Melanocytes are particularly susceptible to cellular stress, due to their exposure to UV irradiation and other environmental stressors, as well as the production of melanin, which generates reactive oxygen species (ROS) and activates the unfolded protein response (UPR). Electron microscopy of cellular substructure revealed that the endoplasmic reticulum (ER) in melanocytes from vitiligo patients was dilated compared to healthy controls [36], a characteristic that implicates cellular stress. Elevated levels of H₂O₂ and oxidative byproducts are present in vitiligo patients’ epidermis compared with controls, suggesting uncontrolled generation of reactive oxygen species (ROS) [37]. In addition the enzyme catalase, which reduces H₂O₂ to O₂ and H₂O and thereby relieves oxidative stress, is decreased in lesional skin [38], which may be either a cause or an effect of increased H₂O₂. Treatment of vitiligo with topical exogenous catalase has been attempted, with variable results [39].

Tyrosinase facilitates a number of steps that convert the amino acid tyrosine to melanin. Tyrosine is a phenol, containing a hydroxyl group attached to a benzene ring. Monobenzone was the first chemical reported to induce vitiligo in a group of factory workers exposed to the chemical through acid-cured rubber gloves. Distant spread of depigmentation to areas beyond the exposure sites (hands and forearms) suggested that monobenzone is not simply directly cytotoxic to melanocytes, but induces autoimmunity during exposure. Monobenzone treatment of vitiligo patients, used to depigment their skin to induce an even tone, also induces depigmentation at sites remote from the site of application, supporting this concept. Like tyrosine, monobenzone and other depigmenting agents are phenols, which act as tyrosine analogs but bind irreversibly to tyrosinase and thereby interrupt the melanin synthesis pathway [40]. This induces cellular stress through the induction of ROS and activation of the UPR, which then results in activation of innate immunity to induce inflammation [40–42]. Many household products contain other chemical phenols, including hair dyes and cleaning products, which have both been reported to induce chemical leukoderma, or chemically-induced vitiligo [28]. It is likely that phenols used as ingredients in these products act in a similar way as monobenzone, producing cellular stress through interaction with tyrosinase.

There is evidence that the initiation of vitiligo requires innate immune activation. Several studies have implicated innate immune sensing of cellular stress, including dendritic cell (DC) activation by stress-induced release of exosomes [40], the heat shock protein HSP70i [43] and an apparent increased recruitment of natural killer (NK) cells into the skin of vitiligo patients [44]. Melanocyte stress is therefore linked to innate immune activation, and thus initiation of inflammation that may lead to adaptive autoimmunity; however, more studies will be required to identify the precise signaling pathways involved [45]. Innate immune gene polymorphisms in vitiligo have been identified through genome-wide association studies, though their functional roles in disease are still being defined (see section “Genetic Contributions”). Environmental triggers that induce melanocyte stress have been identified, and patients may be counseled to avoid these exposures (see section “Melanocyte Stress”).

Vitiligo is driven by CD8⁺ T cell-mediated killing of melanocytes. This has been demonstrated ex vivo using a human skin culture system in which melanocyte-specific T cells isolated from affected skin migrated into an unaffected skin explant from the patient and induced melanocyte apoptosis. CD8⁺ T cells were both necessary and sufficient for this effect [46]. Increased frequencies of melanocyte-specific CD8⁺ T cells in the blood and skin correlate with disease severity [47]. The antigen specificities of the T cell receptors (TCRs) in vitiligo have been identified and are shared with melanoma, including MART-1, gp100, tyrosinase, and tyrosinase-related proteins 1 & 2 (TRP1 & 2) [48]. These T cells produce IFNγ which, in addition to inducing T cell recruitment, may be directly cytotoxic to melanocytes [49]. It is possible that vitiligo results from an overzealous anti-tumor response [50]. Patients with vitiligo are at a three-fold lower risk for melanoma, and those with melanoma who spontaneously regress or who are cured following treatment frequently develop vitiligo [51].

CD4⁺ helper T cell responses may also play a role in vitiligo via IFNγ production, although functional evidence for their role is lacking [52, 53]. There is growing evidence that vitiligo is dependent on IFNγ and downstream genes including IFNγ-inducible chemokines [52, 54]. There are several case studies of patients who were treated with IFNα for hepatitis who developed vitiligo [55–64], suggesting that type I interferons can induce disease, though it is unclear what their role is in spontaneous vitiligo. Several studies have questioned whether T_H17 cells and IL-17 drive pathogenesis, but their functional role in vitiligo remains unclear. It is possible that a subset of vitiligo patients, such as those with inflammatory vitiligo, also have an IL-17 component in their disease pathogenesis, but studies to date have not classified T_H1 versus T_H17 in different vitiligo subtypes. Autoantibodies against melanocytes have been identified in patients [65], though it is uncertain whether they play a direct role in pathogenesis or are merely a biomarker of disease. A role for regulatory T cells (Tregs) have also been implicated in vitiligo through genetic studies [66] and the fact that patients with immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, who lack Tregs, have an increased incidence of vitiligo [67]. However evidence is conflicted regarding whether their numbers are normal or fewer, whether they have difficulty homing to the skin, and whether or not they are functionally impaired [68–72].

Hair depigmentation is not commonly observed in vitiligo, most likely due to hair follicle immune privilege. Immune privilege is defined as tissue that is not readily inflamed, possibly due to limited access by the immune system and/or the expression of immunoregulatory proteins or cell populations [73–75]. Melanocyte stem cells reside in hair follicles, which migrate out into the epidermis to repigment the skin [76]. When repigmentation occurs in patients, it usually begins around pigmented hair follicles and spreads outward, indicating that the stem cell populations in the hair follicles may be maintained through immune privilege. Little is known about the mechanisms of repigmentation, though this is critical for effective therapy (see also section “Future Perspectives into Vitiligo Treatment Strategies”). A summary of vitiligo pathogenesis is presented in Fig. 28.2.

One of the earliest models of vitiligo identified was a spontaneous mouse model caused by a point mutation in the microphthalmia-associated transcription factor (MITF) gene, which caused melanocyte degeneration as the mouse aged. However due to its monogenic nature and lack of autoimmunity [88, 89], this strain (mi^vit) does not appear to accurately model human vitiligo pathogenesis. Several other mouse models have been developed to study vitiligo, including models induced through exposure of the skin to melanocyte antigens plus adjuvant to induce depigmentation [90], or through adoptive transfer of transgenic T cells engineered to react against melanocyte antigens. Early models were developed from animal models of melanoma immunotherapy [91–95] in which TCR transgenic mice responding to gp100 (called PMEL for pre-melanosome protein were used to mediate tumor clearance. The mice spontaneously developed white hair, and several studies have used these PMEL host mice to study spontaneous hair depigmentation [43]. We developed a model through the adoptive transfer of PMEL T cells into hosts that have epidermal melanocytes [95], which resulted in progressive epidermal depigmentation with sparing of the hair (Fig. 28.4a). Studies using this model implicated IFN-γ and the IFN-γ-induced chemokine CXCL10 as required for both disease progression and maintenance [54]. This model is most useful for studying the effector phase of vitiligo, as loss of tolerance through innate immune activation is largely bypassed.

Perhaps the most representative animal model of vitiligo is the Smyth Line (SL) chicken, which develops spontaneous depigmentation of its feathers over time (Fig. 28.4a). Affected brown SL chickens develop white feathers with age and produce anti-melanocyte antibodies. Depigmentation is due to melanocyte loss by apoptosis induced by cytotoxic T cells. Interestingly, the SL chicken also has melanocyte defects, including autophagocytosis of melanosomes and increased generation of ROS. Consistent with the theory that vitiligo pathogenesis is multifaceted, these melanocyte defects are not sufficient for depigmentation in the absence of a functional immune system [96]. While the SL chicken appears to model vitiligo in a way that is most similar to human disease, the paucity of tools and expertise for investigation of chicken cells limits its use. Recent studies determined that IFNγ, IL-21 and IL-10 are expressed in evolving lesions [97], and microarray analysis has been conducted to begin to address gene signatures in the model [98].

Histology and immunohistochemistry were the earliest methods used to study inflammatory infiltrates in vitiligo. Histologic analysis of vitiligo patient biopsies revealed a lack of epidermal melanocytes with accompanying lymphomonocytic infiltrates. Immunohistochemistry revealed infiltrates of CD8 and CD4 T cells that are most often detected perilesionally [3, 99, 100]. CD8 T cells have been observed infiltrating the epidermis and may be found proximal to dying melanocytes. Innate immune populations including CD11b+ CD11c+ inflammatory DCs [43] and NK cells [44] have also been identified in vitiligo lesions.

Van den Boorn et al. used human skin organ culture and autologous T cells to demonstrate that CD8⁺ T cells are necessary and sufficient to induce melanocyte apoptosis in human skin, consistent with a causative role for adaptive immunity in vitiligo [46]. This approach models processes that occur within the skin, and provides the normal 3D architecture and maintains most of the microenvironment. Punch biopsies of lesional skin from vitiligo patients were cultured ex vivo, T cells migrated out of the explant, and were expanded in vitro. Expanded populations can then be analyzed, manipulated, and re-exposed to punch biopsies of normally pigmented skin from the same patient to determine their effects on melanocytes in situ (Fig. 28.4b). Disadvantages of this approach include the inability to model recruitment of T cells from the blood to the tissue, and the fact that over time culture of skin results in the loss of resident immune cell populations through emigration [101].