Role of Neutrophils in Osteoarthritis and Rheumatoid Arthritis and Their Metabolomics

Abstract

Objectives: Fibroblast-like synoviocytes (FLS) plays a function in the development of synovitis in osteoarthritis (OA) and rheumatoid arthritis (RA). Neutrophil-derived microvesicles (NDMV) can protect cartilage and possess anti-inflammatory effects on recipient cells. We investigated the expression of inflammatory cytokines produced by TNFα-stimulated FLS which were pre-treated with NDMV. Methods: FLS isolated from the synovium of patients with OA were characterized by flow cytometry. Neutrophils were isolated from healthy volunteer blood and were characterized by cytospin and flow cytometry. NDMV, generated from TNFα-stimulated neutrophils, were characterized by electron microscopy, nanoparticle tracking analysis and flow cytometry. MicroRNA of NDMV were sequenced by Illumina NextSeq500. MTT assay was used to measure FLS viability after NDMV treatment, while internalisation of fluorescently labelled NDMV was measured by flow cytometry and confocal microscopy. Cytokines levels in FLS cultured supernatants were quantified by the Bio-Plex suspension array system. Results: High purity (≥95%) of FLS at passage 4 were prepared from OA synovium. Isolated neutrophils (>98%) were treated for 20min treatment with 50ng/ml TNFα prior to preparation of NDMV. Isolated NDMV (200 to 500nm) expressed more Annexin V and CD63 than untreated neutrophils. Incubation of FLS with NDMV at a ratio of 1:100 resulted in a time-dependent uptake, with 35% of FLS containing NDMV over a 6-24h time-period, with no significant change in cell viability. 74 and 26 target genes of NDMV-carried microRNAs were predicted to interact with the PI3K-AKT and MAPK signaling pathways (p<0.0001). TNFα (10ng/ml) stimulated the expression of a number of cytokines and chemokines from synoviocytes and NDMV down-regulated this TNFα-induced expression of IL-5, IL-6, IL-8. MCP-1, IFNγ and MIP-1β (p<0.01). Conclusions: NDMV are internalised by FLS and inhibit TNFα-stimulated inflammatory cytokine secretion via NDMV-carried microRNAs negatively regulating post-transcriptional control of PI3K-Akt and MAPK signaling pathways.