The Role Of Complement In Regulating Uveitogenic T Cells In Experimental Autoimmune Uveitis

Objective 1. Recent studies have suggested that reagents inhibiting complement activation could be effective in treating T cell mediated autoimmune diseases.However, whether the complement anaphylatoxin receptors(C3a and C5 a receptors) take part in the pathogenesis of experimental autoimmune uveitis(EAU) remains elusive and controversial. We induced experimental autoimmune uveitis in mice deficient or sufficient in both C3 a and C5 a receptors and rigorously compared manifestations in the eye and function of the uveitogenic T cells to find out the precise role of C3 a and C5 a receptors in the development of EAU. 2. Complement needs to be activated to function. Although the alternative pathway has been found important in regulating T cell responses, the potential roles of the other two complement activation pathways, the classical and the lectin pathways in this process remain unclear. To address this issue, we studied mice deficient of C4(both classical and lectin pathways deficient) or C1q(classical pathway deficient) in the development of experimental autoimmune uveitis(EAU).Methods 1. WT and C3 a R/C5 a R double knockout(DKO) mice(C57BL/6J background) were immunized with an interphotoreceptor retinoid binding protein(IRBP) peptide 651-670 to induce EAU. After immunization, the development of EAU was monitored by indirect ophthalmoscopy, and clinical scores were assigned according to previously published criteria. Two weeks after immunization, mice were also examined by topical endoscopic fundus imaging(TEFI), scanning laser ophthalmoscopy(SLO), spectral-domain optical coherence tomography(SD-OCT), and electroretinography(ERG), followed by histopathological analysis of the eyes. T cell recall assays were performed 2 or 3 weeks after EAU induction. Splenocytes from each of the immunized WT and KO mice were cultured without or with the presence of IRBP 651–670 peptide or the same concentration of a nonrelevant peptide ovalbumin(OVA) 323–339. Culture supernatants were collected after 72 h of incubation for IFN-γ and IL-17 concentration measurement using conventional ELISA. In addition, in the experiments of adoptive transfer EAU, at 14 d after immunization, we injected the same number of in vitro activated T cells from immunized WT or KO donors to na?ve WT recipients and the development of EAU was assessed. 2. Besides,WT, C4 and C1 q knockout(KO) mice were immunized IRBP 651-670 peptide to induce EAU. After immunization, in addition to monitor the development of EAU by clinical scores,retinal imagings,retinal histopathological analysis and evaluate Th1 and Th17 responses by recall assay and IFN-γ, IL-17 assessment, the antigen-specific CD4 positive T cells were compared by tetramer staining. To compare the functions of antigen presenting cells from WT and C4 KO mice, bone marrow derived dendritic cells(BMDCs) were isolated and induced to mature DCs. T cells purified from splenocytes from transgenic OTII mice were cultured with OVA 323-339 pulsed mature BMDCs. 72 hours later, the proliferation of T cells was assessed using CFSE. To test the function of pathogenic T cells in the spleen from WT and C4 KO mice, CD4 postive T cells were isolated from splenocytes using magnetic beads. T cells were induced to differentiate to Th0 and Th1 subsets. On different time points, activation, proliferation, apoptosis, and differentiation assays of CD4 positive T cells were preformed.Results 1. DKO mice developed significantly milder EAU than WT controls as judged by clinical scores, retinal imagings and retinal histopathology scores. The results of ERG suggested that the function of retina is more sensitive in DKO EAU mice than in WT EAU mice. DKO mice also demonstrated reduced IRBP specific Th1 and Th17 responses compared with WT mice in T cell recall assay, showing a significant lower level of IFN-γ and IL-17 in the cell culture supernatant. In addition, adoptive transfer of activated T cells from immunized WT donors induced more severe EAU in na?ve recipients than the same numbers of cells from DKO immunized donors. 2. After immunization, C4 KO mice developed significantly milder EAU than WT controls while C1 q KO mice developed comparable EAU to WT mice. The results of retinal imagings, retinal histopathological scores, and T cell recall assay were consistent with the clinical observations. In addition, C4 KO mice in EAU also showed reduced percentage of IRBP-specific CD4 positive T cells in the spleen comparing to the WT EAU mice. 3. The BMDCs from WT and C4 KO mice did not show significant differences in functions of antigen presentation. However, CD4 positive T cell in the splenocytes of C4 KO mice showed weakened abilities in activation, proliferation, differentiation, while were more susceptible to apoptosis, comparing to their WT controls.Conclusion 1. The complement anaphylatoxin(C3a and C5a) and complement anaphylatoxin receptors(C3a R and C5 a R) play significant roles in the pathogenesis of EAU. 2. C4, taking part in complement classical and lectin pathway of activation, is required for the pathogenesis of EAU. However C1 q, starting the classical pathway, is not important for the development of EAU. The positive role of C4 in the EAU may might result from enhancing the activation, proliferation and differentiation of CD4 positive T cells, at the same time slowing down their apoptosis. 3. Complement activation should be a novel target to suppress pathogenic T cell responses for treating autoimmune uveitis.