OP0292 THE HELPFUL EIGHT: KEY SPLICING MACHINERY ELEMENTS IN LEUKOCYTE SUBSETS MAY IMPROVE THE TYPIFICATION OF THE DISEASE IN RHEUMATOID ARTHRITIS PATIENTS

Background The Splicing, a process of removing introns of RNA to produce mature messenger RNA, is exerted by an intricate macromolecular complex, the spliceosome. The alternative splicing, key mechanism that provides transcription and protein diversity, is dysregulated in various genetic, metabolic and inflammatory diseases. Splicing variants could contribute to the development and severity of pathologies of crucial importance. Objectives The aim of this study was the identification and characterization of alterations in the splicing machinery of leukocyte subsets of Rheumatoid Arthritis (RA) patients, as well as their influence on the activity of the disease and its atherothrombotic profile. Methods Monocytes, lymphocytes and neutrophils from 74 RA patients and 29 healthy donors (HD) were purified, and 45 elements of the splicing machinery were evaluated using a microfluidic qPCR array (Fluidigm). In parallel, the clinical status of the patients and the carotid intima-media thickness (CIMT) were assessed, along with an extensive serological evaluation, including autoantibodies, oxidative stress markers, inflammatory molecules and prothrombotic mediators. Correlation and association studies and logistic models among those clinical and analytical parameters were performed. Finally, leukocytes from HD were incubated with purified ACPAs-IgG from RA patients and changes promoted in both, splicing machinery and leukocyte inflammatory profile, were assessed. Results A significantly altered expression of spliceosome components was found in all the leukocyte subsets: 21, 17 and 27 components were differentially expressed in monocytes, lymphocytes and neutrophils, respectively, in RA patient’s vs HD. The levels of those altered components were associated to ACPA positivity, high disease activity (DAS28>5.1), early atherosclerosis and radiological involvement. Correlations with inflammatory mediators, oxidative stress markers and NETosis were further identified. Interestingly, eight components, belonging to the major spliceosome (SNRNP70, SNRNP200 and U2AF2), minor spliceosome (RNU4ATAC), and splicing factors (RBM3, RBM17, KHDRS1 and SRSF10) were found simultaneously altered in the three leukocyte subtypes. Logistic regression models, built with levels of simultaneously altered spliceosome components, generated several signatures related to RA disease features that were able to: i) discriminate between RA patients and HD; ii) classify patients with high disease activity (DAS28>5.1); iii) recognize patients with radiological involvement; and iv) identify patients showing atheroma plaques. In vitro studies showed that the purified ACPAs of RA patients modulated the expression of these components. Conclusion 1) The splicing machinery is profoundly altered in leukocytes from RA patients and closely related to the activity of the disease, including inflammation, autoimmunity and articular involvement, as well as with the occurrence of early atherosclerosis. 2) Evaluation of the common altered components of the spliceosome in leukocytes subsets might be used to typify the disease, avoiding complementary ultrasound or radiological tests. Acknowledgement MINECO (FJCI-2016-30825) and ISCIII (PI18/00837 and RIER RD16/0012/0015) co-funded with FEDER. Disclosure of Interests None declared