Abstract
Non-segmental vitiligo (NSV) is an autoimmune disorder due to the destruction of melanocytes, where cytokines like interleukin 17 (IL-17) and biomolecules like vitamin D play a theoretical role in pathogenesis. Previous studies in this regard yielded inconsistent results. This study aimed to compare the serum levels of IL-17 and vitamin D between NSV patients and healthy controls and to know the association of these biomarkers with disease activity and extent. This was a case-control study including adult patients with NSV and age and gender-matched healthy controls. Cases and controls with conditions likely to alter the serum levels of IL-17 and vitamin D were excluded. Serum levels of IL-17 were estimated by ELISA and vitamin D levels by chemiluminescence assay. 42 adult patients of NSV and 42 age and sex-matched healthy controls were recruited over a period of eighteen months. The mean value of serum vitamin D levels in the control group was 19.053 ± 5.340 ng/ml, whereas in the case group, it was 17.336 ± 6.931 ng/ml (P > 0.05). The mean value of serum IL-17 levels in the control group was 199.824 ± 51.244 pg/ml and 213.566 ± 69.018 pg/ml in the case group (P > 0.05). These molecules did not show any association with the disease activity and extent. In contrast to the previous studies, we could not establish the role of IL-17 in the pathogenesis of vitiligo. Furthermore, we could not find any association between vitamin D and vitiligo in our study, even though there is an inconsistent association between the two in the available literature.
KEY WORDS: Interleukin 17, non-segmental vitiligo, vitamin D
Introduction
Among various theories of vitiligo pathogenesis, the autoimmune hypothesis has gained wide attention where cytokines help in intercellular signalling and interactions.[1,2] Interleukin-17 (IL-17) produced by CD4 T helper-17 (TH17) cells is proinflammatory, increasing oxidative stress and reducing the functional capability of melanocytes.[3] Vitamin D3 compounds are known for their effects on immunomodulation and upregulation of melanogenesis.[4] There are inflammatory cytokines like IL-17, IL-2, IL-6, IL-8, tumour necrosis factor alpha, and interferon-gamma involved in vitiligo depigmentation whose expression is influenced by vitamin D ligands.[4,5]
Many studies have assessed the role of these biomolecules in non-segmental vitiligo (NSV). IL-17 is significantly elevated in active and widespread NSV in most of the studies. However, there is a considerable paucity of Indian data. Whether a deficit in vitamin D can induce NSV as it does in other autoimmune diseases is not clear. Moreover, no Indian studies could assess a possible regulatory effect of vitamin D on IL-17 in NSV patients. Therefore, this study was conducted to assess the serum levels of IL-17 and vitamin D in NSV patients and to study their association with the extent and activity of vitiligo.
Materials and Methods
This was a case-control study including adult patients of NSV presenting to the Department of Dermatology and age and gender-matched healthy controls over a period of one and a half years after obtaining approval from the institutional ethical committee [ECR/714/Inst/CT/2015/RR-18]. NSV is a clinical subtype of vitiligo that includes acrofacial, mucosal, generalized and universal vitiligo. Patients with a history of receiving systemic therapy for vitiligo in the past two months, phototherapy in the last three months, vitamin D supplements in the last year, severe anaemia, other autoimmune diseases, and pregnant and lactating women were excluded from the study.
Detailed history, clinical examination and relevant investigations were recorded in a preset proforma. Vitiligo disease activity score (VIDA score) and Vitiligo area severity index (VASI) score were calculated. Controls were subjected to clinical examination to rule out NSV. 5 ml venous blood samples were collected in a plain vial from both cases and controls, and serum was separated and stored at −80°C until the time of estimation. Serum levels of IL-17 were estimated by ELISA using commercial kits and serum vitamin D levels were measured using an automated chemiluminescence assay system.
A commercial kit developed for the quantitative measurement of human IL-17 in serum manufactured by Sincere Biotech/Diaclone was used for IL-17 estimation. The ELISA kit estimated IL-17 levels using an antibody specific to human IL-17 coated on wells. Samples were placed into the wells, and IL-17 present in the sample was made bound to the wells by the immobilised antibody. After washing the wells, the biotinylated antihuman IL-17 antibody was added, followed by horseradish-conjugated streptavidin. The wells were again washed, tetramethylbenzidine (TMB) substrate solution was added to the wells, and a yellow colour was developed in proportion to the amount of IL-17 bound. The reaction was terminated by the addition of sulphuric acid solution and the intensity of the colour was measured at 450 nm. Serum 25 hydroxy vitamin D levels were detected by chemiluminescence assay (ADVIA Centaur Vitamin D Assay) using an antifluorescein monoclonal mouse antibody covalently bound to paramagnetic particles, a 25 hydroxy vitamin D monoclonal antibody labelled with acridinium ester and a vitamin D ester labelled with fluorescein. The inverse relationship between the amount of vitamin D present in the patient sample and the number of relative light units detected by the system was utilised for the quantification of 25 hydroxy vitamin D.
The cases were treated as per standard treatment guidelines. The data were entered in Excel sheets and analysed using case-control study statistics, inferential statistics and statistical software SPSS (statistical program for social sciences). The continuous variables were presented as mean standard deviation or median. The results were analysed statistically and were carried out in a 95% of confidence interval with P value < 0.05 considered as significant.
Results
Forty-two adult patients of NSV were recruited as cases and 42 age and sex-matched healthy volunteers as controls. The mean age of the study population was 39.10 ± 12.323 years [Table 1] with 47.62% females and 52.38% males in each group. The mean age of disease onset was 28.79 ± 14.222 years. There was no significant correlation between serum levels of IL-17 or vitamin D levels with age of onset. There was a significant inverse correlation between vitamin D levels and disease duration (P = 0.043) [Figure 1]. Based on the VIDA score, 38.1% of cases (n = 16) had stable vitiligo and 61.9% (n = 26) had unstable vitiligo. There was no statistically significant correlation between serum levels of IL-17 or vitamin D levels with the VIDA score. Around 21.4% (n = 9) cases had a family history of vitiligo of which 55.5% had their first-degree relative with vitiligo. Mean body surface area involvement was 19.9252 ± 33.009% and the mean VASI score was 1372.9074 ± 2983.199. There was no statistically significant correlation between serum levels of IL-17 or vitamin D levels with VASI or the extent of vitiligo. Out of 42 cases, 42.9% (n = 18) had leucotrichia, 11.9% (n = 5) had koebnerisation, 9.5% (n = 4) had trichrome vitiligo, 2.4% (n = 1) had inflammatory vitiligo lesions and 7.1% (n = 3) had confetti-like lesions [Figure 2]. Patients with confetti-like vitiligo lesions had statistically significant lower levels of vitamin D (P = 0.015). The mean value of serum vitamin D levels in the control group was 19.053 ± 5.340 ng/ml, whereas in the case group was 17.336 ± 6.931 ng/ml (P > 0.05) [Table 1]. The vitamin D levels in cases and controls have been categorised into sufficient, insufficient, deficient and severely deficient and have been shown in Figure 3. The mean value of serum IL-17 levels in the control group was 199.824 ± 51.244 pg/ml, whereas in the case group was 213.566 ± 69.018 pg/ml (P > 0.05). There was no significant difference in the serum levels of IL-17 and vitamin D levels between cases and controls. There was no significant correlation between serum levels of IL-17 and vitamin D levels among cases and controls [Figure 4].
Table 1.
Demographic features and biochemical parameters of cases and controls
| Cases (n=42) | Controls (n=42) | |
|---|---|---|
| Mean age | 39.10±12.323 years | 39.10±12.323 years |
| Gender distribution | Males: 52.38% | Males: 52.38% |
| Females: 47.62% | Females: 47.62% | |
| Mean age of disease onset | 28.79±14.222 years | |
| Vitiligo activity | ||
| Stable | 38.1% (n=16) | |
| Unstable | 61.9% (n=26) | |
| Family history of vitiligo | 21.4% (n=9) | |
| Mean VASI score | 1372.9074±2983.199 | |
| Pattern of vitiligo | ||
| Localised | 26.2% (n=11) | |
| Generalised | 73.8% (n=31) | |
| Poor prognostic factors | Leucotrichia: 42.9% (n=18) Koebnerisation: 11.9% (n=5) Trichrome vitiligo: 9.5% (n=4) Inflammatory lesions: 2.4% (n=1) Confetti-like lesions: 7.1% (n=3) | |
| Mean serum IL-17 levels | 213.566±69.018 pg/ml | 199.824±51.244 pg/ml |
| Mean serum vitamin D levels | 17.336±6.931 ng/ml | 19.053±5.340 ng/ml |
Figure 1.
Scatter diagram showing an inverse correlation between vitamin D and duration of vitiligo
Figure 2.
Confetti like lesions of vitiligo in a female patient
Figure 3.
Horizontal bar diagram showing deficient, insufficient and sufficient vitamin D levels in cases and controls
Figure 4.
Scatter diagram showing inter-correlation between serum interleukin 17 levels and serum vitamin D levels in cases
Discussion
Among the different theories proposed for vitiligo pathogenesis, the autoimmunity hypothesis has been studied extensively and has been established as a reliable theory for NSV pathogenesis. The role of IL-17 in NSV has been studied in greater depth based on this assumption as IL-17 produced by Th 17 lymphocytes is believed to mediate autoimmune diseases. Our study could not find any correlation of IL-17 levels with age and gender consistent with the studies conducted by Aly et al.[6] and Metwalli et al.[7]
Since we could not obtain significantly elevated serum levels of IL-17 in vitiligo cases in comparison to controls, the role of IL-17 in disease pathogenesis is being questioned. Our findings are in contrast with most of the previous studies,[8,9,10,11,12,13,14] which have reported significantly higher levels of IL-17 in the patient group than the controls. Sushama et al.[1] observed significantly raised levels of IL-17 in generalised vitiligo. In a recent study by Karagaiah and Budamakuntla,[8] serum IL-17 levels were reported significantly higher in the patient group than in the controls, however, there was no correlation with the disease extent or activity. There are only a few studies that support our findings. Boniface et al.[15] suggested that there is no comparable difference between the levels of IL-17 secreted by vitiligo skin T cells compared to healthy skin and the levels are significantly lower than psoriatic skin T cells suggesting IL-17 may not cause melanocyte disturbance in vitiligo.
Our study could not reveal any correlation of IL-17 levels with disease duration and the results were consistent with studies conducted by Aly et al.[6] and Metwalli et al.[7] However, the study conducted by Bassiouny and Shaker showed a significant positive correlation between serum IL-17 and disease duration (r = 0.42, P = 0.02).[12]
There was no correlation noticed between IL-17 levels and disease activity and these findings were similar to the studies conducted by Aly et al.,[6] Karagaiah and Budamakuntla,[8] Habeb et al.,[10] Bhardwaj et al.[16] and Esmaeili et al.[17] Le et al.[18] found significantly higher IL-17 levels in progressive vitiligo patients. Elela et al.[14] also showed a positive correlation of IL-17A levels with VIDA scores in vitiligo patients.
There was no correlation of IL-17 levels with body surface area involvement or VASI score of vitiligo in our patients consistent with the studies conducted by Sushama et al.,[1] Aly et al.,[6] Basak et al.,[13] Elela et al.,[14] Bhardwaj et al.[16] and Le et al.[18]
Our study could not find any correlation between IL-17 levels with poor prognostic factors. Le et al.[18] has found significantly higher levels of IL-17 in vitiligo patients with leucotrichia.
We did not find any correlation of vitamin D levels with age and gender similar to the results from studies conducted by Aly et al.[6] and Metwalli et al.[7] Our study could not prove any significant differences in the serum levels of vitamin D between cases and controls. Few earlier studies have reported vitamin D deficiency in vitiligo patients as compared to controls.[19,20] The non-association of vitiligo with vitamin D observed by us was similar to the results of a recent study by Karagaiah and Budamakuntla.[8] Even though the small sample size of our study population could have attributed to this, the high frequency of vitamin D insufficiency in India might also explain this finding. Vitamin D levels are universally low in several studies undertaken in diverse geographical locations across the country, across age groups, and sociodemographic strata. This appears counterintuitive at first, given increased exposure to sunlight, as India is a tropical country. It could be explained by the fact that Indian skin of Fitzpatrick phototype IV and V has more melanin content, which acts as a natural sunscreen, as well as considering nutritional factors such as the large proportion of individuals following a vegetarian diet in our study population that is a poor source of vitamin D. In addition, vitamin D deficiency levels are standardised based on western norms. When determining vitamin D levels, ethnicity is usually not taken into account, as should be the case. To be precise, considering the above-mentioned factors, we cannot completely rule out the association of vitamin D levels and vitiligo based on our results.
Our study found a significant inverse correlation between serum vitamin D levels and disease duration. The patients having a longer duration of vitiligo had relatively lower vitamin D levels. The results from the study conducted by Khurrum et al.[5] showed similar findings. It is difficult to infer whether low vitamin D levels are a cause or outcome of prolonged disease duration. One hypothesis could be the existence of vitiliginous skin for a prolonged time resulting in reduced overall vitamin D production as melanised skin plays an important role in vitamin D synthesis in our body. However, it also implies the significance of testing for vitamin D levels in patients with chronic vitiligo.
Our study could not find any correlation between vitamin D levels with disease activity and these conclusions were consistent with studies conducted by Aly et al.,[6] Xu et al.,[21] and Ustun et al.[22] Also, our study could not find any correlation between vitamin D levels and with distribution and extent of vitiligo and these results were consistent with studies conducted by Khurrum et al.[5]
To the best of our knowledge, no prior studies have assessed the correlation between vitamin D levels and poor prognostic factors in vitiligo. Our study found a significant inverse correlation of vitamin D levels with the presence of confetti-like lesions. More research is needed in this regard to establish vitamin D deficiency as a risk factor or poor prognostic factor since mean levels of vitamin D did not show any significant variation between cases and controls. As of now, it can be inferred that the estimation and indicated correction of serum vitamin D levels in patients having confetti-like vitiligo lesions cases could help in managing unstable cases.
Only two previous studies[6,7] have examined the inter-correlation between vitamin D and IL-17 in vitiligo and no Indian studies have assessed such a link so far. This study could not prove vitamin D as a hormone influencing IL-17 cytokine expression. This is similar to the findings reported by Aly et al.[6] and Karagiah and Budamakuntla et al.[8] Metwalli et al.[7] reported inverse inter-correlation between serum IL-17 levels and serum vitamin D levels in vitiligo patients.
Limitation
Our study was limited by the small sample size and lack of tissue-level estimation of IL-17.
Conclusion
The present study could not prove the role of IL-17 in the pathogenesis of NSV as opposed to previous findings. The role of vitamin D supplementation in the management of vitiligo also remains doubtful based on our findings. These were not found to be reliable markers for vitiligo activity or extent either. We found a significant inverse correlation between vitamin D levels with disease duration and the presence of confetti-like lesions. Further studies from India with a larger group of patients and tissue-level measurements are required to support our findings.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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