title: Role of Neutrophils in Osteoarthritis and Rheumatoid Arthritis
Creators: name: Zhan, Dong
Publisher: name: University of Liverpool Repository
Year: 2020

description: Objectives: The synovitis observed in knee osteoarthritis (OA) differs from that typically found in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) may play a function in the development of synovitis by the production of inflammatory cytokines and chemokines or inflammatory mediators in synovial fluid. Neutrophil-derived microvesicles (NDMV) possess the capability of cartilage protection and anti-inflammatory effects. Thus, we investigated the expression of inflammatory cytokines produced by tumour necrosis factor alpha (TNFα)-stimulated FLS which were pre-treated with NDMV. Methods: FLS isolated from the synovium of patients with OA undergoing knee replacement were characterized by flow cytometry for fibroblast markers. 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. MicroRNAs of NDMV were sequenced by Illumina NextSeq500 and analysed by miRWalk 2.0 to predict target genes and possible signaling pathways. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to measure the viability of the FLS after NDMV treatment, while internalisation of fluorescently labelled NDMV was measured by flow cytometry and confocal microscopy. Levels of inflammatory cytokines in FLS cultured supernatants were quantified by the Bio-Plex suspension array system. Results: High purity (≥ 95%) of FLS in sub-culture at passage 4 were prepared from OA synovium. Isolated neutrophils (purity &gt; 98%) were treated for 20min treatment with 50ng/ml TNFα prior to preparation of NDMV. Isolated NDMV (diameter range from 200 to 500nm) expressed more Annexin V and CD63 on their membrane surface than untreated control neutrophils. Incubation of FLS with NDMV at a ratio of 1:100 resulted in a time-dependent uptake, with 35% of synoviocytes containing microvesicles over a 6-24h time-period, with no significant change in cell viability. NDMV contained 159 different microRNAs which were predicted to interact with 7,978 target genes. 74 and 26 target genes of NDMV-carried microRNAs were predicted to interact with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signaling pathways, respectively (p&lt;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 interleukin (IL)-5, IL-6, IL-8. Monocyte chemotactic and activating factor (MCP)-1, interferon (IFN)γ and macrophage inflammatory proteins (MIP)-1β (p&lt;0.01). However, this down-regulation was selective, as NDMV had non-significant effects on TNFα-stimulated expression of granulocyte colony stimulating factor (G-CSF), IL-17, IL-2 or IL-4. Conclusions: NDMV are internalised by FLS and can selectively inhibit TNFα-stimulated inflammatory cytokine/chemokine secretion. This process may occur via NDMV-carried microRNAs negatively regulating post-transcriptional control of PI3K-AKT and MAPK signaling pathways. NDMV therefore may perform an anti-inflammatory role in the regulation of FLS function in synovitis. More research is necessary to identify if NDMV could be a potential therapeutic tool to improve life quality of OA and RA patients.