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Impaired microRNA processing in RA neutrophils
Introduction
Several immune cells including T and B lymphocytes, macrophages, synovial fluid (SF) fibroblast and neu- trophils are known to be relevant in the rheumatoid arthritis (RA) pathogenesis.1 Among them, RA neutrophils are activated cells, characterized by a prolonged lifespam, increased migratory capacity and production of inflamma- tory molecules and reactive oxygen species (ROS). In severe acute inflammation, SF accumulates a great number of these cells in a more activated state, promoting cartilage destruction and joint damage.2
Antibodies to citrullinated protein antigens (ACPA) are currently considered the most specific autoantibodies in RA, being related to the activity of the disease and poorer prognosis.3 ACPA have been shown to be able to induce neutrophils to produce high levels of inflammatory medi- ators, ROS and to generate NETosis.2,4
Epigenetic modifications contribute to the development of RA, affecting disease susceptibility and severity.5,6 Among them, several microRNA (miRNA) have been linked to the chronic inflammation in RA.5 MiRNA are short noncoding RNA present in all multi-cellular organ- isms involved in a broad range of cellular processes. They cause posttranscriptional and posttranslational gene silencing, by recognizing a specific sequence of mRNA, binding to it and inhibiting its translation into protein.7 MiRNA is first transcribed into long primary miRNA of several kb in length (pri-miRNA) and this pri-miRNA is then processed by Drosha into a precusor miRNA (pre- miRNA) of appoximately 70-nucleotide. The pre-miRNA is transported out of the nucleus by exportin 5 (XPO-5) and is then processed by DICER into a mature double stranded miRNA of approximately 22 nucleotides. The RNA-induced silencing complex (RISC) (composed of the transactivation-responsive RNA-binding protein [TRBP] and argonaute [AGO]) removes the complementary strand. DICER then binds to RISC, forming the core of RISC-loading complex. DICER is considered a crucial fac- tor in miRNA processing since its presence is necessary for the stimulation of RNA processing by AGO.8,9 Functional miRNA is able to bind to the 3’-untranslated region (UTR) of the target mRNA, causing mRNA cleavage or transla- tional repression.10
Several studies, mainly conducted on lymphocytes, monocytes, macrophages and SF fibroblasts, have report- ed that the role of various miRNAs in the pathogenesis of RA is critical for the increased expression of inflammatory cytokines and prolonged cell survival.5,11
We undertook this study to evaluate the miRNA profile and the proteins involved in miRNA processing in circulat- ing and SF neutrophils from RA patients, in order to gain an insight of its role in the different activation states of these cells. The effects of ACPA or inflammatory compo- nents and biological therapies on the expression of miRNA in neutrophils was further assessed.
Methods
For details see the Online Supplemental Materials and Methods.
RA patients and healthy donors
Forty RA patients and 40 healthy donors (HD) were included in
this study. RA patients fulfilled at least four 1987 American College of Rheumatology (ACR) criteria and achieved a total score of 6 or greater according to 2010 criteria. The patients were under the following treatment regimes: corticosteroids (50.0%), leflunomide (42.5%), hydroxychloroquine (45.0%), NSAID (80.0%) and methotrexate (65%). All patients were tested for the presence of ACPA and rheumatoid factor (RF) by clinical labora- tory routine analysis. All participants enrolled were Caucasian, recruited at the Department of Rheumatology, and gave their written informed consent approved by the ethical committee of the Reina Sofia Hospital (Cordoba, Spain).
Clinical details of the RA patients and HD are shown in Table 1. Peripheral blood (PB) was withdrawn from all the RA patients and the HD. SF from RA patients was obtained through arthro- centesis. The study design is displayed on a flow chart (Online Supplementary Figure S1).
Isolation of neutrophils from PB and SF
Neutrophils from PB of HD and paired SF and PB samples of RA patients were isolated (after centrifugation to obtain buffy coat and osmotic lysis of the pellet) by immunomagnetic positive selection with human anti-CD15 microbeads (Miltenyi Biotec S.L, Bergisch Gladbach, Germany) using AUTOMACs (Miltenyi Biotec).12
miRNA expression profiling
The nCounter miRNA Assay (NanoString Technology, Seattle, WA, USA) detects simultaneously 800 human miRNA in each sample. 100 ng of RNA, pooled samples of neutrophils from PB of 10 HD, neutrophils from PB of 10 RA patients and neutrophils from SF of 10 RA patients were prepared by ligating a specific DNA tag (miR-tag) onto the 3 end of each mature miRNA fol- lowed by 16-20 hour hybridization (at 65oC) to nCounter Reporter and Capture probes. The rest of the protocol was per- formed following the manufacturer’s recommendations (NanoString Technologies, Seattle, WA, USA). Data were normal- ized by the geometric mean of top 100 miRNA detected using the nSolver software. This miRNA array was performed in pooled samples of the 10 RA patients that best represented the mean val- ues of age, sex, disease activity, evolution time and autoimmunity of the clinical validation cohort.
IgG-ACPA isolation from RA patients
Immunoglobulin G (IgG) from serum of five different RA patients with high titers of ACPA and negative for RF (enriched IgG-ACPA) and 5 HD (IgG- normal human serum [NHS]) were isolated using HiTrap protein G HP columns (GE Healthcare, Chicago, Il, USA).
In vitro treatments of neutrophils
Neutrophils purified from five RA patients (taking Disease-
modifying antirheumatic drugs and not taking any biological ther- apies) were pre-treated with FCRII blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min and subsequent- ly incubated with infliximab (IFX) at 100 g/mL or tocilizumab (TCZ) at 20 g/mL for 6 hours. The selection of these patients allowed the isolation of assumingly activated neutrophils, leading to an increased expression of inflammatory cytokines, in order to demonstrate the effects of miRNA transfection by proving the reduction in expression levels of those molecules.
Neutrophils purified from five HD were treated in vitro with IgG-NHS or enriched IgG-ACPA (500 ug/mL), tumor necrosis fac- tor-a (TNF-a) and interleukin-6 (IL-6) (10 ng/mL) for 6 hours. Samples were processed for RT-PCR analyses.
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