Background/Purpose: To identify specific molecular profiles associated to the enhanced cardiovascular (CV)-risk present in Rheumatoid Arthritis (RA) patients and their modulation by biologic disease modifying anti-rheumatic drugs (b-DMARDs).

Methods: Two hundred and eleven RA patients and 52 healthy donors (HD) were recruited to the study. Serum inflammatory and oxidative stress biomolecules and Netosis-derived products were quantified, and miRNnomes were identified using next-generation sequencing miRNA assay. The CV-risk score was calculated following EULAR recommendations. To evaluate the contribution of molecular profiles to the CV-risk, unsupervised clustering analyses were developed.

In parallel cohorts of 45 and 27 RA patients, respectively, the in vivo effects of biologic drugs [anti-TNFalpha (TNFi) and anti-CD20 (CD20i) inhibitors] were evaluated before and after 6 months of therapy.

Results: A number of circulating biomolecules related to inflammation, NETosis, and oxidative stress were coordinately altered in the serum of RA patients and closely related to the activity of the disease. Besides, more than a hundred circulating miRNAs were found altered in RA patients and linked with the biomolecules found altered.  Unsupervised clustering analyses differentiated 2 clusters representing different molecular profile groups.  Clinically, each cluster was characterized by: 1) RA patients with high CV-risk Score (7.01±8.4), medium disease score (DAS28: 3,37±1,4), and positivity for ACPAS and RF near 50%. 2) RA patients with low CV-risk Score (1.6±3.2), medium-high disease score (DAS28: 4.5±1.5) and positivity for ACPAS and RF around 75%. Interestingly, patients belonging to each cluster displayed a distinctive molecular profile in terms of inflammatory molecules deregulated, so that cluster 1 expressed higher levels of cytokines and chemokines related to cell proliferation, chemotaxis and angiogenesis (i.e. IFNg, IL5, IL6, IL10, IL15, VEGF, MCP1, and eotaxin), while in cluster 2 these molecules were reduced and predominated a different set of cytokines (IL1RA, IL1b, IL2, IL7, IL8, IL12, IL13, IL17, FGFb, MIP1b, TNFa). Hence, a different signature of miRNAs regulating these proteins was altered, so that in cluster 1 miR-106a-5p, miR-199a-5p and miR-148b-3p were reduced and miR-346, miR-299-3p, miR-6816 were elevated in relation to cluster 2. Likewise, NETosis derived products and oxidative stress molecules were increased in cluster 2 in comparison with cluster I. In vivo treatments with TNFi and CD20i reduced disease activity and induced the re-establishment of normal levels in these altered biomolecules in an inhibitor-dependent manner.

Conclusion: 1. Extensive molecular profiling of serum in RA patients might help to define precise CV-risk profiles in RA patients. 2. Specific mediators of autoimmunity, inflammation, oxidative damage and Netosis, along with the miRNAs modulating their expression, coordinately determine a higher CV-risk score in RA patients. 3. Biological drugs, such as TNFi and CD20i re-establish normal levels of these circulating biomolecules, thus reducing the CV risk in RA patients. 

Funded by PI-0285-2017, ISCIII, PI18/00837 and RIER RD16/0012/0015 co-funded with FEDER.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/unsupervised-molecular-profile-clustering-in-the-serum-of-rheumatoid-arthritis-patients-identifies-groups-with-differential-cv-risk-score-modulation-by-biological-therapies/