Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy
Alejandro Ibáñez‐Costa, Carlos Perez-Sanchez, A. M. Patiño-Trives, M. Luque-Tévar, Pilar Font, I. Arias de la Rosa, Cristóbal Román-Rodríguez, M. C. Ábalos‐Aguilera, Carmen Conde, Antonio González, Sergio Pedraza‐Arévalo, Mercedes del Río-Moreno, Ricardo Blázquez‐Encinas, P. Segui, J. Calvo-Gutiérrez, R. Ortega Castro, Alejandro Escudero‐Contreras, Nuria Barbarroja, M. Á. Aguirre, Justo P. Castaño, Raúl M. Luque, Eduardo Collantes‐Estévez, C. López-Pedrera
Abstract
<h3>Objectives</h3> To characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response. <h3>Methods</h3> Leucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed. <h3>Results</h3> An altered expression of several SME was found in RA leucocytes. Eight elements (<i>SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1</i> and <i>SRSF10</i>) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced <i>SNRNP70-</i>overexpression and <i>KHDRBS1</i>-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts. <h3>Conclusions</h3> Overall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.