Enhancement effect of TLR7 agonist CL097 on the activity of Th17 cells in experimental autoimmune uveitis

Toll like receptor 7 (TLR7) has been implicated in the development of autoimmune diseases, but its role in the regulation of T helper cell 17 (Th17) response in experimental autoimmune uveitis (EAU) by dendritic cells (DCs) has not been revealed yet.

This study was to investigate the effect of bone marrow-derived dendritic cells (BMDCs) treated by TLR7 agonist CL097 on the regulation of interphotoreceptor retinoid-binding protein (IRBP)_1-20-specific Th17 cells response in EAU.

BMDCs were isolated and collected from femur and tibia of naive C57BL/6 mice and cultured with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4 (rmIL-4). After cultured for six days, 5 μg/ml CL097 was added into the medium for 8 hours in the CL097 treated group, and PBS was used in the control group. Real-time fluorescence quantitative PCR (RT-qPCR) was employed to detect the relative expressions of IL-6, IL-23, IL-1β and transforming growth factor-β (TGF-β) mRNA in BMDCs. These mice received 1 μg of Bordetella pertussis toxin intra-peritoneally on the day of immunization, C57BL/6 mice were immunized with IRBP_1-20 in complete Freund adjuvant containing heat-killed mycobacterium tuberculosis H37RA after two hours, EAU was evaluated by fundus examination on a scale of 0-4 reported by Caspi. Eyes harvested at 13 days after active immunization were processed for histopathological examination and stained with hemotoxylin and eosin. IRBP_1-20-specific T cells were isolated from spleen cells and lymph node cells on the thirteenth day after immunization and co-cultured with CL097 treated or untreated BMDCs. After co-cultured for 5 days, cells were collected. Then, the relative expressions of IL-17, interferon-γ (IFN-γ), retinoic acid-related orphan receptor-γt (RORγt), T-box 21 transcription factor (T-bet) mRNA expression in T cells were tested by RT-qPCR. Percentages of Th1 cells and Th17 cells were assessed by flow cytometry. The results were compared between the two groups by independent sample t test.

EAU model was successfully established. Compared with the control group, the expressions of IL-6, IL-23, IL-1β mRNA in BMDCs were significantly much higher, but the expression of TGF-β was much lower in the CL097 treated group than those in the control group (t=4.560, P=0.045; t=5.476, P=0.032; t=17.240, P=0.003; t=10.410, P=0.009). After IRBP_1-20-specific T cells co-cultured with CL097-treated BMDCs, the expressions of RORγt mRNA and IL-17 mRNA were significantly higher than those of the control group (t=8.844, P=0.012; t=8.831, P=0.013), but no significant differences were seen in the expressions of T-bet mRNA and IFN-γ mRNA between the CL097 treated group and the control group (t=2.078, P=0.173; t=1.082, P=0.393). The flow cytometry revealed that the average percentages of Th17 cells were (17.750±4.793)% in the CL097 treated group and (10.240±3.173)% in the control group, showing a significant difference between them (t=4.938, P=0.039); while the average percentages of Th1 cells were not significantly different between the two groups ([1.123±0.356]% versus [1.060±0.384]%)(t=2.714, P=0.113).

TLR7 agonist CL097 can stimulate BMDCs to secrete Th17-polarizing cytokines and promote the differentiation of Th17 cells.