1673 - The Role of Microrna-34 and Microrna-22 in Dendritic Cells and Monocyte Activation in Rheumatoid Arthritis

Monday, November 7, 2011: 5:00 PM
W475a (McCormick Place West)
Stefano Alivernini1, Derek S. Gilchrist2, Lynn Crawford2, Lucy Ballantine2, John Hunter3, Derek Baxter2, Barbara Tolusso4, Elisa Gremese4, Gianfranco Ferraccioli4, Iain B. McInnes2 and Mariola Kurowska-Stolarska2, 1University of Glasgow,, Glasgow, United Kingdom, 2University of Glasgow, Glasgow, United Kingdom, 3Gartnavel General Hospital, Glasgow, United Kingdom, 4Division of Rheumatology, Catholic University of the Sacred Heart, Rome, Italy
Presentation Number: 1673


MicroRNAs (miRs) are a novel class of post-transcriptional regulators that have been implicated in the pathogenesis of distinct human diseases, including Rheumatoid Arthritis (RA). We have previously shown that miR-34 family members and miR-22 are overexpressed in synovial fluid (SF) monocytes compared to matched peripheral blood (PB) monocytes in RA patients. The aim of this study was to investigate the functional role of miR-34 and miR-22 in the biology of monocytes and monocyte-derived DCs in the context of their abnormal activation in RA. 

Method: Expression of miR-34a and miR-22 in RA and osteoarthritis (OA) synovial tissues were evaluated by in situ hybridization. To characterize miR-34a positive cells, fluorescent in situ hybridization and immunostaining for CD68 were performed on RA tissues. Expression of miR-34a and miR-22 was evaluated by qPCR on DCs isolated by CD1c and CD304 specific microbeads from matched PB and SF of RA patients (n=3). Monocytes from PB of healthy donors (n=5) were isolated by CD14+ microbeads (AutoMACS) and transfected with miR-34a, miR-22 or control mimics. Cells were subsequently stimulated with LPS (10 ng/ml) or CL097 (1 mg/ml). DCs were generated from PB CD14+ cells stimulated with GM-CSF (100 ng/ml) and IL-4 (20 ng/ml) for 7 days. Once generated, CD14+ derived DCs (n=4) were transfected with miR mimics and stimulated as described above. After 18h of stimulation, supernatants were collected and evaluated for chemokine and cytokines levels (Luminex assay). To identify miR-34/22 targets HumanTargetScan cross-referenced with transcriptomic profile of SF CD14+ cells was employed. Identified targets were experimentally verified by miR luciferase assay and qPCR.

Result: miR-34a and miR-22 are overexpressed in SF DCs compared to matched PB DCs in RA patients. In situ hybridisation showed that miR-34a and miR-22 are widely expressed in RA synovium compared to OA. Double immunofluorescence staining revealed that miR-34a is present in RA synovial tissue myeloid cells. Enforced overexpression of miR-34a but not miR-22 in PB monocytes increased TLR7/8 triggered TNF-alpha production. Overexpression of miR-34a and miR-22 in monocytes-derived DCs increased spontaneous, TLR4 and TLR7/8 triggered TNF-alpha production. In addition, miR-22 but not miR-34a induced production of chemokines and interferon alpha by DCs. Computational target ranking system cross-referenced with transcriptomic profile of RA SF CD14+ cells identified Axl and Tyro3, receptor tyrosine kinases involved in negative feedback mechanism limiting TLRs-induced myeloid cells activation, as potential direct targets for miR-34a and miR-22. Experimental validation confirmed that miR-34a and miR-22 target 3’ UTR of Axl and Tyro3 mRNAs, respectively. Consistently, Axl and Tyro3 levels are downregulated in myeloid cells overexpressing miR-34a and miR-22, respectively. 

Conclusion: This study indicates that overexpression of miR-34a and miR-22 in myeloid cells can lead to the disregulation of their self-regulatory mechanism. Thus, high levels of miR-34a and miR-22 in synovial myeloid cells of RA patients may be responsible for an excessive pro-inflammatory activation of these cells.

Keywords: cytokines, dendritic cells, epigenetics, monocytes and synovitis

Disclosure: S. Alivernini, None; D. S. Gilchrist, None; L. Crawford, None; L. Ballantine, None; J. Hunter, None; D. Baxter, None; B. Tolusso, None; E. Gremese, None; G. Ferraccioli, None; I. B. McInnes, None; M. Kurowska-Stolarska, None.