All procedures were approved by the Institutional Review Board of Jining Medical University and were conducted in accordance with the 1964 Declaration of Helsinki and its later amendments. All participants provided written, informed consent. Healthy individuals (n = 10) and patients with psoriasis (n = 14) were randomly selected and subjected to peripheral blood drawing (6 mL/person). Human PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque (GE Healthcare Bio-Science AB, Uppsala, Sweden). Plasma samples were stored at –80°C for subsequent analysis. PBMCs at a concentration of 1 × 10⁶/mL were maintained in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

C57BL/6 mice (8 weeks old) were administered a daily dose of 62.5 mg/mouse IMQ cream (Mingxin Pharmaceutical Co. Ltd., Chengdu, China) on their shaved back skin for 7 days and the mice were randomly and blindly divided into control and experimental groups. To determine the therapeutic potential of IL-35, we administered IL-35 (5 μg) (Sino Biological, Beijing, China) one day before the establishment of the IMQ-induced psoriasis mouse model, with three additional injections administered every other day (Days 2, 4, and 6) during the establishment of the model. Twenty-four hours after the last injection, the mice were photographed. The mice were euthanized, and serum and psoriatic lesions were collected. The spleen and skin were used for flow cytometry and histological examination. All animal procedures were approved by the Institutional Animal Ethics Committee (approval number: 2018-JC-003).

To evaluate the severity of inflammation in the ears and neck skin of mice, we used an objective scoring system based on the clinical Psoriasis Area and Severity Index (PASI). Erythema, scaling, and thickening were scored independently on a scale of 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked (26).

Mouse ear and dorsal skin tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. Images were acquired using an Olympus BX600 microscope (Olympus Corp., Tokyo, Japan) and assessed using the Baker scoring system (27).

The expression of human C-X-C motif chemokine ligand 8 (CXCL8, Cat: 431507) and IL-6 (Cat: 430507) in the cell culture supernatant and that of other proinflammatory factors, including mouse IL-17A (Cat: 432501), IL-1β (Cat: 432604), and IL-6 (Cat: 431307), in the serum and skin of mice were detected by ELISA purchased from Biolegend (San Diego, CA, USA). Mouse IL-23 ELISA kit (CSB-E-08463m) was provided by CUSABIO (Wuhan, China). The plates were washed with phosphate-buffered saline (PBS) containing Tween-20 (0.05%) and blocked with assay diluent for 1 hour at 37°C. After washing, samples were serially diluted with assay diluent, added to the plates, and incubated for 2 hours at 37°C, followed by the addition of diluted detection antibody at 37°C. The plates were washed again. Avidin-horseradish peroxidase was added and incubated for 30 minutes at 37°C. Tetramethylbenzidine substrate solution was then added and incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μL of acid stop solution. Absorbance was measured at 450 nm within 20 minutes using an ELISA reader (Bio-Rad Laboratories, RRID : SCR_008426).

PBS supplemented with 2% heat-inactivated fetal bovine serum was used as the staining buffer for flow cytometry. PBMCs from healthy individuals and patients with psoriasis were resuspended in staining buffer for extracellular staining of CD4, CD19, CD14, CD15, CD11b, and human leukocyte antigen DR (HLA-DR), as well as intracellular staining of EBI3 and p35. Directly-labelled isotype-matched rat anti-mouse antibodies (BioLegend, San Diego, CA, USA) were used as controls. For intracellular staining, the cells were first stimulated with 50 ng/mL phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO, USA) and 1.0 μg/mL ionomycin (Sigma-Aldrich) for 2 hours and then cultured for 4 hours in the presence of 2 μL monensin (BioLegend). After extracellular staining, the PBMCs were fixed and permeabilized (Fixation/Permeabilization Solution Kit; BD Biosciences, St. Louis, MO, USA), according to the manufacturer’s protocol. EBI3 and p35 staining were subsequently performed.

The spleens of treated mice were collected, homogenized, and suspended as single cells in red blood cell lysis buffer. Skin tissues from treated mice were collected, added with type IV collagenase (2 mg/ml, Cat: 17104019, Gibco) and DNase I (25 U/ml, Cat: SSNP0, SinoBiological) in 1640 medium, with shock digestion for 2 hours at 220 r/min at 37 °C. After filtration and centrifugation, bottom cells were suspended as single cells flow cytometry. We used Zombie Aqua™ (Cat: 423101, Biolegend) for dead cell exclusion as follows: Single-cell suspension was prepared and cells were washed with PBS buffer (no Tris buffer and protein free). Zombie Aqua™ dye was diluted at 1:500 in PBS. Cells at 1×10⁷ were diluted in 100 µl Zombie Aqua™ solution. Then cells were extracellularly stained with CD11b, Gr-1, Ly6G, and Ly6C for 30 minutes at 4°C in the dark. Directly-labelled, isotype-matched rat anti-mouse antibodies (BioLegend) were used as controls. After washing twice with PBS, the cells were resuspended in 200 mL of PBS and analyzed via flow cytometry. For intracellular staining, the cells were stained extracellularly and then fixed and permeabilized (Fixation/Permeabilization Solution Kit; BD Biosciences, Franklin Lakes, NJ, USA) for 30 minutes at 4°C. Finally, the cells were intracellularly stained with anti-iNOS and anti-IL-10.

The following antibodies were used: Fluorescein isothiocyanate (FITC) anti-human CD4, anti-mouse CD11b (BioLegend), anti-human HLA-DR (BD Biosciences). Brilliant Violet 421 (BV421) anti-human CD19, anti-human CD11b (BioLegend). phycoerythrin (PE) anti-human CD15 (BD Biosciences, San Diego, CA, USA), anti-human p35, anti-mouse Ly6G, anti-mouse iNOS, anti-mouse IL-10 (BioLegend). Allophycocyanin (APC) anti-human EBI3, anti-mouse Gr-1, anti-mouse Ly6C (BioLegend).

Bone marrow cells were isolated from mice by flushing their femurs and tibiae, centrifuging the elution at 1,800 rpm for 5 minutes, and resuspending the cells in complete Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Shanghai, China) supplemented with 40 ng/mL IL-6 (Peprotech, Suzhou, China) and 40 ng/mL granulocyte-macrophage colony-stimulating factor (Peprotech). Bone marrow cells were maintained at 37°C in a 5% carbon dioxide humidified atmosphere for 4 days. To investigate the effect of IL-35 on the expansion of MDSCs, IL-35, IL-6, and granulocyte-macrophage colony-stimulating factor were added to the medium simultaneously.

We performed immunofluorescence staining for IL-35 and M-MDSCs in the skin of patients with psoriasis and healthy controls and for MDSCs and iNOS⁺ MDSCs in murine skin lesions. Mice were treated with IL-35; the back skin was obtained the day after the last treatment, frozen, and then stained. Samples were incubated with primary antibodies (rat anti-human p35 and CD14, anti-mouse CD11b and iNOS, rabbit anti-human EBI3⁺ and HLA-DR, anti-mouse Gr-1) (Abcam) at 4°C overnight, followed by directly-labelled IgG anti-rat or rabbit antibodies (ZsBio, Beijing, China) for 1 hour. Tissue sections were examined under a fluorescent microscope (Olympus Optical, Tokyo, Japan). Images were captured at zoom magnification.

For adoptive transfer, 2 × 10⁶ CD11b⁺Gr1⁺ cells isolated from the spleen of IMQ-induced (wild-type [WT] and iNOS knockout [iNOS^-/-]) mice by flow cytometry were washed twice, resuspended in 200 μL PBS and injected into the tail vein. MDSCs were injected the day before the application of IMQ and injected again on the third day after the application of IMQ.

All the results were analyzed using the GraphPad Prism (GraphPad Prism, RRID : SCR_002798). Data are presented as means ± standard error (SEM). One-way analysis of variance (ANOVA) and Student’s t-test were used to analyze the differences between multiple groups and two groups, respectively. Statistical significance was set at P < 0.05.

To investigate the involvement of IL-35 in skin diseases, we examined IL-35 levels in the serum of healthy individuals and patients with psoriasis. We recruited patients (n = 53) and age- and sex-matched healthy controls (n = 20); the demographic characteristics are summarized in Table 1 . ELISA showed that serum IL-35 levels were significantly increased in patients with psoriasis compared to those in healthy controls (P = 0.0089; Figure 1A ). Moreover, as the symptoms of psoriasis worsened (higher PASI score), the expression of IL-35 increased. Specifically, the expression of IL-35 in specimens with a PASI score ≥ 3 was significantly higher than that in those with a PASI score < 3 ( Figure 1A ). Flow cytometry also revealed increased populations of CD4⁺EBI3⁺p35⁺ and CD19⁺EBI3⁺p35⁺ cells in the peripheral blood of patients with psoriasis ( Figures 1B–D ). We demonstrated that IL-35 secreted by both T and B lymphocytes was significantly increased (P < 0.01 and P < 0.05, respectively). Moreover, immunofluorescence staining showed that IL-35 expression was significantly increased in the skin of patients with psoriasis ( Figure 1E ).

We examined whether the number of M-MDSCs was altered in patients with psoriasis. We recruited patients (n = 14) and age and sex-matched healthy donors (n = 10); the demographics are summarized in Table 2 . Flow cytometry confirmed that the number of human M-MDSCs (CD11b⁺CD14⁺HLA-DR^-) was considerably higher in the peripheral blood of patients with psoriasis (P = 0.0065; Figures 2A, B ). Immunofluorescence staining of CD14⁺HLA-DR^- M-MDSCs in the inflamed skin of patients with psoriasis revealed that the infiltration of M-MDSCs in psoriatic skin tissue increased significantly compared to that in healthy controls ( Figure 2C ).

The IMQ-induced psoriasis mouse model is the most common model of psoriasis (28). The experimental procedures and acquisition of images of mouse back skin are shown in Figures 3A, B . Based on PASI scoring of erythema, scaling, and thickness, we determined the cumulative scores for different groups ( Figure 3C ). The IMQ group had higher cumulative scores than the control group. Flow cytometry was used to detect changes in MDSCs populations in the spleen and skin of mice with IMQ-induced psoriasis. The numbers of MDSCs in the spleen and skin of mice in the IMQ group were significantly higher than those in the control group (P < 0.001 and P < 0.05, respectively; Figure 3D ). The results were consistent with those obtained using clinical samples.

We stimulated HaCaT cells with M5 in combination with IL-35 to observe the effect of IL-35 on CXCL8 and IL-6 expression. We found consistently lower levels of CXCL8 and IL-6 in the IL-35-treated group than in the control group, suggesting that IL-35 suppresses the expression of CXCL8 and IL-6 in stimulated HaCaT cells ( Figures S1A, B ).

Next, we determined whether IL-35 has a therapeutic effect in the IMQ-induced psoriasis mouse model. The schedule of IL-35 administration in mice with IMQ-induced psoriasis is shown in Figure 4A . After treatment, the control group had serious inflammation and skin flaking, which had worsened by the end of the experiment ( Figure 4B ). Based on PASI scoring of erythema, scaling, and thickness ( Figure 4C ), we determined the cumulative scores for different groups ( Figure 4C ). The IL-35 group had lower cumulative scores than the control group. H&E staining revealed reduced epidermal thickening in the IL-35 group compared to the control group ( Figure 4D ). The Baker score of the IL-35-treated group was significantly lower than that of the control group (P < 0.001; Figure 4E ).

To further investigate the therapeutic effect of IL-35, we treated IMQ-induced psoriasis mice with p35 neutralizing antibodies. The symptoms in the p35 neutralizing antibody group were not only substantially worse than those in the IL-35-treated group but also worse than those in the IgG control group ( Figure S2 ).

We also explored whether IL-35 could have a therapeutic effect on K14-VEGF-A-Tg mice. K14-VEGF-A-Tg mice (10 weeks old) were injected with 5 μg IL-35 recombinant protein ( Figure S3A ). Mice in the control group showed increased ear thickening; however, mice in the IL-35-treated group were healthy ( Figure S3B ). Pathological features, such as erythema, scaling, and thickness, based on PASI scores, are shown in Figure S3C . The IL-35-treated group had lower cumulative scores than the control group ( Figure S3C ). H&E staining revealed the severity of the disease in the control group ( Figure S3D ; left panel). Conversely, a reversal in ear thickness and reduction in leukocyte infiltration were observed in the dermis and epidermis of the IL-35-treated group ( Figure S3D ; right panel). Moreover, the IL-35-treated group had a lower disease score, based on the Baker scoring system, than the control group ( Figure S3E ).

The above results showed that IL-35 had a therapeutic effect on psoriasis, and MDSCs were expanded in patients with psoriasis and in the mouse model. To test whether IL-35 ameliorates psoriasis by reducing the accumulation of MDSCs, we used flow cytometry to analyze the number of MDSCs in mice with IMQ-induced psoriasis treated with or without IL-35. Compared with that in the control group, the administration of IL-35 reduced the infiltration of MDSCs in the spleen and skin of mice with IMQ-induced psoriasis ( Figures 5A, B ).

Next, we investigated whether G-MDSCs and M-MDSCs populations were altered in response to IL-35 treatment. In the spleen and skin, both G-MDSCs and M-MDSCs populations were substantially reduced ( Figures 5C–E ). Immunofluorescence staining of CD11b⁺Gr-1⁺ MDSCs in inflamed skin showed that IL-35 treatment inhibited the accumulation of MDSCs ( Figure 5F ). These results demonstrated that the immunosuppressive effect of IL-35 on IMQ-induced psoriasis was achieved by reducing the infiltration and proportion of MDSCs. IL-35 can also alter the proportions of MDSCs subtypes. Finally, we examined whether IL-35 directly affects the differentiation and recruitment of MDSCs in vitro using fluorescence-activated cell sorting. IL-35 had little effect on the differentiation of MDSCs ( Figures S4A, B ). IL-35 also did not significantly affect the numbers of G-MDSCs and M-MDSCs ( Figures S4C–E ).

Inflammatory cytokines play an important role in the progression of psoriasis (29). ELISA was used to measure cytokine levels in serum and dorsal skin. Cytokines in supernatants of extracted tissue protein were assayed and are presented as picograms of cytokine per milligram of tissue. IL-17A ( Figure 6A ), IL-23 ( Figure 6B ), IL-1β ( Figure 6C ), and IL-6 ( Figure 6D ) were detected in serum and skin tissue. These results showed that, although there was no significant difference in serum IL-6 levels, other cytokines were significantly different in serum and skin tissue.

To further explore whether the therapeutic effect of IL-35 was related to MDSCs, MDSCs from IMQ-induced mice were adoptively transferred, following the experimental schedule shown in Figure 7A . We found that adoptive transfer of MDSCs from IMQ-induced mice aggravated disease progression. When MDSCs from IMQ-induced mice were transferred to mice in the IL-35-treated group, psoriasis symptoms worsened in these mice compared to those treated with IL-35 alone ( Figure 7B ). We evaluated pathological features, such as erythema, scaling, and thickness, using the PASI scoring system, and found that, compared to the control group, the MDSCs group had higher PASI scores ( Figure 7C ). Similarly, the IL-35+MDSCs group had higher PASI scores than the IL-35-treated group. We determined the cumulative scores for different groups and found that the IL-35-treated group had the lowest cumulative scores ( Figure 7C ).

H&E staining revealed increased epidermal thickening in the MDSCs group compared to the control group. The epidermis in the IL-35+MDSCs group was substantially thicker than that in the IL-35-treated group ( Figure 7D ). Moreover, the Baker score of the IL-35-treated group was the lowest of the four groups ( Figure 7E ).

The above results indicated that IL-35 inhibits the recruitment of MDSCs. Next, we determined whether IL-35 affects the expression of inflammatory mediators in MDSCs. Flow cytometry was used to analyze the expression of iNOS and IL-10, which were secreted by MDSCs in mice with IMQ-induced psoriasis treated with or without IL-35. The results showed that, compared to the control group, the IL-35-treated group had reduced iNOS expression in the spleen and skin ( Figures 8A, B ). However, there was no difference in the level of IL-10 ( Figures 8C, D ). Immunofluorescence staining of iNOS⁺Gr-1⁺ MDSCs in inflamed skin showed that iNOS⁺Gr-1⁺ MDSCs accumulation was inhibited by IL-35 ( Figure 8E ).

To explore the role of iNOS⁺ MDSCs in the pathogenesis of psoriasis, MDSCs from WT or iNOS^-/- IMQ-induced mice were adoptively transferred, following the experimental schedule shown in Figure 7A . The adoptive transfer of WT-MDSCs derived from mice with IMQ-induced psoriasis aggravated disease progression in the PBS group (PBS+WT-MDSCs). However, there was no exacerbation of symptoms when MDSCs from IMQ-induced iNOS^-/- mice were transferred to the PBS group (PBS+iNOS^-/- MDSCs; Figure 9A ). Moreover, when WT-MDSCs were transferred to the IL-35-treated group, psoriasis symptoms were aggravated in these mice compared to those treated with IL-35 alone. There was no significant difference between the IL-35+iNOS^-/- MDSCs and IL-35-treated groups ( Figure 9A ). We evaluated pathological features, such as erythema, scaling, and thickness, using the PASI scoring system, and found that, compared to the PBS group, the PBS+WT-MDSCs group had higher PASI scores ( Figure 9B ). Similarly, the IL-35+WT-MDSCs group had higher PASI scores than the IL-35-treated group. The PBS+iNOS^-/- MDSCs group had similar PASI scores to those of the PBS group. There was no significant difference in PASI scores between the IL-35+iNOS^-/- MDSCs and IL-35-treated groups. We determined the cumulative scores for different groups and found that the cumulative scores were comparable between the PBS+iNOS^-/- MDSCs group and the PBS group, and between the IL-35-treated group and the IL-35+iNOS^-/- MDSCs group ( Figure 9B ).

H&E staining revealed increased epidermal thickening, severe hyperkeratosis, and incomplete keratosis in the PBS+WT-MDSCs group compared to the control group. The PBS+iNOS^-/- MDSCs group had reduced hyperkeratosis and incomplete keratosis compared to the PBS+WT-MDSCs group but similar morphology to that of the PBS group. The epidermis in the IL-35+WT-MDSCs group was substantially thicker than that in the IL-35-treated group; however, the IL-35+iNOS^-/- MDSCs group had similar morphology to that of the IL-35-treated group ( Figure 9C ). Moreover, the Baker score of the IL-35-treated group was the lowest of the six groups ( Figure 9D ). The Baker scores were consistent with the phenotype of each group.