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Background: Ankylosing spondylitis (AS) is an inflammatory autoimmune disease that mostly affects different joints of the body. Macrophages are the predominant cells that mediate disease progression by secreting several pro-inflammatory mediators. Different receptors are involved in macrophages' function including the adenosine receptors (AR). Our main objective in this study was to assess the effect of applying A2A adenosine receptor agonist (CGS-21,680) on the gene expression of inflammatory mediators including bone morphogenetic proteins (BMP)-2, 4 and matrix metalloproteinases (MMP)-3, 8, 9, and 13 on the macrophages from AS patients compared to healthy macrophages.Methods: Monocytes were isolated from the whole blood of 28 individuals (AS patients and healthy controls in a 1:1 ratio). Macrophages were differentiated using macrophage colony-stimulating factor (M-CSF), and flow cytometry was performed to confirm surface markers. CGS-21,680 was used to treat cells that had been differentiated. Using SYBR green real-time PCR, relative gene expression was determined.Results: Activating A2AAR diminished MMP8 expression in healthy macrophages while it cannot reduce MMP8 expression in patients' macrophages. The effect of A2AAR activation on the expression of BMP2 and MMP9 reached statistical significance neither in healthy macrophages nor in the patients' group. We also discovered a significant positive connection between MMP8 expression and patient scores on the Bath ankylosing spondylitis functional index (BASFI).Conclusion: Due to the disability of A2AAR activation in the reduction of MMP8 expression in patients' macrophages and the correlation of MMP8 expression with BASFI index in patients, these results represent defects and dysregulations in the related signaling pathway in patients' macrophages. [ABSTRACT FROM AUTHOR]

Various studies suggest that ankylosing spondylitis (AS) as a chronic inflammatory disease with many disabilities can have impacts on different aspects of patients' life. Despite many quantitative studies, only few qualitative studies have thus far been published on this subject. For the first time, the present study aims at gaining insight into the life experience of Iranian AS patients. We performed a content analysis through semi-structured interviews with twenty-eight patients diagnosed with AS, including three females and twenty-five males with an average age of 38.5 years, to gain insight into their experiences. Whatever the patients expressed was written and transcribed verbatim. Then, we did analysis of the results after each interview. The detailed information completely extracted from the interviews was classified as sub-themes and main themes. Three main themes were identified by the analysis: (i) "Always with pain" describing the effects were found in regard to pain on patients' life, (ii) "The perceived limitation" describing many difficulties that people may face in the society as a result of their disease, and (iii) "Fearing the unknown future" which implies to both patients and their families have concerns about the future and what will happen. Our research findings in line with other qualitative studies showed that AS disease puts a heavy and intolerable burden on patients and their family. It seems that the experiences of people living with AS can be useful to meet challenges caused by the disease and it can enhance their coping with the disease. [ABSTRACT FROM AUTHOR]

Background: Ankylosing spondylitis (AS) is a multifactorial rheumatic disease which mainly involves the axial skeleton. Macrophages and extracellular nucleotides have been shown to contribute to the inflammation process in autoimmune diseases. Membrane‐bound purinergic P2 receptors might be involved in the modulation of immune cells in AS. Therefore, we aimed to analyze the messenger RNA (mRNA) 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 were included in our study. Whole blood‐separated monocytes of study participants were stimulated by macrophage colony‐stimulating factor for 7 days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. SYBR green real‐time polymerase chain reaction was used to measure the relative expression levels of P2RX1, P2RX2, P2RX3, P2RX4, P2RX5, P2RX6, P2RX7, P2RY1, P2RY2, P2RY4, P2RY6, P2RY11, P2RY12, P2RY13, P2RY14, and PANX1 genes. Results: P2RY13 and P2RY6 genes had the highest expression levels in macrophages among P2RY genes. P2RY1 mRNA expression was significantly down‐regulated (−1.75 fold) and P2RY14 was up‐regulated (2.6 fold) in macrophages of AS 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 activity and patients' status. [ABSTRACT FROM AUTHOR]

Ankylosing spondylitis (AS) is a highly heritable immune-mediated arthritis common in Turkish and Iranian populations. Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disease most common in people of Mediterranean origin. MEFV, an FMF-associated gene, is also a candidate gene for AS. We aimed to identify AS susceptibility loci and also examine the association between MEFV and AS in Turkish and Iranian cohorts. We performed genome-wide association studies in 1001 Turkish AS patients and 1011 Turkish controls, and 479 Iranian AS patients and 830 Iranian controls. Serum IL-1β, IL-17 and IL-23 cytokine levels were quantified in Turkish samples. An association of major effect was observed with a novel rare coding variant in MEFV in the Turkish cohort (rs61752717, M694V, OR = 5.3, P = 7.63×10−12), Iranian cohort (OR = 2.9, P = 0.042), and combined dataset (OR = 5.1, P = 1.65×10−13). 99.6% of Turkish AS cases, and 96% of those carrying MEFV rs61752717 variants, did not have FMF. In Turkish subjects, the association of rs61752717 was particularly strong in HLA-B27-negative cases (OR = 7.8, P = 8.93×10−15), but also positive in HLA-B27-positive cases (OR = 4.3, P = 7.69×10−8). Serum IL-1β, IL-17 and IL-23 levels were higher in AS cases than controls. Among AS cases, serum IL-1β and IL-23 levels were increased in MEFV 694V carriers compared with non-carriers. Our data suggest that FMF and AS have overlapping aetiopathogenic mechanisms. Functionally important MEFV mutations, such as M694V, lead to dysregulated inflammasome function and excessive IL-1β function. As IL-1 inhibition is effective in FMF, AS cases carrying FMF-associated MEFV variants may benefit from such therapy. [ABSTRACT FROM AUTHOR]

Background: Ankylosing spondylitis (AS) is a common debilitating rheumatic disease in which the innate immune components especially the Interleukin (IL)-23/IL-17 axis related genes play important role in its pathogenesis. Nucleotide binding oligomerization domain-containing protein (NOD)2, as an innate receptor, is critical for IL-23 production in cells. Therefore, we aimed to stimulate NOD2 signaling and study its effects on cytokine production in peripheral blood mononuclear cells (PBMC) of these patients. Methods: PBMCs from 18 patients with active AS and 18 healthy individuals were separated by Ficoll-Hypaque density gradient centrifugation and cultured in the presence of muramyl dipeptide (MDP), as NOD2 ligand. Quantitative expression analysis of NOD1, NOD2, RIPK2, SLC15A4, NLRP1, NLRP3, IL23A, IL17A, IL1B, and TNFA genes was performed using Real-time polymerase chain reaction (PCR). Finally, protein changes of IL23A and IL17A expression were validated using enzyme linked immunosorbent assay (ELISA). Results: Apart from NOD1 that tend to be downregulated in the controls, all the selected genes showed overexpression in response to MDP in cells from the studied groups. Except RIPK2, all the genes had higher expression changes upon MDP stimulation in the AS population. Overexpression of IL23A and IL17A were confirmed at protein levels using ELISA. The strong positive correlation between NLRP3 and NOD2 was decreased after stimulation but new correlations between NLRP3 and IL1B, RIPK2 and SLC15A4 were observed after treatment. Conclusions: This study indicated that AS PBMCs were hyper-responsive to MDP stimulation. This observation implies an important role of NOD2 in the pathogenesis of inflammatory diseases including AS. [ABSTRACT FROM AUTHOR]

Macrophages play an important role in the ankylosing spondylitis (AS) auto-inflammatory responses and fibrocartilage destruction. Adenosine is a key modulator of inflammatory conditions. The various effects of adenosine are mediated by its interaction with adenosine receptors (AR). In this study, we investigated the mRNA expression of A1, A2A, A2B, and A3 adenosine receptors, ectonucleoside triphosphate diphosphohydrolase-1 (CD39), and ecto-5′-nucleotidase (CD73) in the monocyte-derived macrophages from AS patients in comparison to healthy controls. We also explored the correlation between analyzed gene expression and patients’ clinical manifestations. Whole blood-separated monocytes from 23 healthy controls and 23 active AS patients were stimulated by macrophage colony-stimulating factor (M-CSF) for 7 days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. Analysis of adenosine receptors (ADORA1، ADORA2A، ADORA2B، ADORA3), CD39 and CD73 gene expression was performed by SYBR green real-time PCR. Our results demonstrated monocyte-derived macrophages from AS patients expressed increased level of A2AAR and reduced level of A1, A2BAR, and CD39 mRNA compared to healthy controls. We found an inverse correlation between A2AAR mRNA expression and Bath Ankylosing Spondylitis Functional Index (BASFI) score in AS patients. According to our results, altered expression level of adenosine-relying system would be involved in AS macrophage dysfunction and inflammation and correlated with functional status in AS patients. [ABSTRACT FROM AUTHOR]

Objective: Interleukin (IL)-23/IL-17 pathway involves in the pathogenesis of ankylosing spondylitis (AS). The exact mechanism implicated in overexpression of IL-23 and activation of the IL-23/IL-17 axis is not clear. The aim of the study was to clarify whether macrophages of AS patients undergo unfolded protein response (UPR) and secret increased IL-23. Methods: Peripheral blood monocyte isolated from 10 HLA-B27þ patients and five HLA-B27þ normal subjects were differentiated to macrophages by macrophage-colony stimulating factor (M-CSF) for seven days. Flow cytometry was used to detect monocyte purity and expression of macrophage markers. Analysis of mRNA expression for HLA-B and B27, UPR-associated proteins (BiP, CHOP, MDG1, and XBP1) and IL-23 was performed by RT-qPCR. Results: RT-qPCR data showed a significant overexpression of HLA-B27, UPR genes (BiP, CHOP, and XBP1), and IL-23 in M-CSF-derived macrophages from AS patients compared to healthy controls. Increased expression of MDG1 was not significant. Conclusions: Our data suggest that UPR activation occurs in M-CSF-derived macrophages of AS patients and is accompanied by overexpression of HLA-B27. UPR appears to be associated with overproduction of IL-23 in AS macrophages. [ABSTRACT FROM AUTHOR]

• A 2A AR agonist decreases TNF-α secretion by macrophages of AS patients. • A 2A AR activation increases IL23A mRNA expression by macrophages of AS patients. • A 2A AR activation did not have any influence on macrophages of healthy individuals. Ankylosing spondylitis (AS) is an auto-inflammatory debilitating disorder with a complex pathogenesis. The adenosinergic pathway is an immunologic regulating pathway with a potential role in AS pathophysiology. In the present study, we have aimed to investigate the influence of A 2A adenosine receptor (A 2A AR) activation on tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) expression and secretion by monocyte-generated macrophages of AS patients. Whole-blood separated monocytes were extracted from 14 AS patients and 14 healthy controls. Macrophages were differentiated by macrophage colony-stimulating factor (M-CSF), and surface markers were confirmed by flow cytometer. Cells were treated with CGS-21680 as a known agonist of A 2A AR. Analysis of ADORA2A , TNFA , and IL23A gene expression was performed by SYBR green real-time PCR. The concentration of secreted cytokines was also measured by ELISA kits. Based on our analysis, CGS-21680 significantly decreased TNF-α secretion by monocyte-derived macrophages of AS patients. Moreover, A 2A AR agonist increased the IL23A mRNA expression level in monocyte-derived macrophages of AS patients considerably. Whereas, CGS-21680 did not have any influence on macrophages of healthy individuals. According to our results, it appears that A 2A AR activation can increase IL-23 secretion by monocyte-derived macrophages of AS patients. Although the TNF-α reducing effect of A 2A AR agonists can be a potential target in AS treatment, robust increasing of IL-23 should be considered as the undesirable effect of these agents. [ABSTRACT FROM AUTHOR]

Background: Ankylosing Spondylitis (AS) is a chronic autoinflammatory Spondyloarthropathy (SpA) which is characterized by sacroiliitis, which progresses to the axial skeleton. It seems that non-Human Leukocyte Antigen (HLA) and also HLA-B27 are associated with the susceptibility and pathogenesis of the disease. The recent Genome-Wide Association Studies (GWASs) have reported intergenic rs6759298 to be associated with AS etiology. The aim of this study was investigation of the rs6759298 polymorphism in Iranian AS patients. In addition, probable correlations with clinical indices and manifestations were considered. Methods: This study included 403 patients with AS. The control group consisted of 506 healthy individuals who were matched for sex, age, and ethnicity with AS group. Genotyping of rs6759298 was determined using the Amplification-Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). Results: The GG genotype and G allele were found to be significantly more prevalent in the patient group in comparison to the control group [(p=2x10-6 and 7.44x10-9; OR (95% CI) =2.16 (1.56-2.98) and 1.73 (1.43-2.08)], respectively. Conclusion: No associations were found between patients with three genotypes and any disease manifestations or clinical indices. This investigation confirmed a highly significant association of rs6759298 with disease susceptibility, with no effect on disease progress or clinical presentations. Since rs6759298 belongs to the 2p15 gene desert, further studies would elucidate the exact role of this polymorphism in the pathogenesis of AS. [ABSTRACT FROM AUTHOR]

Objective To evaluate the efficacy, safety and tolerability of β- d -mannuronic acid (M2000) in the treatment of ankylosing spondylitis (AS). Methods The study was a 12-week randomized, double-blind, placebo-controlled, phase I/II clinical trial with 3 treatment arms: placebo, β- d -mannuronic acid and naproxen. Patients who had AS according to the modified New York criteria, with active disease at baseline were eligible for study. Primary outcome measure was the Assessment of SpondyloArthritis international Society (ASAS) 20 response rate at week 12. Results Of the 85 randomized patients, 27 were allocated to receive placebo, 28 naproxen, and 30 β- d -mannuronic acid. There were no statistically significant differences between treatment groups at baseline. Of the patients receiving β- d -mannuronic acid, 57.7% achieved an ASAS20 response at week 12, compared with 59% of the patients in the naproxen group (P > 0.05) and 19% of the patients in the placebo group (P = 0.007). In comparison with patients receiving placebo over the 12-week treatment period, those receiving β- d -mannuronic acid and naproxen demonstrated statistically significantly greater improvement in all secondary endpoints. Interestingly, β- d -mannuronic acid reduced some parameters associated with inflammation more effectively than naproxen and placebo. The incidence of gastrointestinal and other adverse events were higher on naproxen than on β- d -mannuronic acid and placebo. Conclusion The present study demonstrated similar efficacy, but with a more favorable safety profile for β- d -mannuronic acid than naproxen and, therefore, suggest that β- d -mannuronic acid is suitable for the management of AS. Trial registration Iranian registry of clinical trials; www.irct.ir ; IRCT2013062213739N1. [ABSTRACT FROM AUTHOR]