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

Authors: Chen Lin,  Nian Hong,  Wei Ruihua,  Zhang Hongmei,  Zhang Xiaomin,  Xiao Qing,  Li Xue,  Li Xiaorong
DOI: 10.3760/cma.j.issn.2095-0160.2015.04.002
Published 2015-04-10
Cite as Chin J Exp Ophthalmol, 2015,33(4): 294-299.

Abstract                              [Download PDF] [Read Full Text]


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 IRBP1-20 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. IRBP1-20-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 IRBP1-20-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.

Key words:

Toll-like receptor 7/agonist, CL097; Uveitis/immunology; CD4-positive T-lymphocytes/secretion; T helper cell 17; Cell polarity/immunology; Inflammation mediators; Dendritic cells/bone marrow

derived; C57BL/6 mouse

Contributor Information

Chen Lin
Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, Tianjin Medical University College of Optometry, Tianjin 300384, China
Nian Hong
Wei Ruihua
Zhang Hongmei
Zhang Xiaomin
Xiao Qing
Li Xue
Li Xiaorong
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Updated: April 10, 2023 — 9:28 am