Abstract
Background:
Vitiligo is an immune-mediated, chronic skin condition that affects both the innate and adaptive immune systems. Antimicrobial peptide overexpression is one of its defining characteristics. Granulysin (GNLY), an antimicrobial peptide, may play a role in the pathogenesis of various autoimmune diseases.
Objectives:
To estimate the serum GNLY levels in vitiligo patients and to correlate those levels with the severity and activity of the disease.
Materials and Methods:
This case-control study included 60 non-segmental vitiligo patients (Group A) and a control group of 60 people who were matched for age and sex, appeared to be in good health, and were not suffering from vitiligo (Group B). The serum granulysin levels of all subjects were measured using an enzyme-linked immunosorbent assay.
Results:
When compared to the control group, vitiligo patients had significantly higher serum GNLY levels (P = 0.001). When compared to patients with stable disease, those with active vitiligo had significantly higher serum GNLY levels (P = 0.008). Additionally, there was a positive correlation between the serum GNLY levels and the vitiligo area severity index and vitiligo disease activity scores (P = 0.004 and <0.001, respectively).
Limitations:
Study population was relatively small. Evaluation of serum granulysin before and after treatment could have been more beneficial.
Conclusions:
Blood granulysin levels could contribute to the pathogenesis of vitiligo. A higher serum granulysin level may also be a trustworthy predictor of the severity and progression of a disease.
Keywords: Autoimmunity, Granulysin, Vitiligo
Introduction
Vitiligo is an immune-mediated, chronic skin condition that affects both the innate and adaptive immune systems. Despite the scientific progress in understanding the pathogenesis of disease, the cause of vitiligo is still unclear. A few theories based on evidence have been put forth to explain how melanocytes in the epidermis are damaged; however, vitiligo risk is influenced by both genetic and environmental factors. Autoimmune mechanisms provide a more comprehensive explanation for the etiopathogenesis of generalized or non-segmental (NSV) vitiligo.[1,2] Depigmentation is accompanied by an accumulation of auto-reactive CD8+T cells in the skin. The recruitment and activation of T cells may be supported by plasmacytoid dendritic cells, which are a component of the perilesional cellular infiltrate supporting the autoimmune hypothesis. Apoptosis in vitiligo melanocytes has also been shown to support this mechanism.[3]
Granulysin (GNLY) is a protein molecule found in the cytoplasm of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), where it interacts with perforin and granzyme. It is capable of inducing apoptosis in target cells. Granulysin is also a chemotactic and proinflammatory immune effector molecule.[4,5,6,7] It induces T lymphocytes chemoattraction and inflammatory cytokines expression that affect the onset and development of vitiligo.[7] Granulysin is an essential arbitrator in a number of cutaneous inflammatory and autoimmune diseases.[8,9,10,11,12,13] However, its role in vitiligo has not yet been fully studied. In this study, we sought to assess the serum GNLY level in vitiligo patients and relate it to various clinical parameters.
Materials and Methods
Study population
Sixty vitiligo patients were included in this case-control study (Group A). A control group of 60 subjects who were matched for age and sex and appeared to be in good health was also selected (Group B). All patients were chosen from the dermatology outpatient department at Benha University Hospital. The local ethics board for research involving human subjects approved the study (RC 18-12-2022). Each person was asked for their informed consent before inclusion in the study.
Vitiligo patients who were newly diagnosed or had discontinued treatment (topical or systemic) for 4 weeks before the study were recruited and underwent Wood’s lamp examination. Patients with segmental vitiligo, those with infectious, inflammatory, or other autoimmune diseases, those who were taking any systemic medications or receiving phototherapy that might affect vitiligo, pregnant women, and lactating mothers were excluded from the study.
All studied patients provided a detailed history. The Taïeb and Picardo[14] classification was used to classify the studied patients, and the vitiligo area severity index (VASI) score was used to assess disease severity.[15] To assess disease activity, the vitiligo disease activity (VIDA) score was used.[16]
Laboratory investigations
Five milliliters of fresh venous blood were collected from vitiligo patients and controls under complete aseptic conditions by venepuncture on the plain tube for serum separation. The collected samples were left to clot at room temperature for 30 min and were then centrifuged at 1860 g for 10 min. The separated serum samples were kept frozen at -20°C. Serum GNLY was assessed using an enzyme-linked immunosorbent assay kit for human granulysin (human granulysin ELISA kit; Sun Red Biotechnology Company Catalogue No. 201-12-0355).
Statistical analysis
Data was analyzed by SPSS version 25 (IBM Inc., Chicago, IL, USA). Parametric quantitative variables were presented as mean and standard deviation (SD) and compared using Student’s t-test. Non-parametric quantitative variables were presented as median, minimum, and maximum and compared using Mann–Whitney test for comparison between two groups or Kruskal–Wallis test for comparison between three groups. Qualitative variables were presented as frequency and percentage (%). The receiver operating characteristic (ROC) curve was used to determine the serum GNLY cutoff values with the highest sensitivity and specificity for predicting disease activity. Spearman’s correlation analysis was performed to assess the strength of the association between two quantitative variables. A P value of 0.05 was considered significant.
Results
The mean age of the studied vitiligo patients was 41.1 ± 14.7 years. Twenty of the study patients (33.33%) were males, while 40 (66.67%) were females. The control group’s mean age was 39.6 ± 12.8 years, and in regard to sex, 18 (30%) were males while 42 (70%) were females. The basic clinical and laboratory data of the studied patients are presented in Table 1. When compared to the control group, the vitiligo group had significantly higher levels of serum GNLY (P < 0.001) [Table 1 and Figure 1].
Table 1.
Comparison of basic data between studied groups
| Variable | Control (n=60) | Vitiligo (n=60) | P | ||
|---|---|---|---|---|---|
| Age (years) Mean±SD | 39.6±12.8 | 41.1±14.7 | 0.721 | ||
| Sex | |||||
| Males | 18 | 30% | 20 | 33.3% | 0.804 |
| Females | 42 | 70% | 40 | 66.7% | |
| Age of onset (years) | - | 32.5 (16–67) | - | ||
| Duration (years) | - | 11.5 (0.25–84) | - | ||
| Positive family history | - | 8 (26.7%) | - | ||
| Course | |||||
| Stationary | - | 24 (40.0%) | |||
| Progressive | 36 (60.0%) | - | |||
| Site | |||||
| Head and neck | - | 36 (60%) | - | ||
| Upper limb | - | 24 (40.0%) | |||
| Lower limb | - | 20 (33.3%) | |||
| Trunk | - | 14 (23.3%) | |||
| Clinical types | |||||
| Focal | - | 24 (40%) | - | ||
| Vulgaris | - | 16 (26.7%) | |||
| Acrofacial | - | 20 (33.3%) | |||
| VASI | - | - | |||
| Mean±SD (Range) | 9.17±1.95 (0.2–42) | ||||
| VIDA | - | - | |||
| Mean±SD (Range) | 1.17±0.17 (0–3) | ||||
| Serum granulysin level (ng/ml) Median (Range) | 23.9 (2-40.8) | 54.1 (26.4–184.5) | <0.001 | ||
VASI - Vitiligo area severity index; VIDA - Vitiligo disease activity score. Mann–Whitney test was used for the comparison of numerical parameters. Numerical data are expressed as mean and SD, compared by t-test; categorical data are expressed as number and percentage, compared by Chi-square test
Figure 1.
Box plot for serum granulysin level in control and vitiligo groups. The centerline of the box denotes the median value; the extremes of the box are the interquartile range; and the bars are the upper and lower limits of 95% of the data. The circles represent outlying data
Receiver operating characteristic (ROC) analysis revealed that serum granulysin shows 80% sensitivity and 90% specificity in diagnosing vitiligo and 88.9% sensitivity and 66.7% specificity in differentiating active from stable vitiligo at the best cutoff value (41.5 ng/ml) [Table 2, Figures 2 and 3].
Table 2.
Diagnostic performance of serum granulysin in vitiligo
| No. 120 | Cutoff value (ng/ml) | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) | Accuracy (%) | AUC |
|---|---|---|---|---|---|---|---|
| Patients vs controls | 35.3 | 80.0 | 90.0 | 92.3 | 75.0 | 84 | 0.939 |
| Active vs stable | 41.5 | 88.90 | 66.70 | 80.0 | 80.0 | 80.0 | 0.792 |
AUC - Area under ROC (receiver operating characteristic) curve; PPV - Positive predictive value; NPV- Negative predictive value
Figure 2.
ROC curve of serum granulysin level for discrimination between vitiligo cases and control groups
Figure 3.
ROC curve of serum granulysin level for prediction of active vitiligo
When compared to patients with stable vitiligo, those with active vitiligo had significantly higher serum GNLY levels (P = 0.008). Nonetheless, no significant relation or correlation was discovered between serum GNLY levels and other clinical characteristics in vitiligo patients (P > 0.05 for each) [Table 3 and Figure 4]. Serum GNLY levels correlated significantly with VASI and VIDA scores (P = 0.004 and <0.001, respectively) [Table 4].
Table 3.
Comparison of serum granulysin level according to the studied parameters in vitiligo patients
| Variable | Serum granulysin level (ng/ml) | P | ||
|---|---|---|---|---|
|
| ||||
| Median | Minimum | Maximum | ||
| Sex | ||||
| Males | 50.8 | 26.40 | 110.80 | 0.235 |
| Females | 56.9 | 30.00 | 184.50 | |
| Clinical data | ||||
| Course based on history | ||||
| Progressive | 77.0 | 30.00 | 184.50 | 0.220 |
| Stationary | 49.0 | 26.40 | 164.10 | |
| Clinical type | ||||
| Focal | 48.2 | 30.70 | 184.50 | 0.986 |
| Vulgaris | 60.1 | 29.30 | 164.10 | |
| Acrofacial | 130.00 | 57.00 | 190.00 | |
| Activity based on VIDA | ||||
| Stable | 36.6 | 26.40 | 110.80 | 0.008* |
| Active | 76.9 | 32.40 | 184.50 | |
Mann–Whitney test was used for the comparison of numerical parameters between two groups, while Kruskal–Wallis was used for the comparison of numerical parameters between more than two groups. VIDA - Vitiligo disease activity score; *Significant P
Figure 4.
Box plot for serum granulysin level in active and stable vitiligo cases. The centerline of box denotes the median value; the extremes of the box are the interquartile range; and the bars are the upper and lower limits of 95% of the data
Table 4.
Correlations between the level of serum granulysin (GNLY) and different tested variables in the vitiligo patients
| Variable | GNLY level | |
|---|---|---|
|
| ||
| Correlation coefficient (r) | P | |
| Age | 0.055 | 0.828 |
| Age of onset | -0.254 | 0.310 |
| Duration | 0.342 | 0.304 |
| VASI | 0.641 | 0.004* |
| VIDA | 0.684 | <0.001* |
r - Spearman’s correlation coefficient. *Significant P
Discussion
The pathogenesis of vitiligo has been attributed to a number of mechanisms with the leading theory being the autoimmunity theory.[17] A cytolytic protein, granulysin, is produced by activated CTLs. Granulysin levels in the serum accurately indicate the activity of cell-mediated cytotoxic immunity. Consequently, patients with other autoimmune diseases were found to have high serum granulysin levels.[8,9,10,11,12]
In the current study, when compared to the control group, the vitiligo group had significantly higher serum GNLY levels (P < 0.001). Cytotoxic CD8+T cells and secreted cytokines are found to be high in vitiligo patients and play a vital role in vitiligo pathogenesis by destroying melanocytes.[18] Hogg et al.[19] mentioned that IL-15 and IL-21 induce GNLY expression by peripheral blood CTLs. Also, IL-15 and IL-21 were detected to be high in the serum of vitiligo patients and play crucial roles in vitiligo etiopathogenesis, as IL-15 is crucial for the natural killer (NK) cells, neutrophils, and dendritic cells survival and maturation.[20] Granulysin participates in various immune responses with other granular components such as perforin and granzyme.[6,21]
The significant elevation in serum granulysin in vitiligo patients compared to control subjects, as well as its high sensitivity (80%) and specificity (90%) in distinguishing between patients and healthy controls at a cutoff point of 35.3 ng/mL, contributes to a better understanding of vitiligo pathogenesis. Furthermore, its significant elevation in patients with active vitiligo compared to those with stable lesions, combined with its high sensitivity (88.9%) and specificity (66.7%) in differentiating active from stable vitiligo at a cutoff value of 41.5 ng/mL, suggests that serum granulysin is a promising marker to predict vitiligo progression.
Since granulysin is stable in serum and has a longer half-life, measuring its levels is thought to be helpful for tracking in vivo cell-mediated cytotoxic immune responses.[22] Although there are conflicting reports, it has been suggested that apoptotic mechanisms involving the perforin/granzyme B system are responsible for the persistence and severity of some autoimmune diseases in cell-mediated cytotoxicity.[23,24] In vitiligo, chemokine dysregulation results in leukocyte recruitment and the development of melanocyte-specific adaptive immunity.[8,25,26]
Serum GNLY level showed a significant positive correlation with both VASI and VIDA scores (P = 0.004 and <0.001, respectively). According to our knowledge, this study is the first to examine the association between serum GNLY levels and vitiligo based on investigating its levels in other autoimmune and inflammatory diseases. Autoimmune factors have been strongly linked to NSV pathogenesis, and studies have mainly concentrated on CD8+CTLs and shown how crucial is a role they play in vitiligo’s melanocyte destruction.[27] NSV patients’ peripheral blood mononuclear cells and perilesional skin showed significantly higher levels of CD8+CTLs that produce interferon, granzyme B, and perforin.[28,29] The cytolytic granule protein, granulysin, collaborates with perforin and induces melanocytes apoptosis.[6] Granulysin stimulates T lymphocytes chemoattraction and the expression of several inflammatory cytokines that have an impact on the onset and progression of vitiligo.[7,30]
Limitations
The relatively small study population was the current work constraint, so it would be important to conduct a larger-scale study. Further studies evaluating serum GNLY before and after treatment could be more beneficial in assessing the relationship between GNLY, disease pathogenesis, and therapeutic efficacy because samples were not collected both before and after stopping therapy. Further research is advised because our study did not evaluate the GNLY-secreting NK cells in the perilesional/lesional skin of vitiligo patients.
Conclusions
The study’s findings imply that vitiligo may be affected by granulysin levels in the serum, and it could also be employed as a predictor of its severity and activity. The clinical relevance of serum granulysin levels in vitiligo has not been extensively studied. To define the function of serum granulysin as a novel marker for vitiligo prognosis, more research is necessary.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
We are extremely grateful to all patients and volunteers who took part in this study and the research team who collected the data
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