miR-223-3p Inhibits Antigen Endocytosis and Presentation and Promotes the Tolerogenic Potential of Dendritic Cells through Targeting Mannose Receptor Signaling and Rhob

Figure 7

Mannose receptor-mediated antigen endocytosis and presentation were involved in miR-223-3p expression. (a) MR expression in DCs treated with OVA and miR-223-3p mimic or inhibitor was determined by Western blot. (b) Mouse immature DCs were transfected with siRNA-MR or siRNA control (50 nM) for 24 h, and the expression of miR-223-3p was detected by qRT-PCR. (c) Mouse immature DCs were transfected with siRNA-MR, siRNA-Rhob, or siRNA control (50 nM). After 24 h, DCs were stimulated with 100 μg/ml OVA for 24 h. Cell lysates were subjected to Western blot for the mannose receptor, Rhob, and GAPDH. (d–f) Mouse immature DCs were transfected with siRNA-MR or siRNA control at a final concentration of 50 nM for 24 h. (d) DCs were incubated with FITC-OVA for 30 minutes, and the endocytic activity (FITC-OVA uptake) of BMDCs was measured by flow cytometry. For determining the MHC-II level, DCs were stimulated with 100 μg/ml OVA for 24 h and then stained with MHC-II antibody, followed by flow cytometry analysis. (e) The transfected DCs were stimulated with 100 μg/ml OVA for 24 h. Purified CD4+ T cells were cocultured with the transfected DCs at a ratio of 1 : 10 (DC/T cells). After 4 d, cells were collected and stained with anti-CD4-APC, followed by intracellular staining with FITC-conjugated anti-Foxp3, FITC-conjugated anti-IL-17A, PE-conjugated anti-GATA-3, or PE-conjugated anti-T-bet, respectively. The cells were then analyzed by flow cytometry. The bar chart indicated the specific transcription factor of T cells in the gate of CD4+ cells in each group. Data were presented as of three independent experiments. -test, , .

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