Increased Circulatory Interleukin-17A Levels in Patients with Progressive and Leukotrichial Vitiligo

Background Vitiligo is a chronic condition characterized by skin depigmentation. Although not life-threatening, it significantly impacts quality of life. The pathophysiology of vitiligo remains poorly understood, and treatment options are limited. Mounting evidence supports the importance of autoreactive T cells and, particularly interleukin-17A- (IL-17A-) secreting Th17 cells, in vitiligo. IL-17A targeting has been proven successful in various inflammatory dermatological conditions, including psoriasis and lupus erythematosus. Objective We evaluated the relationship between serum levels of IL-17A and the clinicopathological characteristics of Vietnamese vitiligo patients. Methods In this cross-sectional study, we analyzed data from 52 nonsegmental vitiligo patients and 50 age- and sex-matched healthy individuals. Serum levels of IL-17A were measured using an enzyme-linked immunosorbent assay. We evaluated the correlation between IL-17A levels and clinical characteristics including leukotrichia, disease duration, vitiligo activity, and body surface area involvement. Results Patients with progressive vitiligo had significantly higher IL-17A levels than patients with stable vitiligo (P = 0.014) or healthy individuals (P = 0.002). In addition, serum IL-17A levels were higher in vitiligo patients with leukotrichia than in patients without it (P = 0.04). Furthermore, serum IL-17A levels were negatively correlated with age (r = −0.39, P = 0.004) and age of onset (r = −0.33, P = 0.016) in vitiligo patients. Conclusions Higher serum levels of IL-17A in patients with progressive vitiligo and leukotrichia suggest a potential role of IL-17A in melanocyte destruction in the epidermis and the follicular matrix.


Introduction
Vitiligo is characterized by the progressive destruction of pigmentary cells in the epidermis and the hair follicles, which leads to white macules that are challenging to cure. e disease has an estimated prevalence of 0.1-2% of the world's population [1][2][3]. Although not life-threatening, vitiligo significantly impacts patients' quality of life, particularly those with facial lesions. e precise etiology of vitiligo remains unclear although (a) biochemical [4], (b) neural [5], and (c) autoimmune [6,7] mechanisms seem to play a role in its onset. Notably, the immune-related mechanisms underlying nonsegmental vitiligo have been comprehensively investigated.
Changes in CD4+ T cell functions and the presence of autoreactive melanocyte-specific cytotoxic T cells play important roles in vitiligo pathogenesis [8,9]. Importantly, the role of the CD8-CXCR3-CXCL9/10-IFNc axis in the pathogenesis of nonsegmental vitiligo is becoming increasingly evident [10,11]; however, targeting this pathway may lead to long-term adverse effects, such as skin cancer, as it plays a vital role in maintaining the cutaneous immunity [12]. On the contrary, T helper 17 ( 17) cells exert their biological functions by producing interleukin (IL)-17, which may exacerbate autoimmune inflammation in vitiligo. IL-17 also stimulates keratinocytes to produce several chemokines, resulting in recruitment of T cell, neutrophil, macrophage [13], and potential loss of melanocytes [14]. IL-17 is a potent stimulator of chemokine CCL20 production, which drives the migration of cytotoxic CD8+ T cells from systemic circulation into peripheral tissues [15,16]. In mouse models, the migration of CD8+ T cells to the skin results in melanocyte loss [17,18]. Additionally, IL-17 stimulates endothelial cells to express E-and P-selectins and the adhesion molecules ICAM-1 and VCAM-1, resulting in increased neutrophil migration [19].
Nevertheless, the role of IL-17A in vitiligo remains poorly understood. In this cross-sectional study, we evaluated the relationship between serum levels of IL-17A and the clinicopathological characteristics of Vietnamese individuals with nonsegmental vitiligo.

Study Subjects.
We recruited 52 nonsegmental vitiligo patients (Group 1), including patients with focal acrafacial, mucosal, generalized, and universal vitiligo. Subjects who were treated with systemic or topical corticosteroids, or who received any other systemic immunosuppressive therapy in the preceding 2 months of recruitment, were excluded. Pregnant/lactating patients, or patients with a history of Hashimoto thyroiditis, Graves' disease, insulin-dependent diabetes, Addison's disease, alopecia areata, psoriasis, rheumatoid arthritis, or dysfunctional thyroid, were also excluded. We also enrolled 50 age-and sex-matched healthy individuals (Group 2) who did not have any criteria above as well as acute or chronic diseases. In Group 1, we recorded age, sex, duration of the disease, age of onset, family history, Koebner phenomenon, clinical features of the lesions, body area involvement (in quartile percentiles), and disease activity by using the vitiligo disease activity (VIDA) score [20]. e study was approved by IRB of University of Medicine and Pharmacy at Ho Chi Minh City (IRB number: 424/DHYD-HDDD).

Measurement of Serum IL-17A Levels by ELISA.
Serum samples were collected from study subjects. Serum IL-17A levels were measured using an ELISA with a Human IL-17A ELISA Kit (ANOGEN Inc, Ontario, Canada) following the manufacturer's instructions.

Statistical Analysis.
Data are expressed as mean-± standard deviation (SD), median and range, or prevalence.
Mean values were compared using Mann-Whitney U (for two groups) or the Kruskal-Wallis test (for three or more groups). Spearman's rank correlation coefficient was used to investigate the relationship between clinical parameters. P values less than 0.05 were considered statistically significant. Statistical analyses were performed using R packages (RStudio Desktop, version 1.2 for Windows, RStudio Inc., Boston, USA).
In the subgroup analysis of active vitiligo, we also could not find the association between affected body surface area and the concentration of IL-17A (r � −0.15, P � 0.38). Furthermore, serum IL-17A levels were negatively correlated with age (r � −0.39, P � 0.004) and age of onset (r � −0.33, P � 0.016) in vitiligo patients but not in healthy controls (Table 3 and Figure 2).

Discussion
e pathophysiology of vitiligo remains understudied. Changes in humoral and cellular immunity are considered important causes of melanocyte destruction and subsequent vitiligo. Consistently, vitiligo is associated with autoimmune endocrinopathies [2]. Furthermore, the presence of autoantibodies against melanocyte antigens, including tyrosinase and tyrosinase-related protein (TYRP) 1 and 2, in the sera of vitiligo patients suggests an important role of humoral immunity in vitiligo pathophysiology [21]. In addition, high numbers of T-cell in the lesional margins in inflammatory vitiligo indicate the role of cellular immunity in vitiligo [22]. Besides the 1 response and subsequent TNF-α and IFN-c production, 17 cells and L-17A have recently been implicated in the pathogenesis of vitiligo. IL-17 A targets multiple cell types, including fibroblasts, endothelial cells, epithelial cells, keratinocytes, and macrophages. Kotobuki et al. [23] investigated the biological effects of IL-17A and  Dermatology Research and Practice found that expression of MITF, an important transcriptional regulator of melanogenesis, and its downstream genes was reduced by more than 10% in melanocytes treated with IL-17. In addition, melanin production from IL-17-treated melanocytes decreased by approximately 30%, accompanied by profound morphological changes. In addition, IL-17 synergizes with various inflammatory mediators, including IL-1α, IL-6, and TNF-α [24], to inhibit melanocyte proliferation [25].
In this study, we found that serum IL-17A level was significantly increased in patients with nonsegmental vitiligo compared to healthy controls, confirming the findings of studies [26][27][28][29][30]. We also found that serum IL-17A levels were negatively correlated with age in patients with vitiligo    but not in healthy controls. Previously, Kosar Hedayat et al. [31] demonstrated that vitiligo patients under 30 years had a lower quality of life than those aged above 30 years. Interestingly, mental stress is a trigger for vitiligo in 50-65% of vitiligo patients [32,33], likely due to aggravation of innate and adaptive immunity [34]. Additionally, chronic stress polarizes immune response towards 17 rather than 2 response [35,36]. Consequently, we hypothesized that the increased levels in younger vitiligo patients may be due to mental stress. However, further investigations are required to elucidate the relationship between IL-17A levels and age.
We also found a negative correlation between IL-17A levels and age of onset in vitiligo patients. is finding is in agreement with the findings of Basak et al. [25] but contradict those of Zhou et al. [37]. is suggests that IL-17A may contribute to the immune response in early-onset vitiligo. In addition, Arcos-Burgos et al. [38] demonstrated an association of early onset vitiligo with HLA-DR4, which is involved in CD4+ T cell activation and subsequent production of proinflammatory cytokines, including IL-17 and IFN-c [39]. More studies are needed to understand the role of IL-17A in early onset vitiligo.
A correlation between serum IL-17A levels and body surface involvement has been reported in previous studies [25,26,29]. However, we found no significant association between these two factors, consistent with Tembhre et al. [28] and Zhou et al. [37]. Consistent with previous findings [28,37,40,41], we found that IL-17A levels were correlated with progressive vitiligo, suggesting that IL-17A might be involved in vitiligo induction and progression. Zhen et al. [41] reported that while serum IL-17A levels were significant higher in the patients with nonsegmental vitiligo, there were no significant differences in serum levels of IFN-c, IL-4, and TGF-β1. Furthermore, in this study, the numbers of peripheral CD4 + IL-17A + 17 cells were significantly higher in vitiligo patients compared to healthy volunteers; however, no differences were observed in the numbers of peripheral CD4 + IL-4 + 2 or regulatory T cells. ese findings suggest that IL-17 may be crucial in the onset and progression of autoimmune nonsegmental vitiligo. However, regarding the biomarkers of disease activity, circulating cytokines (IL-1β, IL-17, IFN-c, and TGF-β), autoantibodies, oxidative stress markers, soluble CDs (sCD25 and sCD27), and chemokines (CXCL9 and CXCL10) remain competing [42].
Follicular melanocytes serve as a melanocyte reservoir, facilitating repigmentation in vitiligo. e presence of leukotrichia in white patches may be a sign of refractory vitiligo [43], and untreated leukotrichia could severely impact the quality of life [44]. e understanding of follicular vitiligo pathogenesis and the relationship between IL-17A and leukotrichia, in particular, is limited. In the present study, we found that nonsegmental vitiligo patients with leukotrichia had significantly higher serum levels of IL-17A than patients without leukotrichia. Gan et al. [45] reported the presence of perifollicular lymphocytes in the infundibular region of the hair follicles, while melanocytes were absent in the basal layer of the epidermis and the hair follicle. ese findings suggest that both CD8+ and CD4+ T cells could attack hair follicle melanocytes [46]. In addition, Rongioletti et al. [47] reported a repigmentation of follicular melanocytes in psoriasis patients after treatment with secukinumab, supporting the role of IL-17A in the pathogenesis of follicular melanocyte destruction. Further studies are warranted to understand the role of IL-17 in the pathogenesis of vitiligo.
is study had some limitations that need to be addressed. First of all, the concentration of IL-17A was only measured in the serum, but not in the skin or follicles of the vitiligo patients. us, further researches implemented at the lesional sites should be conducted. Secondly, the size sample of our study was modest partly due to the uncommon characteristic of the vitiligo. Finally, this was a cross-sectional study in which a causal relationship could not be established.

Conclusions
In conclusion, this study provides evidence of that IL-17A may play a crucial role in the induction of vitiligo along with other factors, namely, the genetics, environment, and inflammatory cytokines. In addition, we found that serum levels of IL-17A were significantly associated with leukotrichia, suggesting the role of IL-17A in the destruction of follicular melanocytes.

Data Availability
Due to privacy and ethical concerns, neither the data nor the source of the data can be made available.

Conflicts of Interest
e authors declare that there are no conflicts of interest.