Interleukin-9 and soluble tumor necrosis factor-like weak inducer of apoptosis in serum and suction blister fluid of nonsegmental vitiligo patients: Relation to disease severity

Amira Abulfotooh Eid; Yasmine Amr Issa; Amira Naser Mohamed; Fairouz Khalil Badran

doi:10.4103/ds.ds_44_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 39 | Issue : 4 | Page : 192-197

Interleukin-9 and soluble tumor necrosis factor-like weak inducer of apoptosis in serum and suction blister fluid of nonsegmental vitiligo patients: Relation to disease severity

Amira Abulfotooh Eid¹, Yasmine Amr Issa², Amira Naser Mohamed¹, Fairouz Khalil Badran¹
¹ Department of Dermatology, Venereology and Andrology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
² Department of Medical Biochemistry, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission	17-Jul-2021
Date of Decision	24-Sep-2021
Date of Acceptance	10-Oct-2021
Date of Web Publication	14-Dec-2021

Correspondence Address:
Dr. Amira Abulfotooh Eid
Department of Dermatology, Venereology and Andrology, Faculty of Medicine, Alexandria University, Alexandria
Egypt

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ds.ds_44_21

Abstract

Background: Autoimmunity is a key player in nonsegmental vitiligo (NSV). Interleukin-9 (IL-9) and tumor necrosis factor-like weak inducer of apoptosis (TWEAK) are pleiotropic cytokines that are involved in many chronic autoimmune diseases. Objectives: To measure IL-9 and TWEAK in serum and suction blister fluid of NSV patients and study their relation to vitiligo. Methods: Thirty NSV patients and thirty controls were recruited. Following detailed history and clinical examination, the vitiligo area scoring index (VASI) and the vitiligo disease activity score (VIDA) of the patients were calculated. IL-9 and TWEAK were measured in serum of patients and controls and in suction blister fluid of patients. Results: Serum levels of IL-9 and TWEAK were significantly higher in patients than in controls (P < 0.001). Meanwhile, in patients, IL-9 and TWEAK were significantly higher in serum than in blister fluid (P < 0.001). A significant positive correlation of serum IL-9 (P < 0.001) and serum TWEAK (P < 0.001) with VASI was detected. A significant positive correlation between serum IL-9 and TWEAK in patients was also detected (P < 0.001). Blister fluid levels of both cytokines showed no significant correlation with any of the studied parameters. Conclusion: The elevated serum IL-9 and TWEAK in NSV possibly contributes to disease development and influences disease severity. Exploring their potential as possible therapeutic targets is, therefore, recommended.

Keywords: Interleukin-9, pathogenesis, tumor necrosis factor-like weak inducer of apoptosis, vitiligo

How to cite this article:
Eid AA, Issa YA, Mohamed AN, Badran FK. Interleukin-9 and soluble tumor necrosis factor-like weak inducer of apoptosis in serum and suction blister fluid of nonsegmental vitiligo patients: Relation to disease severity. Dermatol Sin 2021;39:192-7

How to cite this URL:
Eid AA, Issa YA, Mohamed AN, Badran FK. Interleukin-9 and soluble tumor necrosis factor-like weak inducer of apoptosis in serum and suction blister fluid of nonsegmental vitiligo patients: Relation to disease severity. Dermatol Sin [serial online] 2021 [cited 2023 Mar 21];39:192-7. Available from: https://www.dermsinica.org/text.asp?2021/39/4/192/332516

Introduction

Nonsegmental vitiligo (NSV) is an acquired depigmenting disease characterized by autoimmune destruction of melanocytes, in which Cytotoxic CD8+ T cells and interferon-γ (IFN-γ) together with its downstream chemokines (CXC chemokine ligand-9 [CXCL9] and CXCL10) are believed to play a pivotal role. The exact etiopathogenesis of the disease has not been fully elucidated; nevertheless, autoimmunity is believed to be a key player. Overexpression of several cytokines and genetic susceptibility were found to contribute to the immune dysregulation detected in vitiligo, in which both innate and adaptive immune responses have been incriminated. In addition, intrinsic melanocyte defects, oxidative stress, and external triggering factors are also involved in disease pathogenesis.^[1],[2]

Interleukin-9 (IL-9) is a multifunctional cytokine that is mainly produced by T helper 9 (Th9) cells and to a lesser extent by other immune cells such as innate lymphoid cells, natural killer T-cells, T helper 17 (Th17), T helper 2 (Th2), regulatory T-cells (T-regs), and cytotoxic CD8+ T-cells.^[3] Th9 cells are produced from naïve CD4 T-cells under the influence of transforming growth factor-β (TGF-β), IL-2, and IL-4. In addition, several members of the tumor necrosis factor (TNF) family were reported to induce Th9 differentiation.^[4] The involvement of IL-9 in many autoimmune diseases such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), and psoriasis has been reported, where IL-9 is believed to enhance chronic inflammation through stimulating Th17 cells differentiation.^[3],[5]

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF family of cytokines, and like other members of this family, it exists in a transmembrane form and a soluble form. It is a pleiotropic cytokine produced mainly from monocytes and macrophages and is involved in the regulation of inflammation, apoptosis, and angiogenesis, in addition to playing a role in controlling cell proliferation and differentiation.^[6] TWEAK levels were reported to be elevated in several chronic autoimmune and inflammatory diseases including RA, SLE, IBD, and psoriasis where a relation between TWEAK and Th17 cells has been described.^[7],[8]

The objective of this work was to measure serum and suction blister fluid levels of IL-9 and TWEAK in patients with NSV and to determine their relation to different disease-and patient-related variables.

Patients and Methods

Using repeated measures analysis of variance power analysis in NCSS and PASS program, the minimum required sample size to evaluate the levels of the tested variables in serum and suction blister fluid in patients with NSV was eighteen patients (each subject measured twice: Once in serum and once in blister fluid) that achieves 80% power with an effect size of 0.5, and a target significance level at 5%.

The current study was conducted on thirty patients with NSV and 30 healthy controls. All patients signed an informed consent before participation, and the study protocol was approved by the local medical ethics committee (approval number: 0105809/December 31, 2018).

Exclusion criteria included pregnant and lactating females, patients with associated autoimmune diseases or skin or systemic diseases known to be associated with an increase in TWEAK level^[6],[7],[8] and/or IL-9 level,^[3],[5] patients already on treatment for vitiligo and those who received topical treatment for vitiligo during the last month or systemic treatment or phototherapy during the last 3 months before recruitment. Twelve months were chosen as the cutoff point for determining disease activity/stability;^[9] accordingly, patients with no development of new lesions and/or expansion of existing lesions for a year or more (vitiligo disease activity score [VIDA] 0 or-1) were considered stable and were also excluded from the study.

All patients were subjected to detailed history taking and examination. For assessment of disease activity, the VIDA was calculated and patients were examined for the presence of ill-defined borders and the Koebner phenomenon. Disease severity was assessed using the vitiligo area scoring index (VASI). IL-9 and TWEAK levels were measured in serum and suction blister fluid of lesional skin in patients and in serum of controls using enzyme-linked immunosorbent assay (ELISA).

Blood sampling

Five ml of venous blood were collected and allowed to clot at room temperature for 30 min; they were then centrifuged for 15 min at ×3000g. The supernatant was withdrawn and stored at −80°C.

Suction blister formation

Following Wood's light examination, suction blisters were performed on the most recent nonphoto exposed lesional skin. The piston of a 10 ml syringe was removed and a latex tube of an intravenous (IV) drip set was connected to the nozzle of the 10 ml syringe on one end and the nozzle of a 50 ml syringe on the other end. Following adequate local disinfection and anesthesia, blisters were formed by inducing negative pressure to the selected area using the base of a 10 ml syringe. The 50 ml syringes were used to create the required negative pressure for blister formation and the negative suction induced was maintained by closing the valve of the IV drip set.^[10] After blister formation, blister fluid was aspirated using a 3 ml syringe, stored in Eppendorf tubes at -80°C. Topical antibiotics and sterile dressing were applied daily to the suction blister site and patients were followed up till complete healing occurred.

Determination of interleukin-9 and tumor necrosis factor-like weak inducer of apoptosis in serum and suction blister fluid

IL-9 and soluble TWEAK were measured using ELISA kits (Elabscience, Wuhan, PRC, catalog number: E-EL-H0180 and E-EL-H3651, respectively). The steps were performed according to the manufacturer's protocol. Optical densities of IL-9 and TWEAK were read at 450 nm, and their respective concentrations were calculated using an automated ELISA reader (ELISA Humareader Single; Human Diagnostics, Wiesbaden, Germany).

Statistical methodology

Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 22 (IBM Corp., Armonk, NY, USA). Categorical variables were described by frequencies and percentages and compared using the Chi-square test.

Quantitative variables were tested for normality using Shapiro–Wilk test and abnormally distributed data were described as Median (interquartile range) and compared using Mann–Whitney test (comparison between two groups) and Wilcoxon signed-ranks test (comparison within the same group). Normally distributed data were expressed as mean ± standard deviation and compared using independent sample t-test. To correlate between two quantitative variables, the Spearman correlation coefficient was used. A P ≤ 0.05 was considered significant. Using Med calc version 17, the receiver operating characteristic (ROC) curve was used to detect the diagnostic ability of serum level of TWEAK and IL-9 in discrimination of NSV.

Results

The study was conducted on 30 patients (15 males and 15 females) and 30 controls (21 males and 9 females). The mean age of the patients was 37.7 ± 14.3, whereas the mean age of the controls was 38.1 ± 3.4. No significant difference was detected between patients and controls regarding gender (P = 0.114) or age (P = 0.88). Disease duration ranged from 1 to 30 years with a median of 9.5 years, mean age at the time of disease onset was 27.9 ± 12.97 years. VASI ranged from 10.5 to 56.6 with a median of 15.05. Meanwhile, the VIDA score ranged from 1 to 4 with a median of 2.5. Family history of vitiligo was reported in 20% of patients (six patients), whereas none of the patients had personal or family history of other autoimmune diseases.

Serum and blister fluid interleukin-9

Serum IL-9 was significantly higher in patients than in controls (P < 0.001). Meanwhile, lesional blister fluid IL-9 was significantly lower than serum IL-9 in patients (P < 0.001) [Table 1].

Table 1: Comparison between cases and control subjects and between serum and blister fluid according to IL-9 and TWEAK levels

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In patients, a significant positive correlation of serum IL-9 with VASI (rs = 0.639, P < 0.001) was detected. In addition, a significant positive correlation was found between serum IL-9 and serum TWEAK (rs = 0.600, P < 0.001) in patients [Table 2] but not in the controls (rs = −0.182, P = 0.336). IL-9 in blister fluid from lesional skin showed no significant correlation with serum IL-9 (rs = 0.015, P = 0.939) or with any of the studied variables [Table 2]. No significant relation of IL-9 (in serum or in blister fluid) with gender, the presence of ill-defined borders or Koebner phenomenon was detected [Table 3].

Table 2: Correlation of interleukin-9 (pg/ml) and TWEAK (pg/ml) with different parameters in patients group

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Table 3: Relation of TWEAK and interleukin-9 levels in serum and in blister fluid to gender and the presence of white hairs in lesions in patients

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Serum and blister fluid tumor necrosis factor-like weak inducer of apoptosis

Serum TWEAK was significantly higher in patients than controls (P < 0.001). In patients, blister fluid TWEAK was significantly lower than serum TWEAK (P < 0.001) [Table 1].

In NSV, a significant positive correlation was detected between serum TWEAK and VASI (rs = 0.992, P < 0.001). TWEAK in blister fluid from lesional skin showed no significant correlation with serum TWEAK, or with any of the tested variables [Table 2]. The nonsignificant relations of serum and blister fluid TWEAK with gender, the presence of ill-defined border, and Koebner phenomenon are shown in [Table 3].

Diagnostic accuracy of tumor necrosis factor-like weak inducer of apoptosis and interleukin-9 in nonsegmental vitiligo

ROC analysis was used to detect the diagnostic ability of serum TWEAK and IL-9 in discrimination of NSV and it revealed that both serum IL-9 and TWEAK have an excellent diagnostic relation with NSV. For serum IL-9, the cutoff point with highest sensitivity and specificity was >32.2 pg/ml (area under curve [AUC] = 1 with 95% confidence interval [CI] [0.94–1] and P < 0.001); Meanwhile, the cutoff point with highest sensitivity and specificity for serum TWEAK was >17.8 pg/ml (AUC = 1 with 95% CI [0.94–1] and P < 0.001) [Table 4].

Table 4: Validity (area under a curve, sensitivity, specificity) for serum TWEAK and serum interleukin-9 to discriminate patients group (n=30) from the control group (n=30)

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Discussion

The immune dysregulation in vitiligo has been attributed to the increased CD4+ and CD8+ lymphocytes, altered Th1, Th17, T regs cytokine levels and to the fact that genes involved in innate and adaptive immunity have been linked to vitiligo pathogenesis. Collectively, this could provide support for the autoimmune theory as the main etiologic theory. Cytokines play a crucial role in autoimmunity and altered expression of several cytokines has been reported.^[1] Gholijani et al. reported increased expression of pro-inflammatory innate immunity, Th1, Th2, and Th17 cytokines in vitiligo patients.^[11] Moreover, IL-17A, IL-1 β, IL-6, and TNF-α were found to decrease melanin production by downregulating microphthalmia-associated transcription factor (MITF) expression in melanocytes.^[12]

IL-9 exhibits intricate relation with immune cells, proinflammatory cytokines, chemokines and different signaling pathways known to be involved in vitiligo.^[3] It promotes differentiation and proliferation of Th17 cells, enhances T reg cells suppressive functions and induces IL-1 β, IL-5, IL-6, IL-13, and TGF-β production from mast cells.^[13] Th9 cells have been shown to augment immune responses by increasing the production of pro-inflammatory cytokines including IFN-γ, IL-13, and IL-17 from Th1, Th2, and Th17 cells, respectively; an effect that was reported to be inhibited by blocking IL-9.^[14] It was hypothesized that in response to tissue insult and stress IL-9 is released, which acts either directly or indirectly through activation of mast cells to ultimately result in inhibition of melanin synthesis and melanocyte apoptosis; suggesting a possible role in vitiligo.^[15] The disturbed balance between Th1, Th17, and T reg cytokines could also contribute to the process.

Results of the present study revealed a significantly elevated serum level of IL-9 in patients with NSV and a significant correlation of those elevated levels with disease severity; hence, pointing to potential involvement of IL-9 in disease development and progression. This was in agreement with recent reports of increased Th9 cells and IL-9 in peripheral blood in vitiligo patients; an elevation that was suggested to highlight the systemic autoimmune nature of the disease.^[16],[17] The present study did not detect a relation between serum IL-9 and disease duration, which was consistent with the findings of Kumar et al.,^[17] who reported no difference in peripheral Th9 levels between early and chronic cases. Taking into consideration, the relation of IL-9 with inflammatory cells, this increase in IL-9 level irrespective of disease duration could help maintain the autoimmune activation and possibly contribute to the chronic nature of the disease.

The relation between IFN-γ and the IL-9/Th-9 axis is rather complex. In vitro studies on healthy melanocytes, revealed increased IL-9 receptor expression following exposure to IFN-γ, and decreased IFN-γ-induced reactive oxygen species (ROS) production following exposure to IL-9, which suggests a possible protective role of IL-9 in vitiligo.^[17] Because functional melanocytes are known to be lacking from lesional skin in vitiligo,^[9] this potential protective effect of IL-9 may be of little value in lesional skin but may help in halting disease progression by protecting melanocytes in nonlesional skin from oxidative damage. This could provide a plausible explanation for the lack of elevation of IL-9 in lesional suction blister fluid detected in the present study. However in vitiligo, given the in vivo interplay of many variables, other than oxidative stress, this potential protective role of IL-9 in vitiligo remains to be proved.

TWEAK is a multifunctional cytokine, which is upregulated in response to stress or tissue damage.^[6] IFN-γ, a key cytokine in vitiligo, has been shown to stimulate monocytes to produce TWEAK.^[18] TWEAK is, in turn, capable of inducing the production of several cytokines and chemokines known to be involved in vitiligo such as IL-6, IL-8, IL-17, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and CXCL-10.^[1],[6],[19] This could provide an explanation for the elevated serum TWEAK levels in vitiligo patients and for the relation with disease severity detected in the current study. Our findings were consistent with the results of El-Taweel et al., who also reported increased serum TWEAK in patients with vitiligo.^[20] However, the level of blister fluid TWEAK in lesional skin in the present study was significantly lower than serum values in patients and showed no correlation with disease severity. These findings possibly point to circulatory cells as the major source responsible for the elevation of TWEAK in NSV patients, which is rather consistent with the systemic nature of NSV.

Apoptotic markers have been detected in residual melanocytes in vitiligo lesions,^[21] and cytotoxic CD8+ T cells in vitiligo have been reported to produce IFN-γ and induce melanocyte apoptosis.^[22],[23] Furthermore, interactions of TNF-α with TNF receptors have been shown to bring about the cytotoxic effector function of CD8+ cells.^[24] Interestingly, TWEAK has been reported to induce TNF-α secretion and to exhibit proapoptotic properties.^[25] Hence, stimulating melanocyte apoptosis might be a possible mechanism by which TWEAK could contribute to vitiligo pathogenesis and influence disease severity. TWEAK could also influence melanocytes in vitiligo by inducing keratinocyte apoptosis, with the resultant decrease in keratinocyte-derived factors necessary for melanocyte survival eventually leading to melanocyte death.^[26]

Moreover, defective Wnt signaling in melanocytes that was ascribed to oxidative stress was detected in vitiligo.^[27] TWEAK was reported to increase ROS production,^[28] and to interfere with Wnt signaling.^[29] Hence, increasing oxidative stress is another possible mechanism by which TWEAK can influence disease severity.

Although several members of the TNF family were found to induce the differentiation of Th9 cells,^[4] no direct relation between IL-9 and TWEAK was previously reported. The significant positive correlation between serum IL-9 and serum TWEAK detected in the current study in patients but not in the control group could suggest the existence of a relation between the two cytokines under pathological conditions. However, the exact nature of such relation remains to be elucidated.

Targeting IL-9 by anti-IL-9 antibodies was found to downregulate Th2, Th9, and Th17 responses and upregulate Treg responses. In addition, anti-IL-9 antibodies were reported to cause a significant reduction in IFNγ.^[30] Meanwhile, targeting the TWEAK-Fn14 system has been proved effective in reducing systemic inflammation and in ameliorating the signs of several immune and inflammatory diseases.^[31] It is thus possible that IL-9 and TWEAK could serve as potential therapeutic targets for vitiligo.

Conclusion

The elevated level of serum IL-9 and TWEAK and their positive correlation with disease severity could suggest the potential involvement of both cytokines in the pathogenesis of NSV with the possible contribution to disease severity. An important limitation of this study is that TWEAK and IL-9 in perilesional skin were not measured. Hence, further studies on a larger number of patients investigating the level of TWEAK and IL-9 in lesional and perilesional skin and exploring in-depth the relation between IL-9 and TWEAK and their value as possible therapeutic targets in vitiligo are recommended.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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