3.3 IL-35 protein therapy relieved the symptoms of psoriasis models
We stimulated HaCaT cells using M5 together with IL-35 to observe the effect of IL-35 on the expression of CXCL8 and IL-6. We found consistent lower levels of CXCL8 and IL-6 in the IL-35-treated group than in the control group (Supplementary Fig. 1A, B). The results suggest that IL-35 suppresses the expression of CXCL8 and IL-6 in stimulated HaCaT cells.
We explored whether IL-35 could exhibit a therapeutic effect in K14-VEGF-A-Tg mice. Therefore, we injected K14-VEGF-A-Tg mice (10 weeks old) with IL-35 (5 μg) recombinant protein, as shown in Supplementary Figure 2A. We found that mice in the control group showed increased ear thickening; however, mice in the IL-35-treated group were healthy (Supplementary Fig. 2B). Pathological features, such as erythema, scaling, and thickness based on PASI scores are shown in Supplementary Figure 2C. Concomitantly, we obtained the cumulative scores of these two groups (Supplementary Fig. 2C). In addition, we observed that the IL-35-treated group had lower cumulative scores than the control group. H&E staining of lesioned tissues revealed the severity of psoriatic disease in the control (PBS) group (Supplementary Fig. 2D, left panel). In contrast, we observed the reversal in ear thickness and reduction in leukocyte infiltration in the dermis and epidermis in the IL-35-treated group (Supplementary Fig. 2D, right panel). Moreover, we demonstrated that the IL-35-treated group exhibited a lower disease score based on the Baker scoring system compared to the control group (Supplementary Fig. 2E).
The IMQ-induced psoriasis mouse model is the most common model of psoriasis (Shih et al., 2020). Experimental procedures and the acquisition of mouse back skin images are shown in Supplementary Figures 3A and 3B. Based on the PASI scoring evaluation of erythema, scaling, and thickness, we examined the cumulative scores for different groups in the IMQ-induced psoriasis mouse model (Supplementary Fig. 3C). In particular, we noticed that the IMQ model group exhibited higher cumulative scores than the control group. We then used flow cytometry to detect any changes in the population of MDSCs in the spleen and skin tissues of mice in the IMQ-induced model; we found that the number of MDSCs in the spleen and skin tissues of mice was significantly higher than that in the control group (Supplementary Fig. 3D, p < 0.001 and p < 0.01, respectively). The results were consistent with those obtained using clinical samples.
We determined whether IL-35 exerts a therapeutic effect on the IMQ-induced psoriasis mouse model. The schedule of the delivery of IL-35 for the therapy of mice with IMQ-induced psoriasis is shown in Figure 3A. After treatment, we found that the control group had serious inflammation and skin flaking, which worsened by the end of the experiment (Fig. 3B. Based on the PASI scoring evaluation of erythema, scaling, and thickness (Fig. 3C), we calculated the cumulative scores of different groups (Fig. 3C). IL-35 group showed lower cumulative scores relative to the control group. In addition, H&E-stained skin sections showed less epidermal thickening in the IL-35 group compared with the control group (Fig. 3D). The Baker system-based disease score in the IL-35 treated group was significantly lower than that in the control group (Fig. 3E, p < 0.001).
3.4 IL-35 regulates inflammatory cytokine production in systemic and local immune microenvironment in IMQ-induced mice .
Inflammatory cytokines play important roles in the progression of psoriasis (Liang, Xu, Peng, Pan & Ye, 2014). ELISA was used to measure the levels of cytokines in serum and dorsal skin. Tissue cytokines from supernatants of extracted tissue protein were assayed and are presented in terms of picograms cytokine per milligram tissue (pg/mg). IL-17A (Fig. 4A), IL-23 (Fig. 4B), IL-1β(Fig. 4C) and IL-6 (Fig. 4D) were detected in serum and skin tissue. Data are representative of three independent experiments. These results showed that, in addition to no significant difference in serum IL-6, other cytokines were significantly different in serum and skin tissue. Columns represent mean; bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.
3.5 Treatment with IL-35