Background/Purpose: 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. Methods: 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. Results: 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.
The Role of Microrna-34 and Microrna-22 in Dendritic Cells and Monocyte Activation in Rheumatoid Arthritis
Gremese E;
2011-01-01
Abstract
Background/Purpose: 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. Methods: 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. Results: 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
