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Activation of adenosine A2A receptor induced interleukin-23 mRNA expression in macrophages of ankylosing spondylitis patients
Cytokine 128 (2020) 154997
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Activation of adenosine A2A receptor induced interleukin-23 mRNA expression in macrophages of ankylosing spondylitis patients
T
Maryam Akhtaria,b, Mahdi Vojdaniana, Ali Javinania, Amir Ashraf-Ganjoueia, ⁎ Ahmadreza Jamshidia, Mahdi Mahmoudia,c, a
Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran Department of Cell & Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran c Inflammation Research Center, Tehran University of Medical Sciences, Tehran, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Interleukin-23 Macrophages Adenosine A2A Receptor Ankylosing Spondylitis Tumor Necrosis Factor-alpha
Background: 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 A2A adenosine receptor (A2AAR) activation on tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) expression and secretion by monocytegenerated macrophages of AS patients. Methods: 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 A2AAR. 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. Results: Based on our analysis, CGS-21680 significantly decreased TNF-α secretion by monocyte-derived macrophages of AS patients. Moreover, A2AAR agonist increased the IL23A mRNA expression level in monocytederived macrophages of AS patients considerably. Whereas, CGS-21680 did not have any influence on macrophages of healthy individuals. Conclusion: According to our results, it appears that A2AAR activation can increase IL-23 secretion by monocytederived macrophages of AS patients. Although the TNF-α reducing effect of A2AAR agonists can be a potential target in AS treatment, robust increasing of IL-23 should be considered as the undesirable effect of these agents.
1. Introduction Ankylosing spondylitis (AS) is an auto-inflammatory disorder of the musculoskeletal system, which predominantly involves the spinal column, sacroiliac and peripheral joints [1]. In addition to enthesis, it also infrequently affects specific extra-articular organs [1]. The pooled prevalence of AS is 0.18 (0.15–0.23) percent and has significant male predominance [2]. Due to the high prevalence, younger age dominance, progressive and incurable nature of the AS; it has a noteworthy burden and cost for the society [3,4]. However, by elucidating the pathogenesis of AS, certain cost-effective biological medications were discovered with higher efficacy in comparison to conventional non-biological treatments [5,6]. The pathophysiology of AS consists of a sequential triad of
inflammation, bone destruction, and subsequent compensatory new bone formation [7]. The inflammatory process is orchestrated by several immune cells in response to environmental factors in a genetically predisposed patient, particularly in mechanically stressed sites including spine, sacroiliac joints, and weight bearing entheses [8,9]. It is known that AS susceptibility is associated with several genes including HLA-B27, ERAP1, IL23R, etc. [10]. In general, it seems that pathogenic proteins coded by genes with certain specific polymorphisms, are contributed to disease pathogenesis by excessive activation of innate immune responses [9,10]. Consequently, the inflammatory environment produced by macrophages and other innate immune cells could form the adaptive immune response and leads to disease progression. Several signaling pathways contribute in the AS pathogenesis. The adenosinergic pathway is one of the evolutionary ancient signaling
⁎ Corresponding author at: Rheumatology Research Center, Tehran University of Medical Sciences, Shariati Hospital, Kargar Ave., P.O. Box: 1411713137, Tehran, Iran. E-mail address: [email protected] (M. Mahmoudi).
https://doi.org/10.1016/j.cyto.2020.154997 Received 27 August 2019; Received in revised form 2 January 2020; Accepted 10 January 2020 1043-4666/ © 2020 Elsevier Ltd. All rights reserved.
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time. Samples were diluted 1:2 in the pH of 7.2 by using phosphate buffered saline (PBS; GIBCO Invitrogen). Peripheral blood mononuclear cells (PBMCs) were extracted using Ficoll (Lymphodex, Inno-Train) density gradient centrifugation and then washed by PBS. To isolate the monocytes, mononuclear cells were incubated with magnetic beads to undergone positive selection for CD14 using MACS CD14 microbeads and magnetic-activated cell sorter columns (all from Miltenyi Biotec). The purity of 92–95% was obtained by flow cytometry analysis [16]. Phycoerythrin (PE)-conjugated anti-CD14 antibody (BD bioscience) was used to immunofluorescence staining of the isolated cells. Afterward, the isolated monocytes were cultured on 24-well plates at a concentration of 500,000 cells per well in complete Roswell Park Memorial Institute (RPMI). The media contained two mM L-glutamine (Biosera), 10% fetal bovine serum (FBS; Gibco BRL), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma). To shift cultured monocytes toward macrophages, 50 ng/ml recombinant human macrophagecolony stimulating factor (M-CSF; eBioscience) was added to culture media for seven days [25].
pathways contributing to diverse mechanisms activated by adenosine. It also plays a significant role in regulating the immune system to upregulate the anti-inflammatory pathways, and its dysregulation contributes noticeably in several autoimmune disorders [11,12]. Adenosine is a degradation product of adenosine triphosphate (ATP) by extracellular ectonucleotidase (including CD39 and CD73), which seems to function as a danger-associated molecular pattern (DAMPs) [12,13]. Adenosine P1 receptors consist of four subtypes A1, A2A, A2B, and A3 that are the members of G-protein coupled receptors family [12]. These receptors are widely expressed on the immune cells including macrophages [14]. Adenosine downregulates classic M1 macrophages activation by A2A adenosine receptor (A2AAR), while it upregulates the alternative M2 macrophages activation via A2B adenosine receptor [14]. Interestingly, M2 macrophages generate an adenosine-rich microenvironment by increasing the expression of CD39 and CD73 enzymes to maintain the anti-inflammatory condition [13]. Additionally, adenosine inhibits the secretion of TNF-α and the production of reactive oxygen species by macrophages, whereas it promotes the anti-inflammatory cytokines generation and production of tissue remodeling mediators including IL-10 and vascular endothelial growth factor (VEGF) [14,15]. At the moment, there is limited evidence regarding the contribution of the adenosinergic pathway in AS pathogenesis. We have previously demonstrated that expression of A2AAR mRNA was increased whereas A1, A2B and CD39 mRNA were decreased in monocyte-derived macrophages of AS patients [16]. Moreover, higher mRNA expression rate, receptor density, and affinity of A2AAR and A3 receptors (A3AR) were shown in the lymphocytes of AS patients [17]. Furthermore, A2AAR and A3AR agonists inhibited the production of TNF-α, IL-1β, and IL-6 by lymphocytes and also reduced the production of matrix metalloproteinase-1 (MMP-1) and MMP-3 by monocytes [17]. These results are so promising, to conduct complementary studies for investigating the therapeutic effects of adenosine receptors agonists. Nevertheless, sound of evidence from other disorders has suggested that adenosinergic pathway significantly promotes Th-17 response, which is the dominant mechanism in AS pathogenesis [18–21]. Taken together, we aimed to investigate the influence of adenosinergic pathway on IL-23 production among AS patients which is the main cytokine in Th-17 response formation [22]. Therefore, we have reported the effect of A2AAR activation on IL-23 and TNF-α production by monocyte-derived macrophages obtained from healthy individuals and AS patients.
2.3. Analysis of macrophage surface marker by flow cytometry After seven days of monocytes incubation with M-CSF, the surface markers of monocyte-derived macrophages (2 × 105) were analyzed by flow cytometry. The surface marker of macrophages including CD163 and CD206 were measured in this study [26]. Cells were incubated for 30 min with fluorescein isothiocyanate (FITC)-conjugated anti-human CD163, PE conjugated anti-human CD206 (BD bioscience), and appropriate isotype-matched antibodies without any light exposure. Then, cell surface markers were analyzed by CyFlow ML flow cytometer (Partec, GmbH, Munster, Germany) and FlowJo software (Tree Star, Ashland, OR, USA) was used to analyze FASC data. In this study, 97 and 95 percent of macrophages expressed CD163 and CD206, respectively [16]. 2.4. Measurement of cytokines and A2AAR gene expression by real-time quantitative PCR Monocyte-derived macrophages were seeded on RPMI, then 10 µM CGS-21680 (Sigma), a well-known agonist of A2AAR, was added to half of the samples [17]. After 24 h, total RNA was extracted using the High Pure RNA Isolation Kit (Roche). The complementary DNA (cDNA) was generated by CellAmp direct RNA prep kit for real-time PCR (Takara bio) from the equal amount of total RNA. The relative expression level of TNFA (TNF-α), IL23A (IL-23p19), and ADORA2A (A2AAR) genes were measured by StepOnePlus™ Real-Time PCR system (Applied Biosystems) and SYBR green master mix (Ampliqon). The endogenous control gene was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The specific primer sequences are shown in Table 1. Comparative CT method (2−ΔΔCT) was used to compare the selected mRNA expression level between A2AAR agonist (CGS-21680) treated cells and control group. It is noticeable to mention that the cytotoxic potential of
2. Material and methods 2.1. Study population In the present study, 14 AS patients were selected with the male to female ratio of 3.6:1 and the mean age of 32 ± 10 years. Patients were recruited from outpatient AS clinic of Rheumatology Research Center, Shariati Hospital, Tehran University of Medical Sciences. All of the patients have fulfilled the modified New York classification criteria [23]. According to the significant influence of methotrexate on the expression of adenosine receptors [24], naïve to treatment patients were selected in this study. Meanwhile, 14 age- and sex-matched healthy individuals were also included with the similar gender distribution to AS patients and the mean age of 32 ± 9.6 years. Control group did not have any personal or familial history of rheumatic diseases, psoriasis, and inflammatory bowel disease. Written informed consent was obtained from all participants. This study was approved by the ethics committee of Tehran University of Medical Sciences.
Table 1 Primer sequences and product size of the studied genes. Gene name
Sequence
Size (bp)
GAPDH
F: 5′-GAGTCAACGGATTTGGTCGT-3′ R: 5′-GACAAGCTTCCCGTTCTCAG-3′ F: 5′-CATGCTAGGTTGGAACAACTGC-3′ R: 5′-AGATCCGCAAATAGACACCCA-3′ F: 5′-CCTGCCCCAATCCCTTTATT-3′ R: 5′-CCCTAAGCCCCCAATTCTCT-3′ F: 5′-GGGACAACAGTCAGTTCTGCTT-3′ R: 5′-TGGGACTGAGGCTTGGAATC-3′
185
ADORA2A TNFA
2.2. Monocyte separation and macrophage cell culture
IL23A (p19)
20 ml of peripheral blood sample of participants were obtained into Ethylenediaminetetraacetic acid (EDTA) containing tubes. All of the samples were possessed for less than five hours from the collection
185 81 210
*GAPDH: glyceraldehyde-3-phosphate dehydrogenase, ADORA2A: adenosine receptor 2A, TNFA: tumor necrosis factor-α, IL23: interleukin 23. 2
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CGS-21680 was assessed before interpreting the results. In the present study, the death-inducing effect of CGS-21680 was evaluated by MTT assay. According to this assay, CGS-21680 did not have any cytotoxic effects and cells viability was entirely similar to untreated cells. 2.5. Measurement of cytokine production levels in monocyte-derived macrophages TNF-α and IL-23 levels were measured in CGS-21680 treated and control cells. After 1 day incubation of cells with 10 µM CGS-21680, the supernatant was used to evaluate the mentioned pro-inflammatory cytokines level. TNF-α and IL-23 were measured by ELISA kit (Mabtech) according to the manufacturer's instruction. 2.6. Statistical analysis All of the variables were checked for normality by Shapiro-Wilk test. Non-parametric tests including Mann-Whitney U test were used to compare non-normal distributed variables. To compare the mRNA expression level and cytokine level between CGS-21680 treated and control cells, the Paired sample t-test and Wilcoxon test were performed. Spearman's correlation test analyzed the correlation between the relative mRNA expression of TNF-α and IL-23 with A2AAR expression level. P values less than 0.05 were considered statistically meaningful. All of the statistical analysis were performed by SPSS version 22 and graphs were designed using GraphPad Prism 6. In addition, post hoc statistical power analysis was performed by PASS 11 software. 3. Results 3.1. Demographic and clinical features The demographic features of the AS patients and control group are shown in Table 2. The clinical scores representing disease activity were also demonstrated for AS patients. Moreover, it is described that none of the patients were under neither biological nor methotrexate treatment.
Fig. 1. The relative mRNA expression (A) and cytokine production level (B) of TNF-α in AS patients and control group before and after treatment by A2AAR agonist. A: The mRNA expression level of TNFA was not different significantly between patients and healthy individuals. The TNFA expression level did not alter after CGS-21680 treatment in either groups. B: The TNF-α production level was not different between untreated cells of AS patient and healthy cases but CGS-21680 significantly diminished the TNF-α secretion by monocyte-derived macrophages of AS patients without any influence on control group macrophages (TNF-α: tumor necrosis factor-α, A2AAR: adenosine receptor A2A).
3.2. A2AAR agonist decreased TNF-α secretion in monocyte-derived macrophages of AS patients The influence of A2AAR activation on TNF-α mRNA expression level and protein secretion was investigated and shown in Fig. 1. The CD163 Table 2 Clinicodemographic features of AS patients and control group. Variable
AS patients (N = 14)
Healthy controls (N = 14)
Gender (Male%) Age (years) Disease duration (years) Smoking (%) HLA-B27 positivity, no. (%) ESR (mm/h) BASMI score BASFI score BASDAI score BASG score PDGA score ASQoL score Biological agents Methotrexate
11 (79%) 32 ± 10 7.5 ± 6 5 (35%) 10 (71%) 37 ± 19 3.7 ± 2.5 4.6 ± 2.7 6.1 ± 1.9 7.2 ± 1.8 7 ± 2.7 9.5 ± 5.6 0 (0%) 0 (0%)
11 (79%) 32 ± 9.6 – 5 (35%) – 5 ± 3 – – – – – – – –
and CD206 positive macrophages of AS patients were compared to healthy individuals. Macrophages were treated with A2AAR agonist (CGS-21680) and compared to control samples within each population. The CGS-21680 decreased the TNF-α production level in monocytederived macrophages of AS patients by −1.1-fold (P < 0.05, Power = 0.65). Nevertheless, the TNF-α secretion level did not altered in macrophages of healthy cases after A2AAR agonist treatment. However, the mRNA expression level and protein secretion were not significantly different between AS patients and healthy individuals either with or without CGS-21680 treatment.
3.3. IL-23 mRNA expression was increased in monocyte-generated macrophages of AS patients by A2AAR agonist The effect of the A2AAR agonist on IL-23 mRNA expression and protein secretion was also determined by the similar process mentioned previously and shown in Fig. 2. CGS-21680 significantly augmented IL23A mRNA in macrophages of AS patients by 1.5-fold (P < 0.05, Power = 0.78) whereas it did not alter IL23A mRNA expression in macrophages of healthy individuals. Therefore, AS macrophages
*AS: ankylosing spondylitis, ESR: erythrocyte sedimentation rate, HLA-B27: human leukocyte antigen (subtypes B*2701-2759), BASMI: bath ankylosing spondylitis metrology index, BASFI: bath ankylosing spondylitis functional index, BASDAI: bath ankylosing spondylitis disease activity index, BAS-G: bath ankylosing spondylitis global score, PDGA: patient’s disease global assessment, ASQoL: ankylosing spondylitis quality of life. 3
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the T helpers by regulating the cytokine milieu [14,15]. In the present study, we have shown that the relative mRNA expression level of IL-23 was significantly increased after A2AAR activation in M-CSF-derived macrophages of AS patients. The A2AAR agonist did not alter the IL-23 expression level among macrophages of healthy subjects. This finding can be possibly explained by the higher expression level of A2AAR mRNA in monocyte-generated macrophages of AS patients compared to the control [16]. In line with our findings, Crean et al. demonstrated the influence of A2AAR activation on IL-23 expression [18]. They showed that IL-23 mRNA was expressed 1.5-fold higher in human primary PBMCs after treatment with non-specific adenosine receptor agonist (59-N-ethylcarboxamidoadenosine, NECA) plus LPS in comparison to cells primed solely by LPS. Moreover, they reported that IL-23 mRNA was increased significantly in monocytic THP-1 cell line by CGS-21680/ LPS activation. However, IL-23 mRNA level did not alter after priming by A2B adenosine receptor agonist (BAY-60-6583). Comprehensively, these findings highlighted the provocative role of A2AAR activation on IL-23 expression. Th-17 cells are a distinct group of lymphocytes that are emerged from CD4 + T helper cells and characterized by the production of IL17. Depending on the primary microenvironment, they transform into two subgroups of effector Th-17 and regulatory Th-17 cells [27]. Effector Th-17 cells are developed by the stimulation of IL-23, IL-6, IL-1β and act as pro-inflammatory cells [27]. Moreover, effector Th-17 cells can emerge from protective regulatory Th-17 cells by the IL-23 stimulation [27]. The crucial role of IL-23 in inducing Th-17 response is reinforced by the promising therapeutic effects of biological medications that target IL-23/IL-17 axis [22]. These therapeutic effects have also shown previously in AS treatment which strengthen the highlighted role of Th-17 response in its pathogenesis [22]. Several studies have suggested the influence of adenosinergic pathway in developing the Th-17 response. Liang and colleagues investigated the role of adenosine receptor activation on the Th-17 response devalopment in a mouse model of autoimmune uveitis [28]. They showed that A2AAR agonist inhibited the Th-17 response in the early phase of disease induction, whereas this effect was inverted when the A2AAR agonist was injected in the late stage of the disease. Moreover, it is shown in another study that the enhanced function of the A2AAR by an allosteric modulator can significantly inhibit the IL-23 secretion by dendritic cells and also decrease IL-17 expression in γδ T cells in a mice model of psoriatic-like dermatitis [29]. In contrast, Liang et al. have demonstrated that A2AAR positive γδ T cells significantly promote the activation of Th-17 autoreactive cells in the pro-inflammatory context [30]. These controversial findings proposed that the effect of the adenosinergic pathway on orchestrating the immune response influenced by disease progression stage and the cytokine milieu. Our study revealed that A2AAR activation reduces TNF-α secretion in monocyte-generated macrophages of AS patients. These results were not replicated in macrophages of healthy subjects. Similarly, it appears that the TNF-α reducing effect of A2AAR agonist in macrophages of AS patients is due to the higher expression level of A2AAR mRNA among these cells compared to control group [16]. In line with our findings, Ravani et al. have reported that A2AAR and A3AR agonists markedly diminished NF-κB level in addition to inhibition of TNF-α release in cultured lymphocytes of AS patients [17]. Similarly, Varani and colleagues have investigated the anti-inflammatory role of A2AAR and A3AR agonists in rheumatoid arthritis (RA) patients. They reported that A2AAR and A3AR activation significantly reduced the nuclear level of NF-κB and TNF-α in lymphocytes of RA patients [31]. Moreover, it is reported previously that therapeutic role of certain types of medications in RA treatment was reversed by the A2AAR antagonist, which reinforces the role of this receptor in anti-inflammatory responses [32]. Interestingly, it is demonstrated that low-dose methotrexate induced adenosine secretion and consequently reduced TNF-α secretion via A2AAR activation [33]. The inhibitory effect of the A2AAR agonist on
Fig. 2. The relative mRNA expression (A) and cytokine production level (B) of IL-23 in AS patients and control group before and after treatment by A2AAR agonist. A: The mRNA expression level of IL23A was significantly increased in M-CSF-generated macrophages of AS patients after CGS-21680 treatment. B: Untreated macrophages of AS patients significantly secreted higher level of IL23 in comparison to control group. However, CGS-21680 did not alter IL-23 production in either groups (IL-23: interleukin-23, A2AAR: adenosine receptor A2A).
expressed significantly higher level of IL23A mRNA after CGS-21680 treatment compared to healthy macrophages (P < 0.05, Power = 0.65). CGS-21680 also increased IL-23 secretion in M-CSFderived macrophages of AS patients, however, it was not significant. Moreover, macrophages of AS patients secreted significantly higher level of IL-23 in comparison to healthy individuals (P < 0.05, Power = 0.75). 3.4. Correlation between the relative mRNA expression of TNFA with A2AAR expression level The correlation between the expression level of TNFA mRNA with ADORA2A (A2AAR) mRNA in monocyte-derived macrophages of AS patients and healthy individuals was also evaluated. We did not find any substantial correlation between expression levels. 4. Discussion The ancient evolutionary adenosinergic pathway has a pivotal role in regulating immune responses and prevents exaggerated inflammatory responses through its anti-inflammatory effects [11,12]. Adenosine can also orchestrate the type of immune response and skew 4
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TNF-α secretion was also shown in human keratinocytes that have a role in psoriasis pathogenesis [34]. Besides, TNF-α secretion was significantly reduced by the A2AAR agonist in the lymphocytes of a mouse model of spontaneous ileitis [35]. Although this finding was in favor of the probable anti-inflammatory role of the adenosinergic pathway in the inflammatory bowel diseases, it was not replicated in mice with dextran sulfate-induced colitis [36]. Furthermore, the inhibitory effect of A2AAR activation on TNF-α release from lymphocytes was also reported in multiple sclerosis and systemic lupus erythematosus (SLE) patients [37,38]. Generally, these studies support the anti-inflammatory role of A2AAR agonists by their TNF-α reducing effects. In conclusion, our present study provides evidence that A2AAR activation induces IL-23 secretion in macrophages of AS patients. Due to the pivotal role of Th-17 response in AS pathogenesis, it is highly probable that A2AAR activation can aggravate this response by stimulating IL-23 production. Meanwhile, substantial evidence has supported the anti-inflammatory role of A2AAR agonists by inhibiting pro-inflammatory cytokines secretion including TNF-α from macrophages. Also, it is mentioned previously that the inflammatory microenvironment significantly influences the A2AAR downstream activation effects on inducing distinct immunological responses. Consequently, further studies should be designed to investigate the therapeutic role of A2AAR agonists on AS treatment regarding disease progression state.
[10]
[11] [12] [13] [14] [15] [16]
[17]
[18]
[19]
CRediT authorship contribution statement
[20]
Maryam Akhtari: Conceptualization, Writing - review & editing, Validation. Mahdi Vojdanian: Writing - original draft, Validation. Ali Javinani: Data curation, Writing - original draft, Validation. Amir Ashraf-Ganjouei: Formal analysis, Writing - original draft, Validation. Ahmadreza Jamshidi: Project administration, Writing - review & editing, Validation. Mahdi Mahmoudi: Conceptualization, Supervision, Writing - review & editing, Validation.
[21]
[22]
[23]
[24]
Declaration of Competing Interest [25]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[26]
Acknowledgment
[27]
We would like to thank all participants for their collaboration. This study was supported by a grant from deputy of research, Tehran University of Medical Sciences (Grant No. 95-04-41-33867).
[28]
[29]
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Activation of adenosine A2A receptor induced interleukin-23 mRNA expression in macrophages of ankylosing spondylitis patients
T
Maryam Akhtaria,b, Mahdi Vojdaniana, Ali Javinania, Amir Ashraf-Ganjoueia, ⁎ Ahmadreza Jamshidia, Mahdi Mahmoudia,c, a
Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran Department of Cell & Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran c Inflammation Research Center, Tehran University of Medical Sciences, Tehran, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Interleukin-23 Macrophages Adenosine A2A Receptor Ankylosing Spondylitis Tumor Necrosis Factor-alpha
Background: 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 A2A adenosine receptor (A2AAR) activation on tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) expression and secretion by monocytegenerated macrophages of AS patients. Methods: 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 A2AAR. 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. Results: Based on our analysis, CGS-21680 significantly decreased TNF-α secretion by monocyte-derived macrophages of AS patients. Moreover, A2AAR agonist increased the IL23A mRNA expression level in monocytederived macrophages of AS patients considerably. Whereas, CGS-21680 did not have any influence on macrophages of healthy individuals. Conclusion: According to our results, it appears that A2AAR activation can increase IL-23 secretion by monocytederived macrophages of AS patients. Although the TNF-α reducing effect of A2AAR agonists can be a potential target in AS treatment, robust increasing of IL-23 should be considered as the undesirable effect of these agents.
1. Introduction Ankylosing spondylitis (AS) is an auto-inflammatory disorder of the musculoskeletal system, which predominantly involves the spinal column, sacroiliac and peripheral joints [1]. In addition to enthesis, it also infrequently affects specific extra-articular organs [1]. The pooled prevalence of AS is 0.18 (0.15–0.23) percent and has significant male predominance [2]. Due to the high prevalence, younger age dominance, progressive and incurable nature of the AS; it has a noteworthy burden and cost for the society [3,4]. However, by elucidating the pathogenesis of AS, certain cost-effective biological medications were discovered with higher efficacy in comparison to conventional non-biological treatments [5,6]. The pathophysiology of AS consists of a sequential triad of
inflammation, bone destruction, and subsequent compensatory new bone formation [7]. The inflammatory process is orchestrated by several immune cells in response to environmental factors in a genetically predisposed patient, particularly in mechanically stressed sites including spine, sacroiliac joints, and weight bearing entheses [8,9]. It is known that AS susceptibility is associated with several genes including HLA-B27, ERAP1, IL23R, etc. [10]. In general, it seems that pathogenic proteins coded by genes with certain specific polymorphisms, are contributed to disease pathogenesis by excessive activation of innate immune responses [9,10]. Consequently, the inflammatory environment produced by macrophages and other innate immune cells could form the adaptive immune response and leads to disease progression. Several signaling pathways contribute in the AS pathogenesis. The adenosinergic pathway is one of the evolutionary ancient signaling
⁎ Corresponding author at: Rheumatology Research Center, Tehran University of Medical Sciences, Shariati Hospital, Kargar Ave., P.O. Box: 1411713137, Tehran, Iran. E-mail address: [email protected] (M. Mahmoudi).
https://doi.org/10.1016/j.cyto.2020.154997 Received 27 August 2019; Received in revised form 2 January 2020; Accepted 10 January 2020 1043-4666/ © 2020 Elsevier Ltd. All rights reserved.
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time. Samples were diluted 1:2 in the pH of 7.2 by using phosphate buffered saline (PBS; GIBCO Invitrogen). Peripheral blood mononuclear cells (PBMCs) were extracted using Ficoll (Lymphodex, Inno-Train) density gradient centrifugation and then washed by PBS. To isolate the monocytes, mononuclear cells were incubated with magnetic beads to undergone positive selection for CD14 using MACS CD14 microbeads and magnetic-activated cell sorter columns (all from Miltenyi Biotec). The purity of 92–95% was obtained by flow cytometry analysis [16]. Phycoerythrin (PE)-conjugated anti-CD14 antibody (BD bioscience) was used to immunofluorescence staining of the isolated cells. Afterward, the isolated monocytes were cultured on 24-well plates at a concentration of 500,000 cells per well in complete Roswell Park Memorial Institute (RPMI). The media contained two mM L-glutamine (Biosera), 10% fetal bovine serum (FBS; Gibco BRL), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma). To shift cultured monocytes toward macrophages, 50 ng/ml recombinant human macrophagecolony stimulating factor (M-CSF; eBioscience) was added to culture media for seven days [25].
pathways contributing to diverse mechanisms activated by adenosine. It also plays a significant role in regulating the immune system to upregulate the anti-inflammatory pathways, and its dysregulation contributes noticeably in several autoimmune disorders [11,12]. Adenosine is a degradation product of adenosine triphosphate (ATP) by extracellular ectonucleotidase (including CD39 and CD73), which seems to function as a danger-associated molecular pattern (DAMPs) [12,13]. Adenosine P1 receptors consist of four subtypes A1, A2A, A2B, and A3 that are the members of G-protein coupled receptors family [12]. These receptors are widely expressed on the immune cells including macrophages [14]. Adenosine downregulates classic M1 macrophages activation by A2A adenosine receptor (A2AAR), while it upregulates the alternative M2 macrophages activation via A2B adenosine receptor [14]. Interestingly, M2 macrophages generate an adenosine-rich microenvironment by increasing the expression of CD39 and CD73 enzymes to maintain the anti-inflammatory condition [13]. Additionally, adenosine inhibits the secretion of TNF-α and the production of reactive oxygen species by macrophages, whereas it promotes the anti-inflammatory cytokines generation and production of tissue remodeling mediators including IL-10 and vascular endothelial growth factor (VEGF) [14,15]. At the moment, there is limited evidence regarding the contribution of the adenosinergic pathway in AS pathogenesis. We have previously demonstrated that expression of A2AAR mRNA was increased whereas A1, A2B and CD39 mRNA were decreased in monocyte-derived macrophages of AS patients [16]. Moreover, higher mRNA expression rate, receptor density, and affinity of A2AAR and A3 receptors (A3AR) were shown in the lymphocytes of AS patients [17]. Furthermore, A2AAR and A3AR agonists inhibited the production of TNF-α, IL-1β, and IL-6 by lymphocytes and also reduced the production of matrix metalloproteinase-1 (MMP-1) and MMP-3 by monocytes [17]. These results are so promising, to conduct complementary studies for investigating the therapeutic effects of adenosine receptors agonists. Nevertheless, sound of evidence from other disorders has suggested that adenosinergic pathway significantly promotes Th-17 response, which is the dominant mechanism in AS pathogenesis [18–21]. Taken together, we aimed to investigate the influence of adenosinergic pathway on IL-23 production among AS patients which is the main cytokine in Th-17 response formation [22]. Therefore, we have reported the effect of A2AAR activation on IL-23 and TNF-α production by monocyte-derived macrophages obtained from healthy individuals and AS patients.
2.3. Analysis of macrophage surface marker by flow cytometry After seven days of monocytes incubation with M-CSF, the surface markers of monocyte-derived macrophages (2 × 105) were analyzed by flow cytometry. The surface marker of macrophages including CD163 and CD206 were measured in this study [26]. Cells were incubated for 30 min with fluorescein isothiocyanate (FITC)-conjugated anti-human CD163, PE conjugated anti-human CD206 (BD bioscience), and appropriate isotype-matched antibodies without any light exposure. Then, cell surface markers were analyzed by CyFlow ML flow cytometer (Partec, GmbH, Munster, Germany) and FlowJo software (Tree Star, Ashland, OR, USA) was used to analyze FASC data. In this study, 97 and 95 percent of macrophages expressed CD163 and CD206, respectively [16]. 2.4. Measurement of cytokines and A2AAR gene expression by real-time quantitative PCR Monocyte-derived macrophages were seeded on RPMI, then 10 µM CGS-21680 (Sigma), a well-known agonist of A2AAR, was added to half of the samples [17]. After 24 h, total RNA was extracted using the High Pure RNA Isolation Kit (Roche). The complementary DNA (cDNA) was generated by CellAmp direct RNA prep kit for real-time PCR (Takara bio) from the equal amount of total RNA. The relative expression level of TNFA (TNF-α), IL23A (IL-23p19), and ADORA2A (A2AAR) genes were measured by StepOnePlus™ Real-Time PCR system (Applied Biosystems) and SYBR green master mix (Ampliqon). The endogenous control gene was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The specific primer sequences are shown in Table 1. Comparative CT method (2−ΔΔCT) was used to compare the selected mRNA expression level between A2AAR agonist (CGS-21680) treated cells and control group. It is noticeable to mention that the cytotoxic potential of
2. Material and methods 2.1. Study population In the present study, 14 AS patients were selected with the male to female ratio of 3.6:1 and the mean age of 32 ± 10 years. Patients were recruited from outpatient AS clinic of Rheumatology Research Center, Shariati Hospital, Tehran University of Medical Sciences. All of the patients have fulfilled the modified New York classification criteria [23]. According to the significant influence of methotrexate on the expression of adenosine receptors [24], naïve to treatment patients were selected in this study. Meanwhile, 14 age- and sex-matched healthy individuals were also included with the similar gender distribution to AS patients and the mean age of 32 ± 9.6 years. Control group did not have any personal or familial history of rheumatic diseases, psoriasis, and inflammatory bowel disease. Written informed consent was obtained from all participants. This study was approved by the ethics committee of Tehran University of Medical Sciences.
Table 1 Primer sequences and product size of the studied genes. Gene name
Sequence
Size (bp)
GAPDH
F: 5′-GAGTCAACGGATTTGGTCGT-3′ R: 5′-GACAAGCTTCCCGTTCTCAG-3′ F: 5′-CATGCTAGGTTGGAACAACTGC-3′ R: 5′-AGATCCGCAAATAGACACCCA-3′ F: 5′-CCTGCCCCAATCCCTTTATT-3′ R: 5′-CCCTAAGCCCCCAATTCTCT-3′ F: 5′-GGGACAACAGTCAGTTCTGCTT-3′ R: 5′-TGGGACTGAGGCTTGGAATC-3′
185
ADORA2A TNFA
2.2. Monocyte separation and macrophage cell culture
IL23A (p19)
20 ml of peripheral blood sample of participants were obtained into Ethylenediaminetetraacetic acid (EDTA) containing tubes. All of the samples were possessed for less than five hours from the collection
185 81 210
*GAPDH: glyceraldehyde-3-phosphate dehydrogenase, ADORA2A: adenosine receptor 2A, TNFA: tumor necrosis factor-α, IL23: interleukin 23. 2
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CGS-21680 was assessed before interpreting the results. In the present study, the death-inducing effect of CGS-21680 was evaluated by MTT assay. According to this assay, CGS-21680 did not have any cytotoxic effects and cells viability was entirely similar to untreated cells. 2.5. Measurement of cytokine production levels in monocyte-derived macrophages TNF-α and IL-23 levels were measured in CGS-21680 treated and control cells. After 1 day incubation of cells with 10 µM CGS-21680, the supernatant was used to evaluate the mentioned pro-inflammatory cytokines level. TNF-α and IL-23 were measured by ELISA kit (Mabtech) according to the manufacturer's instruction. 2.6. Statistical analysis All of the variables were checked for normality by Shapiro-Wilk test. Non-parametric tests including Mann-Whitney U test were used to compare non-normal distributed variables. To compare the mRNA expression level and cytokine level between CGS-21680 treated and control cells, the Paired sample t-test and Wilcoxon test were performed. Spearman's correlation test analyzed the correlation between the relative mRNA expression of TNF-α and IL-23 with A2AAR expression level. P values less than 0.05 were considered statistically meaningful. All of the statistical analysis were performed by SPSS version 22 and graphs were designed using GraphPad Prism 6. In addition, post hoc statistical power analysis was performed by PASS 11 software. 3. Results 3.1. Demographic and clinical features The demographic features of the AS patients and control group are shown in Table 2. The clinical scores representing disease activity were also demonstrated for AS patients. Moreover, it is described that none of the patients were under neither biological nor methotrexate treatment.
Fig. 1. The relative mRNA expression (A) and cytokine production level (B) of TNF-α in AS patients and control group before and after treatment by A2AAR agonist. A: The mRNA expression level of TNFA was not different significantly between patients and healthy individuals. The TNFA expression level did not alter after CGS-21680 treatment in either groups. B: The TNF-α production level was not different between untreated cells of AS patient and healthy cases but CGS-21680 significantly diminished the TNF-α secretion by monocyte-derived macrophages of AS patients without any influence on control group macrophages (TNF-α: tumor necrosis factor-α, A2AAR: adenosine receptor A2A).
3.2. A2AAR agonist decreased TNF-α secretion in monocyte-derived macrophages of AS patients The influence of A2AAR activation on TNF-α mRNA expression level and protein secretion was investigated and shown in Fig. 1. The CD163 Table 2 Clinicodemographic features of AS patients and control group. Variable
AS patients (N = 14)
Healthy controls (N = 14)
Gender (Male%) Age (years) Disease duration (years) Smoking (%) HLA-B27 positivity, no. (%) ESR (mm/h) BASMI score BASFI score BASDAI score BASG score PDGA score ASQoL score Biological agents Methotrexate
11 (79%) 32 ± 10 7.5 ± 6 5 (35%) 10 (71%) 37 ± 19 3.7 ± 2.5 4.6 ± 2.7 6.1 ± 1.9 7.2 ± 1.8 7 ± 2.7 9.5 ± 5.6 0 (0%) 0 (0%)
11 (79%) 32 ± 9.6 – 5 (35%) – 5 ± 3 – – – – – – – –
and CD206 positive macrophages of AS patients were compared to healthy individuals. Macrophages were treated with A2AAR agonist (CGS-21680) and compared to control samples within each population. The CGS-21680 decreased the TNF-α production level in monocytederived macrophages of AS patients by −1.1-fold (P < 0.05, Power = 0.65). Nevertheless, the TNF-α secretion level did not altered in macrophages of healthy cases after A2AAR agonist treatment. However, the mRNA expression level and protein secretion were not significantly different between AS patients and healthy individuals either with or without CGS-21680 treatment.
3.3. IL-23 mRNA expression was increased in monocyte-generated macrophages of AS patients by A2AAR agonist The effect of the A2AAR agonist on IL-23 mRNA expression and protein secretion was also determined by the similar process mentioned previously and shown in Fig. 2. CGS-21680 significantly augmented IL23A mRNA in macrophages of AS patients by 1.5-fold (P < 0.05, Power = 0.78) whereas it did not alter IL23A mRNA expression in macrophages of healthy individuals. Therefore, AS macrophages
*AS: ankylosing spondylitis, ESR: erythrocyte sedimentation rate, HLA-B27: human leukocyte antigen (subtypes B*2701-2759), BASMI: bath ankylosing spondylitis metrology index, BASFI: bath ankylosing spondylitis functional index, BASDAI: bath ankylosing spondylitis disease activity index, BAS-G: bath ankylosing spondylitis global score, PDGA: patient’s disease global assessment, ASQoL: ankylosing spondylitis quality of life. 3
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the T helpers by regulating the cytokine milieu [14,15]. In the present study, we have shown that the relative mRNA expression level of IL-23 was significantly increased after A2AAR activation in M-CSF-derived macrophages of AS patients. The A2AAR agonist did not alter the IL-23 expression level among macrophages of healthy subjects. This finding can be possibly explained by the higher expression level of A2AAR mRNA in monocyte-generated macrophages of AS patients compared to the control [16]. In line with our findings, Crean et al. demonstrated the influence of A2AAR activation on IL-23 expression [18]. They showed that IL-23 mRNA was expressed 1.5-fold higher in human primary PBMCs after treatment with non-specific adenosine receptor agonist (59-N-ethylcarboxamidoadenosine, NECA) plus LPS in comparison to cells primed solely by LPS. Moreover, they reported that IL-23 mRNA was increased significantly in monocytic THP-1 cell line by CGS-21680/ LPS activation. However, IL-23 mRNA level did not alter after priming by A2B adenosine receptor agonist (BAY-60-6583). Comprehensively, these findings highlighted the provocative role of A2AAR activation on IL-23 expression. Th-17 cells are a distinct group of lymphocytes that are emerged from CD4 + T helper cells and characterized by the production of IL17. Depending on the primary microenvironment, they transform into two subgroups of effector Th-17 and regulatory Th-17 cells [27]. Effector Th-17 cells are developed by the stimulation of IL-23, IL-6, IL-1β and act as pro-inflammatory cells [27]. Moreover, effector Th-17 cells can emerge from protective regulatory Th-17 cells by the IL-23 stimulation [27]. The crucial role of IL-23 in inducing Th-17 response is reinforced by the promising therapeutic effects of biological medications that target IL-23/IL-17 axis [22]. These therapeutic effects have also shown previously in AS treatment which strengthen the highlighted role of Th-17 response in its pathogenesis [22]. Several studies have suggested the influence of adenosinergic pathway in developing the Th-17 response. Liang and colleagues investigated the role of adenosine receptor activation on the Th-17 response devalopment in a mouse model of autoimmune uveitis [28]. They showed that A2AAR agonist inhibited the Th-17 response in the early phase of disease induction, whereas this effect was inverted when the A2AAR agonist was injected in the late stage of the disease. Moreover, it is shown in another study that the enhanced function of the A2AAR by an allosteric modulator can significantly inhibit the IL-23 secretion by dendritic cells and also decrease IL-17 expression in γδ T cells in a mice model of psoriatic-like dermatitis [29]. In contrast, Liang et al. have demonstrated that A2AAR positive γδ T cells significantly promote the activation of Th-17 autoreactive cells in the pro-inflammatory context [30]. These controversial findings proposed that the effect of the adenosinergic pathway on orchestrating the immune response influenced by disease progression stage and the cytokine milieu. Our study revealed that A2AAR activation reduces TNF-α secretion in monocyte-generated macrophages of AS patients. These results were not replicated in macrophages of healthy subjects. Similarly, it appears that the TNF-α reducing effect of A2AAR agonist in macrophages of AS patients is due to the higher expression level of A2AAR mRNA among these cells compared to control group [16]. In line with our findings, Ravani et al. have reported that A2AAR and A3AR agonists markedly diminished NF-κB level in addition to inhibition of TNF-α release in cultured lymphocytes of AS patients [17]. Similarly, Varani and colleagues have investigated the anti-inflammatory role of A2AAR and A3AR agonists in rheumatoid arthritis (RA) patients. They reported that A2AAR and A3AR activation significantly reduced the nuclear level of NF-κB and TNF-α in lymphocytes of RA patients [31]. Moreover, it is reported previously that therapeutic role of certain types of medications in RA treatment was reversed by the A2AAR antagonist, which reinforces the role of this receptor in anti-inflammatory responses [32]. Interestingly, it is demonstrated that low-dose methotrexate induced adenosine secretion and consequently reduced TNF-α secretion via A2AAR activation [33]. The inhibitory effect of the A2AAR agonist on
Fig. 2. The relative mRNA expression (A) and cytokine production level (B) of IL-23 in AS patients and control group before and after treatment by A2AAR agonist. A: The mRNA expression level of IL23A was significantly increased in M-CSF-generated macrophages of AS patients after CGS-21680 treatment. B: Untreated macrophages of AS patients significantly secreted higher level of IL23 in comparison to control group. However, CGS-21680 did not alter IL-23 production in either groups (IL-23: interleukin-23, A2AAR: adenosine receptor A2A).
expressed significantly higher level of IL23A mRNA after CGS-21680 treatment compared to healthy macrophages (P < 0.05, Power = 0.65). CGS-21680 also increased IL-23 secretion in M-CSFderived macrophages of AS patients, however, it was not significant. Moreover, macrophages of AS patients secreted significantly higher level of IL-23 in comparison to healthy individuals (P < 0.05, Power = 0.75). 3.4. Correlation between the relative mRNA expression of TNFA with A2AAR expression level The correlation between the expression level of TNFA mRNA with ADORA2A (A2AAR) mRNA in monocyte-derived macrophages of AS patients and healthy individuals was also evaluated. We did not find any substantial correlation between expression levels. 4. Discussion The ancient evolutionary adenosinergic pathway has a pivotal role in regulating immune responses and prevents exaggerated inflammatory responses through its anti-inflammatory effects [11,12]. Adenosine can also orchestrate the type of immune response and skew 4
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TNF-α secretion was also shown in human keratinocytes that have a role in psoriasis pathogenesis [34]. Besides, TNF-α secretion was significantly reduced by the A2AAR agonist in the lymphocytes of a mouse model of spontaneous ileitis [35]. Although this finding was in favor of the probable anti-inflammatory role of the adenosinergic pathway in the inflammatory bowel diseases, it was not replicated in mice with dextran sulfate-induced colitis [36]. Furthermore, the inhibitory effect of A2AAR activation on TNF-α release from lymphocytes was also reported in multiple sclerosis and systemic lupus erythematosus (SLE) patients [37,38]. Generally, these studies support the anti-inflammatory role of A2AAR agonists by their TNF-α reducing effects. In conclusion, our present study provides evidence that A2AAR activation induces IL-23 secretion in macrophages of AS patients. Due to the pivotal role of Th-17 response in AS pathogenesis, it is highly probable that A2AAR activation can aggravate this response by stimulating IL-23 production. Meanwhile, substantial evidence has supported the anti-inflammatory role of A2AAR agonists by inhibiting pro-inflammatory cytokines secretion including TNF-α from macrophages. Also, it is mentioned previously that the inflammatory microenvironment significantly influences the A2AAR downstream activation effects on inducing distinct immunological responses. Consequently, further studies should be designed to investigate the therapeutic role of A2AAR agonists on AS treatment regarding disease progression state.
[10]
[11] [12] [13] [14] [15] [16]
[17]
[18]
[19]
CRediT authorship contribution statement
[20]
Maryam Akhtari: Conceptualization, Writing - review & editing, Validation. Mahdi Vojdanian: Writing - original draft, Validation. Ali Javinani: Data curation, Writing - original draft, Validation. Amir Ashraf-Ganjouei: Formal analysis, Writing - original draft, Validation. Ahmadreza Jamshidi: Project administration, Writing - review & editing, Validation. Mahdi Mahmoudi: Conceptualization, Supervision, Writing - review & editing, Validation.
[21]
[22]
[23]
[24]
Declaration of Competing Interest [25]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[26]
Acknowledgment
[27]
We would like to thank all participants for their collaboration. This study was supported by a grant from deputy of research, Tehran University of Medical Sciences (Grant No. 95-04-41-33867).
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