|Ahead of print publication
Assessment of Serum Semaphorin-3A Level in Systemic Lupus Erythematosus Patients in Suez Canal Region
Alaa Saber Shams1, Nevene Ramsis Wissa1, Mai Mohamed Abdelnaby2, Rania M Saleh1
1 Department of Clinical Pathology, Rheumatology and Rehabilitation, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
2 Department of Physical Medicine, Rheumatology and Rehabilitation, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
|Date of Submission||12-Sep-2021|
|Date of Acceptance||05-Jan-2022|
|Date of Web Publication||06-Jul-2022|
Mai Mohamed Abdelnaby,
Departments of Physical Medicine, Rheumatology and Rehabilitation, Faculty of Medicine, Suez Canal University, Ismailia
Source of Support: None, Conflict of Interest: None
Background: Semaphorin-3A (Sema-3A) is an important immunoregulator protein; it has a role in the maintenance of self-tolerance, so it is thought to be involved in the pathogenesis of many autoimmune diseases such as systemic lupus erythematosus (SLE). The purpose of the study is to assess the possible role of serum Sema-3A level as a potential biomarker for disease activity in patients with SLE and its relation with lupus nephritis.
Patients and Methods: We recruited fifty SLE patients and 25 healthy controls. According to the SLE disease activity index (SLEDAI), patients were divided into two groups; active SLE (n = 25) and inactive SLE (n = 25). Sema-3A level was assessed in the study groups using enzyme-linked immunosorbent assay. Laboratory work included antinuclear antibodies, anti-ds-DNA, C3, C4, C-reactive protein, and erythrocyte sedimentation rate (ESR).
Results: Serum Sema-3A level was significantly lower among SLE patients compared to healthy controls (18.14 ± 5.77 vs. 65.72 ± 38.08, P < 0.001). Moreover, it was lower among active SLE group compared to inactive group (14.96 ± 4.27 vs. 21.32 ± 5.17, P < 0.001). Serum level of Sema-3A negatively correlated with SLEDAI (P ≤ 0.001) and ESR (P = 0.006) where it was correlated positively with C3 (P ≤ 0.001) and C4 (P = 0.017).
Conclusion: SLE activity is associated with decreased serum level of Sema-3A, thus it is suggested that Sema-3A is a candidate to become a useful marker for SLE disease activity.
Keywords: Autoimmune disease, semaphorin-3A, systemic lupus erythematosus, systemic lupus erythematosus disease activity index
| Introduction|| |
Systemic lupus erythematosus (SLE) is one of the most common autoimmune diseases which involves adaptive and innate immune dysregulation. Its pathogenesis is due to interactions between several factors such as environmental, hormonal, and various genetic factors.,, Defects happen in numerous parts of the immune cascade which result in heterogeneity of the clinical presentations such as skin rashes, nephritis, arthritis, leucopenia, and nervous system affection. Immunologically, SLE is characterized by an increase in the reactivity of B cell and autoantibodies production against either nuclear or cytoplasmic components. Persistent generation of autoantibodies leads to immune complex deposition in brain, kidney, and other organs, Delayed diagnosis of SLE leads to an increase in the damage that occurred to these vital organs.
Chronic inflammation plays a crucial role in the pathogenesis of SLE. The imbalance between pro-inflammatory and anti-inflammatory cytokines is the key mechanism in SLE development.,
Furthermore, the accumulation of immune complex in tissues leads to secondary inflammatory reactions. Clinical symptoms of SLE are reflections of tissue damage mediated by the inflammatory reactions of immune system activation and autoimmunity, along with exaggerated repair consequences.
The worldwide incidence of SLE is 0.36–2.5 per 100,000 per year, while its prevalence is 1.89–25.7 per 100,000. SLE is more common in women than in men. It affects women particularly between puberty and menopause. The female-to-male ratio is 3 to 1 in children and about 9-1 between puberty and menopause.,
Semaphorins are a large family of signaling proteins characterized by a conserved cysteine-rich “sema” domain of 500 amino acids in the N-terminal region., They are subdivided into 8 classes of transmembrane, membrane-bound, or secreted proteins with different C-terminal sequence motifs, including an immunoglobulin domain, a thrombospondin domain, and a glycosyl phosphatidylinositol anchor motif.
Various members of semaphorins play a role in exaggeration of the immune response. Whereas others, semaphorins of Class 3 (Sema-3), may control immune functions negatively. Semaphorin-3A (Sema-3A) is a chemorepulser protein, secreted by surrounding tissues. It guides migrating cells and axons in the developing nervous system, which is important for the formation of neurons and vasculature.
Sema-3A is expressed mainly on T regulatory cells (Tregs) and on other immune cells including T and B cells, resulting in the inhibition of T effector cell proliferation and secretion of pro-inflammatory cytokine., It is still unclear how Sema-3A exerts its action on immune cells, but they are likely to involve a receptor complex formed from one of two Neuropilins (NPs), and one of four Plexin A proteins. NPs act as the primary ligand binding sites, and Plexin A proteins act as the signal-transducing components.
It has been demonstrated that semaphorins are involved in the regulation of the immune system and are implicated in the pathogenesis of autoimmune diseases, Immunologists have found Sema-3A family to be regulators of immune cell responses, thus it was called “immune semaphorins.”
Although there are sufficient data about the pathogenesis of SLE, few biomarkers have been validated as biomarkers of disease activity.
Serum C3 and C4 has traditionally been the gold standard of SLE disease activity. However, these proteins have limited utility as prognostic biomarkers, as they considered substrates rather than products of complements activation. Several major weaknesses have been identified. First, there is a wide range of normal values of serum C3 and C4 levels among populations. Second, Systemic inflammation can increase levels of C3 and C4 as an acute-phase response which may balance the increased catabolism of these proteins. Third, hereditary deficiencies C4-null alleles may result in a persistent lowered serum level of C4 because of the decreased synthesis rather than activity.
So the purpose of this study is to assess the possible role of serum Sema-3A level as a potential and specific biomarker for disease activity in patients with SLE and its relation with lupus nephritis.
| Patients and Methods|| |
Fifty SLE patients and 25 healthy controls from the rheumatology clinic at Suez Canal university hospital were included in this cross-sectional study during the period from December 2018 to July 2019. The patients in the SLE group were age and gender-matched to those in the control group. Participants who were < 16-year-old or have autoimmune disease other than SLE, having an allergic condition, on renal dialysis, or having any chronic infections (i.e., tuberculosis, HCV infection, or HBV infection) were excluded. The study was approved by the institutional ethics committee of faculty of medicine Suez Canal University through order n.research3279# dated December 19, 2018. All participants enrolled in the study signed an informed consent, according to the regulations of the research ethics committee.
Patients were divided according to the presence or absence of disease activity into (i) patients in activity (n = 25) and (ii) Patients in remission (n = 25). SLE was diagnosed according to the systemic lupus international collaborating criteria for the classification of SLE. The activity of the disease was defined by the SLE disease activity index (SLEDAI) at the time of the visit or the proceeding 10 days. SLEDAI score ≥10 was defined as high disease activity and <10 as less disease activity.
Remission is diagnosed by the absence of symptoms, signs, or abnormal labs. Patients on antimalarials drugs only called Remission-off-therapy. Remission-on-therapy is characterized when patients are treated with low-dose steroids, antimalarials drugs, maintenance immunosuppressives and/or biologics. Full medical history and clinical Examination was carried out by a rheumatologist. Basic lab tests for SLE were obtained from the patients' records.
Assessment of serum level of Sema-3A
Serum Sema-3A concentrations were obtained (using enzyme-linked immunosorbent assay [ELISA] Kit for Human Semaphorin 3A; Bioassay Technology Cat. No E2078 Hu) according to the manufacturer's instructions. The serum samples were stored at −20° until ELISA evaluation.
Serum sema3A levels of SLE patients' were correlated with SLEDAI scores, renal involvement (proteinuria), grades of lupus nephritis (obtained from records), and laboratory studies including anti-dsDNA and C3-C4 serum levels.
The data was analyzed by using the SPSS. 25 software (IBM SPSS inc., Armonk, NY, USA). Quantitative data were expressed as mean and standard deviation, while Comparisons were performed using t-test and Chi-squared test was used (for qualitative data). Significance was considered at P < 0.05 was expressed as number and percentage. t-test was used for Comparisons of quantitative data and Chi-squared-test for qualitative data. The correlation between serum Sema-3A and other variables was performed using Pearson correlation (r). Receiver operating characteristic (ROC) curves were used to assess sensitivity and specificity of Sema-3A as an indicator of SLE diagnosis and prognosis.
| Results|| |
Study population characteristics
The study included 50 SLE patients and 25 healthy controls. There was a nonstatistically significant difference between the studied groups regarding age and gender (P = 0.07, 0.7 respectively). Demographic and laboratory characteristics of the study population are shown in [Table 1]. SLE patients showed significantly higher C-reactive protein (CRP), Total Leucocytic Count, (TLC) and erythrocyte sedimentation rate (ESR) (P ≤ 0.001, 0.049 and <0.001 respectively), and lower hemoglobin and PLTs (P = 0.004 and 0.050 respectively) compared to normal control.
|Table 1: Demographic and laboratory characteristics of the study population|
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Features of systemic lupus erythematosus activity
SLEDAI of the Active SLE group was 18.56 ± 7.5 (range 8–38). SLE disease activity was assessed according to SLEDAI and is classified into 3 grades Mild, (0–5) Moderate (5−<12), and Severe (>12). Moderate SLE activity was seen in 32% of our SLE active patients, while severe activity was seen in 68% of them; no mild cases were detected. About 64% of patients with active disease had renal involvement, 40% had mucocutaneous disease, 36% had arthritis, and <5% had fever, Neuropsychiatric or Cardiovascular disease. Patients with active SLE showed a significant decrease in their serum C3 and C4 proteins and a significant elevation in ESR compared to SLE inactive patients. While platelet count, TLC, antinuclear antibodies (ANA), and ds-DNA did not differ between active and inactive SLE patients [Table 2].
|Table 2: Characteristics of active versus inactive systemic lupus erythematosus patients|
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Lupus nephritis grading in systemic lupus erythematosus patients with renal affection
About 10 patients (38.5%) have Grade I lupus nephritis, three patients (11.5%) have Grade II, 4 patients (15.4%) have Grade III, 6 patients (23%) have Grade IV, while three patients (11.5%) have Grade V, no one has Grade VI glomerulonephritis [Table 3].
|Table 3: Lupus nephritis grading in systemic lupus erythematosus patients with renal affection and its Correlation with serum Semaphorin 3-A|
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Sema-3A level among study groups
Serum Sema-3A level was significantly lower among SLE patients compared to healthy normal controls (18.14 ± 5.77 vs. 65.72 ± 38.08, P < 0.001) [Table 1] and [Figure 1]b. Moreover, it was lower among active SLE group compared to inactive group (14.96 ± 4.27 vs. 21.32 ± 5.17, P < 0.001) [Table 2] and [Figure 1]a.
|Figure 1: (a) Simple box-plot of Semaphorin-3A levels in active systemic lupus erythematosus patients and Inactive systemic lupus erythematosus patients (b) Simple box-plot of Sema-3A levels in patients and healthy control|
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The association between the sema-3A serum levels and lupus nephritis
Sema-3A serum levels and the presence of lupus nephritis
It was found that about 72% of patients with a sema3A level below 20 ng/ml had kidney involvement. However, in those with Sema-3A levels above 20 ng/ml, only 33% had kidney involvement, so serum Sema-3A can be potentially used as a prognostic marker for renal affection in SLE patients.
Also we found that the serum level of sema3A was dramatically decreased in Grade III, IVand V compared by its level in both Grade II and III lupus nephritis [Table 3].
Serum sema-3A level as a potential indicator for systemic lupus erythematosus disease and systemic lupus erythematosus activity
The ROC curve was plotted to investigate the best cutoff vaue of 25.5 ng/mL of serum Sema-3A that indicates the presence of SLE disease with 90% sensitivity and 80% specificity (P < 0.001).
Another ROC curve was plotted for serum Sema-3A to indicate the presence of SLE Activity sets a threshold value of 18.5 ng/mL with 76% sensitivity and 68% specificity (P < 0.001).
Clinical correlation of serum sema-3A level in systemic lupus erythematosus patients
Serum level of Sema-3A was significantly positively correlated with serum C3 (r = 0.53, P < 0.001), serum C4 (r = 0.33, P = 0.017) and duration of SLE disease (r = 0.28, P = 0.048), While it was significantly negatively correlated with SLEDAI (r = −0.67, P < 0.001) and ESR (r = −0.38, P = 0.006). Also, there was a significant negative correlation with lupus nephritis grades (r = −0.637, P < 0.001). Regarding the clinical symptoms of SLE, serum level of Sema-3A was significantly negatively correlated with renal affection (proteinuria) [Table 4].
|Table 4: Correlation of systemic lupus erythematosus patients' various parameters with serum Semaphorin 3-A level|
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| Discussion|| |
Sema-3A was found to be involved in immune-mediated diseases. Being an immunoregulator, Sema-3A may play an important role in the diagnosis and prognosis of SLE disease. Thus, serum Sema-3A level may be used as a marker for the disease activity.
We found that serum Sema-3A in our study was significantly lower in SLE patients compared to healthy controls. The results of the present study are in agreement with those conducted by Vadasz et al., who studied the serum level of Sema-3A in 32 SLE patients. They found that serum levels of Sema3A were found to be decreased in SLE patients compared to those in the control group. They found that Sema-3A plays a key role in the pathogenesis of SLE by modulating the autoimmune properties of B cells as well as affecting humoral responses in SLE.
In harmony with the present study is the study of Gao et al., which investigated the expression of Sema-3A on B regulatory cells. They discovered that the serum of Sema-3A was significantly lowered in SLE patients when compared to the control group also the Sema-3A mRNA level in SLE patients was lower than that in healthy controls.
Wang et al. investigated the plasma semaphorin 3A and semaphorin 7A levels in SLE patients, they found that both Sema3A and Sema7A levels were decreased in SLE patients and increased in RA patients; decreased Sema7A level, increased Sema3A level were found in Systemic sclerosis (SSc). However, no significant differences were found between SSc and RA groups concerning Sema3A. Furthermore, there were no significant differences between SSc and SLE groups concerning Sema7A.
Another study by Catalano et al. had shown overexpression of Sema-3A in a mouse model of collagen-induced arthritis which resulted in reduced incidence, articular inflammation, and disease severity.
It was observed in our study that serum concentration of Sema-3A level was significantly lower among active SLE patients than inactive ones. Vadasz et al. also reported similar results where an increase in serum Sema-3A level was observed when the disease activity was decreased, which indicated that Sema-3A plays a role in controlling the disease activity of SLE.
There was a significantly negative correlation between serum Sema-3A level in SLE patients and SLEDAI and lupus nephritis grading. These findings match with those found by Vadasz et al. and Gao et al., who both found an inverse correlation between serum Sema-3A levels and disease activity in SLE patients.,
Results of the present study also showed a significant association of lupus nephritis with low levels of Sema-3A. Sema-3A levels was <20 ng/ml in 72% of lupus nephritis patients. However, only 33% had renal involvement in those with Sema-3A levels above 20 ng/ml. These findings were supported by Vadasz et al. who found an inverse correlation between the level of serum Sema-3A and renal affection. They also found that Sema-3A levels were <50 ng/ml in 73% of lupus nephritis patients, whereas only 5% of SLE patients with serum level of Sema-3A above 50 ng/ml had kidney involvement. They also reported that Sema-3A was expressed strongly in the tubules in lupus glomerulonephritis (LGN), suggesting that it can work as a diagnostic marker for renal damage (Gao, 2017 #26; Vadasz, 2012 #22).
In another study, Vadasz et al. demonstrated that kidney biopsies from LGN patients showed stronger staining with anti-Semaphorin 3A and antibodies when compared to either biopsy from patients with primary nephropathy and proteinuria or normal biopsies.
A study that assessed the level of the excreted sema3A in the urine of SLE patients with and without renal involvement compared to rheumatoid arthritis patients as control group discovered that Urinary excretion of Sema 3A was lowered in SLE patients with renal involvement with a negative correlation with SLEDAI and proteinuria.
Vadasz et al. (2012) used a pharmaceutical composition containing isolated Sema-3A for use for treating SLE. They found that treatment improvement was seen in at least one clinical parameter as renal function, a decrease in both anti-Cardiolipin antibody concentration and anti-dsDNA antibody concentration in the serum. Furthermore, they discovered an increase in serum concentration of complement factor C3 and C4 after administration of Sema-3A.
A statistically significant positive correlation was found in this study between serum Sema-3A levels in SLE patients and the serum C3 and C4, while it was significantly negatively correlated with SLEDAI and ESR. These findings are in line with those reported by Vadasz et al. and with Gao et al.,
Gao et al. found a significant negative correlation between CRP levels and ANA titre with serum Sema-3A level, while PLT counts and Hb levels were significantly positively correlated with serum Sema-3A levels. The present study recorded similar results, but they did not reach a statistically significant level.
When ROC curve was done for serum Sema-3A as a diagnostic tool for SLE, the area under the curve was 93.2%. The cut-off value for the diagnosis of SLE was ≤25.5 ng/ml with sensitivity and specificity of 90% and 80%, respectively. Gao et al. reported a cut-off value of serum Sema-3A (for diagnosis of SLE) ≤63.11 ng/ml with sensitivity and specificity of 80.6% and 77.5%. The area under the curve was 87.6%. The above results indicated the validation of Sema-3A as a diagnostic tool for SLE. In the current study, another ROC curve was done for serum Sema-3A as a prognostic tool for SLE. At a cutoff point of ≤18.5 ng/ml of serum Sema-3A, the area under the curve was 81.4%. The sensitivity and specificity were 76% and 68%, respectively.
| Conclusion|| |
Sema-3A is significantly low in SLE patients compared with controls, in patients with activity than inactive patients, and is negatively correlated with disease activity and renal damage. It is thus suggested that Sema-3A is a candidate to become a useful marker for SLE disease activity and renal affection and a new therapeutic target contributing to the treatment of SLE.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]