miR-22 inhibits DCs from expressing IL-6 and driving Th17 cells differentiation.

<p>(A) Based on the facts that p38 had a stimulatory influence on the transcription of IL-6 gene in bone marrow derived DCs (BMDCs) and that miR-22 could prohibit p38 mRNA from being translated, it was inferred that miR-22 mimics and inhibitor would hinder and promote the expression of IL-6 respectively. This was confirmed by the real-time RT-PCR result of the BMDC groups loaded with different reagents after 24 h of LPS stimulation. The result also illustrated the effect of miR-22 appeared as early as the transcription event occurred. (B) Dose effects of miR-22 mimics and inhibitor were examined and the results further confirmed the negative effect of mimics and positive effect of inhibitor on IL-6 mRNA transcription. (C) Inhibitory effect of miR-22 on IL-6 expression could also be detected on the protein level. BMDCs were grouped and treated as mentioned above and their culture supernatants were collected to determine the IL-6 protein concentrations using enzyme linked immunosorbent assay (ELISA). (D) Dose series of miR-22 mimics and inhibitor were integrated into the BMDC transfection procedure and IL-6 protein levels were examined by ELISA. The results further confirmed the inhibitory effect of miR-22 on IL-6 protein expression. (E) miR-22 inhibitor promoted the ability of DCs to induce Th17 cells generation <i>in vivo</i>. C57BL/6 mice bearing B16 melanomas were given DC immunotherapy using differentially transfected BMDCs, and then the T cell populations in the tumor microenvironment were detected by flow cytometry. All the cells shown in this figure were gated from CD4⁺ lymph cells and it was obvious that compared with the control and miR-22 mimics groups, mice injected intraltumorally with miR-22 inhibitor loaded DCs exhibited a higher percentage of CD4⁺ IL-17A⁺ Th17 in their inguinal lymph nodes. All the real-time RT-PCR and ELISA experiments included in this figure were performed at least 3 times. The bar plots are expressed in the form of mean±SD. * means a significant decrease appears when compared with the control group (p-value < 0.05, t test), while ** represents a significant increase relative to the control (p-value < 0.05, t test).