Introduction
Multiple organs and systems are affected by systemic lupus erythematosus (SLE), which is a chronic autoimmune inflammatory disease affecting primarily young adults, especially women of childbearing age.1 Epidemiological data indicated that SLE prevalence ranges between 0 to 241 per 100,000 people worldwide, with regional and racial variations.2 The exact pathogenesis that causes SLE development remains unclear.3 Lupus develops because of abnormalities in the innate and adaptive immune system as well as environmental and genetic factors.4 Recently, it was found that numerous loci are prone to SLE development.5
Tumor necrosis factor (TNF) superfamily member 15 (TNFSF15), a member of the TNF-α superfamily, is a proinflammatory cytokine also known as vascular endothelial growth inhibitor or TNF-like ligand 1A (TL1A).6 It can serve as an essential modulator of inflammation and vascular homeostasis.7 Binding to its receptor, death receptor 3 (DR3, TNFRSF25), which is predominantly expressed in lymphocytes, TNFSF15 triggers the proliferation of T effector cells and cytokine production by these cells.8 Effective T-cell immune responses in T-cell-mediated autoimmune and inflammatory illnesses, cell proliferation and death, angiogenesis, and tumor metastasis depend on TNFSF15-NFRSF25 signaling.9 The TNFSF15 gene, which has four exons and three introns and is found on chromosome 9 (9q32), encodes the TNFSF15 cytokine.10,11
The TNFSF15 gene is polymorphic, and its variants induce the production of altered TNFSF15, aiding in the development of autoimmune and inflammatory disorders.12 Many disorders, including tumors, Crohn’s disease, ulcerative colitis, and other autoimmune diseases, have been linked to TNFSF15 variations.11,13 An earlier study in the Chinese population has found a strong association between the TNFSF15 gene polymorphism and susceptibility to SLE.14
The substitution variant TNFSF15 rs4979462 C>T has thymine instead of cytosine in the first intron of the TNFSF15 gene. The rs4979462 variant lies in the transcription regulatory elements of the TNFSF15 gene; consequently, the affinity for binding of the transcription factors could vary from the major to the minor sequence variants.15
We aimed to assess the role of the TNFSF15 rs4979462 variant and the TNFSF15 cytokine in SLE in Egyptian patients.
Results
Two hundred and twenty subjects (118 SLE patients and 102 healthy control volunteers with matching age and sex) participated in this study. The mean age of the SLE patients, which included 16 men (13.6%) and 102 women (86.4%), was 32.9 ± 9.6 years. The healthy control group included 94 females (92.2%) and eight males (7.8%), with an average age of 32.6 ± 8.6 years. The descriptive data of patients with SLE is shown in Table 1.
Table 1Descriptive data of patients with SLE
Feature | SLE patients, n = 118 |
---|
Age in years | 32.9 ± 9.6 |
Male sex, n (%) | 16 (13.6) |
Clinical manifestations, n (%) | |
Malar rash | 60 (50.8) |
Vasculitic rash | 25 (21.2) |
Discoid rash | 9 (7.6) |
Alopecia | 54 (45.8) |
Oral ulcers | 50 (42.4) |
Photosensitivity | 46 (39.0) |
Arthritis | 72 (61.0) |
Serositis | 46 (39.0) |
Vasculitis | 24 (20.3) |
Thrombotic manifestations | 31 (26.3) |
Neurological manifestations | 55 (46.6) |
Nephritis | 73 (61.9) |
SLEDAI | 18 (12–27) |
High disease activity, SLEDAI >20 | 50 (42.4) |
Renal SLEDAI | 4 (0–8) |
Laboratory findings | |
Hb in g/dL | 10.9 ± 2.1 |
WBC count in cell/mm3 | 7.2 ± 3.1 |
Lymphocytes in cell/ mm3 | 26.5 ± 11.9 |
Platelet count in platelets/mm3 | 263.8 ± 108.2 |
ESR in mm/h | 55.0 ± 32.7 |
Albumin in g/dL | 3.4 ± 0.7 |
AST in IU/L | 20 (15–29) |
ALT in IU/L | 19 (14–26) |
Urea in mg/dL | 38 (25–68) |
Creatinine in mg/dL | 0.9 (0.7–1.3) |
Protein in urine in g/day | 1.1 (0.4–2.6) |
TGs in mg/dL | 140 (100–247) |
Cholesterol in mg/dL | 204 ± 74 |
LDLc in mg/dL | 132 ± 49 |
HDLc in mg/dL | 48 (42–63) |
Urinary findings, n (%) | |
Hematuria | 33 (28) |
Proteinuria | 67 (56.8) |
Pyuria | 43 (36.4) |
Casts | 12 (10.2) |
Low complement, n = 105 | 86 (91.9) |
Antinuclear antibodies, n = 113 | 108 (95.6) |
Anti-ds-DNA antibodies, n = 84 | 65 (77.4) |
Antiphospholipid antibodies, n = 71 | 23 (32.4) |
Distribution of TNFSF15 rs4979462 in the groups being studied
The TNFSF15 rs4979462 variant was in Hardy–Weinberg equilibrium. The minor allele frequency was 0.10 in the patients’ group and 0.05 in the control group.
There were no significant differences in genotypes and allele distribution between SLE patients and healthy control groups (Table 2). However, the frequencies of the combined (CT+TT) genotypes and T-allele were significantly higher in female SLE patients than in female control subjects [19.6% vs. 8.5%, odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.1–6.3, p = 0.027; 10.8% vs. 4.3%, OR = 2.7, 95% CI = 1.2–6.3, p = 0.015, respectively; Table 3]. In contrast, the distribution of the TNFSF15 rs4979462 variant did not significantly differ in the male group.
Table 2Distribution of the TNFSF15 rs4979462 variant in the studied groups
Genotype | SLE, n = 118 | HC, n = 102 | p-value | OR (95% CI) |
---|
CC | 96 (81.4%) | 91 (89.2%) | 0.17 | |
CT | 20 (16.9%) | 11 (10.8%) | | |
TT | 2 (1.7%) | 0 (0.0%) | | |
CC | 96 (81.4%) | 91 (89.2%) | | 1.00 |
CT-TT | 22 (18.6%) | 11 (10.8%) | 0.10 | 1.9 (0.9–4.1) |
C (Major allele) | 212 (90%) | 193 (95%) | | 1 |
T (Minor allele) | 24 (10%) | 11 (5%) | 0.06 | 1.9 (0.9–4.2) |
Table 3TNFSF15 rs4979462 variant distribution in the female subjects
Genotype | SLE, n = 102 | HC, n = 94 | p-value | OR (95% CI) |
---|
CC | 82 (80.4%) | 86 (91.5%) | 0.060 | |
CT | 18 (17.6%) | 8 (8.5%) | | |
TT | 2 (2.0%) | 0 (0.0%) | | |
CC | 82 (80.4%) | 86 (91.5%) | | 1.00 |
CT-TT | 20 (19.6%) | 8 (8.5%) | 0.027 | 2.6 (1.1–6.3) |
C (Major allele) | 182 (89.2%) | 180 (95.7%) | | 1 |
T (Minor allele) | 22 (10.8%) | 8 (4.3%) | 0.015 | 2.7 (1.2–6.3) |
Association of the TNFSF15 rs4979462 variant with the clinical and laboratory characteristics of SLE
The frequency of the combined genotypes (CT + TT) of TNFSF15 rs4979462 was significantly higher in patients with SLE presenting with serositis and thrombotic manifestations (59.1% vs. 34.4%, OR = 2.8, 95% CI = 1.1–7.1, p = 0.032; 45.5% vs. 21.9%, OR = 2.9, 95% CI = 1.1–7.8, p = 0.023, respectively; Figs. 3 and 4). However, there was no significant association between TNFSF15 rs4979462 and other clinical phenotypes of SLE.
Regarding the laboratory characteristics of patients with SLE, there was no significant association between the TNFSF15 rs4979462 genotypes and any laboratory SLE finding.
TNFSF15 serum levels in the groups being studied
The median TNFSF15 serum levels were significantly higher in patients than in the healthy control subjects [11.4 (10.0–14.6) vs. 10.5 (9.6–12.2) ng/mL, p = 0.023] (Fig. 5).
The associations and correlation of TNFSF15 serum levels with the clinical phenotypes, disease activity, and laboratory features of SLE were further analyzed.
The TNFSF15 serum levels were significantly higher in patients with high disease activity who had SLEDAI scores of more than 20 than in those with lower activity [13.7 (10.3–15.2) vs. 10.8 (10.0–13.3) ng/mL, p = 0.03] (Fig. 6). In addition, there was a significant positive correlation between TNFSF15 serum levels and the total SLEDAI score of patients with SLE (r = 0.230, p = 0.012; Table 4).
Table 4Correlation of TNFSF15 serum levels with clinical and laboratory parameters of SLE
Characteristic | TNFSF15 concentration
|
---|
r | p-value |
---|
Age of onset in years | −0.045 | 0.626 |
ESR in mm/h | 0.014 | 0.888 |
WBC count in cell/mm3 | 0.139 | 0.140 |
Lymphocytes in cell/mm3 | 0.061 | 0.588 |
Hb in g/dL | −0.043 | 0.650 |
Platelet count as platelet count/mm3 | 0.175 | 0.063 |
ALT in IU/L | −0.008 | 0.935 |
AST in IU/L | −0.015 | 0.879 |
Albumin in g/dL | −0.169 | 0.164 |
Creatinine in mg/dL | 0.025 | 0.801 |
Urea in mg/dL | 0.088 | 0.413 |
Protein in urine in g/day | 0.066 | 0.544 |
TGs in mg/dL | 0.077 | 0.630 |
Cholesterol in mg/dL | 0.326 | 0.040 |
LDL in mg/dL | 0.348 | 0. 040 |
HDL in mg/dL | 0.095 | 0.618 |
TOTAL SLEDAI | 0.230 | 0.012 |
Renal SLEDAI | 0.228 | 0.013 |
The patients with SLE who presented with hematuria and urinary casts had higher TNFSF15 serum levels than those without such urinary abnormalities [14.0 (10.5–17.2) vs. 10.8 (10.0–13.6) ng/mL, p = 0.008; 14.6 (12.8–15.5) vs. 10.95 (10.0–14.2) ng/mL, p = 0.019, respectively] (Figs. 7 and 8). In the same context, the TNFSF15 serum levels were significantly correlated with the renal SLEDAI score of patients with SLE (r = 0.228, p = 0.013; Table 4).
Furthermore, there were significant positive correlations between TNFSF15 serum levels and both the total and LDL cholesterol serum levels (r = 0.326, p = 0.040; r = 0.348, p = 0.040, respectively; Table 4).
Discussion
SLE is an autoimmune disease with an affection of multiple organs, its etiology and pathogenesis remain unknown.19 The development of SLE was found to be influenced by multiple genetic factors.20 Single nucleotide variations have become a vital tool to detect disease susceptibility genes, clarifying the pathogenic mechanisms of SLE and discovering new therapeutic approaches.21
We studied the role of the TNFSF15 rs4979462 variant and the TNFSF15 cytokine in SLE in an Egyptian population.
There was no significant difference between SLE patients and healthy controls in the distribution of the TNFSF15 rs4979462 variant. However, when our study subjects were accordingly stratified by sex, combined genotypes (CT + TT) genotypes and T-allele were significantly higher in female patients with SLE than in female healthy subjects (19.6% vs. 8.5%, OR = 2.6, 95% CI = 1.1–6.3, p = 0.027; 10.8% vs. 4.3%, OR = 2.7, 95% CI = 1.2–6.3, p = 0.015, respectively). In contrast, there was no such significant difference in the male subjects of our study population. This suggested that the TNFSF15 rs4979462 variant is associated with a higher risk of SLE development in the female subjects of the Egyptian population.
In agreement with our results, Wang and Tu’s study in 2018 has reported a significant association between the combined (CT + TT) genotypes and T-allele of TNFSF15 rs4979462 variant and a higher risk of SLE in the Chinese population.14
In our study, the association between the TNFSF15 rs4979462 variant and the clinical phenotypes of SLE was further analyzed. There was a significant association between the T-variant and the development of serositis and thrombotic manifestations in SLE patients’ group (OR = 2.8, 95% CI = 1.1–7.1, p = 0.032; OR = 2.9, 95% CI = 1.1–7.8, p = 0.023, respectively). In Wang and Tu’s study,14 the authors found a significant association between the TNFSF15 rs4979462 variant and butterfly rash, serositis, renal nephritis, and arthritis.
In this study, the median TNFSF15 serum levels were significantly higher in patients with SLE than in the healthy control subjects [11.4 (10.0–14.6) vs. 10.5 (9.6–12.2) ng/mL, p = 0.023]. The same results were found by Xu et al. in studies conducted in 2015 and 2019 on the Chinese population.22,23 In the same context, Wang and Tu reported in their study a significantly higher level of TNFSF15 mRNA in the SLE group than in the healthy group.14
The association and correlation of TNFSF15 serum levels with the clinical phenotypes, disease activity, and laboratory features of SLE were further analyzed. There was a significant association between high TNFSF15 serum levels and renal impairment in our patients with SLE, whereas patients with hematuria and urinary casts had higher TNFSF15 serum levels [14.0 (10.5–17.2) vs. 10.8 (10.0–13.6) ng/mL, p = 0.008; 14.6 (12.8–15.5) vs. 10.9 (10.0–14.2) ng/mL, p = 0.019, respectively]. Moreover, there was a significantly positive correlation between TNFSF15 serum levels and the renal SLEDAI score (r = 0.228, p = 0.013).
In line with our findings, Al-Lamki et al. reported that TNFSF15 plays a significant role in renal tubular inflammation and injury.24
In this study, a significant association was found between TNFSF15 serum levels and SLE disease activity, where there was a significant positive correlation between TNFSF15 serum levels and the total SLEDAI scores of SLE patients (r = 0.230, p = 0.012). In addition, the TNFSF15 serum levels were significantly higher in patients with SLEDAI scores >20 than in those with lower SLEDAI scores [13.7 (10.3–15.2) vs. 10.8 (10.0–13.3) ng/mL, p = 0.03]. This comes hand in hand with the results of Xu et al. where the TNFSF15 serum levels were significantly higher in the newly diagnosed SLE patients with high disease activity and SLEDAI scores than in those with lower disease activity.22 In addition, they reported a significant positive correlation between TNFSF15 serum levels and the SLEDAI score of their SLE patients.
The interesting finding in our study was that a significant positive correlation between TNFSF15 serum levels and both the total cholesterol and LDL cholesterol serum levels was found (r = 0.326, p = 0.040; r = 0.348, p = 0.040, respectively). As mentioned previously, the T-variant of TNFSF15 rs4979462 was significantly associated with the thrombotic manifestations of our SLE patients (OR = 2.9, 95% CI = 1.1–7.8, p = 0.023). Recent studies proved that both arterial and venous thrombosis share the same risk factors, and dyslipidemia is presenting one of the most important.25–27 Accordingly, our results suggest that both the TNFSF15 protein and TNFSF15 rs4979462 variant play significant roles in developing dyslipidemia and thrombotic events. This could be through the effect of the rs4979462 variant on the TNFSF15 gene function and transcription level.
In harmony with our results, in a recent study by Della Bella et al. investigated the underlying pathogenesis of unprovoked venous thromboembolism (uVTE),28 the authors discovered the upregulation of TNFSF15 and its receptor TNFRSF25 (DR3) in the endothelial colony-forming cells isolated from the peripheral blood of patients with uVTE. In addition, the TNFSF15 levels were elevated in the sera of patients with uVTE. The authors further conducted functional analysis through blocking experiments; they proved that upregulation of the TNFSF15-TNFRSF25 axis impairs endothelial repair by minimizing the survival and proliferation of endothelial colony-forming cells and thus contributing to the pathogenesis of uVTE. According to the results of our study and that of Della Bella et al.,28 it was postulated that TNFSF15 and its gene variant rs4979462 are involved in the development of dyslipidemia, atherosclerosis, and thromboembolism. However, further studies are needed to elucidate the exact role of TNFSF15 and its gene variants in the pathogenesis of thromboembolism and to confirm the efficacy of the therapeutic use of recombinant anti-TNFSF15 in treating thrombotic disorders.
In the present study, the TNFSF15 serum levels did not significantly differ between the different TNFSF15 rs4979462 genotypes. In Wang and Tu’s study,14 the T-variant of TNFSF15 rs4979462 was associated with higher levels of TNFSF15 mRNA expression. We suggest that TNFSF15 rs4979462 increases the TNFSF15 gene expression; however, it seems that there are multiple factors in addition to gene transcription that affect the TNFSF15 serum protein levels, like binding to various cell receptors, leakage in urine, and distribution through the body cells and fluids. In addition, the presence of TNFSF15 gene variants other than rs4979462 could also affect the TNFSF15 transcription and expression levels. Consistent with our results and hypothesis, Hitomi et al. have found no significant difference in TNFSF15 serum levels concerning different TNFSF15 rs4979462 genotypes15; however, the authors reported that the T-variant of TNFSF15 rs4979462 was associated with significantly higher TNFSF15 mRNA expression levels.
TNFSF15, TL1A, is a transmembrane protein included in vascular and immune homeostasis. It is expressed by most immune cells as monocytes, dendritic cells, T cells, fibroblasts, macrophages, and endothelial cells.7,29,30 TNFSF15 interacts with two types of receptors: TNFRSF25 (DR3) and TNFRSF6B (DcR3). DcR3 lacks the transmembrane and cytoplasmic domains, so it inhibits the immunological function of TNFSF15 by competing with DR3 for binding to it. The presence of DR3 and DcR3 is essential for maintaining the natural balance of the physiological functions of TNFSF15.6,31–33.
The binding of TNFSF15 to TNFRSF25 (DR3) is essential to perform its immunological function as TNFSF15-TNFRSF25 (TL1A-DR3) enhances the production of stimulatory signals and the recruitment of adapter proteins and stimulates the production of inflammatory cytokines.6,29 DR3 is extensively expressed by T helper (Th) 17 cells; TNFSF15-TNFRSF25 stimulates the activation and proliferation of Th17 cells and enhances the production of Th17 cytokines like interleukin (IL)-17 and IL-21.34–37
Xu et al. reported that TNFSF15 serum levels are significantly correlated with those of IL-17 and IL-21, providing proof that TNFSF15 controls the immune response and contributes significantly to the pathogenesis of autoimmune diseases through the regulation of Th17 cells and their cytokine production.36 Th17/IL-17 dysregulation causes neutrophil accumulation and autoantibody production, thus increasing the risk of SLE development.38
TNFSF15 is one of the worthful cytokines that could be a therapeutic target for autoimmune disease. According to Xu et al.,36 TNFSF15 serum levels were comparable to those of the healthy control participants and considerably lower in anti-TNF-treated patients than in anti-TNF-naive patients.
TNFSF15 gene variants have been involved in many autoimmune disorders such as primary biliary cirrhosis,39 rheumatoid arthritis,40 Crohn’s disease,11 and Graves’ disease.41 Hitomi et al. used in vitro functional analysis to detect the causal variants of the TNFSF15 gene and the molecular mechanisms of TNFSF15 responsible for the development of primary biliary cirrhosis in the Japanese population.15 They found that the TNFSF15 rs4979462 risk allele variant generates a novel nuclear factor-1 (NF-1) binding site, which increased the expression levels of TNFSF15 because of the binding of the transcription factor NF-1 to the novel binding site. Increased TNFSF15 expression resulted in hyperactivation and proliferation of the Th17 cells with excessive production of the inflammatory cytokines and, finally, the development of autoimmune diseases.
The findings of our work and those of prior research studies suggest that TNFSF15 rs4979462 changes the expression and the effector function of the TNFSF15 gene and hence modulates the natural balance of Th17 effector cells and their related cytokines, IL-17 and IL-21, hence causing the dysregulation of the natural immune response and provide a key for developing SLE.
To our knowledge, only one study has investigated the role of the TNFSF15 rs4979462 variant in SLE (in the Chinese population). Our study provides a step to improving the understanding of the pathogenesis and the underlying molecular mechanisms implicated in developing SLE in the Egyptian population. Larger-scale studies of various ethnicities are required to understand the exact role of TNFSF15 and its genetic variants in developing autoimmune diseases, especially SLE.