Clinic Rev Allerg Immunol (2018) 54:52–67 DOI 10.1007/s12016-017-8622-7 Vitiligo: Focus on Clinical Aspects, Immunopathogenesis, and Therapy Katia Boniface 1 & Julien Seneschal 1,2 & Mauro Picardo 3 & Alain Taïeb 1,2,4 Published online: 6 July 2017 # Springer Science+Business Media, LLC 2017 Abstract Vitiligo is an acquired chronic depigmenting disorder of the skin, with an estimated prevalence of 0.5% of the general population, characterized by the development of white macules resulting from a loss of epidermal melanocytes. The nomenclature has been revised after an extensive international work within the vitiligo global issues consensus conference, and vitiligo (formerly non-segmental vitiligo) is now a consensus umbrella term for all forms of generalized vitiligo. Two other subsets of vitiligo are segmental vitiligo and unclassified/undetermined vitiligo, which corresponds to focal disease and rare variants. A series of hypopigmented disorders may masquerade as vitiligo, and some of them need to be ruled out by specific procedures including a skin biopsy. Multiple mechanisms are involved in melanocyte disappearance, namely genetic predisposition, environmental triggers, metabolic abnormalities, impaired renewal, and altered inflammatory and immune responses. The auto-immune/ inflammatory theory is the leading hypothesis because (1) vitiligo is often associated with autoimmune diseases; (2) most vitiligo susceptibility loci identified through genome-wide association studies encode immunomodulatory proteins; and (3) Katia Boniface and Julien Seneschal equally contributed to this work. * Alain Taïeb [email protected] 1 INSERM U1035, ATIP-AVENIR, Université de Bordeaux, Bordeaux, France 2 Department of Dermatology and Paediatric Dermatology, National Centre for Rare Skin disorders, Saint-André and Pellegrin Hospital, Bordeaux, France 3 San Gallicano Institute, Rome, Italy 4 Department of Dermatology and Pediatric Dermatology, St André Hospital, Bordeaux University Hospitals, 1 Rue Jean Burguet, 33075 Bordeaux, France prominent immune cell infiltrates are found in the perilesional margin of actively depigmenting skin. However, other studies support melanocyte intrinsic abnormalities with poor adaptation of melanocytes to stressors leading to melanocyte instability in the basal layer, and release of danger signals important for the activation of the immune system. Recent progress in the understanding of immune pathomechanisms opens interesting perspectives for innovative treatment strategies. The proof of concept in humans of targeting of the IFNγ /Th1 pathway is much awaited. The interplay between oxidative stress and altered immune responses suggests that additional strategies aiming at limiting type I interferon activation pathway as background stabilizing therapies could be an interesting approach in vitiligo. This review covers classification and clinical aspects, pathophysiology with emphasis on immunopathogenesis, and promising therapeutic approaches. Keywords Vitiligo . Review . Pathophysiology . Immunopathology . Therapy Abbreviations CRT Calreticulin CTL Cytotoxic T cells ER Endoplasmic reticulum HSP Heat shock protein IFN Interferon IL Interleukin NK Natural killer pDCs Plasmacytoid dendritic cells PRR Pathogen recognition receptors ROS Reactive oxygen species SV Segmental vitiligo TLR Toll-like receptors Clinic Rev Allerg Immunol (2018) 54:52–67 TNF Tregs UPR VGICC VETF Tumour necrosis factor Regulatory T cells Unfolded protein response Vitiligo Global Issues Consensus Conference Vitiligo European task force Introduction Vitiligo is the most common skin depigmenting disorder resulting from a selective loss of epidermal melanocytes, and affects around 0.5% of the world population [1, 2]. Both sexes are equally affected, and there are no apparent differences in rates of occurrence according to phototype or race. Twentyfive percent of cases are children with disease onset before the age of 10, the age of onset in paediatric series varies from 4 to 8 years. Very early onset, as young as 3 months, is acknowledged. The existence of true ‘congenital vitiligo’ remains controversial. In fair-skinned individuals, vitiligo patches are usually detected only after the first exposure of the skin to sunlight, following the first summer of life. The percentage of segmental vitiligo (SV) is higher in children compared to adults, whatever the ethnic background, suggesting a mosaic skin developmental predisposition . The prevalence of SV in childhood varies from 4.6 to 32.5% in published reports . Vitiligo is a complex disease, associating genetic and environmental factors together with metabolic and immune alterations. Abnormalities leading to impaired melanocyte regeneration and/or proliferation suggest a primary defect of melanocytes . However, a major role of silent inflammation and autoimmunity is demonstrable, in particular during the progressive phase of the disease [5–10]. Since neither immune nor non-immune mechanisms in isolation can sufficiently explain all parts of this complex disease, a convergence of combined biochemical, environmental, and immunological factors in genetically predisposed patients has been proposed as a unifying background to the pathophysiology of vitiligo . This review summarizes classification, clinical aspects, and current as well as promising novel therapeutic approaches. The role of the immune response in vitiligo pathogenesis is particularly emphasized because of its therapeutic implications in relation to current drug development. 53 increasing in size progressively or during flares with time, corresponding histologically to a substantial loss of functioning epidermal pigment cells and, usually in a second time, of hair follicle melanocytes. Two other subsets of vitiligo are SV, and unclassified/undetermined vitiligo, which corresponds to focal disease and rare variants. A series of hypopigmented disorders may masquerade as vitiligo, and some of them need to be ruled out by specific procedures including a skin biopsy [1, 2]. Clinical Aspects Vitiligo Subsets Generalized Common Vitiligo This most common form of vitiligo (Fig. 1a) is characterized by milky-white macules involving multiple parts of the body, most often in a symmetrical pattern. Skin hypopigmentation is usually asymptomatic, but a minority of patients mention preceding mild pruritus. The disease can start at any site of the body, but the fingers, hands, and face are frequently the initial sites. Clinical markers of progression are important to detect such as pinpoint depigmentation and fuzzy limits of white macules (Fig. 1b). Obtaining a validated comprehensive list of validated clinical markers of progression is currently an international goal, because history-based scores such as VIDA  are considered as grossly inaccurate. Depigmentation of scars (Fig. 1c) is a common manifestation of the Koebner’s phenomenon (mechanical induction of the disease, also by friction or chronic pressure by clothing or daily activities). Koebner’s phenomenon is usually contemporary of disease flares . Stable lesions are well demarcated (Fig. 1d). Mixed vitiligo is a recently described, mostly paediatric subtype, with segmental involvement preceding typical generalized vitiligo , now included as a subset of vitiligo in the VGICC classification. The presence of leukotrichia and halo nevi have been noted as predictors of passage to mixed vitiligo in patients with SV. Mixed vitiligo may exist in adults but is probably frequently masked by widespread bilateral lesions. Classification Acrofacial Vitiligo The nomenclature has been revised after an extensive international work within the Vitiligo Global Issues Consensus Conference (VGICC) . Vitiligo is now a consensus umbrella term for all forms of generalized vitiligo (formerly designated in the international nomenclature as non-segmental vitiligo) defined as an acquired chronic pigmentation disorder characterized by white patches, most often symmetrical, In acrofacial vitiligo, the involved sites are usually limited to face, head, hands, and feet. A distinctive feature is depigmentation of the distal fingers and facial orifices. It may later include other body sites, resulting in typical generalized vitiligo. Acrofacial vitiligo was shown to be more frequent in adult onset cases of vitiligo in a large series studies using latent class analysis . 54 Clinic Rev Allerg Immunol (2018) 54:52–67 Fig. 1 Clinical features of vitiligo. a Vitiligo/non-segmental vitiligo. b Confetti lesions and fuzzy borders in rapidly progressive vitiligo. c Koebner’s phenomenon on abdominal surgical scars, in a case of progressive vitiligo. d Stable vitiligo with well-demarcated macules. e Extensive vitiligo progressing towards universal vitiligo. f Segmental vitiligo, large blaschkolinear pattern. g Segmental vitiligo, checkerboard pattern, with evidence of follicular repigmentation after phototherapy. h Vitiligo ‘ponctué’/leucoderma punctate. i Hypochromic vitiligo (Courtesy Dr. Silvia Moretti, Florence). j Follicular vitiligo Vitiligo Universalis Segmental Vitiligo Vitiligo universalis (Fig. 1e) is a rare presentation of vitiligo. It is the most extensive form of the disease and generally occurs in adulthood. ‘Universalis’ is generally used when depigmentation is virtually universal (80–90% of body surface), but some pigmentation may be still present, and hairs partially spared. Whereas this diagnosis is easy in dark-skinned individuals, it may be more challenging in fair-skinned individuals. Mono-segmental vitiligo is the most common form of SV, referring to the presence of one or more white depigmented macules distributed on one side of the body, usually respecting the midline (although some lesions may partly cross the midline), early follicular involvement (leukotrichia), and rapid development over a few weeks or months, and overall protracted course, but secondary extension remains possible in a given segment sometimes years after. The shape of SV Clinic Rev Allerg Immunol (2018) 54:52–67 lesions may recapitulate some developmental patterns such as large band Blaschko lines (Fig. 1f). The long held neural theory based on possible dermatomal distribution is challenged by many exceptions. The aetiology of the SV pattern remains overall elusive. Rarely, multiple segmental lesions occur simultaneously or not distributed either unilaterally or bilaterally. A clear segmental distribution of the lesions with midline demarcation, together with the associated features described in mono-segmental cases (leukotrichia, protracted course), distinguishes this diagnosis versus vitiligo in bilateral cases. As in vitiligo, repigmentation may occur, usually under combined therapy (see below), according to a marginal, follicular, or mixed pattern (Fig. 1g). Unclassified and Rare Variants The VGICC classification suggests that focal cutaneous or mucosal vitiligo (defined as small isolated patch that does not fit a segmental distribution, and which has not evolved into vitiligo after a period of at least 2 years) should be until better consensus on nature and course left within the category undetermined⁄unclassified vitiligo. Some rare variants are discussed below. Vitiligo ponctué/punctata Lesions present as sharply demarcated depigmented punctiform 1- to 1.5-mm macules involving any area of the body (Fig. 1h), and has to be distinguished histopathologically from guttate hypomelanosis, a common condition with no loss of melanocytes situated on chronically sun exposed sites such as the legs and forearms. Vitiligo Minor/Hypochromic Vitiligo This disease seems to affect only dark-skinned individuals (Fig. 1i). ‘Minor’ refers to a partial defect in pigmentation. The relation to true vitiligo comes from pathology and coexistence with more typical vitiligo macules. Cutaneous T cell lymphoma needs to be ruled out by repeated biopsies with molecular studies of clonality, and this diagnosis cannot be made without a long-term follow-up . Follicular Vitiligo This designation refers to a form recently described in a young black patient of generalized vitiligo that primarily involved the pigment cell follicular reservoir with limited skin involvement, contrasting with marked generalized hair whitening  (Fig. 1j). As noted later in a small series of patients, contrary to what occurs in common vitiligo, cutaneous depigmentation follows hair depigmentation which is situated not only in 55 vitiliginous areas but also in areas with clinically normalappearing skin . Immunopathogenesis of Vitiligo Intrinsic Abnormalities of Melanocytes and Keratinocytes Several in vitro and in vivo studies have revealed an altered redox status, with the presence of oxidative stress in cultured melanocytes coupled with an increased susceptibility to prooxidant agents [20, 21]. Elevated levels of reactive oxygen species (ROS) have been observed in lesional and nonlesional skin of vitiligo. The increased ROS production by melanocytes could result from an external stress, such as ultraviolet (UV) radiation exposure or chemical damage (monobenzone or other phenols). A wide range of metabolic pathways leads to the uncontrolled generation of ROS, and several lines of evidence suggest that mitochondria could be the main source of ROS in vitiligo [22, 23]. ROS can also create a pro-inflammatory environment that will contribute to the activation of the immune system . Moreover, vitiligo melanocytes, probably as a consequence of increased intracellular oxidative stress, present abnormalities of signal transduction pathways compatible with a condition of stressinduced premature senescence-like phenotype. This is characterized by the production of many proteins among the senescence-associated secretory phenotypes, including the production of pro-inflammatory cytokines as interleukin (IL)-6, matrix metalloproteinase 3 (MMP3), cyclooxygenase-2, insulin-like growth factor-binding protein 3 (IGFB3), and IGFBP7 . In addition, recent observations showed that melanocyte exposure to chemical agents (4-TBP—4-tertiary butyl phenol—and MBEH—monobenzyl ether of hydroquinone) known to trigger vitiligo induces the disruption of the folding machinery of the endoplasmic reticulum (ER), leading to the accumulation of immature proteins and resulting in the activation of the unfolded protein response (UPR) . UPR activation results in transient attenuation of protein synthesis, increased capacity for protein trafficking through the ER, protein folding transport, and increased protein degradative pathways, including ER-associated degradation (ERAD) and autophagy. If these adaptive mechanisms cannot resolve the protein-folding defect, as in vitiligo, cells enter apoptosis. The UPR comprises three parallel signalling branches: PRKR-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eiF2α) pathway, activating transcription factor 6α (ATF6α), and the inositol-requiring enzyme-1 (IRE1)–X-box-binding protein 1 (XBP1). Genome-wide linkage analysis followed by a sequencing study in a Chinese population with vitiligo identified XBP1 as a candidate gene for vitiligo predisposition, which was then confirmed in a vitiligo Caucasian cohort [27–29]. More recently, He et al. 56 Clinic Rev Allerg Immunol (2018) 54:52–67 demonstrated that vitiligo melanocytes are more sensitive to oxidative stress and autophagy, through the impairment of the nuclear factor E2-related factor 2 (Nrf2) protein, a critical transcription factor protecting cells from oxidative stress  (Fig. 2). It is also important to note that Nrf2 is involved in the UPR activation and acts as a direct PERK substrate. Fig. 2 Immunopathogenesis of vitiligo. In a predisposed patient, melanocytes present abnormalities that will lead to the release of DAMPs and inflammatory cytokines that will contribute to activation of the innate immune response and subsequently to adaptive T cell responses, through activation of TRM cells that reside in the skin and recruitment of TEM expressing CXCR3 and/or CCR6. These T cell subsets will produce soluble factors (in particular TNFα and IFNγ) involved in melanocyte loss and development of white patches Clinic Rev Allerg Immunol (2018) 54:52–67 Therefore, stress melanocytes can release pro-inflammatory cytokines but also chemokines important for the recruitment of immune cells, such as CXCL12 or CCL5 . Intrinsic abnormalities of melanocytes are also associated with decreased expression of adhesion molecules on melanocytes, such as E-cadherin. We have recently demonstrated that vitiligo melanocytes in normal-appearing skin of vitiligo patients have impaired expression of E-cadherin, inducing their detachment from the basal layer of the epidermis under stress conditions . Nonetheless, several lines of evidence suggest that melanocyte is not the only epidermal cell involved in the induction of the immune response in vitiligo. Recent studies highlighted that vitiligo keratinocytes could also be an important actor through the release of chemokine ligands important for the recruitment of T cells. Under oxidative stress, UPR activation in keratinocytes induced the production of CXCL16 by stress keratinocytes, leading to the recruitment of CXCR6+ CD8 T cells . CXCL12 and CCL5 are additional chemokines that are likely involved in T cell homing to the skin in vitiligo . Activation of the Innate Immune Response Through Damage-Associated Molecular Patterns (DAMPS) and Other Pro-Inflammatory Signals Increased oxidative stress, impairment of the UPR activation, or pathways leading to cell death in both melanocytes and keratinocytes can induce production of pro-inflammatory cytokines and activation of signals important for the activation of the immune system in vitiligo. Indeed, activation of the innate immune system through the activation of pathogen recognition receptors (PRRs)  is an important mechanism in host defence against pathogens but could be impaired under some circumstances and could induce autoimmunity in a genetically predisposed patient. These receptors are originally specialized in the detection of pathogens through pathogen-associated molecular patterns (PAMPs). Host-derived self-DNA and/or self-RNA from damaged cells recognized by these nucleic acid receptors could lead to skin inflammation such as that found in vitiligo. The view of the machinery of nucleic acid recognition became more complex with the recent identification of several new nucleic acid receptors . Nucleic acid receptors are commonly divided in two subgroups based on their endosomal or cytosolic distribution. In the endosomes, these receptors include Toll-like receptors (TLR) including TLR3, TLR7, TLR8, and TLR9. In the cytosol, nucleic acid receptors embrace the retinoic acid-inducible gene I (RIG-I)like receptors, the cytosolic DNA sensors and receptors are involved in inflammasome formation. In endosomes, TLR9 senses the presence of CpG-containing DNA whereas TLR7 and TLR8 recognize single-strand (ss) RNA, and TLR3 recognizes double-strand (ds) RNA, leading to the transcription of pro-inflammatory cytokines like tumour necrosis factor 57 (TNF)-α, IL-6, or type I interferon (IFN) α/β [36–38]. Cytosolic receptors like the gamma-IFN-inducible protein IFI16 and the IFN-inducible protein AIM2 (absent in melanoma-2) sense dsDNA and recruit the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD) to form an inflammasome complex that activates caspase 1, leading to the processing and release of IL-1β and IL18 [39, 40]. Several inflammasomes have been described and are defined by the NOD-like receptor proteins that they contain: the NLRP1 (NALP1) inflammasome, the LRP3 (NALP3) inflammasome, and the IPAF (NLRC4) inflammasome . Our group has previously reported an increased expression of NLRP1 and IL-1β in perilesional skin of vitiligo that was associated with disease progression  (Fig. 2). More recently, a major focus of interest has been the stimulator of interferon gene (STING), which is a transmembrane protein of the endoplasmic reticulum, which upon activation induces type I IFN production . Several DNA sensors that include IFI16, the DNA-dependent activator of IFNregulatory factors (DAI), and cyclic GMP-AMP synthase (cGAS) can activate the STING pathway. Beyond the recognition of dsDNA, dsRNA are also recognized by other cytosolic recognition receptors such as RIG-like receptors (RLRs) which belong to a superfamily including RIG-I, melanoma differentiation-associated protein 5 (MDA-5) also called IFIH1, and laboratory of genetics and physiology 2 (LGP2). These sensors, upon recognition of RNA, induce activation of the nuclear factor κB (NF-κB) pathway, leading to the production of pro-inflammatory cytokines . As already suggested, in a genetically predisposed patient, a self-molecular pattern released during sterile inflammation in damaged cells can lead to the release of pro-inflammatory cytokines and induce skin inflammation as shown in vitiligo [45, 46]. Again, recent genome-wide association studies (GWAS) have revealed multiple genetic risk factors for vitiligo associated with these PRRs . For example, it has been reported an association of vitiligo with genes encoding NLRP1, IFIH1, the Toll-like receptor adaptor molecule 1 (TICAM1), important for TLR signalling, and caspase 7, a protein activated by the inflammasome complex. Recently, Spritz’s group has shown that variants of IFIH1 associated with loss of function could protect against vitiligo . In addition to nucleic acid recognition, the NLRP3 inflammasome is activated in response to excessive ROS production and mitochondrial stress . Heat shock proteins (HSPs) are protein-folding chaperones induced in response to cellular stress and UPR activation. They can activate TLR2, TLR4, and other PRRs . Inducible HSP (HSPi) is induced in chemical-induced stress in melanocytes and in vitiligo. Interestingly, it was recently demonstrated that HSP70i induces vitiligo in a mouse model of the disease, and accelerates disease progression [45, 46]. 58 Recently, we have found that HSP70i released by epidermal cells can potentiate the production of IFNα by plasmacytoid dendritic cells (pDCs) that have been shown to play a crucial role in the induction of the disease . IFNα can induce the production of CXCL9 and CXCL10 by epidermal cells, inducing an initial signal for the recruitment of effector T cells during the initiation of the disease. High-mobility group box 1 (HMGB1) is an important DNA-binding protein that is present in the nucleus of cells, where it regulates DNA accessibility to transcription factors [51, 52]. During stress and cell death (necrosis), HMGB1 is released into the extracellular environment, can act as an alarmin, and can interact with multiple immune sensors and receptors such as advanced glycation end-products (RAGE) as well as TLR2, TLR4, and TLR9, leading to activation of NF-κB and production of IL-6 and TNFα. Moreover, HMGB1 could act directly on melanocytes leading to their loss in vitiligo  but also binds free DNA and HMGB1-DNA complexes which can induce type I IFN production by pDCs. Another candidate for sensing the immune system in vitiligo is calreticulin (CRT), which is a ubiquitous protein localized predominantly in the endoplasmic reticulum and that plays a central role in intracellular Ca2+ homeostasis . However, in response to stress, CRT can also localize at the surface of cells such as immune cells, affecting antigen presentation, complement activation, and clearance of apoptotic cells. Moreover, CRT can also translocate to the melanocyte surface when these cells undergo H2O2-mediated oxidative stress, increasing melanocyte immunogenicity and providing a new link between oxidative stress, cell death, and immune reactions. CRT also induces the expression of pro-inflammatory cytokines like IL-6 and TNFα, enhancing the immunogenic potential of melanocytes  (Fig. 2). Surface CRT directs the contact of ROS-stressed melanocytes to dendritic cells and Langerhans cells, followed by the activation of downstream adaptive immune responses, leading to the development of the disease. Lastly, S100B is a DAMP protein expressed by melanocytes that has been recently found to be upregulated in vitiligo patients and could be involved in disease development . Local inflammation can also activate natural killer (NK) cells, found to be increased in vitiligo skin . NK cells respond to various cellular signals released by cells under stress, and can be activated through ligands binding to their receptors NKG2D. Besides their cytotoxic function, NK cells influence antigen presentation; stimulate function, survival, and maturation of DCs through the release of proinflammatory cytokines; and drive the adaptive immune response. Adaptive Immunity Several lines of evidence have pointed out the involvement of T cells and their related cytokines in development of vitiligo. Clinic Rev Allerg Immunol (2018) 54:52–67 GWAS studies identified an association of both major histocompatibility complex (MHC) class I and II loci with vitiligo [29, 57, 58], and CD8 and CD4 T cells are consistently found at the edge of actively depigmenting skin of vitiligo patients [5, 59]. In addition, suppression of immune tolerance in vitiligo likely involves an altered proportion and/or function of effector and regulatory T cells (Tregs) . CD4 T Cell Subset Involvement in Vitiligo The involvement of autoreactive CD4 T cells in melanocyte loss is still unclear. CD4 T cells play a major role in coordinating the immune response and seem important for the generation of cytotoxic CD8 T cells . Studies using melanocyte-specific T cell receptor (TCR) transgenic mouse models suggest that CD4 T cells are involved in depigmentation [62–64] and that melanocyte loss in such models involve Fas-Fas ligand (FasL)-induced signalling . Dysregulation of adaptive immune responses with excessive production of Th1-, Th2-, Th17-, Th22-, and/or Th9-related cytokines is a common feature of a wide range of inflammatory and autoimmune diseases, including skin inflammatory disorders (e.g., psoriasis, atopic dermatitis, alopecia areata). Early reports revealed that both CD4 and CD8 T cells produce mainly IFNγ and TNFα in vitiligo , which is the signature of a Th1/Tc1 cell polarization (this aspect will be further developed in the ‘CD8 T Cell-Mediated Immune Response’ section) (Fig. 2). The precise role of Th17 cells in melanocyte disappearance in vitiligo remains to be fully determined. Serum IL-17 levels are positively correlated with the extent of depigmentation . IL-17 expression is increased both in blood and in perilesional skin of vitiligo patients, and T cells appear as the main source of this cytokine [9, 67–69]. In addition, the upregulation of IL-1β observed in perilesional skin of vitiligo patients may act as an inducer of Th17 cells [9, 42]. However, the impact of IL-17 on depigmentation has not been yet extensively studied. Results from the literature suggest that IL17 on its own has little effect on melanogenesis and would rather act indirectly on pigmentation by enhancing the effects of TNFα, IL-1β, or IL-6 to inhibit melanocyte function and/or survival directly or indirectly though upregulation of proinflammatory chemokine production [70, 71]. Hence, if inhibition of the Th17 pathway has already proven efficacy in autoimmune and inflammatory disorders like psoriasis, whether targeting this pathway would be relevant in vitiligo still needs further investigation. The balance between inflammatory and immunoregulatory factors is critical to prevent tissue damage during infection and in the control of detrimental responses to self-antigen. The VITGEN consortium has identified genetic polymorphisms of FOXP3, CTLA-4, IL-10, and TGFβ receptor in vitiligo [29, 47, 60], and several studies reported alterations in Treg number and/or function in vitiligo patients . The number Clinic Rev Allerg Immunol (2018) 54:52–67 of Tregs expressing transcription factor FOXP3 is significantly reduced in the skin of vitiligo patients [72, 73], contrasting with the presence of functional Tregs in the circulation of vitiligo patients . Impairment of Treg function is likely involved in the loss of tolerance in vitiligo since lower suppressive activities of Tregs towards CD8 T cells together with an increase in IFNγ and TNFα production have been reported in patients [74, 75]. Adoptive transfer of Tregs or treatment with rapamycin (known to promote expansion of Tregs) can efficiently halt the depigmentation process in disease-prone mice . CD8 T Cell-Mediated Immune Responses in Vitiligo Accumulating evidence highlights the major role of CD8 T cells in melanocyte loss in vitiligo. CD8 T cells in the skin are more abundant in patients with active disease compared to patients with stable disease or healthy controls; nonetheless, such infiltration remains lower than the one observed in cutaneous lupus . These cells produce Tc1-related cytokines like IFNγ and TNFα. CD8 T cells specific for melanocyte antigens, such as MelanA/MART-1, gp100, and tyrosinase, have been detected in the peripheral blood and perilesional skin of patients with vitiligo, although conflicting results were reported [65, 77–82]. The progression of the disease could be directly related to circulating MelanA-specific CD8 T cells [77, 78], although the functionality of these cells in vitiligo was raised . The frequency of circulating CD8 T lymphocytes expressing granzyme B, perforin, and IFNγ is higher in vitiligo patients than in healthy controls . Such activation of CD8 T cells correlates with impairment of Tregs. In addition, it was reported that Tregs can render self-reactive CD8 T cells hypoproliferative and cytokine hypoproducing. Anergic CD8 T cells reactive with MelanA antigen were also identified in the blood of healthy individuals . Upon self-antigen stimulation and in the absence of appropriate Treg number and/or function, as observed in vitiligo, such cells may become activated and may contribute to the development of abnormal immune responses. CD8 T cells, also known as cytotoxic T lymphocytes (CTL), can directly induce cytolysis of target cells through two distinct pathways: the release of soluble cytotoxic molecules like granzyme B and perforin, and the FasL/Fas interaction, which triggers apoptosis of target cells. The first direct evidence of cytotoxic T cell responses causing skin depigmentation came from melanoma patients, where the majority of T cells infiltrating melanoma tumours are reactive to MelanA and gp100 [84, 85]. CD8 T cells from perilesional skin of vitiligo patients recognize melanocyte antigens; express granzyme B and the proinflammatory cytokines IFNγ, TNFα, and IL-17; and can induce autologous melanocyte apoptosis in normally pigmented skin in vitro [65, 82, 86, 87], suggesting the involvement of a cytotoxic response to melanocytes. The association of 59 autoreactive CTL with depigmentation is also supported in vivo in disease-prone mouse models of vitiligo [61, 88–90]. Yet, in vivo studies performed in similar mouse models showed that depigmentation in mice depends on IFNγ, while cytotoxic mediators like perforin or granzyme were dispensable [88, 90, 91]. Most of the existing mouse models of vitiligo focus on depigmentation of the hair rather than the epidermis and thus may not fully reflect human disease. Those models also feature sensitization steps which have not been validated in naturally occurring vitiligo, and may be more relevant to melanoma-associated depigmentation or vitiligo-like depigmentations associated with immunotherapy used for treating metastatic melanoma . In addition, we have shown that despite a higher granzyme B expression in the skin of patients with active vitiligo compared to patients with stable disease or healthy controls, its expression remained significantly lower than in lupus, the archetype of a cytotoxic-mediated disease . It appears therefore critical to compare the phenotype and function of skin CD8 T cells populating human vitiligo skin to other cutaneous inflammatory diseases, which has never been reported so far. Indeed, studies performed on human vitiligo samples mostly assessed peripheral blood cells, and studies that analysed the skin T cell infiltrate in vitiligo were mainly performed by immunohistochemistry, which is a hurdle to fully define the phenotype of infiltrated T cells. T cells homing to tissues involve chemokine ligandmediated attraction of T cells expressing their cognate receptors. Recently, as suggested above, release of the chemokine ligands CXCL16 by keratinocytes and CXCL12 and CCL5 by melanocytes in vitiligo under oxidative stress could be important for the recruitment of T cells [31, 33]. Moreover, accumulating evidence is pointing out the critical role of the CXCR3/ CXCL9-CXCL10 axis in vitiligo pathogenesis. The expression of CXCR3 and its ligands CXCL9 (also known as monokine induced by IFNγ, MIG) and CXCL10 (also known as IFNγ-inducible protein-10, IP-10) is increased in the skin of patients with vitiligo [10, 93]. These chemokines are induced by IFNγ in various cell types, including immune and epithelial cells. Analysis of the transcriptional profile of lesional vitiligo skin revealed an IFNγ-specific signature . IFNγ is produced by both CD8 and CD4 T cells in vitiligo, although expression by other immune cells and skin cells is not excluded and has to be assessed. In vivo studies performed in mouse models of vitiligo also support the critical role of the IFNγ/CXCR3/CXCL10 pathway [76, 88, 90, 93, 94]. Importantly, accumulation of CD8+ T cells in mouse skin is dependent on IFNγ and CXCR3. Hence, in vitiligo skin, the increased expression of IFNγ and TNFα will induce CXCL9 and CXCL10 secretion together with the initial role played by IFNα, leading to the recruitment of T cells bearing CXCR3 in the skin, therefore amplifying inflammation (Fig. 2). These inflammatory cytokines also directly impact melanocyte function. IFNγ can inhibit 60 melanogenesis and induce melanocyte apoptosis and senescence [95, 96], although high concentrations of IFNγ have to be used to obtain a significant effect. IFNγ also seems important for maintaining epidermal pigmentation homeostasis . An increased expression of TNFα in vitiligo patients’ skin has been consistently observed , and both CD8 and CD4 T cells have been identified as potent producers [65, 82]; yet, keratinocytes, fibroblasts, and even melanocytes could be other sources of this cytokine under inflammatory conditions. In vitro studies revealed direct inhibitory effects of TNFα on melanogenesis and melanocyte function [99, 100]. TNFα, together with IL-17, upregulates production of melanoma mitogens CXCL1 and IL-8 in melanocytes while suppressing genes of the pigmentation pathway . Skin Resident T Cells Human skin contains 20 billion T cells, representing twice more than in the entire blood volume, and most of them are resident T cells . Human skin is populated by distinct populations of both recirculating and resident T cells that protect our organism against invading pathogens [102, 103]. Resident memory cells rapidly respond to pathogen or foreign antigens that attempt to breach skin epithelium, independently of T cell recruitment from the circulation. These skin-resident T cells are nonrecirculating T cells that display an effector memory phenotype and express high levels of skin-homing receptors such as cutaneous lymphocyte antigen (CLA) and the chemokine receptor CCR4, and they are characterized by expression of CD69 (also known to be a T cell activation marker) and CD103 (also known as αE integrin). Inadvertent activation of these skin TRM must be tightly controlled, and we have shown in a different context that a skin resident population of Tregs can locally proliferate and dampen skin effector memory T cell responses . In a predisposed patient, dysregulation of TRM could be very damaging in the context of inflammatory disorders, such as vitiligo. To date, whether TRM cells are involved in melanocyte loss in vitiligo remains unknown. Very recently, Cheuk et al. reported that CD49a defines a specialized subset of tissue-resident CD8 T with cytotoxic potential that could be involved in vitiligo . In addition, vitiligo often recurs on the same anatomic sites, in favour of a local memory immune response. Therefore, investigating the possible contribution of autoreactive skin TRM cell subsets in vitiligo would add a better understanding of the specific immune memory response in this disease and is important to determine the optimal treatment strategy for the patient. Management and Therapy Overview Several issues are important for managing appropriately a patient with vitiligo. Many patients are concerned by Clinic Rev Allerg Immunol (2018) 54:52–67 disfigurement and difficulties in social interaction. The treatment plan should take into consideration the patient’s priorities in this respect. The risk of transmission of vitiligo and its association with other diseases are also questioned by parents and patients. Concerning the transmission of vitiligo, the fear of the disease explains the referral of young children in vitiligo families for minor lesions such as pityriasis alba or nevus depigmentosus, and reassurance is needed. Data on the heritability of vitiligo among most races studies indicate a risk of vitiligo in first-degree relatives of 4–6%, a marked increase over the 0.5% assumed prevalence of vitiligo . Common genetic variants associated with the disease have been identified, and provide insights into biological pathways and reveal possible novel drug targets . Some rare family trees show an impressive aggregation of cases of vitiligo and autoimmune disorders, and may justify a more comprehensive autoimmune screen. Consanguinity is clearly a risk factor, as shown in Middle East studies . In general, thyroid autoimmunity and function are prescribed as a baseline test. The need to reiterate tests on a regular basis has been advocated in female patients and patients with longer duration of disease and greater body surface involvement which are more likely to present with autoimmune thyroid disease . An atopic diathesis is common in childhood onset vitiligo, but generally the sites of involvement are distinct and Koebnerization due to atopic dermatitis lesions is not an important issue . Most patients receive a strong warning against sun exposure to prevent skin cancer, and recommending phototherapy for treatment may be difficult. Recent studies analysing the risk of skin cancer have been reassuring, suggesting that vitiligo provides a better surveillance against skin cancer, not only melanoma . Overall, there is no evidence indicating that vitiligo per se changes one individual’s longevity. Vitiligo treatments have so far been analysed using the proportion of treated patients who achieve a specified degree of repigmentation, usually starting at >50% for a ‘good’ response. ‘Good’ for this level of skin repigmentation is indeed debatable in a patient-oriented view because satisfaction needs, in addition to stopping disease progression, complete restoration of pigmentation, especially in visible areas. This point has been recently examined in depth by the VGICC . The VETF has proposed a simple method of assessment which combines analysis of extent, grading of depigmentation, and progression . Clinical photographs and, if possible, UV light photographs are needed for accurate monitoring of repigmentation. For stopping progression, besides UV therapies, systemic steroids have been evaluated mostly in open studies and seem to arrest disease progression. Commonly used repigmentation therapies for vitiligo that are supported by data from randomized controlled trials (RCT) include UV light (whole body irradiation or UV targeted to lesions) and topical agents (corticosteroids, calcineurin inhibitors, calcipotriol). Clinic Rev Allerg Immunol (2018) 54:52–67 Camouflaging and depigmenting (in widespread disease) are the other current options. Table 1 outlines a stepwise treatment approach according to type of vitiligo and extent, which needs to be modulated by visibility, age, and coping. A ‘zero’ line is always possible, meaning no treatment if the disease is not bothering the patient. Intermittent tacrolimus applications twice weekly on facial lesions prevent recurrence of lesions and can be recommended as a maintenance treatment . Systemic regimens to maintain disease stabilization such as methotrexate for psoriasis or low-dose corticosteroids for lupus have been tried on a small-scale in vitiligo, and more work is needed to recommend their routine use . Overall, the level of evidence for recommendations in vitiligo is low because of the variability of assessment methods and scarcity of good randomized controlled studies. The need to combine phototherapy with other agents to trigger repigmentation explains also the difficulty to design good studies . UV Treatments The currently preferred treatment in adults and compliant children with NSV is narrow-band UVB (NB-UVB), which delivers peak emission at 311 nm. The colour match of repigmented skin is excellent, but the response rate remains low based on patients’ expectations. With twice weekly NB UVB treatments for 1 year, a majority of patients with NSV repigment 75% of the affected areas. At least 3 months and preferably 4 months of treatment is warranted before identifying a patient as a non-responder, and approximately 9– 12 months of treatment is usually required to achieve the maximal repigmentation. Unfortunately, access to this treatment is frequently limited in some areas due to low availability or long Table 1 General outline of management for vitiligo in adults 61 distances to treatment centres. Home phototherapy devices are thus more convenient and suitable for small-extent disease . The optimal dose may differ at different sites, and an option is shielding the area with the lower MED after reaching its optimal dose, while continuing to expose higher MED areas until optimal dosing. There is no apparent relationship between the degree of initial depigmentation and the response to NB UVB treatment, but the duration of the disease is inversely correlated with the degree of treatment-induced repigmentation. The best results are achieved on the face, followed by the trunk and limbs. The poorest outcomes have been noted for hand and feet lesions that at best show a moderate response. Relapses are common at all sites; around 60– 70% of patients resume depigmentation in areas repigmented by treatment within 1 year whatever the regimen PUVA or NB-UVB, justifying more research on maintenance and stabilizing therapies. Responses of segmental vitiligo to narrow-band UVB are at best limited when the same NB-UVB therapy is applied for 6 months, but combined treatment with oral minipulses of corticosteroids seems promising . Overall, earlier and more aggressive treatment schemes are thus advisable. The use of targeted high-fluency UVB (excimer laser or monochromatic excimer lamp, both at 308 nm) which may reach deeper targets such as amelanotic melanocytes of the hair follicle, and also avoid irradiation of uninvolved skin, may improve outcomes, as well as combined approaches. Results are also promising in cases of vitiligo involving limited areas. The safety of UV treatments is overall good because of the generalized use of narrow-band UVB treatments and targeted high-fluency therapies. The risk of sunburn needs to be prevented by a phototype-adapted regimen. PUVA therapy, Type of vitiligo Usual management Segmental and limited vitiligo <2–3% body surface involvement First line: Avoidance of triggering factors, local therapies (corticosteroids, calcineurin inhibitors) combined with localized NB-UVB therapy, especially excimer monochromatic lamp or laser, or home NBUVB phototherapy. Vitiligo >3% BSA Second line: Consider surgical techniques if repigmentation cosmetically unsatisfactory on visible areas. First line: Usual initial combination total body NBUVB with systemic/topical therapies, including reinforcement with localized UVB therapy (see text) Second line: Consider surgical techniques in non-responding areas especially with high cosmetic impact. Third line: Consider depigmentation techniques in non-responding widespread (>50%) or highly visible recalcitrant facial/hands vitiligo A no-treatment option (zero line) can be considered in patients with a fair complexion after discussion. For children, phototherapy is limited by feasibility in the younger age group and surgical techniques rarely proposed before prepubertal age. There is no current recommendation validated to the case of rapidly progressive vitiligo, not stabilized by UV or combined therapy. Camouflage, psychological support, and maintenance treatment are indicated in all cases. UV ultraviolet, NB-UVB narrowband UVB phototherapy 62 which led to more concerns for skin cancer risk after longterm treatment for psoriasis, has been abandoned in most European departments, but is still in use in countries with a majority of darker phototypes . Clinic Rev Allerg Immunol (2018) 54:52–67 UV results in repigmentation of at least 70% of the treated area. Camouflage and Depigmentation Topical Therapies and Combined Therapies Topical therapies are not suitable for widespread NSV, but may be effective in cases with more localized disease (including SV). Combined treatments are frequently considered when phototherapy alone does not show efficacy after 3– 4 months, or in an attempt to accelerate response and reduce cumulative UV exposure. As compared with PUVA, which determines a predominant perifollicular pattern of repigmentation, topical corticosteroids (and topical calcineurin inhibitors—TCI—such as tacrolimus and pimecrolimus) exhibit a diffuse repigmentation type, which is faster but less stable. Class 3 (potent) topical corticosteroids achieve more than 75% repigmentation in more than 50% of patients. TCI are preferred for face and neck lesions, because they do not cause skin atrophy. The efficacy of TCI is enhanced by exposure to UV radiation delivered by high-fluency UVB devices but not so clearly by conventional NB-UVB. The concerns about the risk of cutaneous or even extracutaneous cancer provoked by topical calcineurin inhibitors have been disproportionate . Calcineurin inhibitors can promote repigmentation without immunosuppression . The combined use of UV and calcineurin inhibitors is not yet fully approved, but recommended by the VETF/EDF/EADV/ UEMS guidelines . Topical corticosteroids can also increase the efficacy of UVB . Surgery Surgical methods such as minigrafting, which consists in transplanting punch grafts from an autologous donor site— currently less used when other options are possible—or cellular transplantation using autologous epidermal cell suspensions containing melanocytes or ultrathin epidermal grafts or a combination of cellular transplantation and ultrathin grafting, are used in cases of focal/segmental vitiligo if medical approaches fail. Several variants have been recently developed. UV irradiation is frequently associated. Patients with vitiligo are considered good candidates for surgical techniques depending on availability and cost, if their disease is stable (over the preceding year)  and has a limited extent (2–3% of body surface area). Contrary to SV, in which the grafted cells come from apparently disease-free areas, the survival of living but potentially abnormal transplanted melanocytes is less predictable in vitiligo. Koebnerization limits efficacy (hands in particular). In case of strict preoperative selection for disease stability in vitiligo, cellular transplantation plus Topical camouflaging products mask aesthetic skin disfigurement on a transient, semi-permanent, or permanent basis (tattoos). Benefits can be obtained by the skilled use of corrective cosmetics. For dihydroxyacetone (DHA), the most used selftanner, the higher the concentration, the better the response observed particularly in darker phototypes. Chemical or laser depigmentation can definitely be a choice in a small subset of carefully selected patients, but results are variable both in efficacy and duration. Psychosocial Aspects and Counselling A low self-esteem and high levels of perceived stigma seem to be important factors for quality-of-life impairment in vitiligo patients. A simple visual analogue scale from 0 to 10 with the notice ‘How much does your skin disease bother you currently?’ helps to measure coarsely how patients feel about their disease independently of medical findings. Patients with high perceived severity are the best targets of psychological interventions. Cognitive-behavioural therapy might help improve the quality of life, self-esteem, and perceived body-image . Sunscreens are needed in case of real risk of sunburn on non-photoprotected skin, but not on a routine basis, because moderate sun exposure (heliotherapy) is a good substitute to UV therapies. Photoadaptation exists in depigmented vitiligo skin. Repeated frictions for applying sunblockers without real sunburn risk may be more detrimental than beneficial. It is important to make time available to discuss photoprotection issues vs therapy by UV light in the vitiligo clinic, because this is an important source of confusion for the patients. Evidence-Based Guidelines General guidelines for adults and children have been elaborated by the British Association of Dermatologists  and guidelines for surgery by the Indian Association of Dermatologists, Venereologists and Leprologists (IADVL) Dermatosurgery Task Force . A treatment algorithm has been also proposed based on evidence-based medicine principles . In addition to the Indian and Japanese guidelines, the European VETF/EDF guidelines have been published in 2013  and are currently undergoing revision. Beyond guidelines, personalized/stratified strategies have been proposed . Clinic Rev Allerg Immunol (2018) 54:52–67 Emerging Targeted Therapies in Vitiligo Therapeutic options for patients with vitiligo have overall limited efficacy, and it remains critical to identify new therapeutic strategies that would reduce direct and indirect costs of this disease and potentially benefit other autoimmune conditions associated with vitiligo. Blocking the initiation of the disease by inhibiting the impact of DAMPs on the activation of the disease, such as HSP70i, could be a promising therapy to prevent relapse of the disease. Moreover, targeting IFNα or inhibiting the activation of pDCs, as it was suggested in other chronic inflammatory diseases like lupus or psoriasis, could be another option to dampen the inflammatory response important for disease initiation. Regarding the adaptive immune response, the CXCR3/ CXCL9-10/IFNγ pathway is the most extensively studied in vitiligo and appears to be important in the development of vitiligo. Therapies targeting this pathway through inhibition of CXCR3, its ligand, or downstream-induced signalling could thus represent an attractive strategy in this disease. Two case reports revealed that the use of the JAK inhibitors tofacitinib (a JAK1/3 inhibitor) or ruxolitinib (a JAK1/ 2 inhibitor) might be effective in inducing repigmentation in vitiligo [127, 128]. In addition, a proof-of-concept clinical trial just showed that topical application of ruxolitinib provided significant improvement in facial vitiligo in a small cohort of patients . A phase II pilot interventional study assessing the efficacy of topical application of ruxolitinib in vitiligo is ongoing (clinical trial Gov identifier: NCT02809976). Biological effects of a number of soluble inflammatory mediators, like IFNγ, IFNα, IL-2, or IL-15, rely on activation of the JAK-STAT pathway. Therefore, we can foresee that JAK inhibition would halt the inflammatory and immune signals that are important for T cell survival and depigmentation, and may represent a promising strategy in vitiligo. Anti-TNF therapies are commonly used in a number of autoimmune and inflammatory disorders, in particular in the field of dermatology, rheumatology, and gastroenterology. However, despite the apparent major role of TNFα in vitiligo, the limited number of studies that assessed efficacy of antiTNFα in vitiligo suggests that blocking this cytokine is effective at best in halting disease progression but does not induce significant repigmentation [91, 130]. Inhibition of the immune response in vitiligo appears as a promising strategy. However, repigmentation of vitiligo lesional skin needs repopulation of the epidermis with differentiated melanocytes. The WNT signalling, which has been shown to be repressed in depigmented skin of vitiligo patients, is an important pathway for melanocyte differentiation. Ex vivo treatment of vitiligo skin explants with WNT agonists or GSK3β inhibitors triggered the differentiation of melanoblasts . Therefore, the use of pharmacological agents that 63 would activate the WNT signalling could be an interesting approach in vitiligo. The safety and cost of emerging targeted therapies is currently questioned for their use in a non life-threatening disease. However, the development of therapies in dermatology has been hampered by this kind of argument, and physicians know that vitiligo can be a devastating disorder. The example of psoriasis and the recent development of biologics in atopic dermatitis will fortunately pave the way for the specific development of targeted therapies in vitiligo. The specific risk of increased skin cancer risk for all therapies modulating immune pathways will however remain a strong concern for vitiligo . Conclusion Vitiligo, despite its prevalence, remains a disease with limited therapeutic options for patients. However, recent progress in the understanding of immune pathomechanisms opens interesting perspectives for innovative treatment strategies. The proof of concept in humans of targeting of the IFNγ/Th1 pathway is much awaited. The interplay between oxidative stress and altered immune responses suggests that additional strategies aiming at limiting the type I interferon activation pathway as background stabilizing therapies could be an interesting approach in vitiligo. Hopefully, the status of vitiligo is currently changing from a neglected non-medical problem to a big challenge at the high end of modern translational medicine with real perspectives for cure via specific drug development. 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