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Background and Objectives Monocytes can give rise to different cell types including osteoclasts (OC), which play an important role in maintaining bone homeostasis by resorbing bone matrix. Circulating monocytes consist of at least two main functional subsets of immune cells, Ly6C high and Ly6C low monocytes, arising from a common progenitor in the bone marrow. Excessive and prolonged activation of inflammatory LyC6 high monocytes is a hallmark of many inflammatory diseases including arthritis. Among key molecular rheostats, micro (mi) RNAs are a class of regulatory RNAs that control basic biological functions and orchestrate inflammatory responses. Among master miRNAs of innate immunity, miR-146a exerts a negative retro-control on inflammation transduction signals and inhibits osteoclastogenesis. Despite aberrant increased expression in rheumatoid arthritis (RA), miR-146a is unable to properly down regulate inflammation, leading to prolonged TNF production and increased OC, two arthritis hallmarks. Here, we investigated whether miR-146a was differentially regulated in both Ly6C high and Ly6C low monocyte subsets in healthy and arthritic conditions. Moreover, we developed techniques to deliver siRNA to LyC6 high monocytes in vivo and thus determined the specific impact of miR-146a overexpression in this particular monocyte subset on inflammation and osteoclastogenesis, in the context of arthritis. Materials and Methods Subset monocytes were isolated from peripheral blood of arthritic and healthy mice by FACS sorting following membrane stainings. Transcriptomic analyses for miRNA expression were performed (n = 6) and differential miRNA expression levels were validated on individual samples using multiplex RT-qPCR (n = 13). Collagen-induced arthritic (CIA) mice were injected intravenously at disease onset with lipoplex containing either control or miR-146a mimic (0.5 mg/kg). Arthritis severity was monitored and OC differentiation was performed by stimulating bone marrow-derived monocytes (BMDM) with M-CSF and RANK-L miR-146a expression levels measured during OC differentiation. TRAP activity, OC numbers and nuclei per OC were quantified. Results Transcriptomic analyses showed higher expression of miR-146a in Ly6C low monocytes when compared to Ly6C high monocytes, in both healthy and arthritis mice. In arthritis mice, expression of miR-146a in Ly6C high monocytes is down-regulated as compared to healthy controls. During differentiation of BMDM into OC, miR-146a expression levels are down-regulated on day 2. Enforced expression of miR-146a in Ly6C high monocytes upon intravenous injection of miR-146a-containing lipoplex leads to decreased bone erosion in mouse CIA. This effect was associated with a decreased number of mature OC and TRAP activity, as well as a reduced number of nuclei per OC. Conclusions Overall, our results show that specific over-expression of miR-146a in Ly6C high monocytes alters OC differentiation and decreased bone erosion in mouse CIA. These data also suggest that Ly6C high monocytes might be the monocyte subset precursor of OC, and that targeting this specific monocyte subset might represent a therapeutic option in the context of arthritis to inhibit bone loss.

Background and objectives The inflammatory monocyte subset is essential in innate immunity but it also contributes to the pathogenesis of many inflammatory and autoimmune disorders including rheumatoid arthritis (RA). Strategies to manipulate this cell subset are thus of importance both scientifically and therapeutically. The pre-B cell colony enhancing factor (PBEF/visfatin/Nampt) is an essential enzyme in the NAD biosynthetic pathway that exerts a key role in the persistence of inflammation through the induction of the expression of the tumour necrosis factor α (TNFα) and IL-6 pro-inflammatory cytokines and is highly expressed in patients with a variety of autoimmune disorders. Here the authors aimed at evaluating whether the precise targeting of PBEF within the inflammatory monocytes could be therapeutically beneficial in RA. Materials and methods Using an optimised lipid nanoparticle formulated with a PBEF-silencing short interfering (si) RNA or a non-targeting fluorescent siRNA (0,5mg/kg), and systemic administration to collagen-induced arthritic mice (CIA), fluorescence and PBEF expression were monitored in various leucocyte populations including Ly6C high monocytes within blood, spleen, and inflamed joints by cytometry. Clinical and biological features of CIA were monitored as previously described. Results Upon intravenous injection, siRNA-containing liposomes are engulfed by circulating Ly6C high inflammatory monocytes and provide efficient down-regulation of PBEF expression as well as of PBEF-induced pro-inflammatory cytokines (TNF and IL-6) in this subset but not in the non-inflammatory subset, leading to decreased number of infiltrating leucocytes in arthritic joints and reduced count of effector T cells. Importantly, when the liposome formulation is optimised for translation to the clinic, siRNA lipoplexes efficiently silence PBEF in human CD14 + CD16 - inflammatory monocytes ex vivo. Conclusions Delivery into the inflammatory mouse Ly-6C high monocyte population of a siRNA against PBEF is able to reduce molecular and cellular markers of inflammation in a preclinical model of RA, both systemically and locally, inhibiting disease progression.