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THU0007 Microrna-155/PU.1 Axis as an Epigenetic Regulator of B-Cell Activation in Rheumatoid Arthritis
  1. S. Alivernini1,
  2. B. Tolusso1,
  3. M. Kurowska-Stolarska2,
  4. S. Canestri1,
  5. R. Benvenuto3,
  6. A. Mangoni3,
  7. A.L. Fedele1,
  8. L. Petricca1,
  9. E. Gremese1,
  10. I.B. McInnes2,
  11. G. Ferraccioli1
  1. 1Division Of Rheumatology, Institute Of Rheumatology And Affine Sciences, Catholic University Of The Sacred Heart, Rome, Italy
  2. 2Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
  3. 3Division of Pathology, Catholic University Of The Sacred Heart, Rome, Italy

Abstract

Background MicroRNA-155 (miR-155) has been shown to be a key regulator of B cell biology. The regulation of the transcription factor PU.1 by miR-155 is required for high-affinity IgG1 production and B cell maturation. However, the role of miR-155 in the activation of B in Rheumatoid Arthritis (RA) has not been explored.

Objectives To investigate miR-155 expression in RA B cells and its association with B cell activation and synovial inflammation.

Methods 53 RA patients underwent synovial (ST) biopsy. ST samples were categorized on a basis of immunostaining for CD68, CD21, CD3 and CD20 cells as diffuse or aggregate pattern. MiR-155 expression in RA ST was evaluated by in situ hybridization (ISH)(n=15). B cells from peripheral blood (PB) and matched synovial fluid (SF) of RA (n=19) and PB of healthy controls (HC)(n=10) were isolated by CD19+ microbeads. B-cell subsets were determined through Flow-Cytometry. IL-6 and BAFF levels in PB and SF were measured by ELISA. MiR-155 and PU.1 expression was determined by qPCR in B cells from PB and SF of RA (n=10) and on ST samples of osteoarthritis (OA)(n=3), RA diffuse (n=8) and RA aggregate (n=8) patients. Finally, HC PB B cells (n=5) were cultured with or without IL-6 (30ng/ml) or BAFF (20ng/ml) and miR-155 and PU.1 expression was assessed by qPCR.

Results 23 (43,4%) RA patients had an aggregate pattern of ST biopsy. RA with an aggregate pattern were more likely anti-CCP+ compared to RA with diffuse pattern (p=0.04).Moreover, anti-CCP plasma levels directly correlated with the synovial aggregate grade (r=0.39; p=0.01). IL-6 and BAFF levels were higher in SF than in PB of RA regardless of the synovial infiltrate pattern (p=0.001 for both). ISH showed that miR-155 was expressed at significantly higher levels in ST of aggregate RA compared to diffuse RA (p=0.03). qPCR further confirmed that miR-155 was significantly increased in ST of aggregate RA compared to diffuse RA (p=0.03) and OA (p=0.03) respectively. Consistently, PU.1 staining was found to be lower within synovial lymphoid aggregates in RA patients. On the single cell level, miR-155 was expressed significantly higher in RA PB B-cells compared to HC (p=0.0002). In addition, miR-155 was over-expressed in SF B-cells compared to matched PB B-cells (p=0.05) in RA. Consistently, PU.1 expression was lower in SF B-cells compared to matched PB B-cells (p=0.001). Moreover, anti-CCP+ RA showed higher miR-155 expression in PB B-cells compared to anti-CCP- RA (p=0.02) and HC (p=0.001). CD19+/IgD-CD27- cells were significantly over-represented in PB of aggregate compared to diffuse RA (p=0.04). Finally, IL-6 and BAFF in vitro stimulation of HC B-cells induced miR-155 overexpression (p=0.04 and p=0.03) whereas PU.1 was significantly down-regulated (p=0.01 and p=0.03).

Conclusions MiR-155 is over-expressed in B-cells of RA and is associated to anti-CCP positivity, an aggregate synovial pattern and a PU.1 lower expression. IL-6 and BAFF are significantly increased in the SF environment and induce in vitro miR-155 expression in B-cells. Thus, miR-155 may represent a key regulator of B-cells in RA patients with a memory phenotype.

Disclosure of Interest None declared

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