Abstract
Purpose: :
Experimental Autoimmune Uveitis (EAU) is a model for human non-infectious intraocular inflammation (uveitis). This CD4+ T cell mediated disease can be induced with peptides from the retinal autoantigen retinol binding protein-3 (RBP-3 or interphotoreceptor retinol binding protein; IRBP). RBP-3 is a conserved protein made up of four subunits, each of which contains approximately 300 amino acids. Previous work has demonstrated that leukocyte populations infiltrate the retina at different disease stages during RBP-3 peptide induced EAU. Whilst T cell epitopes influence effector cells and inflammation, only few uveitogenic epitopes have been identified from the whole protein in the C57BL/6 model. Therefore, the purpose of this study was to analyse the population dynamics and disease kinetics induced by protein subunits of RBP-3 in the C57BL/6 EAU model.
Methods: :
Recombinant RBP-3 (rRBP-3) subunit proteins were expressed in bacteria and purified via a histidine tag. C57BL/6 mice were immunised with the rRBP-3 subunits or RBP-3 1-20 peptide. Disease severity was assessed by topical endoscopic fundal imaging (TEFI), cellular infiltrate by flow cytometry and structural damage by histology.
Results: :
All four rRBP-3 subunits were pathogenic and able to induce EAU. rRBP-3 subunit 3 caused the most severe inflammation compared to EAU induced with RBP-3 1-20 peptide or the other subunits. In addition, onset of peak disease differed in rRBP-3 subunit 3 immunised mice with severe inflammation observed by day 16. The CD45+ cellular infiltrate was the highest with subunit 3, with the corresponding severe structural damage evident from histological analysis. At day 25 post immunisation, the extent of CD4+ and CD11b+ cell infiltrate was similar in both subunit and peptide-induced EAU. Further, animals immunised with the rRBP-3 subunits and not RBP-3 1-20 peptide, showed an overall increase in retinal CD8+ cell population but no difference was observed in the proportion of Ly6G+ infiltrating cells at peak disease.
Conclusions: :
Severity of rRBP-3 subunit 3 EAU may indicate multiple pathogenic epitopes. All major leukocyte types are represented within the cellular infiltrate, but distinct population differences and relative numbers alter depending upon initiating peptide/protein.
Keywords: autoimmune disease • inflammation • flow cytometry