Figure legends
Fig. 1 Expression of IL-35 increased in psoriatic serum and peripheral blood specimens . (A) Levels of expression of IL-35 in the serum of patients with psoriasis (n = 49). Psoriasis Area and Severity Index (PASI) < 3 (n = 19), PASI ≥ 3 (n = 19), or control (n = 20) were measured by ELISA. Obtained values from individual controls and patients are plotted as dots. (B) The number of CD4+EBI3+p35+ and CD19+EBI3+p35+cells in the peripheral blood of patients with psoriasis was detected by flow cytometry. (C-D) Flow cytometry proportional analysis results in B. (E) Immunofluorescence staining of IL-35 (p35+EBI3+) in patients (n = 7) and control (n = 5) skin lesions. Red represents anti-p35 Ab, green represents anti-EBI3 Ab, yellow represents p35 and EBI3 merged, and blue represents 4′,6-diamidino-2-phenylindole (DAPI). Original magnification 400×. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 2 Expansion of monocytic myeloid-derived progenitor cells(MDSCs) in the blood of patients with psoriasis. (A) Peripheral blood mononuclear cells (PBMCs) freshly isolated from blood samples of healthy donors (control) or patients with psoriasis (psoriasis) were analysed for the expression of HLA-DR and CD14, with CD11b+CD14+HLA-DRno/lowcells being gated (shown by a small window) (B) Mononuclear MDSCs (M-MDSCs) among PBMCs are summarised in scatter graphs, (C) Immunofluorescence staining of M-MDSCs (CD14+HLA-DR-) in patients (n = 7) and control (n = 5) skin lesions. Red represents anti-CD14 Ab, green represents anti-HLA-DR Ab, yellow represents CD14 and HLA-DR merged, and blue represents 4′,6-diamidino-2-phenylindole (DAPI). Original magnification 400×. **p < 0.01.
Fig. 3 Therapy with IL-35 recombinant protein weakens the inflammatory process in the imiquimod (IMQ)-induced psoriasis mouse model . (A) Schedule of the delivery of IL-35 for the treatment of mice with IMQ-induced psoriasis. (B) Phenotype of murine skin with IMQ-induced psoriasis after therapy (n = 6). (C) Individual psoriasis area and severity index (PASI) scores of erythema, scaling, and thickness as well as cumulative PASI scores. (D) Hematoxylin and eosin (H&E)-stained skin sections from IMQ-treated mice, 200× and 400× (the 400× image is the enlarged image in the box in the 200× image). (E) Pathological scores of skin sections using the Baker scoring system. Columns represent the mean, and bars represent the standard deviation (SD). ***p < 0.001. This experiment was repeated three times.
Fig. 4 IL-35 inhibited secretion of inflammatory cytokines in serum and local lesions of imiquimod (IMQ)-induced mice. Several proinflammatory cytokines were detected by ELISA in serum and skin tissue after IL-35 treatment. Tissue cytokines from supernatants of extracted tissue protein were assayed and are presented in terms of picograms cytokine per milligram tissue (pg/mg). (A) IL-17A, (B) IL-23p40, (C) IL-1β, and (D) IL-6. Data are representative of three independent experiments. Columns represent mean, and bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.
Fig. 5 IL-35 suppresses the infiltration of myeloid-derived progenitor cells (MDSCs) in mice with imiquimod (IMQ)-induced psoriasis. (A) Representative fluorescence-activated cell sorting (FACS) plots of MDSCs (CD11b+Gr-1+) in the spleen and skin tissues (gated on CD45 events) at 24 h after the last delivery of IL-35. (B) Statistics of flow cytometry results in A. (C) Representative FACS plots of granulocytic MDSCs (G-MDSCs) (CD11b+Ly6G+Ly6Clow) and mononuclear MDSCs (M-MDSCs) (CD11b+Ly6G-Ly6Chigh) in the spleen and skin tissues (gated on CD45+events). (D-E) Quantification analyses of flow cytometry results in c. (F) Immunofluorescence staining of infiltrated CD11b+Gr-1+ MDSCs in murine skin lesions. Green represents anti-CD11b Ab, red represents anti-Gr-1 Ab, yellow represents CD11b and Gr-1 merged, and blue represents 4′,6-diamidino-2-phenylindole (DAPI). Original magnification 400×. n = 6, Columns represent the mean, and bars represent the standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.
Fig. 6 Adoptive transfer of myeloid-derived progenitor cells(MDSCs) weakens the effect of treatment of imiquimod (IMQ)-induced psoriasis with IL-35. MDSCs from mice with IMQ-induced psoriasis were adoptively transferred, following the experimental schedule (A). (B) The representative phenotype of the murine skin with IMQ-induced psoriasis after the adoptive transfer of MDSCs. (C) Individual psoriasis area and severity index (PASI) scores of erythema, scaling, and thickness as well as cumulative PASI scores. (D) Hematoxylin and eosin (H&E) stain was used to stain the skin sections from all four groups, 200× and 400× (the 400× image is the enlarged image in the box in the 200× image). (E) Scores of each group are based on the Baker scoring system. n = 6. Columns represent the mean, and bars represent the standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.
Fig. 7 IL-35 inhibited the expression of inducible nitric oxide synthase (iNOS) in myeloid-derived progenitor cells (MDSCs).IL-35 protein was used to treat imiquimod (IMQ)-induced psoriasis mouse model, and the expression of iNOS and IL-10 in MDSCs was detected. Fluorescence-activated cell sorting (FACS) analysis of iNOS and IL-10 expression in the spleen were shown in A and C (gated on CD11b+Gr-1+ events), and the results of their expression analysis in the skin were shown in (B) and (D) (gated on CD45+CD11b+Gr-1+events). E, Immunofluorescence staining of infiltrated iNOS+Gr-1+ MDSCs in skin lesions. Green represents anti-iNOS Ab, red represents anti-Gr-1 Ab, yellow represents iNOS and Gr-1 merged, and blue represents 4′,6-diamidino-2-phenylindole (DAPI). Original magnification 400×. n = 5. Columns represent the mean, and bars represent standard deviation (SD). *p < 0.05, **p < 0.01. ns, no significance.
Fig. 8 Adoptive transfer of inducible nitric oxide synthase (iNOS)-/- myeloid-derived progenitor cells(MDSCs) have no effect on the function of IL-35 in mice with imiquimod (IMQ)-induced psoriasis. MDSCs from WT and iNOS-/- mice with IMQ-induced psoriasis were adoptively transferred. (A) The representative phenotype of the murine skin with IMQ-induced psoriasis after the adoptive transfer of WT-MDSCs and iNOS-/--MDSCs. (B) Individual psoriasis area and severity index (PASI) scores of erythema, scaling, and thickness as well as cumulative PASI scores. (C) Hematoxylin and eosin (H&E) stain was used to stain the skin sections from all six groups, 200× and 400× (the 400× image is the enlarged image in the box in the 200× image). (D) Scores of each group are based on the Baker scoring system. n = 5. Columns represent the mean, and bars represent standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.
Fig. 9 Schematic summary of possible mechanisms of IL-35 amelioration of psoriasis. This mechanism is associated with IL-35 can inhibit inflammatory factors in the skin microenvironment of psoriasis, and these inflammatory factors can induce the differentiation and recruitment of MDSCs, so that IL-35 can indirectly inhibit the recruitment and secretion of iNOS of MDSCs.