POS0218 DECREASED miR-122-5p IN NEUTROPHIL-DERIVED EXOSOMES ATTENUATED IMMUNOREGULATORY FUNCTION ON MACROPHAGES BY TARGETING IRF5 EXPRESSION IN BEHCET’S DISEASE

Abstract

BackgroundBehçet’s disease (BD) is a chronic systemic vasculitis characterized by the overactivation of neutrophils and macrophages. Exosomes are membrane-derived vesicles that mediate intercellular communications and neutrophil-derived exosomes account for the major portion of serum exosomes in BD. However, the role of neutrophil-derived exosomes in BD remains unknown.Objectives1) To investigate the production of exosomes by BD neutrophils; 2) To elucidate the regulation of macrophage by BD neutrophil-derived exosomes; 3) To explore the mechanism of immunoregulatory functions of BD neutrophil-derived exosomes.MethodsBD and healthy control (HC) neutrophil-derived exosomes were extracted and quantified. Human monocyte-derived macrophages (HMDM) were stimulated with BD and HC neutrophil-derived exosomes, and TNF-α and IL-6 production were examined. Differently expressed miRNAs in BD neutrophil-derived exosomes were analyzed using miRNA sequencing. LPS-induced HMDM were treated with miRNA mimics or inhibitors, and TNF-α and IL-6 production were detected. miRNA was overexpressed in macrophages, and RNA sequencing was performed to analyze regulating pathways. Dual-luciferase assays were performed to confirm miRNA-mRNA interaction.ResultsBD neutrophils produced a significantly lower level of exosomes than HC ones. Both BD and HC neutrophil-derived exosomes suppressed TNF-α and IL-6 production by macrophages, but to a lesser extent by BD neutrophil-derived exosomes. Six downregulated miRNAs were presented in BD neutrophil-derived exosomes, including miR-122-5p. miR-122-5p mimics inhibited IL-6 and TNF-α production while miR-122-5p inhibitor promoted IL-6 and TNF-α production by HMDMs. Overexpression of miR-122-5p attenuated TLR4 and IFN-β signaling. miR-122-5p directly targeted 3’UTR of IRF5, the TF regulating TLR4 pathway and autocrine of IFN-β, and downregulated IRF5 expression confirmed by dual luciferase assay. Knocking down IRF5 dampened IL-6 and TNF-α production in HMDMs.Figure 1.(A) Decreased production of BD neutrophil-derived exosomes. (B) Reduced suppression of macrophage activation by BD neutrophil-derived exosomes. (C) Differentially expressed miRNAs (downregulated) in BD neutrophil-derived exosomes. (D) miR-122-5p suppressed TLR4 and JAK-STAT signaling in HMDM. (E) miR-122-5p inhibited activation of HMDM. (F) miR-122-5p inhibited IRF5 expression in HMDM.ConclusionOur findings suggested the reduced production and immunoregulatory function of BD neutrophil-derived exosomes, mediated by lower levels of miR-122-5p in neutrophil-derived exosomes. Impaired BD neutrophil-derived exosomes might be implicated in the overactivation of macrophages in BD.