Plasma Levels of Interleukin-17, Interleukin-23, and Transforming Growth Factor-β in Sudanese Patients with Vitiligo: A Case-Control Study.

BACKGROUND: Vitiligo is the most common pigmentary skin disorder. It is a multifactorial polygenic disease with epidermal melanocyte destruction. The cytokines profile found in vitiliginous patients was not fully elucidated. AIMS: We sought to assess the autoimmune nature of vitiligo by comparing plasma levels of interleukin (IL)-17, IL-23, and transforming growth factor beta (TGF-β) in adult Sudanese vitiligo patients with matched control individuals. SUBJECTS AND METHODS: Case-control study was conducted in Khartoum Dermatologic Teaching Hospital, in the period between July and December 2013. The cases were 42 adult Sudanese vitiligo patients matched with 43 control individuals. The cytokines were measured in the plasma by the quantitative "sandwich" ELISA.
RESULTS: Patients showed a significant lower median (25-75(th) inter-quartile) of TGF-β than control (0.042 [0.041-0.044] vs. 0.047 [0.042-0.049]; P ≤ 0.001). Both IL-17 and IL-23 showed no significant difference between cases and controls. IL-17 showed a significant inverse relationship when correlated with TGF-β (r = -0.24; P = 0.026) while showing direct relationship when correlated with age (r = 0.28; P = 0.009).
CONCLUSION: The positive findings detected in this study coincide with the important immunoregulatory role of the TGF-β, and support the autoimmune nature of the disease.

What was known?

The autoimmune nature of vitiligo is well-established, but the full-blown profile of the associated cytokines was not fully elucidated.

Introduction

Vitiligo is an acquired pigmentary disorder of unknown etiology. Etiologically, there is a loss of epidermal melanocytes, and clinically, there is the development of white macules related to the selective loss of melanocytes. Worldwide prevalence of the disease ranges from <0.1% to >8% in some areas, like India.[1] It is slightly more common in women. Half of all patients develop the disease before 20 years of age. Generalized vitiligo is the most common clinical presentation and often involves the face and acral regions. The course of the disease is unpredictable, and the response to treatment varies. Depigmentation may be the source of severe psychological distress, diminished quality of life, and increased risk of psychiatric morbidity.[2]

The autoimmune hypothesis of vitiligo proposes that an immune system disorder results in the destruction of melanocytes. It is first supported by the frequent observation that several autoimmune disorders are associated with vitiligo, particularly thyroid dysfunction, and/or thyroid antibodies.[3]

It is likely that both humoral and cellular immunity co-operate in the destruction of melanocytes. Concerning cellular immunity, an important role has been given to the infiltrate underlying the depigmented lesional skin, where CD4 and CD8 positive T-cells were detected, also expressing activation molecules.[4] A substantial number of infiltrating T-cells express the cutaneous lymphocyte antigen (CLA) typical of skin homing T-cells,[4] and a recent study localized CLA positive cytotoxic T-cells in apposition to disappearing melanocytes in the peri-lesional skin.[5] In vitiligo patients, high frequencies of Melan-A/Mart1 (a melanosomal antigen) specific CD8 positive T-cells were detected in peripheral blood.[6] Interestingly, these T-lymphocytes were identified in inflammatory lesions of melanocyte destruction following the infusion of Melan-A/Mart1 specific CD8 positive T-cell clones in melanoma patients. This finding gives direct evidence of T-cell-mediated vitiligo.[7]

Interleukin-17A (IL) is a secreted protein produced by T-helper (Th) 17 cells, which form a distinct subset of the CD4 + T-cell lineage. It stimulates the production of IL-1 β, TNF-α, and IL-6.[89] In the past decade, Th17 cells have been identified in autoimmune skin inflammatory disorders such as psoriasis and atopic dermatitis.[1011]

IL-23 is a cytokine secreted by activated dendritic and phagocytic cells and by keratinocytes. It is also expressed by dermal Langerhans cells. It induces autoimmune inflammation and thus may be responsible for autoimmune inflammatory diseases.[12]

Transforming growth factor beta (TGF-β) is a multifunctional protein that controls proliferation, differentiation and other functions in many cell types. It is produced by the T-regulatory CD4 + cell subset (Treg), wherein they play a pivotal role in the control of T-cell homeostasis by suppressing the proliferation of effector T-cells, thus maintaining self-tolerance.[13] TGF-β has been reported to play a role in the suppressive function of T-regulatories (Tregs).[14] It was suspected that dysregulation of Tregs might be one of the factors that can break tolerance to melanocyte self-antigens and contribute to the pathogenesis of vitiligo.[15]

Subjects and Methods

Forty-two adult vitiligo patients and 43 age and sex-matched control individual, free of the disease, were selected by simple random selection from the out-patient clinic of the Khartoum Dermatologic Teaching Hospital, in the period between July and December 2013. Depigmentation in the selected patients was not due to chemicals, burns or any other traumatic cause. The patients also were free of any concomitant autoimmune disease such as diabetes and thyroiditis. The diagnosis of vitiligo was made by experienced dermatologists, and it was on clinical base. A brief history and a thorough clinical examination with vitiligo type determination were done for every vitiligo patient.

After the selection, a venous blood sample of 5 ml was collected in ethylenediaminetetra-acetic acid containing tubes from every participant in both groups. The sample was centrifuged at 3000 rpm and the plasma separated within <1 h from sample collection and then stored at –80°C until ELISA investigations.

All lab investigations were done in the molecular biology lab in the “Institute of Endemic Diseases,” University of Khartoum/Sudan. The 80 stored plasma samples were allowed to reach room temperature (20–25°C) once before analysis, to avoid repeated melting/freeze cycles. The quantitative “sandwich” (indirect) ELISA method was performed to measure the cytokines’ levels in both groups. The WKEA company kits were used. These kits can recognize all cytokines’ isoforms.

Ethics

This study received ethical clearance from Khartoum University Faculty of Medicine Research Ethics Review Board. All participants provided signed informed consent.

Statistical analysis

All data were entered to a computer using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL, USA) and double-checked before analysis. Abnormally distributed data were expressed as median (25–75th inter-quartile), and the median of cases and control were compared by using Mann–Whitney U-test. Spearman correlation test was used to detect any correlation between the cytokines and patients age and disease duration. Tests were considered to be significant when P < 0.05.

Results

The male:female ratio in both studied groups was nearly similar, equaling 1:3.5. Mean (standard deviation) age of patients was about 42 (5) years, while that of control individuals was about 41 (8) years. The most common vitiligo type detected in the patients was the generalized type (68.5%) while the least common one was the localized type (5.5%). The acrofacial and peripheral types were nearly equal (14.8% and 11.2%, respectively). The mean duration of the disease in the patient group was found to be 12 years.

The median (25–75th inter-quartile) of the TGF-β were significantly lower in cases than control (0.042 [0.041–0.0044] vs. 0.047 [0.042–0.049]; pgm/ml P ≤ 0.01). Plasma IL-17 and IL-23 were lower in patients (41.5 [35.8–47.2] vs. 45.8 [37.2–64.4] pgm/ml; P = 0.09; 0.044 [0.044–0.046] vs. 0.045 [0.045–0.047] pgm/ml; P = 0.19) than in control, but statistically not significant, respectively [Table 1]. When the patients’ cytokines levels were correlated with the ages and disease duration, significant direct correlation has only been detected between IL-17 and the patients’ ages, (r = 0.28; P = 0.009). The inter-correlation between the patients’ cytokines revealed a significant inverse correlation only between TGF-β and IL-17 (r = −0.24; P = 0.026) [Table 2 and Figure 1].

Table 1

IL-17, IL-23, and TGF-β levels in plasma of Sudanese vitiligo patients and control individual

Table 2

Correlation between various clinical and biochemical measures including cytokines IL-17, IL-23, and TGF-β of Sudanese patients with vitiligo, KDH, 2012

Figure 1

Correlation between levels of transforming growth factor beta and interleukin-17 in plasma of Sudanese vitiligo patients

Discussion

Th17 cells and Tregs exist in a counter-balance, so in the absence of Tregs, unopposed Th17 cells activity amplifies inflammatory cascades initiated by glycoprotein I/anti-glycoprotein I immune complexes in involved skin of patients with vitiligo.[16] Therefore, IL-17 imbalance may suggest its involvement in the pathogenesis of vitiligo and supports the hypothesis that skewing of the immune system toward Th1 or Th17 and away from Tregs and Th2 may confirm the autoimmune nature of vitiligo.

No significant difference in IL-17 levels in plasma of vitiligo patients and control group has been depicted in this study. A reverse result was detected by Khan et al., in India when they found that IL-17 levels in the patients were significantly higher than in the control subjects.[16] Esmaeili et al. in Iran investigated the gene expression of IL-17 and found no significant differences in IL-17 gene expression in lymphocytes of the vitiligo patients compared with the control.[17] Two separate studies enrolled in Egypt confirmed the significant association between IL-17 levels and vitiligo.[1819] Moreover, the first study had noticed a significant positive correlations between the disease duration and IL-17 levels.[18] Basak et al., on the other hand, found no significant difference between the patient and control groups in mean levels of IL-17, despite the positive correlation of IL-17 with the size of the affected skin area.[20] By exploring the IL-17 assaying results in the vitiliginous patients in this study and in the above comparative ones, it can be so easily deduced that the role of the Th17 and regulatory T-cells in the pathogenesis of vitiligo was not yet clear cut. More studies, therefore, in this aspect of the pathogenesis of vitiligo are definitely required.

No significant difference in IL-23 levels has been detected in this study between vitiligo patients and control group. The levels of this cytokine were not so extensively studied in vitiligo patients worldwide. Wang et al. reported significant higher IL-23 levels in the patients than in the healthy controls.[21] Again, more studies are needed to relate IL-23 with the pathogenesis of vitiligo.

The low levels of TGF-β in vitiligo patients can be used as a marker of Tregs dysregulation, and hence support the autoimmune hypothesis of the disease. In this study, the kit used for detection of the TGF-β is nonspecific and can recognize all the cytokine's isoforms. TGF-β has been found to be significantly lower in the vitiligo patients compared with the control group. This finding was strengthened by Tu et al., who found that the TGF-β1 levels were significantly lower in the active vitiligo group when compared with the control group. They concluded that TGF-β might play a role in the pathogenesis of nonsegmental vitiligo related to the suppressive function of Tregs.[22] Basak et al. reported that plasma TGF-β1 levels were significantly decreased in patients with vitiligo and consequently concluded that this might facilitate the occurrence of vitiligo.[20] El-Komy, et al. in Egypt examined the degree of expression of TGF-β1 in serum and tissue of 20 vitiligo patients and 10 matched control individuals, and they related this expression with the disease development, progression, and severity. They found that serum and tissue levels of TGF-β1 were significantly lower in patients than the controls (P = 0.001 and P < 0.001, respectively). However, they detected no significant difference between the lesional and nonlesional TGF-β1 levels in the skin of patients (P = 0.634). In addition, they did not find any significant relationship between serum and tissue TGF-β1 levels on one hand, and the age, sex, age of onset, disease duration, disease activity, family history, concomitant autoimmune disease, clinical disease subtypes, or extent of skin affection on the other hand.[23] Yun et al. worked differently, that is, at the genes level, precisely on TGF-β receptor II (TGFBR2) polymorphisms and their association with nonsegmental vitiligo in the Korean population. They found that all of the single nucleotide polymorphisms were significantly different between the vitiligo patients and controls. As a result, they stated that TGFBR2 might be related to nonsegmental vitiligo via its ligand TGF-β. In brief, the TGF-β levels in vitiligo patients found in this study hardening the role of T regulatory CD4+ (Treg) cells in the pathogenesis of vitiligo, particularly the autoimmune hypothesis of the disease.

Inter-correlation between levels of TGF-β and IL-17 in plasma of Sudanese vitiligo patients in this study revealed a significant reverse relationship, despite the insignificant difference in IL-17 levels between patients and controls. This TGF-β IL-17 reverse relationship coincides with the expected roles of these cytokines in vitiligo, since the action of Th17 cells and Tregs is counterbalanced, as can be deduced from the above reviewed studies. Accordingly, this finding supports more the autoimmune hypothesis of the disease. However, a single study contradicting our findings was enrolled by Zhou et al. in USA.[24] They described a positive relationship between IL-17A and TGF-β1 in sera of patients with nonsegmental vitiligo when compared to a control group. This contradiction may be partially related to the specific isoforms they worked on, though, their solitary study needs to be supported by more studies.

What is new?

The crucial immunoregulatory role of the transforming growth factor beta in the pathogenesis of vitiligo had been strengthened, and can be targeted in the treatment of this disease.