Background Rheumatoid arthritis (RA) is a multifactorial disease, which is likely associated with the effects of environmental and genetic factors. The PTPN22 rs2476601 variant has been unambiguously identified as a RA risk genetic factor. PTPN22 encodes the lymphoid specific phosphatase Lyp that affects the responsiveness of T and B cell receptors. However, to date, its role in the activation of neutrophils NADPH oxidase remains undetermined.
Objectives To study the role of Lyp in neutrophils, particularly the activation and the priming, of NADPH oxidase in healthy donors.
Methods Neutrophils will be isolated from the blood of healthy donors using the Dextran-Ficoll gradient techniques. Lyp mRNA will be analyzed by quantitative real time PCR. Expression and localization of Lyp will be detected by SDS-PAGE, Western Blot techniques and confocal microscopy. The effects of the TNFα will be investigated. The signaling pathways implicated in activation of the NADPH oxidase will be analyzed by SDS-PAGE and Western Blots techniques. The protein targets of LYP will be analyzed by co-immunoprecipitation. The role of Lyp in reactive oxygen species (ROS) production by neutrophils will be determined by using two Lyp inhibitors. ROS production will be measured by luminol-amplified chemo-luminescence, NADPH activation will be measured by the cytochrome c reduction assay.
Results Lyp is expressed in resting human neutrophils. With TNFα, Lyp expression increase and Lyp translocates to the membrane. Two Lyp inhibitors inhibit the production of ROS and anion superoxyde (O2°-) in human neutrophils stimulated with fMLF alone and when they are primed by TNFα. They have no effect on degranulation. Lyp inhibitors also inhibit the phosphorylation of ERK1/2 after stimulation with TNFα, and the phosphorylation of p47phox induced by fMLF.
Conclusions The tyrosine phosphatase Lyp could have a NADPH oxidase activator role in human neutrophils. Lyp is involved in the activation of ERK1/2 and p47phox phosphorylation. Lyp inhibitors could have anti-inflammatory effects in RA.
Disclosure of Interest None declared