Effect Of TLR7 Agonist CL097 On Th17 Cells In Experimental Autoimmune Uveitis

Objective Autoimmune uveitis is one of the serious inflammatory diseases of visual impairment.Recently, T helper 17(Th17) cells have been closely related to the occurrence and development of autoimmune diseases and Toll like receptor 7(TLR7) has been affected Th17 cells differentiation in some autoimmune diseases, but its role in the regulation of Th17 cells response in experimental autoimmune uveitis(EAU) by dendritic cells(DCs) has not been revealed yet. This study is to investigate the effect of bone marrow-derived dendritic cells(BMDCs) treated by a TLR7 agonist, CL097,on the interphotoreceptor retinoid-binding protein(IRBP)1-20-specific Th17 cells response in EAU and discuss the possible molecular mechanism.Methods BMDCs were collected from naive C57BL/6 mice and cultured with recombinant mouse granulocyte-macrophage colony-stimulating factor(rm GM-CSF) and recombinant mouse interleukin-4(rm IL-4). Then the purity of BMDCs was identified by flow cytometer. After cultured for six days, BMDCs were treated with 5μg/ml CL097 for 8 hours and untreated with CL097 as control group. Real-time fluorescent PCR(RT-PCR) was used to detect the relative m RNA expressions of IL-6, IL-23,IL-1β and transforming growth factor(TGF)-β in BMDCs. C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein(IRBP) 1-20 in complete Freund adjuvant(CFA) containing heat-killed Mycobacterium tuberculosis H37 RA.At thirteenth day after active immunization, the eyes of C57BL/6 mice was examined clinical signs of uveitis by indirect ophthalmoscope and scored by Caspi’s criteria,then the eyes harvested prepared for the pathological examination by hemotoxylin-eosin staining. Besides, IRBP1-20-specific T cells were isolated from spleen cells and lymph node cells and co-cultured with CL097 treated or untreated BMDCs. After co-cultured for five days, cells were collected. Then, the relative expresstion of IL-17, interferon(IFN) –γ, retinoic acid receptor-related orphan receptor(ROR) γt, T-box expression in T cells(T-bet) m RNA were tested by RT-PCR. Average percentages of Th1 cells and Th17 cells were assessed by flowcytometry. In addition, BMDCs cultured for six days were treated with CL097. At 0h,0.25 h, 0.5h, 1h, 3h, 6h point, p-p38, p-ERK1/2 and p-JNK were examined by Western blot, respectively.Results1 、 On the days 6 of culture, the purity of BMDCs was 90% identified by flow cytometer.2、Compared with control group, RT-PCR revealed the CL097-treated BMDCs,showed much higher expression of IL-6, IL-23, IL-1β(t=4.560, P=0.045; t=5.476, P=0.032; t=17.24, P=0.003), while much lower expression of TGF-β(t=10.41, P=0.009).3、At thirteenth day after immunization, EAU model was successfully established.4、Flow cytometer showed that the average percentage of Th17 cells significantly increased from(10.240±3.173)% to(17.750±4.793)%(t=4.938, P=0.039), while the average percentage of Th1 cells showed no significant change between two groups( [1.123±0.356]% versus [1.060±0.384]%)(t=2.714, P=0.113).5、RT-PCR revealed IRBP1-20-specific T cells co-cultured with CL097-treated BMDCs showed significantly higher expression of RORγt and IL-17 m RNA than the control group(t=8.844, P=0.013; t=8.831, P=0.013), but the expressions of IFN-γ and T-bet m RNA have no statistically difference(t= 2.078, P=0.173; t=1.082, P= 0.393).6、Western blot showed that the expression of p-p38 protein was only significantly increased at 0.25 h point, p-ERK1/2 protein level was significantly increased at 0.25 h,0.5h point and then decreased to 0h point level, p-JNK protein level was significantly increased at 0.25 h, 0.5h, 1h point and then decreased to 0h point level.Conclusions1、In vitro, TLR7 agonist CL097 can stimulate BMDCs to secrete Th17-polarizing cytokines and indirectly promote the differentiation of Th17 cells.2、TLR7 agonist CL097 can activate MAPKs signaling pathway(p38, ERK1/2 and JNK) in BMDCs to indirectly promote the differentiation of Th17 cells.