Macrophages play an important role in host defense pathways and are also involved in a variety of diseases, including atherosclerosis (1, 2). The key role of macrophages in the development of atherosclerosis has made this cell type a versatile in vitro model of this disease (3).Transfection of macrophages is a powerful tool to study their function, and a number of methods have been described to achieve high levels of expression of different genes through transient transfection (4-6). These levels of expression are sufficiently high to study synthetic processes, when proteins are tagged or otherwise distinguished from host proteins. However, studying cell functions such as growth, lipoprotein binding, lipid uptake, and efflux requires not only high levels of gene expression but also for the gene to be expressed in a majority of cells, a highefficiency transfection. High efficiency of transfection is also critical for a multiple gene transfection, as it requires that all transfected genes be expressed in the same population of cells. Viral and stable transfections offer adequate efficiency of DNA transfer; however, they are often laborintensive and time-consuming. High-efficiency transient transfection of macrophages has proved to be difficult.Here, we describe a method for high-efficiency transient transfection of RAW 264.7 mouse macrophages. We fortuitously found that the low efficiency of expression of transfected genes in macrophages is a consequence of methylation-mediated silencing of transfected genes rather than of low uptake of DNA into cells. To maximize the efficiency of macrophage transfection, we evaluated the DNA methylation inhibitor, 5-azacytidine, an epigenetic modifier often used to reactivate methylation-dependent transcriptionally silent genes (7). We demonstrated by methylationspecific PCR that 5-azacytidine prevents methylation of the promoter of transfected genes, and for the first time we achieved transient expression of a reporter protein in 80-100% of macrophage cells. The method was then used for the high-efficiency transient transfection of RAW 264.7 macrophages with sterol 27-hydroxylase (CYP27A1), which led to the stimulation of cholesterol efflux from these cells.Abbreviations: apoA-I, apolipoprotein A-I; CMV, cytomegalovirus; CYP27A1, sterol 27-hydroxylase; GFP, green fluorescent protein; LXR, liver X receptor.

Background:micro-RNAs (miR) are strong regulators of gene expression. Their involvement in RA key cytokines pathway regulation entitles them as important players in RA pathophysiology. The miR 17-92 cluster has been widely studied in cancer as they regulate cell apoptosis.Objectives:The aims of this study were to screen miR 17-92 cluster’s expression in different RA phenotypes (erosive and non erosive), further elucidate the mechanisms and direct targets involved in miR-17-5p anti-inflammatory role and to investigate miR-17-5p therapeutic effect in arthritis.Methods:A miR array was performed in synovial tissue from naïve erosive and non-erosive RA patients. Intra-articular delivery of miR-17 lipoplex was performed in collagen induced arthritis model in mice. Clinical, histological and structural effects were studied over the course of arthritis. In depth studies of miR-17 mechanisms of action were performed in primary RA-FLS isolated from RA synovial tissue.Results:Among others, miR-17-5p expression was reduced in erosive RA. miR-17 transfection in arthritic paws significantly reduced clinical inflammation. Moreover, synovial B cells, T cells, macrophages and polynuclear neutrophils infiltrates were significantly reduced. Structural damage was also decreased as shown by a reduction in the number of osteoclasts and erosion score by CT analysis. Pro-inflammatory cytokines of the IL-6 family, STAT3 target genes and IL-1β expression were also significantly reduced, but not TNF-alpha. miR17 directly targeted the 3’-untranslated region of STAT3 and JAK1. STAT3 and JAK1 mRNA and protein expression were reduced in RA-FLS following miR-17 transfection. STAT3 and JAK1 mRNA and activation of STAT3 as assessed by immunohistochemistry were also reduced in injected paws.Conclusion:We demonstrate an anti-inflammatory and anti-erosive role of miR-17 in vivo. This effect involves the suppression of the IL-6 family autocrine amplifying loop through the direct targeting of JAK1 and STAT3 as shown in RA-FLS.FigureEffect of miR-17 lipoplex injection in Collagen induced arthritis (CIA) mice’s ankles.Disclosure of Interests:None declared