Dynamic expression and mechanism of γδ T cells in the spleen of mouse with experimental autoimmune uveitis

Background

Previous studies showed that the pathogenesis of uveitis is related to γδ T cells.However, it remains unclear that how these cells are involved in experimental autoimmune uveitis (EAU).

Objective

This study aimed to observe the dynamic changes of γδ T cells in EAU and explore the role of γδ T cells in the pathological process of EAU.

Methods

Forty-five C57BL/6(B6) mice were assigned to the normal control group (six mice) and EAU model group (thirty-nine mice). The mice were immunized subcutaneously at 6 spots on the footpads, tail base, and flank with emulsion containing human interphotoreceptor retinoid binding protein_1-20 (IRBP_1-20) emulsified in complete Freund’s adjuvant.After immunization, the mice were examined for clinical signs of EAU by using a Genesis-D camera.The changes of histopathology were compared by hematoxylin and eosin staining.Mouse lymphocytes were isolated and purified from the spleens of IRBP_1-20-immunized or normal B6 mice by using a γδ T-cell isolation kit.Flow cytometry was used to detect the changes of intracellular expression of interleukin-17A (IL-17A), and then transferred the activated γδ T cells into EAU models to analyze the changes of clinical signs and histopathology of EAU.Experimental study program as well as the use and feeding of the animals were authorized by the Animal Management and Use Committee of Shandong Traditional Chinese Medicine University.

Results

The inflammatory symptoms in mouse eyes appeared on day 12 after modeling.The initial changes were fundal blood vessel thickening and minimal inflammatory cell infiltration.Then, multifocal chorioretinal lesions, serious vasculitis and linear lesions were observed on days 16-20, along with abundant lymphocyte infiltration in the vitreous and retinal disorganization.The inflammation symptom scores and the pathological inflammation scores at different time points after modeling had statistically significant differences (F=51.399, P=0.000; F=47.342, P=0.000). The inflammatory symptoms in the eyes began to abate from day 28 onwards.The number of γδ T cells was obviously increased during the inflammation phase of EAU at day 16-20 after modeling, with the number of γδ T cells was (5.67±0.49)% and (5.78±0.55)%, respectively, which was significantly higher than (1.53±0.14)% before modeling, with significant differences between them (both at P<0.05), meanwhile CD69 levels and the integrin lymphocyte function-associated antigen-1 (LFA-1) and secreted IL-17A were elavated.The secretion level of IL-17A was (13.40±0.50)% and (17.80±2.37)% on day 16 and day 20 after modeling, respectively, which was significantly higher than (1.53±0.19)% before modeling, with significant differences between them (P=0.000, 0.001). The activated γδ T cells were transferred into EAU model, the inflammation symptom scores were 1.00 (1.00, 2.00) after activated γδ T cells were transferred into EAU model, which was significantly higher than 0.75 (0.05, 1.00) of the untransferred group (Z=27.00, P=0.03), and the symptoms of EAU were aggravated.

Conclusions

The proportion of γδ T cells reaches peak in inflammation of EAU, and the cells are activated.The activated γδ T cells in the EAU model play a immune regulation role by secreting IL-17A.