Impacts of the A20 protein polymorphism, rs2230926, in human B cells

Abstract

Abstract Patients with autoimmune diseases, such as systemic lupus erythematosus (SLE), often have skewed B cell population ratios, accumulation of autoantibodies, and an increased amount of proinflammatory cytokines aiding in disease development. Genome-wide association studies (GWAS) have strongly linked polymorphisms in A20, encoded by TNFAIP3, to autoimmune diseases including SLE. A20 functions to inhibit the activation of nuclear factor kappa B (NF-κB) through ubiquitination and deubiquitylation of key signaling proteins and has been extensively studied in epithelial cells in response to TNF. Given the role of autoantibodies in SLE and the importance of NF-κB in regulating B cell fate, the role of SLE linked polymorphisms in A20 and their effects on both the canonical and noncanonical NF-κB pathways in human B cells is poorly understood. Our lab has identified an A20 polymorphism, rs2230926 (F127C), that is enriched in a cohort of African American females with SLE. Herein, we investigate the impact of rs2230926 on B cell function. We employed CRISPR/Cas9 genome-editing and generated a TNFAIP3knockout lymphoblastoid Raji human B cell line. To determine how the polymorphism rs2230926 affects the NF-kB pathway, we re-introduced WT A20, A20-rs2230926, and a truncated version of A20 into the TNFAIP3-KO cell line. Using a combination of qRT-PCR, western blotting, and flow cytometry, we determined how rs2230926 influences the expression of proinflammatory cytokines, response to various B cell stimuli, and ultimately NF-κB activity. Dissecting A20’s role in NF-kB signaling will help to understand the dysfunction and regulation of B cells in autoimmunity. Supported by grants from NIH to CDS (R01 AI148471)