Generalized Vitiligo Research Articles

HLAB27 may confer protection to COVID-19 in generalized vitiligo patients from South Gujarat population

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), while generalized vitiligo(GV) is an autoimmune disease that causes the loss of functional melanocytes, resulting in white patches all over the body. Human Leukocyte Antigen (HLA) plays a crucial role in immune response to pathogens. Studies assessing the link between GV and COVID-19 are lacking; therefore, our current study was aimed to establish the association between GV and HLAB27 by genotyping the HLAB27 allele in 150 GV patients and 150 controls from South Gujarat population through polymerase chain reaction-sequence-specific primers (PCR-SSP) method. Additionally, we assessed the correlation of GV with COVID-19 and the influence of HLAB27 on COVID-19 development. Interestingly, our study suggested that the HLAB27 allele was prevalent in GV patients as compared to controls (52% vs 35.33%; p = 0.0051). Moreover, the occurrence of COVID-19 was significantly lower in GV patients than in controls (10% vs 32.66%; p < 0.0001). Disease activity-based analysis suggested that COVID-19 occurrence was significantly lower in active vitiligo (AV) patients as compared to stable vitiligo (SV) patients(6.87% vs 31.57%; p = 0.0045). Furthermore, COVID-19 development was significantly reduced in HLAB27 positive individuals as compared to HLAB27 negative individuals (p = 0.0025). Overall, our study suggests, for the first time, that HLAB27 allele might be a genetic risk factor for GV susceptibility, and an ongoing immune response in GV patients, more specifically in AV patients, might protect against COVID-19 infection in South Gujarat population. Additionally, our study highlighted the likely role of HLAB27 in protection against COVID-19 development.

Altered Regulatory T cell-mediated Natural Killer Cells Suppression may lead to Generalized Vitiligo

Generalized vitiligo(GV) is characterized by white patches due to autoimmune loss of melanocytes. Regulatory T cells(Tregs) maintain immune homeostasis, while NK cells eliminate pathogens and tumors. Increased NK cell frequency and reduced Treg frequency and suppressive capacity are observed in vitiligo patients. However, studies assessing Treg-mediated suppression of NK cell functions in GV are lacking. Therefore, our study aimed to assess in vitro Treg-mediated suppression of NK cells function over K562 and SK-Mel-28 cells in 31 GV patients and 30 controls using the BrdU-cell proliferation assay. We found decreased Treg-mediated suppression of NK cell function in GV patients(p=0.0289). Moreover, increased NK cell-mediated K562 and SK-Mel-28 cells’ suppression was observed in GV patients(p=0.0207,p=0.0419). Disease activity-based analysis, suggested reduced Treg-mediated suppression of NK cell function and increased NK cell function in active vitiligo patients(p=0.03,p=0.0436). Interestingly, age-based analysis suggested decreased Treg-mediated suppression of NK cell function in 1-20 and 21-40 years age groups compared to 41-60 years age group of GV patients(p=0.005,p=0.0380). Overall, our study, for the first time, suggests that decreased Treg-mediated suppression of NK cells may lead to increased destruction of melanocytes in GV, and this knowledge may help in developing effective therapeutics based on Tregs and NK cells for GV.

Genetic association of nuclear factor of activated T cells’ 3′UTR and structural polymorphisms with susceptibility to generalized vitiligo in Gujarat population

Generalized vitiligo (GV) is an autoimmune skin depigmenting disease characterized by loss of functional melanocytes. Nuclear factor of activated T cells (NFATs) play a key role in regulatory T cells’ (Tregs) activation and function. Our previous studies have highlighted the role of reduced NFATs expression and activity in impaired Tregs suppressive capacity, leading to GV pathogenesis. 3′UTR region and structural single nucleotide polymorphisms(SNPs) could lead to reduced NFAT expression and activity. Therefore, we studied the association of NFATs 3′UTR [NFATC2 rs4811198 (T > G) &NFATC4 rs11848279 (A > G)] and structural [NFATC1 rs754093 (T > G) &NFATC2 rs12479626 (T > C)] SNPs in 427 GV patients and 415 controls from Gujarat population by Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). Additionally, we carried out genotype-phenotype correlation and in silico analysis to assess the effect of NFATs SNPs on NFATs expression and structure. NFATC2 rs4811198 (T > G) 3′ UTR &NFATC2 rs12479626 (T > C) structural SNPs were significantly associated with GV (p < 0.0001). Interestingly, for NFATC2 rs4811198 (T > G) SNP, there was a significant difference in the TT vs GG genotypes’ frequencies (p = 0.0034; Table 2), and for NFATC2 rs12479626 (T > C) SNP there was a significant difference between TT vs TC and CC genotypes’ frequencies (p < 0.0001 & p = 0.0002) between GV patients and controls. Furthermore, Odds ratio suggested that the susceptible alleles for NFATC2 rs4811198 (T > G) &NFATC2 rs12479626 (T > C) SNPs increased the risk of GV by 1.38 & 3.04 fold. However, the NFAT 3′ UTR [NFATC2 rs4811198 (T > G)] and structural [NFATC1 rs754093 (T > G)] SNPs were not significantly associated with GV. Interestingly, the genotype-phenotype correlation suggested that the susceptible ‘G’ allele of NFATC2 rs4811198 (T > G) &NFATC4 rs11848279 (A > G) 3′ UTR SNPs lead to reduced NFATC2 and NFATC4 expression (p < 0.0001). Furthermore, in silico analysis suggested that hsa-miR-3183 & hsa-miR-6720-3p miRNAs specifically bound to ‘G’ allele of NFATC2 rs4811198 SNP and has-miR-4652-3p miRNA specifically bound to ‘G’ allele of NFATC4 rs11848279 SNP. Overall, our study suggests that NFATC2 rs4811198 (T > G) 3′ UTR &NFATC2 rs12479626 (T > C) structural SNPs may be associated with GV susceptibility in Gujarat population. Moreover, the susceptible alleles for the 3′ UTR SNPs could lead to reduced NFATs levels, which may further possibly, affect the Treg suppressive function leading to GV.

Decreased GZMB, NRP1, ITPR1, and SERPINB9 Transcripts Lead to Reduced Regulatory T Cells Suppressive Capacity in Generalized Vitiligo Patients

Generalized vitiligo (GV) is an autoimmune skin disease characterized by bilateral white patches over the entire body. Regulatory T cells (Tregs) maintain peripheral tolerance; however, they are found to be reduced in numbers and function in vitiligo patients. The exact mechanism for reduced Treg suppressive capacity is unknown. Therefore, we aimed to assess transcript levels of Tregs-associated immunosuppressive genes (GZMB, NRP1, PDCD1, FASLG, and TNFRS18), regulatory molecules of Tregs suppressive function (SERPINB9, ITPR1, and UBASH3A), and Treg-associated transcription factors (GATA2, GATA3, RUNX1, STAT3, and STAT5) in 52 GV patients and 48 controls by real-time PCR (qPCR). We found significantly reduced GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs compared to controls (p = 0.03, p = 0.023, p = 0.0045, and p < 0.0001, respectively). There were 0.44-, 0.45-, 0.32-, and 0.54-fold decrease in GZMB, NRP1, SERPINB9, and ITPR1 transcripts in GV Tregs. Additionally, disease activity and severity-based analyses revealed significantly decreased GZMB (p = 0.019 and 0.034), SERPINB9 (p = 0.031 and p = 0.035), and ITPR1 (p = 0.0003 and p = 0.034) transcripts in active vitiligo and severe GV patients' Tregs. Interestingly, we found a positive correlation for ITPR1 with GZMB (r = 0.45, p = 0.0009) and SERPINB9 (r = 0.52, p = 0.001) transcripts in GV Tregs. Moreover, we found positive correlation for percentage Treg mediated suppression of CD4+ and CD8+T cells with ITPR1 (r = 0.54; r = 0.49), GZMB (r = 0.61; r = 0.58), NRP1 (r = 0.55; r = 0.52), and SERPINB9 (r = 0.56; r = 0.48) in GV Tregs. Further, calcium treatment of Tregs resulted into significantly increased ITPR1, SERPINB9, and GZMB transcripts in GV Tregs (p = 0.023, p = 0.0345, p = 0.02). Overall, our results for the first time revealed the crucial role of GZMB, NRP1, SERPINB9, and ITPR1 transcripts in decreased Treg suppressive capacity leading to GV pathogenesis, progression, and severity. In addition, our study highlighted that ITPR1 might be linked with decreased GZMB and NRP1 expression in GV Tregs. Moreover, our study for the first time suggest that increased SERPINB9 transcripts may lead to endogenous granzyme B-mediated Tregs apoptosis, and calcium treatment of Tregs may improve the Treg suppressive capacity. These findings may further aid in development of Treg-based therapeutics for GV.

Calcium controlled NFATc1 activation enhances suppressive capacity of regulatory T cells isolated from generalized vitiligo patients.

NFATs and FOXP3 are linked with impaired regulatory T-cells (Tregs) in generalized vitiligo (GV). To elucidate calcium mediated NFATc1 signalling pathway and its effect on Treg suppressive capacity in GV. Calcium levels, calcineurin, NFATc1 and GSK-3β activity and cell proliferation were assessed in 52 GV patients and 50 controls by calcium assay kit, calcineurin phosphatase assay kit, TransAM NFATc1 kit, GSK-3β ELISA and BrdU cell proliferation assay. Transcripts (CNB, CAM, GSK3B, DYRK1A and calcium channel genes) and protein (IFN-γ, IL-10 and TGF-β) expressions were assessed by qPCR and ELISA, respectively. Reduced plasma and intracellular Tregs calcium levels and ORAI1 transcripts suggested altered calcium homeostasis in GV Tregs (p=0.00387, p=0.0048, p < 0.0001), which led to decreased calcineurin and NFATc1 activity in GV Tregs (p=0.0299, p < 0.0001). CNB and CAM transcripts were reduced in GV Tregs (p < 0.0001, p=0.0004). GSK-3β activity, GSK3B and DYRK1A transcripts significantly increased in GV Tregs (p=0.0134, p < 0.0001 and p < 0.0001). Plasma (p=0.0225, p=0.032) and intracellular Treg (p=0.0035, p=0.005) calcium levels, calcineurin (p=0.001) and NFATc1 (p=0.001, p < 0.0001) activity and ORAI1 (p=0.0093, p < 0.0001), CAM and CNB (p=0.0214) transcripts significantly decreased in active vitiligo (AV) and severe GV (sGV) Tregs. Calcium treatment significantly increased intracellular calcium and ORAI1 transcripts in GV Tregs (p=0.0042, p=0.0035). Moreover, calcium treatment enhanced calcineurin and NFATc1 activity in GV Tregs (p=0.0128, p < 0.0001). Remarkably, calcium treatment increased Treg mediated suppression of CD4+ and CD8+ T-cells (p=0.015, p=0.006) in GV and increased Tregs associated cytokines: IL-10 (p=0.0323, p=0.009), TGF-β (p=0.0321, p=0.01) and decreased IFN-γ production (p=0.001, p=0.016) by CD4+ and CD8+ T-cells. Intracellular calcium levels positively correlated with calcineurin (r=0.83; p < 0.0001) and NFATc1 (r=0.61; p < 0.0001) activity, suggesting the enhanced Treg immunosuppressive capacity after calcium treatment. Our study for the first time suggests that reduced plasma calcium and ORAI1 transcripts are linked to calcium uptake defects in Tregs, which leads to reduced calcineurin and NFATc1 activation, thereby contributing to decreased Tregs immunosuppressive capacity in GV. Elevated GSK-3β activity and GSKB and DYRK1A transcripts are involved in reduced NFATc1 activity in GV Tregs. Overall, the study suggests that calcium-NFATc1-signalling pathway is likely to be involved in defective Tregs function and can be implicated for development of effective Treg mediated therapeutics for GV.

Genetic variations in NLRP1 and Glutathione S-Transferase are associated with Generalized Vitiligo of Phlegmatic Clinical Phenotypes in Unani Medicine Philosophy of South Indian Population

Background: Generalized vitiligo is a common, multifaceted, polygenic condition in which autoimmune loss of melanocytes results in depigmented skin patches, overlying hair and mucous membranes. NLRP1 has been proposed to be implicated in the susceptibility of a broad variety of autoimmune disorders, including generalized vitiligo (GV). Genetic polymorphisms in the NLRP1 encoding gene (formerly known as NALP1) have previously been found to be linked with GV and there is uncertainty as to their role in the modulation of NLRP1 expression. Oxidative stress is a significant pathogenesis theory for vitiligo. Glutathione S-transferases (GSTs) are enzymes active in the defense of cells against chemical toxicity and stress.This study validates some of the Unani concepts of humors or temperaments (Phenotypes), with regard to Vitiligo, Where vitiligo is regarded as a phlegmatic disease. We selected Vitiligo subjects with Phlegmatic Clinical Phenotype for our study, with an aim to determine its association with the genetic biomarkers- NLRP1, GSTM1 and GSTT1 null genotypes and other biochemical parameters. Methods: The Unani clinicians randomly selected 100 vitiligo patients with a phlegmatic Clinical Phenotype who were attending NRIUMSD for treatment and 100 healthy volunteers belonging to Phlegmatic (Phlegmatic clinical Phenotype). Besides looking at temperaments/ humors as susceptibility factors – we included a genetic factor- NLRP1, GSTM1- and GSTT1-null genotypes to our investigation. We have genotyped the NLRP1, GSTM1- and GSTT1-null genotypes by PCR-RFLP and by Multiplex PCR, GST protein level estimation by ELISA method. Results: NLRP1 rs2670660 polymorphism was shown to be in significant association with GV, with the presence of minor alleles in active GV. We found that the frequencies of GSTM1 null genotype and GSTT1 null genotype in vitiligo patients were significantly high compared to the controls (OR= 1.47, 95% CI=0.765--2.861), (OR = 4.75, 95% CI = 2.131-10.63), respectively. In combination analysis with both genes, the results suggested significant association of vitiligo risk with both GSTM1\GSTT1 null genotypes (OR=4.83, 95% CI=1.523– 15.32).We observed a significant decrease (p&lt;0.001) in GST protein levels. Conclusion: Our findings indicate that NLRP1 rs2670660 polymorphism may be genetic risk factor for susceptibility to GV and the null genotypes of GSTM1 and GSTT1 of both genes increase the risk of the disease. A significant decrease (p&lt;0.001) in GST protein levels appeared to be a key feature in Vitiligo subjects, Therefore, detection of antioxidant enzyme levels can be effective biomarkers for early detection of the disease. We believed that GSTM1-and GSTT1-null genotype polymorphisms were associated with an increased risk of vitiligo. This is the first study of its kind along with Clinical Phenotype as per Unani Philosophy.

Comment on: The Difference in Expression of Autophagy-Related Proteins in Lesional and Perilesional Skin in Adult Patients with Active and Stable Generalized Vitiligo-A Cross-Sectional Pilot Study.

Comment on: The Difference in Expression of Autophagy-Related Proteins in Lesional and Perilesional Skin in Adult Patients with Active and Stable Generalized Vitiligo-A Cross-Sectional Pilot Study.

Dendritic cells and their associated pro-inflammatory cytokines augment to the inflammatory milieu in vitiligo skin

Vitiligo is a progressive, autoimmune, hypomelanotic acquired disorder of skin which is characterized by depigmentation. The initial immunological events of this diseases are still at enigma that includes breach of immune tolerance, and defect in antigen presentation. Hence, we aimed to explore role of Dendritic cells (DCs) and its associated cytokines in the pathogenesis of generalized vitiligo (GV) patients. For this case-control study, 20 active patients and controls were enrolled. Phenotypic characterization of myeloid and plasmacytoid DCs (mDCs, pDCs) were done by flow-cytometry. Primary culture of DCs was done by monocyte differentiation supplemented with rIL-4 and rGM-CSF. Functional analysis DCs related cytokines and co-stimulatory molecules (CD80, CD40) was done by ELISA and qPCR respectively. Tissue localization of DCs was evaluated by immunohistochemistry. The frequency of mDCs (0.3715% v/s 0.188%) and pDCs (0.2331% v/s 0.1156%) were elevated in patients as compared to controls. Circulatory level of IL-12, TNF-α were significantly higher whereas IFN-α was decreased in patients than controls. Similar results were obtained in the culture supernatants of patients. Relative mRNA expression profiling of co-stimulatory molecules (CD80, CD40) were found to be up regulated in patient's skin. Tissue localization of Langerhans cells (Langerin, CD1a+) were found to be significantly higher in patients. Elevated frequency of mDCs and pDCs along with elevated levels of IL-12, TNF-α and CD80, CD40 may contribute in defective antigen presentation of DCs. Altered pro-inflammatory and anti-inflammatory cytokines along with tissue localization of Langerhans cells might be involved in the pathogenesis of GV. These DCs associated cytokines can be explored as a therapeutic target in future.

Association of FOXP3 and GAGE10 promoter polymorphisms and decreased FOXP3 expression in regulatory T cells with susceptibility to generalized vitiligo in Gujarat population.

Alterations in regulatory T (Treg) cells have been observed in generalized vitiligo (GV) patients and decreased Forkhead Box P3 (FOXP3) has been implicated in the disease pathogenesis. The present study examined FOXP3 rs3761547(A>G), rs3761548(C>A), rs2232365(A>G) and GAGE10 rs11798415(A>T) promoter single nucleotide polymorphisms (SNPs) in 419 GV patients and 429 controls from Gujarat population using PCR-RFLP and ARMS-PCR. Real-time PCR and flow cytometry were used for assessment of FOXP3 mRNA and protein levels respectively in 96 GV patients and 90 controls. The frequency of genotypes (p<0.001) and alleles (p=0.012 & p=0.040) for rs3761547(A>G) and rs11798415(A>T) SNPs significantly differed between GV patients and controls. FOXP3 mRNA and protein levels were significantly decreased (p<0.001) in GV Tregs compared to controls. Active vitiligo (AV) Tregs showed significantly reduced FOXP3 mRNA and protein levels compared to that of stable vitiligo (SV) (p=0.02 & p=0.039). The correlation of genotype-phenotype of FOXP3 SNPs suggested reduced FOXP3 mRNA (p=0.019, p<0.001 & p<0.001) and protein (p=0.028, p<0.001 & p=0.022) levels in patients with susceptible GG, AA and GG genotypes respectively. The GAGT, GCGT & ACGT haplotypes were prevalent in GV patients (p=0.004, p=0.004 & p=0.016); however, GAGT & GCGT were overrepresented in patients with AV (p=0.044 & p=0.024). The susceptible GAGT and GCGT haplotypes in patients exhibited reduction in FOXP3 mRNA (p=0.014 & p=0.019) and protein (p=0.024 & p=0.028). DNA-protein docking analysis revealed reduced binding for transcription factor C/EBP to the susceptible allele 'G' (rs3761547) compared to A allele. For the first time, the study suggests significant association of FOXP3 rs3761547(A>G) & GAGE10 rs11798415(A>T) SNPs with susceptibility to GV in Gujarat population. In addition, the likely role of these SNPs in altered FOXP3 expression of Tregs may possibly affect Treg suppressive function in GV.

Consensus on the treatment of vitiligo – Brazilian Society of Dermatology

BackgroundVitiligo is a muco-cutaneous, autoimmune, localized, or disseminated disease, which manifests through hypochromic or achromic macules, with loss in quality of life. The prevalence of vitiligo in Brazil was determined to be 0.54%. There is no on-label medication for its treatment. To date, no Brazilian consensus on the treatment of vitiligo had been written. ObjectivesThe objective of this group of Brazilian dermatologists with experience in the treatment of this disease was to reach a consensus on the clinical and surgical treatment of vitiligo, based on articles with the best scientific evidence. MethodsSeven dermatologists were invited, and each was assigned two treatment modalities to review. Each treatment (topical, systemic, and phototherapy) was reviewed by three experts. Two experts reviewed the surgical treatment. Subsequently, the coordinator compiled the different versions and drafted a text about each type of treatment. The new version was returned to all experts, who expressed their opinions and made suggestions for clarity. The final text was written by the coordinator and sent to all participants to prepare the final consensus. Results/ConclusionThe experts defined the following as standard treatments of vitiligo: the use of topical corticosteroids and calcineurin inhibitors for localized and unstable cases; corticosteroid minipulse in progressive generalized vitiligo; narrowband UVB phototherapy for extensive forms of the disease. Surgical modalities should be indicated for segmental and stable generalized vitiligo. Topical and systemic anti-JAK drugs are being tested, with promising results.

Decreased suppression of CD8+ and CD4+ T cells by peripheral regulatory T cells in generalized vitiligo due to reduced NFATC1 and FOXP3 proteins.

Regulatory T cells (Tregs) are involved in the suppression of activated T cells in generalized vitiligo (GV). The study was aimed to investigate Tregs functional defects in Treg:CD8+ and Treg:CD4+ T cells' co-culture systems of 55 GV patients and 45 controls. CD8+ and CD4+ T-cell proliferation was assessed by BrdU assay; production of IL-10, TGF-β and IFN-γ cytokines was assessed by ELISA; and FOXP3, CD25, NFATC1 and CD44 proteins were measured by flow cytometry. Generalized vitiligo patients showed reduced suppression of CD8+ and CD4+ T cells (P=.0384, P=.0084), increased IFN-γ (P<.0001, P=.0019), decreased IL-10 and TGF-β (P<.0001) and decreased FOXP3, CD25 and NFATC1 proteins (P<.0001). Active vitiligo (AV) patients showed reduced suppression of CD8+ & CD4+ T cells (P=.006, P=.015), increased IFN-γ (P=.036, P=.045), decreased IL-10 (P=.009, P=.021), FOXP3 (P=.0244) and NFATC1 (P=.019). Severe GV (50%-75% VASI) patients showed reduced suppression of CD8+ and CD4+ T cells (P=.0003, P=.001), increased IFN-γ (P=.0029, P<.0001), decreased IL-10 (P=.0057, P=.0017), FOXP3 (P=.002) and NFATC1 (P=.0347). VASI score was positively correlated with the suppression of CD8+ and CD4+ T cells (P=.0006, P<.0001), IL-10 (P=.0096, P=.029), FOXP3 (P=.0008) and NFATC1 (P=.043), whereas it was negatively correlated with IFN-γ (P=.0029, P=.0017). Early age of onset patients' Tregs demonstrated decreased suppression of CD8+ and CD4+ T cells (P=.0156, P=.0074), decreased TGF-β (P=.0212, P=.0083) and NFATC1 (P=.0103). NFATC1 was positively correlated with FOXP3 in Tregs (P<.0001). Our results suggest impaired Tregs suppressive function in GV patients due to decreased NFATC1, FOXP3, CD25, IL-10 and TGF-β resulting into increased CD8+ and CD4+ T-cell proliferation and IFN-γ production. For the first time, decreased NFATC1 levels were correlated with decreased FOXP3, thereby altering Treg cell function in GV patients. Additionally, decreased Treg cell function also affected onset, activity and severity of GV.

Altered expression of nuclear factor of activated T cells, forkhead box P3, and immune‐suppressive genes in regulatory T cells of generalized vitiligo patients

The study was aimed to analyze expression of nuclear factor of activated T cells (NFATs), forkhead box P3 (FOXP3), and their associated genes (sCTLA4, flCTLA4, IL10, TGFB, IL2, IL4, CD25) in regulatory T cells (Tregs) of 48 generalized vitiligo (GV) patients and 45 unaffected controls. The transcripts of NFATC1 to NFATC4, FOXP3, IL10, flCTLA4 (p<.0001), NFAT5 (p=.0003), sCTLA4 (p=.001), and FOXP3 protein in Tregs and plasma IL-10 levels were reduced significantly (p<.0001) in GV Tregs compared to controls. The FOXP3 promoter polymorphisms [rs3761548(C>A), rs3761547(A>G), and rs2232365(A>G)] revealed significantly decreased FOXP3 protein levels in patients' Tregs with susceptible AA, GG, and GG genotypes (p<.0001, p=.028, p=.022, respectively). The active vitiligo Tregs showed reduced levels of NFATC3, NFATC4, NFAT5, FOXP3, TGFB, and flCTLA4 transcripts (p=.0005, p=.0003, p=.0002, p=.020, p<.0001, p=.006, respectively) and FOXP3 and TGF-β proteins (p=.0394 and p=.0013) compared to stable vitiligo. Early-onset patients (1-20years) demonstrated decreased IL-10, sCTLA-4, flCTLA-4, TGFB, and FOXP3 transcripts and FOXP3 protein as compared to late-onset patients (41-60years) (p=.001, p=.003, p=.009, p=.005, p=.038, p=.0226, respectively). Overall, our results for the first time suggest a likely role of NFATs and FOXP3 together with Treg immune-suppressive genes in GV pathogenesis and disease progression, warranting additional investigations.

The role of IFIH1 gene rs1990760 and rs2111485 single-nucleotide polymorphisms in generalized vitiligo predisposition

Background/aim Interferon-induced helicase (IFIH1) is a gene locus that has been recently defined as a candidate for susceptibility to generalized vitiligo (GV). The objectives of this study were to assess the association of IFIH1 gene, rs2111485, and rs1990760 single-nucleotide polymorphisms (SNP) with susceptibility to GV and the autoimmune diseases accompanying GV.Materials and methods We prospectively studied GV patients and frequency-matched healthy controls by age and sex. The genotypes of the participants were determined for rs1990760 and rs2111485 SNPs of IFIH1. Dominant, recessive, and additive models were evaluated for each SNP adjusted for age and sex. Results The patients and their controls were observed to be in the Hardy–Weinberg equilibrium for SNP1 (2q24.2, rs1990760, IFIH1, T/C) and SNP2 (2q24.2, rs2111485, IFIH1, G/A), respectively (all P > 0.7). For SNP1, every T allel addition was significantly associated with 1.53 times protectiveness in terms of vitiligo risk (P = 0.033). As for SNP2, every G allel addition was associated with 1.42 times protectiveness, close to statistical significance (P = 0.100). Conclusions: We detected that for SNP1, each T allel and for SNP2, each G allel are protective in terms of vitiligo development. Hereby, we confirmed that IFIH1 gene locus has a role in GV susceptibility.

The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual

BackgroundVitiligo is a common acquired depigmentation skin disease characterized by loss or dysfunction of melanocytes within the skin lesion, but its pathologenesis is far from lucid. The gene expression profiling of segmental vitiligo (SV) and generalized vitiligo (GV) need further investigation. ObjectiveTo better understanding the common and distinct factors, especially in the view of gene expression profile, which were involved in the diseases development and maintenance of segmental vitiligo (SV) and generalized vitiligo (GV). MethodsPeripheral bloods were collected from SV, GV and healthy individual (HI), followed by leukocytes separation and total RNA extraction. The high-throughput whole genome expression microarrays were used to assay the gene expression profiles between HI, SV and GV. Bioinformatics tools were employed to annotated the biological function of differently expressed genes. Quantitative PCR assay was used to validate the gene expression of array. ResultsCompared to HI, 239 over-expressed genes and 175 down-expressed genes detected in SV, 688 over-expressed genes and 560 down-expressed genes were found in GV, following the criteria of log2 (fold change)⿥0.585 and P value<0.05. In these differently expressed genes, 60 over-expressed genes and 60 down-expressed genes had similar tendency in SV and GV. Compared to SV, 223 genes were up regulated and 129 genes were down regulated in GV. In the SV with HI as control, the differently expressed genes were mainly involved in the adaptive immune response, cytokine-cytokine receptor interaction, chemokine signaling, focal adhesion and sphingolipid metabolism. The differently expressed genes between GV and HI were mainly involved in the innate immune, autophagy, apoptosis, melanocyte biology, ubiquitin mediated proteolysis and tyrosine metabolism, which was different from SV. While the differently expressed genes between SV and GV were mainly involved in the metabolism pathway of purine, pyrimidine, glycolysis and sphingolipid. ConclusionsAbove results suggested that they not only shared part bio-process and signal pathway, but more important, they utilized different biological mechanism in their pathogenesis and maintenance. Our results provide a comprehensive view on the gene expression profiling change between SV and GV especially in the side of leukocytes, and may facilitate the future study on their molecular mechanism and theraputic targets.

Relationship between autophagy of melanocytes in patients with vitiligo and clinical types

To evaluate autophagy of melanocytes and its mechanism in patients with vitiligo and to analyze its correlation with clinical types of vitiligo. Nine cases of segmental vitiligo (SV) and 11 cases of generalized vitiligo (GV) were recruited in Hangzhou Third Hospital between May 2014 and June 2015. Six people with healthy skin were recruited as controls. Epidermal melanocytes were obtained from the normal colour skin around the white spot area in SV and GV patients and from foreskin in controls for culture in vitro. Cultures for each group contained negative control and rapamycin (30 nmol/L) sub-groups. The autophagy was observed using transmission electron microscopy (TEM) and immunofluorescence laser scanning confocal microscope (LSCFM). Protein expressions of microtube-associated protein light chain 3 (LC3Ⅱ) and microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase related protein (TYRP)1, and TYRP2 were detected by Western blot. (1)Autophagy of melanocytes was observed under TEM and LSCFM in both SV and control groups before rapamycin treatment, but not in GV patients. The expressions of autophagosome and LC3Ⅱ protein were increased in melanocytes in SV, GV and control groups after autophagy induction(SV group 0.58±0.10 vs 0.37±0.06; GV group 0.57±0.16 vs 0.22±0.08; control group 0.67±0.09 vs 0.46±0.12), and the autophagy intensity was higher in the GV group compared to the SV group and the control group. (2)Before autophagy induction, the expressions of MITF, TYR, TYRP1, and TYRP2 protein were lower in the GV and SV patients compared with the control group; after autophagy induction, the expressions of MITF, TYR, and TYRP1 statistically significantly increased in melanocytes in all the three groups(all P<0.05), while TYRP2 protein expression was not significantly changed (all P>0.05). Autophagy of melanocytes may be present in vitiligo and affect the expression of functional molecules, and is related with clinical type of vitiligo.

Hydroxychloroquine protects melanocytes from autoantibody-induced injury by reducing the binding of antigen-antibody complexes.

Vitiligo is a polygenic autoimmune disorder characterized by loss of pigmentation due to melanocyte destruction. Hydroxychloroquine (HCQ) is an effective immunosuppressant widely used in the treatment of autoimmune disorders. As generalized vitiligo (GV) is commonly considered to be a Tcell and autoantibody-induced immune disorder, the present study aimed to determine whether HCQ protects melanocytes from autoantibody‑induced disruption. Anti‑melanocyte antibodies were obtained from the serum of patients with progressive GV and the effects of HCQ on prevent the autoantibody‑induced disruption of melanocytes was observed. Cell‑based ELISA, indirect immunofluorescence and western blotting were used to analyze the autoantibody content of sera samples obtained from 32patients with progressive GV. The cytotoxicity of HCQ was detected by MTT assay, and 1µg/ml HCQ was applied to human primary melanocytes (HMCs) to examine whether it could exert protective effects against autoantibody‑induced immune injury. Flow cytometry was used to measure autoantibody binding to the surface of HMCs. Complement‑dependent cytotoxicity (CDC) and antibody‑dependent cell‑mediated cytotoxicity (ADCC) were monitored by MTT and lactate dehydrogenase‑releasing assays. The concentration of autoantibodies in sera samples taken from GV patients was significantly higher than in controls, particularly in patients who had >10% of their body surface affected by vitiligo. The majority of the autoantibodies presented in the HMCs and human keratinocytes (HKCs) and were predominantly localized to the cell surface and cytoplasm. The molecular weights of the autoantigens were identified as 30, 37‑39, 42, 53, 60‑75, 90, 100, 110, and 126kDa; the 30kDa protein was observed only in HMCs. The addition of HCQ at a concentration of 1µg/ml produced no significant cytotoxicity in HMCs and was demonstrated to reduce the binding of GV immunoglobulinG (IgG) to the surface of HMCs. HCQ also significantly decreased the effects of ADCC and CDC that were mediated by GV IgG. The present study provides evidence that HCQ dissociates autoantibody-antigen complexes on the surface of HMCs and reverses ADCC and CDC activity invitro. Thus, in addition to its effectiveness as an antimalarial therapeutic agent, HCQ may also be a promising potential treatment for patients with vitiligo.

Alteration in regulatory T cells and programmed cell death 1-expressing regulatory T cells in active generalized vitiligo and their clinical correlation.

Vitiligo is an autoimmune depigmentation disease, and defects in regulatory T cells (Tregs) have been proposed in the pathogenesis of generalized vitiligo (GV). However, the role of programmed cell death (PD)1(+) Tregs has not been studied. To investigate the status of Tregs, PD1(+) Tregs and associated parameters in active GV (aGV) during the first episode of disease attack and to establish the clinical correlation. The percentages of circulating Tregs, PD1(+) Tregs and CD3(+) CD4(+) PD1(+) T cells were evaluated in 50 patients with aGV and 51 controls. Expression levels of FOXP3, TGFB1, CTLA4 and genes for chemokine receptors (CCR4, CCR7) and their ligands (CCL21, CCL22) were quantified in peripheral blood and in lesional, perilesional, nonlesional and normal skin sections. The corresponding proteins were immunolocalized in tissue of aGV. The percentage of Tregs was decreased (P=0·001) and that of PD1(+) Tregs increased (P=0·001) in peripheral blood of patients with aGV compared with controls. The abundance of TGFB1 and CCL21 mRNA was significantly decreased in the peripheral blood of patients with aGV. Significant differences in forkhead box P3, transforming growth factor-β and CCL21 protein expression were found in skin sections. Deficiency in Treg frequency and decreased expression of Treg-associated parameters (TGFB and CCL21) suggested a possible defect in Tregs that may alter their suppression function and skin homing in aGV. The increased PD1(+) Tregs suggests that the PD1/PD ligand pathway may be involved in aGV and may have a role in Treg exhaustion. Further study is required to delineate the effect of PD1 in regulating Treg function in aGV.

Genetic susceptibility to vitiligo: Recent progress from genome-wide association studies

Generalized vitiligo (GV) is a complicated disease in which patchy depigmentation results from the autoimmune loss of melanocytes from affected regions. It may follow a pattern of polygenetic or multifactorial inheritance. Previously, a number of genetic susceptibility factors have been identified through linkage and candidate gene studies, such as HLA , PTPN22 , NALP1 , and XBP1 . Recently, a series of genome-wide association studies have been carried out in different populations to further explore the susceptibility variants for GV. More than 30 robust susceptibility loci have been identified and confirmed. Most of these associated genes encode components of biological pathways reaching from the immune cell to the melanocytes. In this review, we summarize the advances of vitiligo epidemiology and genetics, and highlight recent findings from genome-wide association studies, emphasizing susceptibility loci and comparing the susceptibility loci between Chinese people and Caucasians. These genetic studies may help in providing an insight into the pathogenesis of this disease and initiating the feasibility of genetic diagnosis and personalized treatment for patients with GV in the future.

Association ofNLRP1genetic variants and mRNA overexpression with generalized vitiligo and disease activity in a Gujarat population

It has been suggested that NLRP1 is involved in susceptibility to a wide range of autoimmune diseases including generalized vitiligo (GV). Genetic polymorphisms in the gene encoding NLRP1 (previously known as NALP1) have previously been shown to be associated with GV and there is speculation about their involvement in the regulation of NLRP1 expression. To explore NLRP1 polymorphisms and investigate their association with NLRP1 mRNA expression and disease activity in patients with GV. Polymerase chain reaction (PCR)-restriction fragment length polymorphism and TaqMan single nucleotide polymorphism (SNP) genotyping techniques were used to genotype NLRP1 A/G (rs2670660), T/C (rs6502867) and A/T (rs12150220) polymorphisms in 537 patients with GV and 645 controls in Gujarat. NLRP1 mRNA levels were measured in the whole blood of 122 patients with GV and 175 controls using real-time PCR. The NLRP1 rs2670660 and rs6502867 polymorphisms were found to be in significant association with GV, minor alleles of these SNPs being prevalent in active cases of GV. The rs12150220 polymorphism was found have a marginal association with GV. The frequency of susceptible haplotype 'GCT' was significantly higher in patients with GV and increased the risk of vitiligo twofold. A significant increase in NLRP1 mRNA expression was observed in patients with GV and patients with active GV. NLRP1 mRNA expression was increased in patients with GV with the susceptible GG (rs2670660) and CC (rs6502867) genotypes. Patients with the susceptible GG (rs2670660) and CC (rs6502867) genotypes had early age of onset of GV. Moreover, patients in the age at onset group of 1-20years showed increased expression of NLRP1 mRNA compared with the older age groups. Female patients showed a significant increase in NLRP1 mRNA and early age at onset of GV compared with male patients. Our results suggest that NLRP1 rs2670660 and rs6502867 polymorphisms may be genetic risk factors for susceptibility to and progression of GV. The upregulation of NLRP1 mRNA in patients with susceptible genotypes advocates the crucial role of NLRP1 in GV.

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.