Background/Purpose:  MicroRNAs (miRNA) have recently emerged as a new class of modulators of gene expression, regulating inflammation, degradation of extracellular matrix and invasive behavior of the resident cells in rheumatoid arthritis (RA). Objectives: 1) To investigate the miRNA expression profile in synovial and blood neutrophils in RA and its role in the pathogenesis of this disorder. 2) To study the effects of biological therapies on the miRNA profile in neutrophils.

Methods:  Neutrophils were isolated from peripheral blood of 20 healthy donors and 20 RA patients. Synovial neutrophils were isolated from paired synovial fluid of 10 RA patients. nCounter microRNA Assay was used to detect simultaneously 800 human microRNAs in each sample. Altered miRNAs were analyzed for potential mRNA targets using Ingenuity pathways analysis (IPA) software. Several mRNA targets of these miRNAs were analyzed by RT-PCR. Healthy neutrophils were treated in vitro with anti-CCPs isolated from RA patients. mRNA expression of DICER, Ago1, Ago2, exportin was evaluated through RT-PCR. DICER protein expression was analyzed by western blot. Synovial and peripheral blood RA neutrophils were treated in vitro with tocilizumab and infliximab.

Results:  Among 800 miRNAs analyzed, 121 were detected in neutrophils from peripheral blood or synovial fluid. Using a 2 fold change cut off, 97 miRNAs were altered in peripheral blood neutrophils from RA patients compared to healthy donors. Most of them were downregulated (94 downregulated and 3 upregulated). Accordingly, RA neutrophils showed a downregulation of the proteins participating in miRNA biogenesis, including DICER, Ago1, Ago2, exportin 5. In addition, treatment of healthy neutrophils with IgGs anti-CCPs isolated from RA patients decreased the expression of these proteins, thus pointing at these autoantibodies as main modulators of microRNA neutrophil expression. Functional IPA analysis showed that altered miRNAs regulated cellular functions such as development, growth, proliferation and movement and were mainly involved in immunological disease. Among the miRNAs found deregulated blood, 34 miRNAs were found even more significantly reduced in synovial neutrophils compared to paired blood neutrophils from RA patients. These miRNAs were mainly involved in inflammatory response, suggesting the abnormal activation of this cell subtype in the joint. In vitro treatment of RA neutrophils with infliximab or tocilizumab reversed the expression of miRNAs and genes involved in their biogenesis.

Conclusion:

1) RA neutrophils exhibit a defect in the miRNAs processing, showed by a decrease in most of the detected miRNAs and the concomitant downregulation of proteins involved in their biogenesis. This defect seems to be modulated, at least partially, by anti-CCPs antibodies. 2) miRNA downregulation is even more pronounced in synovial resident neutrophils, which may contribute to the high inflammatory profile of these cells in the joint. 3) Tocilizumab and infliximab reverse the altered miRNA profile and the defect in the biogenesis machinery, reducing the proinflammatory pattern in these cells. Funded by CTS7940, PI2013-0191, CP15/00158, PI15/001333

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/altered-microrna-expression-pattern-in-synovial-and-blood-neutrophils-in-rheumatoid-arthritis-reveals-the-pathogenic-profile-of-these-cells-effect-of-biological-therapies/