Identification of Tribbles Homolog 2 as an Autoantigens in Uveitis

Abstract

Abstract: : Purpose: Several autoantigens have been well characterized in uveitis animal models. However, it is unknown whether these autoantigens are pathogenic targets in uveitis patients and how many other autoantigens are yet to be identified. Using a novel system of subtractive phage biopanning for screening ocular autoantigens, we identified tribbles homolog 2 (TRB2) as a candidate autoantigen from a uveitic patient’s serum. Herein we further characterize the immunoreactivity and ocular expression of TRB2. Methods: TRB2 was identified from a human eye cDNA phage–display library by subtractive phage biopanning using purified IgG from a patient with anterior/intermediate uveitis. Binding affinity and patient specificity of TRB2–expressing phage clone (TRB2–phage) was analyzed by phage–based binding assay, ELISA, dot blot and Western blot. TRB2–specific Ab activity in patient and control sera was characterized by ELISA. Recombinant TRB2 was expressed as glutathione–S–transferase fusion protein (GST–TRB2) and purified. Anti–TRB2 antibody was affinity purified from patient serum using immobilized TRB2 resin. TRB2 expression in ocular tissues was characterized by immunohistochemistry. Results: Phage binding assay demonstrated that TRB2–phage has a 388–fold increase in binding affinity and 170–fold increase in patient specificity. These results were further verified by phage–ELISA (p<0.001) and phage–dot blot. Western blot analysis with purified GST–TRB2 indicated that TRB2 was recognized by uveitic patient IgG, but not by control IgG. Moreover, TRB2–specific antibody was detected in the sera of three out of ten patients with anterior/intermediate uveitis, while none of the control sera had anti–TRB2 activity. Anti–TRB2 antibody titer (mainly IgG1 isotype) among the three positive patients was as high as 1:10,000. TRB2 expression was detected in the ciliary body by immunohistochemistry using affinity purified TRB2–specific antibody. Conclusions: We have identified TRB2 as a candidate autoantigen by subtractive phage biopanning and demonstrated its immunoreactivity and patient specificity. Furthermore, we have characterized TRB2 expression in uveitis–susceptible ocular tissue by immunohistochemistry using affinity purified anti–TRB2 autoantibody.

Keywords: autoimmune disease • uveitis-clinical/animal model • uvea