reduced the number of recruited MDSCs
The above results showed that IL-35 had a clear effect in treating psoriasis and MDSCs were significantly expanded in patients with psoriasis and the mouse model. To test whether IL-35 ameliorates psoriasis by restricting the accumulation of MDSCs, we used flow cytometry to analyse the number of MDSCs in mice with IMQ-induced psoriasis with or without treatment of IL-35. The results showed that compared with that in the control group, administration of IL-35 reduced the infiltration of MDSCs in the spleen and skin tissues of mice (Fig. 5A and B). Then, we investigated whether the populations of G-MDSCs and M-MDSCs were altered in response to this therapy stress. We found that both G-MDSC and M-MDSC populations were significantly reduced in both the spleen and skin tissues (Fig. 5C-E). Finally, immunofluorescence staining of CD11b+Gr-1+ MDSCs in the inflamed skin showed that the MDSC accumulation was inhibited by IL-35 treatment (Fig. 5F). These results demonstrated that the immunosuppressive effect of IL-35 on the IMQ-induced psoriasis model was achieved by reducing the infiltration and proportion of MDSCs, and it can adjust the proportion of MDSC subtypes. Next, we examined whether IL-35 directly affects the differentiation and recruitment of MDSCs in vitro using fluorescence-activated cell sorting (FACS). IL-35 had little effect on the differentiation of MDSCs (Supplementary Fig. 3A, B IL-35 also did not significantly affect the numbers of G-MDSCs and M-MDSCs cells (Supplement Fig. 3C-E).
3.6Adoptive transfer of MDSCs weakened the effect of IL-35 in mice withIMQ-induced psoriasis
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 (Fig. 6A). We found that the adoptive transfer of MDSCs derived from IMQ-induced mice aggravated disease progression. Moreover, when these MDSCs were transferred to the IL-35-treated group, the psoriasis symptoms were aggravated in these mice compared with those in mice treated with IL-35 alone (Fig. 6B). We evaluated pathological features in mice, such as erythema, scaling, and thickness, in different groups using the PASI scoring system (Fig. 6C). We observed that compared with the control (PBS) group, the PBS + MDSC group exhibited a higher PASI score. Similarly, the score of the IL-35 + MDSC group was significantly higher than that of the group treated with IL-35 alone. Furthermore, we calculated the cumulative scores from different groups and found that the IL-35-treated group exhibited the lowest cumulative scores than the other groups (Fig. 6C).
H&E-stained skin sections from these groups exhibited increased epidermal thickening in PBS + MDSC group relative to that in the control group. We also found that the skin epidermis in IL-35 + MDSC group was significantly thicker than that in the IL-35-treated group (Fig. 6D). In addition, the Baker system-based score of the IL-35-treated group was the lowest of these four groups (Fig. 6E).
3.7IL-35 inhibited the expression of iNOS in MDSCs in mice with IMQ-induced psoriasis
The above results indicated that IL-35 inhibits the recruitment of MDSCs. Next, we investigate whether IL-35 affects the expression of inflammatory mediators in MDSCs. We used flow cytometry to analyse the expression of iNOS and IL-10, which were secreted by MDSCs in mice with IMQ-induced psoriasis with or without treatment of IL-35. The results showed that compared with the control group, the IL-35-treated group exhibited reduced expression of iNOS in the spleen and skin tissues (Fig. 7A and B). However, there was no difference in the level of IL-10 (Fig. 7C and D). Finally, immunofluorescence staining of iNOS+Gr-1+ MDSCs in the inflamed skin showed that the iNOS+Gr-1+ MDSC accumulation was inhibited by IL-35 (Fig. 7E).