Background and objectives Elevated miR-155 expression levels mediate adaptive and inflammatory events in Rheumatoid Arthritis by inhibiting the translation of key mRNAs involved in germinal centre and anti-inflammatory signalling. miR-155-deficient mice are protected from collagen-induced arthritis due to impaired auto-antibody production, Th17 differentiation and macrophage activation. However, active miR regulation as a therapeutic modality has not been attempted in arthritis models before and is a critical next step towards evaluating the translational potential of these pathways. We therefore investigated active neutralisation of miR-155 using in vitroand in vivo models with a novel miR-155 inhibitor.
Materials and methods For in vitro experiments, bone marrow was isolated from wild-type mice and differentiated into macrophages with M-CSF (50 ng/ml) for 6 days. Mature macrophages were transfected with miR-155 inhibitor (125, 250 or 500 nM; Regulus) or control inhibitor 24 h before stimulation with LPS (10 ng/ml). TNF-α production was measured by ELISA 24 h post-stimulation. miR-155 levels were measured by RT-qPCR. In vivo, CIA was induced using type II collagen with Complete Freund’s Adjuvant on day 1, and re-challenging with collagen on day 21. Mice were treated therapeutically with miR-155 inhibitor, control inhibitor or PBS on days 22, 29, 36 and 41 at the dose of 25 mg/kg. Clinical changes were scored every second day and concentration of serum IgG anti-collagen antibodies measured by ELISA.
Results Treatment of macrophages with miR-155 inhibitor significantly reduced TNF-α production in response to LPS (p = 0.007; n = 3 experiments), across a range of inhibitor concentrations. The therapeutic treatment with miR-155 inhibitor in vivo ( n = 12 mice/group) reduced onset of CIA and significantly reduced the severity of arthritis compared to control inhibitor at day 42 (clinical score 5.1+/-1.8mm versus 2.3+/-0.9mm, p < 0.001). There was no difference between control inhibitor and miR-155 inhibitor treatment in levels of anti-collagen antibodies.
Conclusions These experiments highlight the therapeutic potential of a miR-155 inhibitor to ameliorate severity of CIA in vivo. The in vitro mechanistic data demonstrates that the anti-inflammatory effect occurs inhibition of macrophage TNF-α production in response to LPS stimulation. The therapeutic effect of miR-155 inhibition during the effector phase of arthritis provides proof-of-concept that miR-155 is crucial in driving articular inflammation.
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