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P2 Receptors Mrna Expression Profiles in Macrophages from Ankylosing Spondylitis Patients and Healthy Individuals

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P2 Receptors Mrna Expression Profiles in Macrophages from Ankylosing Spondylitis Patients and Healthy Individuals Received: 14 August 2019 | Revised: 2 December 2019 | Accepted: 6 December 2019 DOI: 10.1111/1756-185X.13783 ORIGINAL ARTICLE P2 receptors mRNA expression profiles in macrophages from ankylosing spondylitis patients and healthy individuals Maryam Akhtari1,2 | Seyed Jalal Zargar1 | Mahdi Vojdanian2 | Amir Ashraf-Ganjouei2 | Ali Javinani2 | Elham Hamzeh2 | Alireza Rezaiemanesh3 | Ahmadreza Jamshidi2,4 | Mahdi Mahmoudi2,4 1Department of Cell & Molecular Biology, School of Biology, College of Science, Abstract University of Tehran, Tehran, Iran Background: Ankylosing spondylitis (AS) is a multifactorial rheumatic disease which 2 Rheumatology Research Center, Tehran mainly involves the axial skeleton. Macrophages and extracellular nucleotides have University of Medical Sciences, Tehran, Iran 3Department of Immunology, School of been shown to contribute to the inflammation process in autoimmune diseases. Medicine, Kermanshah University of Medical Membrane-bound purinergic P2 receptors might be involved in the modulation of Sciences, Kermanshah, Iran immune cells in AS. Therefore, we aimed to analyze the messenger RNA (mRNA) 4Inflammation Research Center, Tehran University of Medical Sciences, Tehran, Iran expression of P2 receptors in the macrophages of AS patients and healthy controls. Methods: Twenty-three AS patients and 23 age- and sex-matched healthy individuals Correspondence Mahdi Mahmoudi, Rheumatology Research were included in our study. Whole blood-separated monocytes of study participants Center, Tehran University of Medical were stimulated by macrophage colony-stimulating factor for 7 days and differenti- Sciences, Shariati Hospital, Kargar Ave., Tehran, Iran. ated to macrophages. Monocyte and macrophage markers were analyzed by flow Email: [email protected]. cytometry. SYBR green real-time polymerase chain reaction was used to measure Seyed Jalal Zargar, School of Biology, the relative expression levels of P2RX1, P2RX2, P2RX3, P2RX4, P2RX5, P2RX6, P2RX7, College of Science, University of Tehran, P.O. Box: 141556455, Tehran, Iran. P2RY1, P2RY2, P2RY4, P2RY6, P2RY11, P2RY12, P2RY13, P2RY14, and PANX1 genes. Email: [email protected]. Results: P2RY13 and P2RY6 genes had the highest expression levels in mac- Funding information rophages among P2RY genes. P2RY1 mRNA expression was significantly down- Deputy of Research, Tehran University of regulated (−1.75 fold) and P2RY14 was up-regulated (2.6 fold) in macrophages of AS Medical Sciences, Grant/Award Number: 94-02-41-28991 patients compared to healthy individuals. P2RX4 gene had the highest expression in monocyte-derived macrophages, followed by P2RX7 and P2RX1 genes. There was no significant difference in P2X receptor mRNA expression level between macrophages of AS patients and healthy individuals. Conclusions: Our results indicate that AS patients show altered expression levels of P2 receptor genes. Moreover, these changes might be associated with disease activ- ity and patients’ status. KEYWORDS ankylosing spondylitis, extracellular nucleotides, macrophages, P2 receptors 1 | INTRODUCTION joint, alongside involvement of the peripheral joints.1 Although, the environment has a role in the pathogenesis of AS, studies show that Ankylosing spondylitis (AS) is a complex rheumatic disease mainly char- genetic factors provide up to 90% of the susceptibility to the disease; acterized by the involvement of the axial skeleton including sacroiliac half of them are attributable to major histocompatibility complex Int J Rheum Dis. 2019;00:1–8. wileyonlinelibrary.com/journal/apl © 2019 Asia Pacific League of Associations for | 1 Rheumatology and John Wiley & Sons Australia, Ltd 2 | AKHTARI ET AL. genes such as HLA-B27.2 Several studies have focused on the adaptive sex-matched healthy individuals without any familial history of rheu- immune response in AS,3 but due to the polygenic auto-inflammatory matic diseases were included in our study. The included patients had nature of the disease, innate immunity has been proposed to play a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) ≥4. Mean significant role as well.4 ages for patient and control groups were 32 ± 10 and 35 ± 11 years, re- Macrophages are one of the main cells taking part in the innate spectively. Clinical characteristics of AS patients were collected at the immune response and have several tasks such as antigen presenta- time of sample collection. The ethics committee of Tehran University tion, phagocytosis, cytokine production, and so on. Based on their of Medical Sciences approved the study and we obtained written in- microenvironment, macrophages can be transformed into either M1 formed consents from patients and healthy individuals. or M2 subtypes.5 In the presence of pro-inflammatory cytokines such as interferon (IFN)-γ or tumor necrosis factor (TNF)-α, M1 phenotype has emerged. Macrophage colony-stimulating factor (M- 2.2 | Monocyte separation and CSF), interleukin (IL)-4 and IL-13 promote the development of M2 macrophage derivation macrophages.5 M2 macrophages and M2-type polarized monocytes have been shown to be predominant in synovial membrane and pe- Using ethylenediaminetetraacetic acid (EDTA) tubes, 20 mL of pe- ripheral blood of AS patients, respectively. Moreover, the M2/M1 ripheral blood was collected from each individual. All samples were ratio is correlated with inflammatory biomarkers and disease activity processed within the first 5 hours. Adding phosphate-buffered saline scores.6,7 Studies have demonstrated that macrophages are one of (PBS; GIBCO Invitrogen); samples were diluted with a ratio of 1:2 at the main cells contributing to the inflammation process in AS, es- pH 7.2. Subsequently, peripheral blood mononuclear cells were ex- pecially in joints.8 Therefore, investigating the macrophage function tracted using Ficoll (Lymphodex, Inno-Train) density gradient centrifu- and gene expression in patients with AS would be valuable. gation. PBS was used to wash mononuclear cells, and then they were While adenosine triphosphate (ATP) is mostly known for its central incubated with magnetic beads. Monocytes were isolated based on role in energy metabolism, extracellular ATP can affect several biolog- being positive for CD14, using magnetic-activated cell sorter (MACS) ical processes such as activation and migration of immune cells.9 ATP columns and MACS CD14 microbeads (Miltenyi Biotec). Isolated cells is released to the extracellular space through Pannexin 1 (Panx1) pores were stained by phycoerythrin (PE)-conjugated anti-CD14 antibody 10 that is induced by P2X7 purinergic receptor activation. Extracellular (BD Bioscience). Flow cytometry analysis revealed that isolated cells ATP and related nucleotides act through membrane-bound purinergic have 92%-95% purity.18 Using 24-well plates, monocytes were cul- P2 receptors.11 P2 receptors include G-protein coupled P2Y recep- tured at 500 000 cells per well for 7 days in complete Roswell Park tors and ligand-gated ion channel P2X receptors. Macrophages have Memorial Institute media containing 2 mmol/L L-glutamine (Biosera), been shown to be affected by P2 receptors and rapidly change their 100 U/mL penicillin, 10% fetal bovine serum (Gibco BRL), 50 ng/mL 12 function. The P2X7 was the first member of this family discovered. recombinant human macrophage colony stimulating factor (M-CSF; It affects intracellular calcium concentration in both human and mu- eBioscience) and 100 μg/mL streptomycin (Sigma). rine macrophages.13,14 It is demonstrated that alveolar macrophages have all subtypes of P2 receptors except P2Y12, P2X2, P2X3 and P2X6. However, only P2X7, P2Y1, P2Y2 and P2Y11 could increase the intracel- 2.3 | Flow cytometry analysis of macrophage lular calcium level.15 ATP can also affect the production of inflamma- surface markers 16 tory cytokines such as TNF-α by macrophages, which is proven to have a prominent role in AS. Therefore, P2 receptors could contribute After 7 days of stimulation with M-CSF, monocyte-derived mac- to the altered balance of the immune system in AS patients. rophages (2 × 105) were stained with fluorochrome-labeled anti- As was mentioned, P2 receptors are involved in the inflamma- bodies. Subsequently, cells were incubated for 30 minutes with tory processes. However, their role in AS disease pathogenesis is not PE-conjugated anti-human CD206, fluorescein isothiocyanate clear yet. Therefore, we aimed to investigate the mRNA expression (FITC)-conjugated anti-human CD163 (both from BD Bioscience) of P2 receptors and Panx1 hemi-channel in the macrophages of AS and suitable isotype-matched antibodies, avoiding light exposure. patients and healthy controls. Moreover, we have explored the cor- Macrophages were analyzed using a CyFlow ML flow cytometer relation between P2R family mRNA expression and clinical features (Partec, GmbH) and the data were analyzed by FlowJo software of the disease. (Tree Star). Cells were 97% and 95% positive for CD163 and CD206 macrophage markers, respectively.18 2 | MATERIALS AND METHODS 2.4 | Analysis of gene expression using real-time 2.1 | Patients and controls quantitative polymerase chain reaction (PCR) Twenty-three AS patients (4 female and 19 male) who fulfilled The High Pure RNA Isolation Kit (Roche) was used to extract total the modified New York classification criteria17 and 23 age- and RNA from macrophages after 7 days of stimulation with M-CSF. AKHTARI ET AL. | 3 TABLE 1 Primer sequences and Gene name Sequence Size (bp) product size of the selected genes GAPDH F: 5′-GAGTCAACGGATTTGGTCGT-3′
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