The Intervention And Mechanisms Of Colquhoumai Root Tablet In Experimental Autoimmune Uveoretinitis (EAU) And Establishment Of Recurrent EAU

Background and ObjecctiveUveitis are a group of potentially blinding intraocular autoimmune diseases, with chronic recurrent ocular complications and complex pathogenesis. Previous studies showed that exogenous infection microbial antigens and ocular retinal antigens contributed to the destruction of ocular immune microenvironmen tolerance and the aberrant acitivation of CD4+ T cells and were involved in the pathogenesis of autoimmune uveitis. However, the precise pathogenesis of uveitis is not fully understood[1,2].Experimental autoimmune uveitis(EAU) is an organ-specific autoimmune disease mediated by T cells responses to retinal antigens. The EAU in B10.RⅢ mice were induced by interphotoreceptor retinoid-binding protein161-180 peptides emulsified by complete Freund’s adjuvant, and was similar to human uveitis in pathological features and pathogenesis for the study of human uveitis as a classic animal model[3,4].Dendritic cells(DCs) is a professional antigen-presenting cells(APCs) which link the innate and adaptive immune responses with important roles in defense against pathogens and immune tolerance. Mature DCs play major roles for controlling the proliferation and differentiation of T cells via cytokine secretion and for regulating the proliferation and differentiation of T cells via direct contact. Large amount of studies have found differentiation, mature and subsequent functional properties of DCs played an important role in autoinflammatory or autoimmune disease. Moreover, studies showed that intravenous injection of mature DC induced a more severe uveitis, compared with intravenous injection of immature DC in EAU[5].Autoimmune disease were clinically treated by colquhoumai root tablet(CRT). The drops of CRT have therapeutic effects on endotoxin-induced uveitis in rats[6,7,8], whether CRT treatment EAU or subsequent affecting DC differentiation, maturation and function related with uveitis was not fully understood.EAU in B10.RⅢ mice is a classical animal model for studying human uveitis. However, it is self-limited in the course of disease, which could not reproduce the nature of chronic recurrent human uveitis. The induction and features of EAU model in sensitive B10.RⅢ mice were of great significance in exploring the recurrent mechanism, and prevention as well as control methods of human uveitis.Based on the background, we have conducted the following studies. EAU was induced by IRBP161-180, and its clinical and pathological features were evaluated in the occurrence, development and outcome of inflammation; The intervention and mechanisms of CRT in EAU were also studied, to evaluate the features of relapsing EAU in EAU B10.RⅢ mice.PartⅠMethods: The B10.RIII mice in experimental group were immunized with 50μg interphotoreceptor retinoid-binding protein(IRBP)161-180 peptides in 100μl complete Freund,s adjuvant(CFA), mice in control groups were immunized with CFA alone. Two groups of animals were observed for clinical findings and histopathology at weekly intervals for 5 weeks after immunization.Results: The inflammations signs were observed in the 7th day, and rose to a peak in the 14 th day, which clinical findings in mice were ciliary injection, corneal opacity, fibrinous exudates in the anterior chamber, and lesions of the pupil, the prominent histopathologic lesions included inflammatory cellular infiltration in the retinal and choroidal, retinal vasculitis and granuloma, disorganization and loss of photoreceptors, and subretinal exudates, followed by a gradual regression, and disappeared in the 35 th days after immunization.Conclusion: B10.RIII mice were immunized with IRBP161-180-CFA, had a similar clinical manifestations and histopathological features with uveitis.PartⅡMethods: To investigate whether CRT have preventive and therapeutic roles in EAU induction, we injected IRBP into groups of mice and pretreated and treated some with 20ml/kg of CRT(postimmunization day-2~14, 7~14). Control mice treated with PBS. Clinical scores and histological scores were assessed with a slit lamp and an optical microscope in EAU mice and CRT-treated EAU mice in both prevention and effector phases. CRT-treated EAU mice in both prevention and effector phases were labeled and incubated with CD40-PE and MHC Ⅱ-APC antibody to evaluate the expression of DC surface molecules. ELISA was used to evaluate the levels of pro-inflammatory cytokines TNF-α, IL-6 in EAU mice and CRT-treated EAU mice in both prevention and effector phases. Proliferation was detected by cell-counting kit-8 in EAU mice and CRT-treated EAU mice in both prevention and effector phases.Results: CRT reduced the clinical and histological scores in EAU mice. The frequencies of CD11c+CD40+DC was upregulated while CD11c+MHCⅡ+DC was not changed in CRT-treated EAU mice in both prevention and effector phases. In addition, CRT reduced the levels of pro-inflammatory cytokines TNF-α and IL-6, and effectively inhibited the proliferation. There were no significant differences in CRT-treated EAU mice between prevention and effector phases.Conclusion: CRT had therapeutic effect on uveitis in B10.RIII mice by DC pathway. There were no differences in CRT-treated EAU mice between prevention and effector phases.Part ⅢMethods: The animals were given a secondary immunization with IRBP-CFA or CFA at the resolution of inflammation, to induce and evaluate the onsets and features of relapsing EAU in B10.RIII mice.Results: The primary inflammations signs were observed in the 7th day, and rose to a peak in the 14 th day, were followed by a gradual regression, and disappeared in the 35 th days after primary immunization. The animals were obtained a secondary immunization in the 35 th day after primary immunization. The relapsing inflammation signs were observed in the 36 th day, and then rose to a climax in the 42 th day. The mice were found to disease resolution in the 50 th day but about 1/5 of the mice still maintained inflammation signs in the 96 th day after primary immunization. The main clinical findings in mice were ciliary injection, corneal opacity, fibrinous exudates in the anterior chamber, and lesions of the pupil. The prominent histopathologic lesions included inflammatory cellular infiltration in the retinal and choroidal, retinal vasculitis and granuloma, disorganization and loss of photoreceptors, and subretinal exudates.Conclusion: This relapsing mouse EAU model was characterized with chronic course, clinical and histopathologic findings similar to human uveitis, and promises to as a suitable model for study of human uveitis.Summary: The EAU in the B10.RIII mice immunized with IRBP161-180-CFA, show similar clinical manifestations and histopathological features as human uveitis. CRT have preventive and therapeutic effects on uveitis in B10.RIII mice by DC pathway. The recurrent EAU model in mice is characterized with chronic course, with similar clinical and histopathologic findings to human uveitis, and is considered as a suitable model for study of human uveitis.