Enhanced effect of microRNA-338-3p in bone marrow-derived dendritic cells on IRBP1-20-specific T helper 17 cells activation in experimental autoimmune uveitis

Objective

To investigate the role of microRNA-338-3p (miR-338-3p) in regulating the generation and function of interphotoreceptor retinoid-binding protein (IRBP)_1-20-specific T helper 17 (Th17) cells in experimental autoimmune uveitis (EAU).

Methods

Bone marrow cells were flushed from the femurs and tibiae of wild-type C57BL/6 mice and cultured in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)to differentiate into bone marrow-derived dendritic cells (BMDCs). On day 5 after induction, immature BMDCs were collected and divided into miR-338-3p mimics transfection group and mimics negative control transfection group, then transfected with miR-338-3p mimics or negative mimics according to grouping.Twenty-four hours after transfection, the BMDCs were stimulated with 100 ng/ml of lipopolysaccharide to mature.Relative expression levels of miR-338-3p, IL-6, IL-23 and IL-1β mRNA in BMDCs of the two groups were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The EAU model was established with IRBP_1-20, incomplete Freund adjuvant and mycobacterium tuberculosis (H37Ra) in mice.On day 13 after modeling, T cells were isolated from the mice spleen or draining lymph nodes and co-cultured with miR-338-3p mimics or negative control mimics-transfected BMDCs under Th17-polarizing conditions.Concentration of IL-17 in the supernatant was detected by ELISA.Relative expression levels of retinoic acid receptor-related orphan nuclear receptor γt (RORγt) and IL-17 mRNA were analyzed by qRT-PCR.The proportion of IL-17⁺ cells among T cells co-cultured with BMDCs was assessed by flow cytometry.To further verify the role of miR-338-3p in dendritic cells on Th17 cells, BMDCs transfected with miR-338-3p inhibitor or control inhibitor were co-cultured with T cells isolated from spleen or draining lymph nodes of EAU mice.Concentration of IL-17 in the supernatant was detected by ELISA.The use and care of the animals complied with Regulations for the Administration of Affairs Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (No.TJYY2019110117).

Results

Relative expression level of miR-338-3p in BMDCs was significantly increased in the miR-338-3p mimics transfection group than the mimics negative control group (t=6.861, P=0.002). In T cells co-cultured with miR-338-3p mimics-transfected BMDCs, the relative expression levels of RORγt and IL-17 mRNA were 1.34±0.16 and 1.33±0.16, which were significantly higher than 1.00±0.01 and 1.00±0.01 in the mimics negative control group (t=3.632, P=0.022; t=3.681, P=0.021). ELISA showed that the concentration of IL-17 in the supernatant was (5 941.00±452.40)pg/ml in the miR-338-3p mimics transfection group, which was significantly higher than (4 299.00±348.30)pg/ml in the mimics negative control group (t=4.979, P=0.008), and IL-17 concentration in the supernatant was (3 092.00±200.90)pg/ml in the miR-338-3p inhibitor transfection group, which was lower than (4 063.00±131.50)pg/ml in the inhibitor negative control group (t=7.005, P=0.002). The proportion of IL-17⁺ cells among T cells was (8.03±1.35)% in the miR-338-3p mimics transfection group, which was significantly higher than (4.52±0.73)% in the mimics negative control group (t=3.968, P=0.017). The relative expression levels of IL-6, IL-23, and IL-1β mRNA were 2.23±0.21, 2.21±0.56, 2.32±0.43, respectively in the miR-338-3p mimics transfection group, which were significantly higher than 1.00±0.06, 1.00±0.07, 1.01±0.15 in the mimics negative control group (t=10.290, P=0.001; t=3.747, P=0.020; t=5.280, P=0.006).

Conclusions

Overexpression of miR-338-3p in BMDCs can promote the IRBP_1-20-specific Th17 cell response by increasing the expression of Th17-polarizing cytokines including IL-6, IL-1β and IL-23.

Th17 cells/immunology; Experimental autoimmune uveitis; MicroRNA 338-3p; Bone marrow-derived dendritic cells