THU0381 Autoantibodies Specific to D4GDI Isolated from SLE Patients “unlock” RHO Small Gtpases and Affect Actin Remodeling in T Lymphocytes

Background T lymphocytes from patients with Systemic Lupus Erythematosus (SLE) display multiple abnormalities, including increased cell activation, abnormal apoptosis and impairment of cytoskeleton remodeling (1,2). In this regard the Rho GTPase family has been described as a “molecular switch” able to regulate many aspects of actin network dynamics (3). Objectives In this study we investigated the possible implication of specific autoantibodies (Abs) in the pathogenetic mechanisms of SLE. Methods Patients. Sixty-seven consecutive patients with SLE and 95 age- and sex-matched healthy donors were enrolled in this study. Identification of D4GDI and purification of specific autoantibodies from patients. T lymphocytes-surface membrane proteins were separated by 2DE, transferred onto nitrocellulose membrane and analyzed with a pool of sera from patients with SLE. A strongly immunoreactive spot was analyzed by mass spectrometry and, after protein identification, recombinant D4GDI was cloning, expressed in E. coli cells and affinity purified. Anti-D4GDI antibody serum levels were tested by ELISA. For purification of Abs, recombinant D4GDI was spotted onto a nitrocellulose membrane and incubated with sera from SLE patients. Bound Abs were, finally, eluted with 100 mM glycine pH 2.5. Determination of Rho protein activity and evaluation of cytoskeleton organization. T lymphocytes were treated with purified human anti-D4GDI Abs and analyzed with the Rho/Rac/Cdc42 Activation Assay Combo Kit (Cell Biolabs). After cell lysis, the activation of Rho GTPases RhoA, Rac1 and Cdc42 was assessed by western blotting using specific mAbs. The cytoskeleton remodeling was evaluated by immunofluorescence assay and flow cytometry for detection of total actin and filamentous actin. Results In the present study we reported the presence of specific Abs to D4GDI, a small GTPase family inhibitor, in a significant percentage (46%) of SLE patient sera. These antibodies appeared as capable of binding D4GDI at the surface of lymphocytes triggering a series of subcellular events, including Rho GTPase activation (Rac1 and RhoA) and cytoskeleton remodeling by increasing actin polymerization. Conclusions We hypothesize that autoantibodies specific to D4GDI, blocking its inhibitory activity, by the intracellular activation of Rho family small GTPases and remodeling of cytoskeleton, may affect many T lymphocyte functions, including cell polarity and migration. References Rahman A, Isenberg DA. Systemic lupus erythematosus. N Engl J Med 2008;358:929-939. Perl A. Pathogenic mechanisms in systemic lupus erythematosus. Autoimmunity. 2010;43:1-6. Garcia-Mata R, Boulter E, Burridge K. The “invisible hand”: regulation of RHO GTPases by RHOGDIs. Nat Rev Mol Cell Biol 2011;12:493-504. Disclosure of Interest None declared