Background/Purpose: 1) To identify and characterize the alterations present in the splicing machinery in leukocyte subsets of Rheumatoid Arthritis (RA) patients, and 2) To evaluate their influence on the activity of the disease and its atherothrombotic profile.

Methods: The study was conducted in monocytes, neutrophils and lymphocytes purified from 74 RA patients and 29 healthy donors (HD). By using a microfluidic qPCR array, a set of 45 elements of the splicing machinery were evaluated, including the complete major and minor spliceosome components, and splicing factors with potential pathological role. In parallel, extensive clinical/serological evaluation were carried out. Leukocyte subsets from RA patients were transfected with expression plasmids of selected splicing components, and their effects on cell activity were evaluated. Lastly, leukocyte subsets from HD were incubated with serum from high-activity RA patients and changes promoted in splicing machinery and leukocytes activity were assessed.

Results: A global reduction of spliceosome components in the three leukocyte subsets from RA patients was observed, except for a key minor spliceosome component, RNU4ATAC, which was consistently over-expressed. The levels of those altered components were associated to ACPA positivity, disease activity, and radiological involvement. Correlations with inflammatory mediators, oxidative stress markers and netosis were also demonstrated.

Interestingly, eight spliceosome components, including two small nuclear RNA (snRNU) of the major spliceosome (RNU1, RNU5 and U2AF2), the snRNA of the minor spliceosome, RNU4ATAC, and the splicing factors RBM3, RBM17, KHDRS1 and SRSF10 were simultaneously altered in the three leukocyte subtypes. Logistic regression and ROC analyses, including the eight spliceosome components simultaneously altered, identified several signatures as biomarkers of specific RA disease features, being 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.

Mechanistic in vitro studies demonstrated the involvement of several spliceosome components in the altered inflammatory profile of RA leukocytes. Likewise, splicing machinery was deregulated by effect of inflammatory/autoimmune mediators present in the serum of RA patients with high disease activity.

Conclusion: 1) We have identified specific alterations in the splicing machinery of leukocytes from RA patients, associated with the activity of the disease, as well as with its inflammatory and atherothrombotic profile. 2) Several signatures involving common altered components of the spliceosome in leukocytes subsets may be used as novel biomarkers for the typification of the disease, avoiding complementary ultrasound or radiological tests. 3) The altered expression of the splicing machinery in leukocytes of RA patients might derive from their autoimmune and proinflammatory profile, which might, in turn, contribute to the clinical shape of the disease.

Funding: ISCIII (PI15/01333 and RIER RD16/0012/0015) co-funded with FEDER

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/alterations-of-the-splicing-machinery-in-leukocyte-subsets-of-rheumatoid-arthritis-patients-modulate-their-inflammatory-autoimmune-and-atherothrombotic-profiles/