Mitesh Dwivedi

Vitiligo: interplay between oxidative stress and immune system

Vitiligo is a multifactorial polygenic disorder with a complex pathogenesis, linked with both genetic and non‐genetic factors. The precise modus operandi for vitiligo pathogenesis has remained elusive. Theories regarding loss of melanocytes are based on autoimmune, cytotoxic, oxidant–antioxidant and neural mechanisms. Reactive oxygen species (ROS) in excess have been documented in active vitiligo skin. Numerous proteins in addition to tyrosinase are affected. It is possible that oxidative stress is one among the main principal causes of vitiligo. However, there also exists ample evidence for altered immunological processes in vitiligo, particularly in chronic and progressive conditions. Both innate and adaptive arms of the immune system appear to be involved as a primary event or as a secondary promotive consequence. There is speculation on the interplay, if any, between ROS and the immune system in the pathogenesis of vitiligo. The article focuses on the scientific evidences linking oxidative stress and immune system to vitiligo pathogenesis giving credence to a convergent terminal pathway of oxidative stress–autoimmunity‐mediated melanocyte loss.

Decreased regulatory T-cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4+/CD8+ ratio and CD4+CD25hiFoxP3+ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4+/CD8+ ratio and Tregs in 82 patients and 50 controls. CD8+ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8+ T‐cell counts compared with stable GV patients (p = 0.001). The CD4+/CD8+ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4+/CD8+ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.

Increased Tumor Necrosis Factor (TNF)-α and Its Promoter Polymorphisms Correlate with Disease Progression and Higher Susceptibility towards Vitiligo

Abstract Tumor Necrosis Factor (TNF)-α, is a paracrine inhibitor of melanocytes, which plays a critical role in the pathogenesis of several autoimmune diseases including vitiligo, as abnormal immune responses have frequently been observed in vitiligo patients. Moreover, vitiligo patients show higher lesion levels of TNF-α. Genetic polymorphisms in the promoter region of TNF-α are involved in the regulation of its expression. The present study explores TNF-α promoter polymorphisms and correlates them with TNF-α transcript and protein levels in vitiligo patients and controls of Gujarat along with its effect on disease onset and progression. PCR-RFLP technique was used for genotyping of these polymorphisms in 977 vitiligo patients and 990 controls. TNF-α transcript and protein levels were measured by Real time PCR and ELISA respectively. The genotype and allele frequencies for the investigated polymorphisms were significantly associated with vitiligo patients. The study revealed significant increase in TNF-α transcript and protein levels in vitiligo patients compared to controls. In particular, haplotypes: AATCC, AACCT, AGTCT, GATCT, GATCC and AGCCT were found to increase the TNF-α levels in vitiligo patients. Analysis of TNF-α levels based on the gender and disease progression suggests that female patients and patients with active vitiligo had higher levels of TNF-α. Also, the TNF-α levels were high in patients with generalized vitiligo as compared to localized vitiligo. Age of onset analysis of the disease suggests that the haplotypes: AACAT, AACCT, AATCC and AATCT had a profound effect in the early onset of the disease. Moreover, the analysis suggests that female patients had an early onset of vitiligo. Overall, our results suggest that TNF-α promoter polymorphisms may be genetic risk factors for susceptibility and progression of the disease. The up-regulation of TNF-α transcript and protein levels in individuals with susceptible haplotypes advocates the crucial role of TNF-α in autoimmune pathogenesis of vitiligo.

HLA Alleles and Amino-Acid Signatures of the Peptide-Binding Pockets of HLA Molecules in Vitiligo

Vitiligo is a depigmenting disorder of the skin that is characterized by the loss of functional melanocytes from the lesional sites. Although the exact etiology is not understood, autoimmunity is thought to be a crucial deterministic factor. A recurring theme of several autoimmune disorders is the aberrant presentation of self-antigens to the immune system, which triggers downstream perturbations. Here we examine the role of alleles of HLA class I and class II loci to delineate vitiligo manifestation in two distinct populations. Our studies have identified three specific alleles, HLA-A*33:01, HLA-B*44:03, and HLA-DRB1*07:01, to be significantly increased in vitiligo patients as compared with controls in both the initial study on North Indians (N=1,404) and the replication study in Gujarat (N=355) cases, establishing their positive association with vitiligo. Both generalized and localized vitiligo have the same predisposing major histocompatibility complex alleles, i.e., B*44:03 and DRB1*07:01, in both the populations studied, beside the differences in the frequencies of other alleles, suggesting that localized vitiligo too may be an autoimmune disorder. Significant differences in the amino-acid signatures of the peptide-binding pockets of HLA-A and HLA-B α-chain and HLA-DR β-chain were observed between vitiligo patients and unaffected controls.

Interleukin-4 genetic variants correlate with its transcript and protein levels in patients with vitiligo

Background  Vitiligo is an acquired pigmentary disorder resulting from loss of melanocytes. Interleukin (IL)‐4 has been shown to stimulate B‐cell proliferation, to regulate immunoglobulin class switching (IgG1 and IgE) and to promote T‐cell development. Polymorphisms in the IL4 gene are known to increase its expression, thereby implicating its role in vitiligo susceptibility.

The ACE gene I/ D polymorphism is not associated with generalized vitiligo susceptibility in Gujarat population.

Dear Sir, Vitiligo is a hypomelanotic disease characterized by circumscribed depigmented macules and affects 0.5–2% of the world population (Passeron and Ortonne, 2005). However, in India Gujarat has the highest prevalence i.e., approximately 8.8% (Valia and Dutta, 1996). Vitiligo is hypothesized to be of autoimmune origin and genetic factors may also involve in precipitating this disease. Our earlier study suggests that 13.7% of Gujarat vitiliginous patients have at least one firstdegree relative affected (Shajil et al., 2006). Angiotensin-converting enzyme (ACE), a key enzyme in the renin–angiotensin system, plays an important role in the physiology of the vasculature, blood pressure and inflammation. ACE has also been found to be associated with several autoimmune disorders (Scholzen et al., 2003; Vuk-Pavlovic and Rohrbach, 1988). The ACE gene is located on chromosome 17q23.3 and an insertion ⁄ deletion (I ⁄ D) polymorphism of a 287-base pair repetitive sequence in intron 16 of the ACE gene (NCBI: AF118569; repeat region 14094–14381) is reported to be associated with the development of vitiligo (Jin et al., 2004). The I ⁄ D polymorphism of the ACE gene accounts for the variability of serum ACE activity, D ⁄ D genotype having the highest and I ⁄ I genotype having the lowest ACE activity (Rigat et al., 1990). The aim of this study was to investigate the distribution of ACE I ⁄ D genotypes in generalized vitiligo patients and in healthy controls of Gujarat population to find the relationship between ACE I ⁄ D polymorphism and vitiligo. A total of 125 generalized vitiligo patients and 156 ethnically matched healthy controls of Gujarat population were examined for ACE I ⁄ D polymorphism. The patients selected for this study had no other associated diseases. The DNA was prepared from blood samples after written informed consent was obtained. Oligonucleotide primers used for ACE intron 16 PCR amplification were 5¢-CTGGAGACCACTCCCATCCTTTCT 3¢-(forward primer) and 5¢-GATGTGGCCATCACATTCGTCAGAT 3¢ (reverse primer). PCR amplification was performed in 25 ll reaction system containing 50 ng of genomic DNA and 20 pmol of each primer under the following conditions: 94 C for 10 min, 94 C for 30 s, 58 C for 1 min, 72 C for 1 min and final extension at 72 C for 10 min. Amplified PCR products were analyzed on 2% agarose gel electrophoresis with 100 bp DNA ladder to identify the three genotype patterns: I ⁄ I (a 480 bp fragment), D ⁄ D (a 193 bp fragment) and I ⁄ D (two fragments, i.e., 480 and 193 bp). The distribution of the ACE I ⁄ D genotypes for patients and control subjects were compared by using the chi-square test with 2 · 2 and 3 · 2 contingency tables and P-values less than 0.05 were considered statistically significant. The statistical power of the current study was determined by using the G * Power software (Faul et al., 2007). The observed allele and genotype frequencies for the ACE I ⁄ D polymorphism in controls and patient population are shown in Table 1. No significant difference in the genotype frequencies of I ⁄ I, I ⁄ D and D ⁄ D genotypes was observed between vitiligo patients and control subjects (P = 0.459) (Table 1), suggesting that there is no association of the ACE I ⁄ D polymorphism with vitiligo. The observed ACE allele frequencies for I and D alleles were 0.56 and 0.44 in controls; and 0.61 and 0.39 in vitiligo patients, respectively (Table 1). The allele frequencies of ACE I ⁄ D polymorphism did not differ significantly between the controls and patient population (P = 0.252) (Table 1). The observed genotype frequencies of the vitiligo patients did not show significant difference as predicted by the Hardy–Weinberg equation (P < 0.964). Also the control population did not deviate from Hardy– Weinberg equilibrium (P < 0.905). This study has 86% statistical power for the effect size 0.2 to detect

Involvement of superoxide dismutase isoenzymes and their genetic variants in progression of and higher susceptibility to vitiligo.

Oxidative stress has been implicated as the initial triggering event in vitiligo pathogenesis leading to melanocyte destruction. Here, we report a significant increase in oxidative stress in vitiligo patients as evidenced by high lipid peroxidation levels suggesting an imbalance in the antioxidant enzyme system as reported in our previous studies. This study examined the role of the enzymatic antioxidant SOD, which converts the pro-oxidant superoxide into H2O2, in vitiligo pathogenesis. The activity of three isoforms of SOD, i.e., SOD1, SOD2, and SOD3, was significantly higher in vitiligo patients. To identify the underlying mechanism for the increase in activities of SOD isoforms, we explored the SOD1, SOD2, and SOD3 genes for their genetic variations and transcript levels. The SOD2 Thr58Ile (rs35289490) and Leu84Phe (rs11575993) polymorphisms were significantly associated with vitiligo patients, and the Val16Ala (rs4880) polymorphism was associated with active vitiligo patients. Interestingly, SOD2 activity was contributed by these polymorphisms along with its increase in transcript levels in patients. SOD3 activity was associated with the Arg213Gly (rs8192291) polymorphism. The SOD3 transcript levels were also increased in patients, which might contribute to the increased SOD3 activity. However, we could not establish the genotype-phenotype correlation for SOD1 as we could not detect any novel or reported SNPs in SOD1. In addition, both transcript and protein levels of SOD1 were unchanged between patients and controls, though SOD1 activity was increased in patients. Activities of SOD isoforms also correlated with progression of the disease as the activity was higher in active cases of vitiligo compared to stable cases. Here, we report that SOD2 and SOD3 polymorphisms may be genetic risk factors for susceptibility and progression of vitiligo and hence the genetic makeup of an individual may form a basis for the effective treatment of the disease. Overall, our results suggest that increased activity of SOD isoforms under the influence of genetic factors may lead to accumulation of H2O2 in cytoplasmic, mitochondrial, and extracellular compartments resulting in oxidative damage to the melanocytes.

Role of oxidative stress and autoimmunity in onset and progression of vitiligo

Vitiligo is an acquired depigmentation disorder characterized by the loss of functional melanocytes from the epidermis. Two major theories of vitiligo pathogenesis include autoimmunity and oxidative stress‐mediated toxicity in melanocytes. The present study aimed to evaluate both the hypotheses in vitiligo patients and to investigate their role in the disease onset and progression. Antimelanocyte antibody levels and lipid peroxidation (LPO) levels were evaluated in 427 patients and 440 controls; antithyroid peroxidase (TPO) antibody levels were estimated in 102 patients and 72 controls. Patients showed a significant increase in LPO and antimelanocyte antibody levels compared to controls. Antimelanocyte antibody and LPO levels were higher in active vitiligo compared to stable. Only 9.8% of patients showed the presence of anti‐TPO antibodies in their circulation. Oxidative stress may be the initial triggering event to precipitate vitiligo in Gujarat population, which is exacerbated by contributing autoimmune factors together with oxidative stress.

Involvement of interferon-gamma genetic variants and intercellular adhesion molecule-1 in onset and progression of generalized vitiligo.

Interferon-gamma (IFN-γ) is a paracrine inhibitor of melanocytes and genetic variability due to intron 1 polymorphisms in IFNG has been reported to be associated with increased risk for several autoimmune diseases. The aim of present study was to determine whether intron 1 +874A/T (rs2430561) and CA microsatellite (rs3138557) polymorphisms in IFNG are associated with generalized vitiligo (GV) susceptibility and expression of IFNG and intercellular adhesion molecule-1 (ICAM1) affects the disease onset and progression. Here we report that IFNG CA microsatellite but not +874A/T may be a genetic risk factor for GV; however, +874T allele plays a crucial role in increased expression of IFNG mRNA and protein levels which could affect the onset and progression of the disease. Active GV patients showed increased IFNG levels compared to stable GV patients. The genotype-phenotype analysis revealed that IFNG expression levels were higher in patients with +874 TT genotypes and 12 CA repeats. Patients with the early age of onset showed higher IFNG expression and female GV patients showed higher IFNG and ICAM1 expression implicating gender biasness and involvement of IFN-γ in early onset of the disease. Moreover, the increased IFN-γ levels in patients lead to increased ICAM1 expression, which could be a probable link between cytokines and T-cell involvement in pathogenesis of GV.

Association of generalized vitiligo with MHC class II loci in patients from the Indian subcontinent.

TO THE EDITOR Generalized vitiligo is a disease in which patches of depigmented skin and overlying hair result from autoimmune destruction of melanocytes in involved regions (Spritz, 2012). Clinic-based studies cite high prevalence of vitiligo in India, up to 8.8% (e.g. Handa and Kaur, 1999), though population-based surveys report much lower prevalence, 0.46% in Calcutta (Das et al., 1985) and 1.79% in South Gujarat (Mehta et al., 1973). Vitiligo is a distressing cosmetic problem in individuals of dark skin phototypes, due to striking contrast between lesions and unaffected skin. This may explain the reported high prevalence of vitiligo in India and negative impact on perceived quality of life in this population (Parsad et al., 2003). Indeed, vitiligo has long been recognized in India (Singh et al., 1974), the specific use of ultraviolet light treatment was pioneered in India (Menon, 1945), and some of the earliest genetic studies of vitiligo were carried out there: of ABO blood groups, α1-antitrypsin, and haptoglobin, and subsequent candidate gene studies, including GCH1, ACE, CAT, CTLA4, GPX1, IL4, MBL2, and PTPN22, most yielding negative or conflicting results. Recently, Singh et al. (2012) tested genetic association of vitiligo in Indian patients with HLA–A, -B, -C in the MHC class I region and HLA-DRB1 in the class II region, identifying primary genetic association with HLA-DRB1* 07:01. Here, we describe a more comprehensive genetic association study of generalized vitiligo on the Indian subcontinent, utilizing the Immunochip® (Cortes and Brown, 2011) to screen 196,524 SNPs in 128 loci previously implicated in autoimmune and inflammatory diseases, including 9441 SNPs spanning the extended major histocompatibility complex (MHC) on chromosome 6p. Our results suggest there are at least two independent association signals in the MHC class II region, one located upstream of HLA-DRA and the other located between HLA-DRB1 and HLA-DQA1, generally similar to what we previously found in a genomewide association study of vitiligo in European-derived whites (EUR) (Jin et al., 2010). Our initial study group consisted of 255 patients with generalized vitiligo and 377 unrelated non-vitiligo controls of Indian subcontinent (Pakistan, India, Sri Lanka, Bangladesh) derivation. After quality control procedures, data for 120,724 remaining SNPs from 251 remaining cases were compared to those from 349 remaining controls. Suggestive association signals were considered as clusters of nearby SNPs with trend P-values <10−5. The International Immunochip Consortium has agreed on a genomewide significance criterion of P<5 × 10−8 for studies utilizing the Immunochip (Cortes and Brown, 2011). As shown in Figure 1a and Supplementary Table S1, the only highly suggestive association signals were in the MHC class II gene region (Figure 1b), from rs3134942 (chr6:32168770) to rs2856674 (chr6:32659644), spanning the upstream part of NOTCH4 through HLA-DQB1. The principal region of association encompassed c6orf10--BTNL2--HLA-DRA--HLA-DRB5--HLA-DRB1--HLA-DQA1 (Figure 1b), with extensive LD through this region in this population (Figure 1c). One SNP, rs482044, located towards the centromeric end of the region, between HLA-DRB1 and HLA-DQA1, achieved genomewide significance (G allele; P=1.94 × 10−8, OR=1.93; Table 1), remaining significant (P = 4.86 × 10−8) even after correction for the observed genomic inflation factor λ = 1.06. Figure 1 Immunochip association results for generalized vitiligo Table 1 MHC class II region SNPs genotyped in Immunochip screening and in replication studies To determine which SNPs in the MHC class II region represent primary association with vitiligo versus are signals secondary to LD, we applied a backward regression procedure, comparing a model including the seven most significant MHC class II SNPs to alternative models in which each SNP was removed one by one. This analysis suggested that this region contains two independent associated loci, one represented by rs482044-G (located between HLA-DRB1 and HLA-DQA1) and the other represented by rs3129859-C (located 6680 nt upstream of HLA-DRA). Forward regression analysis of these two SNPs showed that the model composed of rs3129859 was significantly (P=4.4 × 10−5) improved by adding rs482044, and that the model composed of rs482044 was significantly improved (P=6.0 × 10−4) by adding rs3129859. In contrast to our previous findings in EUR (Jin et al., 2010), we observed no apparent association of vitiligo with SNPs in the MHC class I region in this Indian-Pakistani population (Figures 1a and 1b). Furthermore, considering loci represented on the Immunochip that have been reported to be associated with vitiligo in previous candidate gene studies from India, no SNPs in the ACE (3 SNPs), CTLA4 (505 SNPs), or IL4 (103 SNPs) gene regions showed even nominal association in the present study. To confirm association of generalized vitiligo with MHC class II region SNPs in the Indian subcontinent, we carried out a replication study of rs3129859 and rs482044, as well as the third most significant Immunochip SNP, rs3096691 (located just upstream of NOTCH4) (Fig. 1b). These three SNPs were genotyped in 685 unrelated generalized vitiligo cases and 774 unrelated controls from Gujarat state, India. All three were in Hardy-Weinberg equilibrium in the controls, and all three achieved at least nominal significance in the replication study (Table 1). Most significant association in the replication study was observed for rs3129859-C (P=9.48 × 10−9), with no significant heterogeneity of OR between the two studies (PBreslow-Day=1.15 × 10−1). Cochran-Mantel-Haenszel meta-analysis of the rs3129859 data from the Immunochip screen and replication study likewise yielded strongest overall association (P=4.30 × 10−14, OR=1.67; 95% C.I. 1.46–1.91). Association was also confirmed in the replication study for rs482044 (P= 1.11 × 10−4), with only nominal association for rs3096691 (P=2.32 × 10−2), although both of these SNPs exhibited heterogeneity of OR. Both rs482044 (P=1.58 × 10−2) and rs3129859 (P = 1.20 × 10−6) remained significant when each was conditioned on the other. Overall, our findings thus generally confirm association of vitiligo with at least two independent loci in the MHC class II region. In a previous genomewide-association study of generalized vitiligo in EUR subjects, we found that both vitiligo susceptibility (Jin et al., 2010) and age of onset (Jin et al., 2011) are likewise associated with at least two independent loci in the MHC class II region. To assess whether the MHC class II loci observed in the Indian subcontinent and EUR populations might correspond ancestrally, we carried out trans-ethnic meta-analysis using MANTRA (Morris, 2011), which indicated that the MHC association signal represented by rs482044 in the Indian subcontinent population apparently corresponds to the MHC signal represented by rs532098 in EUR (Jin et al., 2010) (Table S2). In contrast, rs3129859 is not significantly associated with vitiligo in EUR (Jin et al., 2010), and correspondence between the association signals upstream of HLA-DRA observed in both populations remains uncertain. Our findings thus highlight both similarity and differences of vitiligo MHC genetic associations in subjects from different major world populations. On the Indian subcontinent, this study and that of Singh et al. (2012) support association of vitiligo with loci in the MHC class II region, but show no primary association in the MHC class I region. Similarly, in the EUR population, vitiligo is also associated with multiple signals in the MHC class II region, at least one of which, between HLA-DRB1 and HLA-DQA1, appear to correspond to one in the Indian subcontinent population. However, in the EUR population vitiligo shows primary association with HLA-A in the distal class I region (Jin et al., 2010); specifically, HLA-A*02:01 (Jin et al., 2011). In addition, studies in Chinese show principal MHC association in the class III region (Quan et al., 2010) and in the proximal class I region, between HLA-B and HLA-C (Liu et al., 2012). Together, these similarities and differences of principal MHC genetic associations with generalized vitiligo among different populations may in part underlie differing prevalence of this autoimmune disease in different groups around the world.