|Year : 2017 | Volume
| Issue : 1 | Page : 2-6
Correlation between serum semaphorin 3A and inflammatory disorder in ankylosing spondylitis: Potential function of immunoregulation
Bang-ping Qian, Jin Qian, Yong Qiu, Zezhang Zhu, Zhen Liu, Zhe Qu, Hu Jun, Benlong Shi
Department of Spine Surgery, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China
|Date of Web Publication||29-May-2017|
Department of Spine Surgery, Nanjing Drum Tower Hospital, Medical School of Nanjing University, Zhongshan Road, 321, Nanjing 210008
Source of Support: None, Conflict of Interest: None
Objective: The objective of this study was to verify whether semaphorin 3A (Sema3A) participate in the immunoregulation of T helper 17 cells related inflammatory process and to identify novel serum markers in the immunoregulation in ankylosing spondylitis (AS). Methods: A total of 59 untreated male AS patients and 61 age-matched male normal controls were recruited for the study. The study was approved by the Institutional Review Board. The venous blood of all participants was drawn from just above the elbow with patients' consent. Serum Sema3A, tumor necrosis factor-α (TNF-α), and interleukin-21 (IL-21) levels were evaluated using enzyme-linked immunosorbent assay analysis in both AS patients and normal controls. The optical density was assessed at 450 nm using a microtiter plate reader. Then, the concentration of the target serum marker was measured on the constructed standard curve. Results: We found that serum levels of Sema3A, TNF-α, and IL-21 were all higher in AS patients than normal control (47.5 ± 5.3 vs. 40.2 ± 4.0, 182.6 ± 38.1 vs. 47.5 ± 7.7, 190.3 ± 29.8 vs. 93.3 ± 21.0, P < 0.01). Serum level Sema3A was demonstrated to be statistically significantly positive correlated with both TNF-α and IL-21. Serum levels of Sema3A, TNF-α, and IL-21 were all higher in AS patients than normal control. Conclusion: Sema3A was a potential novel putative factor in the immunoregulation of inflammatory response in AS.
Keywords: Ankylosing spondylitis, immunoregulation, interleukin-21, semaphorin 3A, tumor necrosis factor-α
|How to cite this article:|
Qian Bp, Qian J, Qiu Y, Zhu Z, Liu Z, Qu Z, Jun H, Shi B. Correlation between serum semaphorin 3A and inflammatory disorder in ankylosing spondylitis: Potential function of immunoregulation. J Spinal Stud Surg 2017;1:2-6
|How to cite this URL:|
Qian Bp, Qian J, Qiu Y, Zhu Z, Liu Z, Qu Z, Jun H, Shi B. Correlation between serum semaphorin 3A and inflammatory disorder in ankylosing spondylitis: Potential function of immunoregulation. J Spinal Stud Surg [serial online] 2017 [cited 2020 Sep 28];1:2-6. Available from: http://www.jsss-journal.com/text.asp?2017/1/1/2/207212
| Introduction|| |
Ankylosing spondylitis (AS) is the prototype of spondyloarthritis, which involves the axial joints and bilateral sacroiliac joints, leading to functional impairments and worse quality of life. Clinical diagnosis is generally based on the modified 1984 New York criteria. The pathological changes of AS patients include: systematic inflammatory response, infiltration of T-cell at the sites of axial joints and enthesitis, and development of new syndesmophytes. Therefore, AS is considered as an immune-mediated inflammatory disorder that eventually leads to the spinal kyphotic deformity. The pathogenesis has long been the most concerned issue for the AS patients., The interleukin (IL)-23/IL-17 axis is deemed a major inflammatory pathway that initiates the systematic inflammatory cascade of AS. Recently, various cytokines are proven to participate in the IL-23/IL-17 pathway, including IL-23, IL-21, transforming growth factor (TGF)-β, IL-17, and tumor necrosis factor-α (TNF-α).,,,, However, mechanism of autoimmune regulation remains unknown and relevant insight provides potential targets for the treatment of AS.
T helper (Th) cells are considered to play a major role in the pathogenesis of AS. The Th1 subsets were the first one to be described. Its cytokine signatures are interferon-γ (IFNγ) and TNF-α. The recent studies on Th17 cells have showed that the pathogenesis of AS are attributable to CD4+ Th17 T-cells and the IL-23/IL-17 axis.,,, The Th17 cells are a subset of T-cells featured by its production of IL-17, developmentally distinct from Th1 and Th2 cells. The cytokine profiles of the IL-23/IL-17 axis include IL-23, IL-17A, IL-17F, IL-22, IL-26, and TNF as effectors, IL-21, TGF-β, IL-1 β, and IL-6 as regulators.,,,, IL-17, and IL-23 are considered the most specific to the Th17 response. IL-23 is expressed by activated myeloid cells, endothelial, and epithelial cells. Regulated by IL-21, IL-17 induces Th17 cell development. The expanded Th17 cell produces Th17 cytokines, such as IL-17, IL-6, IL-22, IL-26, IFN-γ, and TNF-α. These Th17 cytokines subsequently enhance T-cell priming and stimulate macrophages, fibroblasts, and endothelial and epithelial cells. These effectors and regulators have also been reported in the pathogenesis of other autoimmune disease.,,
The semaphorin family, which is of signature by their cysteine-rich semaphorin protein domains, was originally considered to play a vital role in neurodevelopment, angiogenesis, tumor development, and metastasis.,,, Moreover, an increasing amount of data has pointed out its unique effect in the immunoregulation. Its regulatory role is found in many immune diseases, such as collagen-induced arthritis, psoriasis, and multiple sclerosis.,,, Semaphorin 3A (Sema3A) is expressed by activated T-cell, endothelial cell, and dendritic cell. It has been considered as a promising serum-signaling regulator for disease activity and a potential putative disease-modifying treatment in autoimmune disease, like systemic lupus erythematosus.
Sema3A has been demonstrated to downmodulates T-cell activation in collagen-induced arthritis. The overexpression of Sema3A can remarkably attenuate the inflammatory disorder in arthritis. The underlying mechanism proves to be that Sema3A shows its influence on anticollagen IgG, simultaneously suppressed the release of proinflammatory cytokines, such as IFN-γ and IL-17. In the rheumatoid arthritis model, expression of Sema3A is compromised in CD4+ T cell while its receptor neuropilin-1 (NP-1) is detected. The CD4+NP-1+ T-cells are a subgroup of T-cell that produces significant elevated serum IL-10, exerts the regulatory function on CD4+ T-cell. Sema3A selectively acts on the CD4+ T cell subset which is characterized by the high level of NP-1 and IL-10. The purpose of the present study is to explore whether similar immunoregulation role of Sema3A exists in AS and how Sema3A interacts with the IL-23/IL-17 inflammatory cascade, supporting the therapeutic potential of Sema3A.
| Methods|| |
Serum sample of 59 untreated male AS patients and 61 age-matched male normal controls were collected from January 2010 to January 2012. All AS patients were recruited from the outpatient clinic of the Department of Spine Surgery and the normal controls were enrolled from the medical control center for the healthy population at the hospital. Inclusion criteria for AS group were the diagnoses fulfilled the modified 1984 New York criteria  while the normal controls were healthy controls with no autoimmune or musculoskeletal disease. AS patients who had spinal fractures and pseudarthrosis were excluded from the study. This study was approved by the Institutional Review Board. All participants gave written informed consent.
The venous blood was drawn from just above the elbow. A serum separator tube was used and samples were allowed to clot for 30 min before centrifugation for 10 min at approximately 3000 ×g. Serum and assay were removed immediately and samples were stored at − 80°C. All reagents were prepared before starting assay procedure. Standard 50 μl was added to the standard well. Testing sample 10 μl was added followed by 40 μl of sample diluent to the testing sample well. The binding was detected using anti-human Sema3A, anti-TNF-α, and anti-IL-21 antibody conjugated with horseradish peroxidase (HRP) (Yunhanbio, CK-E92455H, CK-E10110H, and CK-E90663H, respectively). A volume of 100 μl of HRP-conjugate reagent was added to each well, then covered with an adhesive strip and incubated for 60 min at 37°C. Each well was aspirated and subsequently washed with 400 μl of wash solution. After the last wash, remaining wash solution was removed by aspirating or decanting. The plate was inverted and blotted against clean paper towels. A volume of 50 μl chromogen solution A and 50 μl chromogen solution B were added to each well, then gently mixed and incubated for 15 min at 37°C. The enzymatic reaction was protected from light. The reaction was stopped with 50 μl stop solution when the color in the wells changed from blue to yellow. The optical density (O.D.) was read at 450 nm using a microtiter plate reader Infinite M200Pro (TECAN Instrument, Switzerland) within 15 min. The mean O.D. value was calculated for each standard and sample. All O.D. values were subtracted by the mean value of the zero standard before result interpretation. Then, the standard curve was constructed. To determine the amount in each sample, the O.D. value on the Y-axis was located and a horizontal line was extended to the standard curve. At the point of intersection, a vertical line to the X-axis was drawn and the corresponding concentration was read.
Student's t-test was used in comparing the distribution of variables in two groups. Pearson's correlation analysis was applied in the correlation analysis of Sema3A and cytokines in the IL-17/23 axis. Statistical analyses were performed using the SPSS (20th Edition; IBM Corporation, New York, NY, USA) statistical package. The value of P = 0.01 was considered statistically significant.
| Results|| |
The average age of the AS patients was 36.1 ± 9.6 years (range, 20–60 years) and that of normal controls was 38.3 ± 7.4 years (range, 25–58 years). A total of 56 AS patients (94.9%) were human leukocyte antigen-B27-positive. All participants recruited were male. For patients in AS group, average duration of disease was 3.1 ± 2.1 years, average erythrocyte sedimentation rate (ESR) was 40.8 ± 26.1 mm/h, and average C-reactive protein (CRP) was 32.9 ± 30.6 mg/L. Serum markers Sema3A, TNF-α, and IL-21 demonstrated statistical significance between AS group and normal control [Figure 1] and [Figure 2]. The average value of three biochemical variables was all higher in AS group than in normal control. The demographic, clinical, and biochemical variables data of AS patients and controls are shown in [Table 1].
|Figure 1: Serum semaphorin 3A levels of ankylosing spondylitis patients and control group. Semaphorin 3A indicates semaphorins 3A. *Indicates statistical significance of P < 0.05.|
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|Figure 2: Serum tumor necrosis factor-α and interleukin-21 levels of ankylosing spondylitis patients and control group. Tumor necrosis factor-α indicates tumor necrosis factor-α, IL-21 indicates interleukin-21. *Indicates statistical significance of P < 0.05.|
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|Table 1: Demographic, clinical parameters, and biochemical variables data of ankylosing spondylitis and control group|
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In terms of correlation analysis, the serum level of Sema3A was statistically, significantly positive strong correlation with both TNF-α and IL-21 [Table 2]. R-value of correlation coefficient between Sema3A and TNF-α was 0.797, whereas the correlation between Sema3A and IL-21 was 0.902.
|Table 2: Correlations between semaphorin 3A and cytokines in interleukin-17/interleukin-23 pathways|
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| Discussion|| |
Serum markers have always been a major resort in the assessment of AS ,,,,,,,,, IL-23/IL-17 pathway is widely recognized to be the most important mechanism in the pathogenesis of AS.,, Considerable amount of cytokines have been identified as triggers and drivers of the systematic immune cascade. In the early stage of the cascade, activated dendritic cell produces IL-23, regulated by IL-21, and inducing the development and expansion of Th17 cell. The th17 cell then excretes Th17 cytokines, including TNF-α and IL-17, which result in the enhancement of T-cell priming and stimulation of macrophages, fibroblasts, endothelial, and epithelial cells, consequently, start the systematic immune response in AS., Immunoregulation to this inflammatory disorder leads to relief of clinical and biological signs in active AS patients. However, a few researchers have addressed this issue.
Sema3A is attracting increasing interest due to its roles in modulating inflammatory conditions in many inflammatory disorder diseases.,, Until now, there has been a lack of discussions on regulatory properties of Sema3A in AS patients. In this study, the serum level of Sema3A was found to be elevated [Table 1]. This could be explained by its interactions with NP-1 on dendritic cells and peripheral T-cells, which might lead to peripheral T-cell/dendritic cell communications. Specifically, NP-1 seems to be a part of the immunological synapse formation and colocalized with the T-cell receptor on T-cells, when in contact with the dendritic cell. Lepelletier et al. already showed that Sema3A blocks the anti-CD3/CD28-stimulated proliferation of T-cells. In the light of significantly elevated serum levels of TNF-α and IL-21, this research sought to explore whether similar regulation effect occurred in the setting of early stage of IL-23/IL-17 axis systematic immune response in AS. Compared with normal control, the patients in active AS group exhibit significant higher level of Sema3A [Table 1]. In addition, strong correlations were found between Sema3A and two essential cytokines in the IL-23/IL-17 pathway. The interaction between Sema3A and T-cell infiltration has been documented in other immune-mediated inflammatory skeletal disease. Takagawa et al. showed that expression of Sema3A decreased in the joint synovium of rheumatoid arthritis patients than those with osteoarthritis. Relevant mechanism is that Sema3A affects the receptor polarization of T-cell and interfere with signaling of T-cell priming accordingly. In this study, R-value of correlation coefficient between TNF-α, IL-21, and Sema3A was 0.797 and 0.902, respectively. TNF-α is the center inflammatory cytokine in the IL-23/IL-17 axis and IL-21 plays a modulatory role in the Th17 cell expansion. The result of this study implied that Sema3A might execute its regulation function in the IL-23/IL-17 systematic immune response in the AS patients.
Several limitations of this study should be addressed. First, the serum markers levels in samples obtained from patients peripheral blood might not truly represent levels at the lesion site. Further researches are needed to determine whether the level of Sema3A and IL-21 are elevated at the lesion site to establish a direct relationship. Second, the patients included might be in different disease phases, this variance of immune status is a potential source of bias. Finally, although evaluation of CRP, ESR, TNF-α, and IL-21 proved patients in AS group were in active inflammatory phase and strong correlations were demonstrated, direct immunoregulation mechanism was to be explored in further study.
| Conclusion|| |
Compared with normal controls, AS patients showed elevated serum levels of Sema3A, TNF-α, and IL-21. Serum levels of TNF-α and IL-21 in AS patients were in strong correlation with Sema3A. This study suggested that Sema3A might be involved in the immunoregulation of AS patients.
This work was supported by funding from Jiangsu Province's Key Medical Center (ZX201107), the Fourth Period of “333” High-level Personnel Training Program of Jiangsu Province, China and the National Nature Science Foundation of China (81372099); all authors listed meet the authorship criteria according to the latest guidelines of the International Committee of Medical Journal Editors; all authors are in agreement with the manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]