208 A NOVEL ROLE FOR THE GAS3/PMP22 FAMILY MEMBER EMP2 IN THE REGULATION OF INFLAMMATION

Introduction Cardiovascular disease such as atherosclerosis is currently the leading cause of death by noncommunicable diseases worldwide. Development of atherosclerosis which is considered a chronic inflammatory disease has been associated with a number of pro-inflammatory cytokines including interleukin-1 (IL-1). IL-1 has been associated with atherosclerotic plaque formation as well as plaque rupture. Previous reports using ApoE −/− /IL1-β −/− mice have described a significant decrease in atherosclerotic area further highlighting the importance of this apical cytokine. IL-1β is released by monocytes and macrophages following P2X7 receptor activation by ATP, however the exact mechanism by which release occurs is poorly understood. We have previously identified the GAS3/PMP22 family member, epithelial membrane protein-2 (EMP2), as a P2X7 C-terminus interacting protein. Blocking EMP2 has been shown to reduce early Chlamydia trachomatis infectivity and modify cytokine secretion; however the function of EMP2 in this role is not described. The purpose of this study was to establish the role of EMP2 in P2X7 receptor dependent IL-1β release. Methods THP1 monocytic cells were transfected with non-targeting control siRNA, siRNA specific for EMP2 or the fluorescent indicator SiGLO, using the reagent Dharmafect Duo with an optimised protocol for THP-1 cells. Transfection efficiency was determined by flow cytometry and the efficiency of the knock-down was assessed by real-time PCR. THP1 cells were then treated with PMA (500 nM) for 3 hours to promote differentiation to a more macrophage like phenotype. Differentiated THP1 cells were stimulated with 1 µg/ml of LPS with and without a P2X7 receptor antagonist (A438079 hydrochloride), followed by BzATP (300 µM, P2X7 agonist) for 20 minutes. Cell supernatants were collected and IL-1β release was measured by ELISA; cytotoxicity was determined by lactate dehydrogenase (LDH) release. Results BzATP treatment of THP1 cells significantly enhanced IL-1β release compared with LPS stimulation alone. P2X7 receptor dependent IL-1β release was almost completely inhibited by pre-treatment with the receptor antagonist. SiRNA knockdown of EMP2 was approximately 40%, as confirmed by real-time PCR, and significantly enhanced P2X7 receptor dependent IL-1β release compared with controls. There was no significant difference in LDH release following stimulation with LPS or BzATP, suggesting that the increase in IL-1β release was not due to cytotoxicity. Conclusions EMP2 contributes to the regulation of P2X7 receptor dependent IL-1β release by differentiated THP1 cells.