-way ANOVA was employed. (n = 3 for each group), * for p 0.05 and *** for p 0.001. KD, Knockdown.

To determine the therapeutic efficacy of IDO in the psoriatic model, we treated the psoriatic mice with Scr-KD-IMSCs and IDO-KD-IMSCs (Fig. 5A). We found that the therapeutic efficacy of IMSCs were impaired in the IDO-KD-IMSCs group (Fig. 5B). Furthermore, T cell infiltration in the skin lesions was recovered, and PASI scores were increased after IDO knockdown (Fig. 5C–E). These findings suggested that the therapeutic efficacy of IMSCs was diminished by IDO knockdown. Together, our results indicated that IMSCs could treat psoriasis through restraining T cell infiltration by IDO.

Fig. 5.

The therapeutic effects were abolished after IDO knockdown in IMSCs. (A) The establishment of the experiment. (B) H&E staining of the lesion. The images were captured at 20$\times$ magnification (Scale bars: 50 µm); Quantification of epidermal thickness. (C,D) The T cell percentage was examined when IDO was knocked down in the indicated group. (E) PASI scores of the psoriatic mice. Two-way ANOVA was employed (n = 5 mice for each group), * for p 0.05 and ** for p 0.01. PASI, psoriasis area and severity index.