Journal Pre-proof

New insight into the role of exosomes in vitiligo

Pui Mun Wong, Lili Yang, Lingli Yang, Huali Wu, Wen Li, Xin
Ma, Ichiro Katayama, Huimin Zhang

PII: S1568-9972(20)30239-1
DOI: https://doi.org/10.1016/j.autrev.2020.102664
Reference: AUTREV 102664

To appear in: Autoimmunity Reviews

Received date: 25 April 2020

Accepted date: 30 April 2020

Please cite this article as: P.M. Wong, L. Yang, L. Yang, et al., New insight into the
role of exosomes in vitiligo, Autoimmunity Reviews (2020), https://doi.org/10.1016/
j.autrev.2020.102664

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Article type: Review article

Title: New Insight into the Role of Exosomes in Vitiligo

Pui Mun Wong a1, Lili Yang a1, Lingli Yang b1, Huali Wu c, Wen Li a, Xin Maa, Ichiro
Katayama b*, Huimin Zhang a**
a
Department of Dermatology, Shuguang Hospital affiliated with Shanghai University of
Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New Area, Shanghai 201203,
Shanghai, China.
b
Department of Pigmentation Research and Therapeutics, Graduate School of Medicine, Osaka
City University 545-0051, 1-10-2 Asahimachi, Abeno-ku, Osaka, Japan.
c
Department of TCM Chemistry, School of Pharmacy, Shanghai University of Traditional

of
Chinese Medicine, 1200 Cailun Road, Pudong New Area, Shanghai 201203, Shanghai,
China.

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*Correspondence to: Department of Pigmentation Research and Therapeutics Graduate School
of Medicine, Osaka City University 545-0051, 1-10-2 Asahimachi, Abeno-ku, Osaka, Japan.

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** Correspondence to: Department of Dermatology, Shuguang Hospital affiliated with
Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New
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Area, Shanghai 201203, Shanghai, China.
Email addresses: katayama@derma.med.osaka-u.ac.jp (I. Katayama), zhanghm@shutcm.edu.cn
(H. M. Zhang)
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1
These authors contributed equally to this work and should be considered first authors.
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ABSTRACT
Exosomes are nanosized extracellular vesicles that originate from endosomes and are secreted by
most cells into the extracellular space. They serve as mediators of intercellular communication
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and have been implicated in the regulation of several physiological and pathological processes.
Vitiligo is a depigmentation skin disease caused by progressive destruction of autologous
epidermal melanocytes. Autoimmune intolerance is one of the leading theories proposed for
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melanocyte destruction in vitiligo via CD8+, regulatory T (Treg) and T helper 17 (Th17) cell
imbalance in adaptive immunity. In this review, we investigate the association of exosomes with
vitiligo and emphasize the role of exosomes in immune regulation, melanocyte–keratinocyte
interactions, and melanogenesis. The exosomal pathway is necessary for the regulation of CD8+,
Treg and Th17 cells in both pathological and physiological conditions. Exosomes derived under
pathological conditions can influence CD8+, Treg and Th17 cell balance in the disease
microenvironment, which may contribute to disruption of autoimmune tolerance in vitiligo. In
addition, exosomes serve as mediators of communication between keratinocytes and
melanocytes in the melanogenesis pathway and may also be involved in melanosome transport.
They also regulate melanocyte survival and the protein expression of enzymes such as tyrosinase
(TYR), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein-2 (TYRP2) and
microphthalmia-associated transcription factor (MITF) in melanogenesis, which suggests that
melanin production is associated with exosomes. An improved understanding of the role of
exosomes in immune regulation and melanogenesis may help to elucidate the pathogenesis of
vitiligo and lead to the development of potential diagnostic markers and therapeutic options.
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Keywords: Exosome, Vitiligo, Autoimmune Response, Melanogenesis, Melanocytes,

Keratinocytes.

1. Introduction
Exosomes are extracellular vesicles that contain protein, nucleic acid and lipid molecules
and [1] are derived from endosomes in numerous eukaryotic cells [2, 3]. They were first
discovered by Johnstone in 1983 in sheep reticulocyte maturation and were initially thought to be
a mechanism for discarding redundant cellular components [4]. A role of exosomes as
intercellular mediators in many biological activities has been described [5]. Recent studies have
reported that exosomes are associated with skin physiology and are involved in several
pathological processes associated with skin diseases, such as regulating the secretion of pro-

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inflammatory cytokines [6], promoting angiogenesis [7], depositing collagen in skin defects [8]
and regulating the proliferation and differentiation of skin fibroblasts [9] in the cutaneous

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microenvironment. Exosomes serve as intercellular mediators in the microenvironment of skin
lesions by delivering their cargo to target cells, thus promoting the occurrence of psoriasis [10],
scleroderma [11], melanoma [12] and other skin diseases. Therefore, exosomes may act as potent

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drug carriers [13] that can selectively infiltrate skin lesions for the treatment of various skin
diseases. They have the potential to lead to the development of novel biomarkers for diagnosis
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and prognosis evaluation in various diseases [1] and next-generation therapies for the prevention
and treatment of skin diseases [14].
Vitiligo is a depigmentation skin disease caused by progressive autoimmune destruction
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of epidermal melanocytes [15], resulting in the appearance of disfiguring white patches [16]. The
pathogenesis of vitiligo has not been fully elucidated, and multiple theories have been described
for the development of this disease, including genetic inheritance [17], oxidative stress [18],
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environmental factors [19] and disruption of autoimmune tolerance [15]. Autoimmune

intolerance is the leading theory proposed for vitiligo, and involves both innate and adaptive
immunity [20]. A robust evidence for autoimmune intolerance in vitiligo is that vitiligo often
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coexists with other autoimmune disorders, particularly Hashimoto’s thyroiditis [21]. The
immune cells implicated in melanocyte destruction via adaptive immunity are CD8+ cytotoxic T
lymphocytes [15], regulatory T (Treg) cells [22] and T helper 17 (Th17) cells [23], and
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imbalance of these cells has been reported [20]. Destruction of melanocytes caused by high
levels of cytotoxic CD8+ T cells has been frequently described in studies of vitiligo [24]. Treg
cells in vitiligo reduce their suppressive capacity towards melanocyte-specific CD8+ T cells [24].
Studies have shown that vitiligo severity is strongly associated with an elevated frequency of
Th17 cells [25]. In addition, autoreactive and/or aberrantly activated tissue-resident memory T
cells has been recently described in pathogenesis of vitiligo [26]. Therefore, modulate immune
response is consider as therapeutic target for management of vitiligo [27].
Research on exosomes in immunity has attracted much attention, and an interaction
between exosomes and melanocytes has been reported recently. Thus, the purpose of this review
is to highlight the current understanding of exosomes in vitiligo, discuss recent insights linking
exosomes to immunity and melanogenesis, and discuss how exosomes may contribute to vitiligo.
The first section will briefly describe the characteristics, composition and formation of exosomes
and their function in immune regulation. This will be followed by an overview of the role of
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exosomes in melanocyte–keratinocyte interactions and melanogenesis. We propose future

avenues for the development of exosomes as diagnostic biomarkers and therapeutics for vitiligo.

2. What Are Exosomes?

Exosomes are extracellular vesicles of endosomal origin that are characterized by a cup-
shaped morphology [28], a phospholipid bilayer membrane [1] and a diameter of 30-150 nm [29].
They are released into the extracellular space by a variety of cell types and widely present in
numerous bodily fluids, such as urine, blood [28] and sweat [30]. The composition of exosomes
is largely based on the cell type from which they originate [31], and they generally carry lipids,
proteins and nucleic acids. Exosomes are formed by lipids, such as cholesterol, ceramide,
sphingolipids, glycerophospholipids, prostaglandins [32], glycosylceramides and diglycerides
[31], and bioactive lipids, such as prostaglandins and leukotrienes [31]. There are two types of
proteins contained in exosomes: conserved proteins and exosome-specific proteins [32].

of
Conserved proteins are similar proteins that appear in each exosome, including cluster of
differentiation (CD)63, CD9, CD81, CD82, heat shock protein (Hsp)60, Hsp70, Hsp90, ALIX,

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and tumour susceptibility gene 101 (TSG101) [31]; these proteins serve as common protein
markers for exosome detection. The composition of exosome-specific proteins depends on the
cellular origin of the exosomes and may be altered based on physiological changes and stimuli

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acting on the cell [31]. For example, major histocompatibility complex (MHC) class I and II
proteins specifically appear on exosomes secreted by antigen presenting cells (APCs). In
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addition, the cellular components of exosomes are enriched in nucleic acids, including mRNAs
and other noncoding RNAs [31].
The biogenesis of exosomes includes formation, cargo sorting, secretion and uptake [31],
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and these processes occur via the classic or direct pathways [33]. The classic pathway of
exosome biogenesis involves the endocytosis pathway. In contrast, the direct pathway is a rapid
mechanism that leads to direct synthesis of exosomes from the plasma membrane and mostly
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occurs in T cells [34]. The classic pathway is mainly divided into the following steps:
1) Formation: Inward budding of the endosomal membrane [35] is followed by invagination
of intraluminal vesicles (ILVs) into the endosomal membrane to form multivesicular
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bodies (MVBs) [35]. Exosomes from MVBs are released into the extracellular space as
ILVs through exocytosis [35].
2) Cargo Sorting: The generation of exosomes requires sorting of cellular components into
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the endosomal membrane and delivery into early ILVs [36]. This process is regulated by
endosomal sorting complexes required for transport (ESCRT)-dependent or ESCRT-
independent pathways [33, 36]. The key subunit of ESCRT proteins is required for protein
sorting via the ESCRT-dependent pathway. Proteins from the trans-Golgi network start
with the binding of ubiquitin to their cytosolic domains to direct them towards ESCRT on
the endosomal membrane [36]. Then, the ubiquitylated proteins bind to the ESCRT
protein complex on the endosomal membrane and are loaded onto the exosome membrane
[33]. However, not all proteins require ubiquitinylation for vesicle targeting [33, 36]. In
ESCRT-independent pathways, MVBs are generated via the ceramide pathway [33, 36].
3) Secretion: The secretion of exosomes from MVBs is regulated by various small Rab
GTPases, as they can either be degraded by lysosomes or fuse with the plasma membrane
to be excreted [3, 37]. The Rab proteins implicated in the movement of MVBs towards the
plasma membrane for exocytosis of exosomes are Rab11, Rab35 and Rab27 [37].
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4) Uptake: Exosomes are endocytosed by a target cell and release their exosomal contents
into the cell [1, 38]. Thus, they function as crucial mediators of intercellular
communication to induce a variety of biological actions in both physiological and
pathological processes.
Exosomes have been reported to be involved in several physiological processes, such as the
inflammatory response [39], angiogenesis [40], coagulation [41], cutaneous wound healing [42],
bone fracture healing [43], the immune response [44], transneuronal transport [45] and tissue
homeostasis [46]. In addition, exosomes have been proposed to play roles in skin immunity [30]
and melanogenesis to maintain skin homeostasis [35]. They are strongly implicated in the
microenvironment of cancer [47-49] and in the pathogenesis of other diseases, such as insulin
resistance in diabetes [50], Alzheimer’s disease [51], Parkinson’s disease [52], atherosclerosis
[53], multiple sclerosis [54], liver disease [55], autoimmune disease [56] and skin disease [6, 12].
The exosomal contents carried by exosomes reflect the physiological or pathological conditions

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of their cellular sources. Many studies have reported that the RNA profile of exosomes derived
from pathological cells or diseases is distinct from that of exosomes derived from healthy cells

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[11, 57-59]; thus, exosomes have been proposed as potential diagnostic biomarkers and
treatments for a variety of diseases [1]. Exosomes can also serve as indicators for monitoring
treatment efficacy [60] and predicting disease subtype [59].

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Fig.1. Biogenesis and composition of exosomes.

Exosomes are synthesized through either classic or direct pathways. The direct pathway
induces exosomes synthesized from the plasma membrane, whereas exosomes formed in the
classic pathway begin with endocytosis. Exosomes originate from endosomes and form
within MVBs as ILVs. Cargo sorting of intraluminal vesicles is regulated by ESCRT-
dependent or ESCRT-independent pathways. Certain proteins from the trans-Golgi network
require ubiquitinylation for targeting ESCRT complexes on the endosomal membrane to
load onto the exosome membrane. Ceramide is required for the formation of ILVs in the
ESCRT-independent pathway. MVBs can either undergo degradation by lysosomes or are
released into the extracellular space through exocytosis, which is mediated by Rab11, Rab35
and Rab27.

3. Exosomes and the Immune Response

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Exosomes possess immunomodulatory properties in both physiological and pathological

conditions [61], as they are involved in antigen presentation, immunological synapse formation
[62], and surveillance of invading pathogens and tumour cells through the intercellular
communication system in immune cells, consequently leading to immune activation and
suppression [63]. Exosomes derived from normal immune, nonimmune or pathological cell types
can affect immune homeostasis. A breakthrough related to the role of exosomes in immunity was
made in 1996 by Graqa Raposo, as exosomes were first discovered to be involved in antigen
presentation as mediators of B lymphocyte release of MHC class II [64]. In addition, exosomes
derived from dendritic cells express MHC class I and II T cell costimulatory molecules in
tumour peptide culture, followed by activation of CD8+ cytotoxic cells, resulting in cytotoxicity
for murine tumour cells [65]. Therefore, MHC complexes and costimulatory molecules on
exosomes are capable of stimulating T cells after internalization by APCs [62]. They may be
transported to the surface of APCs [62], forming immunologic synapses between APCs and T

of
cells as a consequence of T lymphocyte activation [62]. However, a recent study showed that
activation of T lymphocytes does not require MHC molecules on exosomes [66].

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Recent studies reported that mesenchymal stromal cell (MSC)-derived exosomes
obtained from bone marrow suppress the release of the pro-inflammatory factors TNF-α and IL-
1β, whereas they promote the secretion of the anti-inflammatory factor TGF-β [67]. In addition,

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exosomes may induce the differentiation of type 1 T helper cells into type 2 T helper cells and
reduce the differentiation of T cells into interleukin 17-producing effector T cells (Th17 cells)
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[67]. Moreover, they increase the number of Treg cells and the levels of cytotoxic T lymphocyte-
associated protein 4 [67]. These authors proposed that MSC-derived exosomes exerted a
modulatory effect on immunity [67]. Therefore, exosomes serve as intercellular mediators that
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regulate immunity under physiological conditions.

The role of autoimmune intolerance as a cause of melanocyte destruction during the
development of vitiligo is a popular research topic [19]. A variety of immune cells contribute to
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the development of autoimmune intolerance in vitiligo. Studies have shown that CD8+, Treg and
Th17 cell imbalance is involved in adaptive immunity in vitiligo, but this conclusion is still
controversial, and the underlying mechanism is unclear [20]. For example, blood phenotyping in
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vitiligo patients shows high frequencies of CLA+/CLA- Th1/type 1 cytotoxic T cell polarization
and Th2, Th9, Th17, and Th22 levels, whereas total Treg cell counts are low in vitiligo [68]. The
severity of vitiligo is associated with the levels of several cytokines, and the duration of disease
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is related to the levels of IFN-γ and IL-17 [68]. Melanocyte destruction in the epidermis is
mediated mainly by cytotoxic CD8+ T cells, but the involvement of Treg and Th17 cells in this
process has also been reported [20]. CD8+ T cell-induced apoptosis of autologous melanocytes
has been demonstrated in vivo, and the cytotoxicity of CD8+ T cells isolated from vitiligo
perilesional skin is higher than that of CD8+ T cells isolated from peripheral blood [69]. A study
showed a significant increase in circulating CD8+ T cells and a decrease in CD4+CD25+CD127-
Treg cells in the peripheral blood in generalized vitiligo patients compared to healthy individuals
[24]. The suppressive effect of Treg cells on CD8+ T cells is significantly lower in generalized
vitiligo patients than in normal healthy individuals [24]; thus, Treg cells fail to suppress the
abnormal killing of melanocytes by CD8+ T cells. In addition to the destruction of melanocytes,
CD8+ T cells can inhibit melanin production by expressing IFN-γ [70]. IFN-γ reduces the
expression of tyrosinase and microphthalmia-associated transcription factor (MITF) in a dose-
dependent manner and directly induces melanocyte apoptosis [70].
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In addition, compared to that of healthy controls, peripheral blood from vitiligo patients
shows high expression of mRNAs related to Th17 cells [23]. Increased circulating Th17 cell
expression was found in non-segmental vitiligo as a result of elevated serum TGF-β1 and IL-21
levels [71]. In addition to the role of CD8+ cell-mediated melanocyte destruction in autoimmune
vitiligo, a role of the Th17 cell-related cytokine environment in depigmentation has also been
reported [72]. Th17 cell-related cytokines, such as interleukin (IL)-17A, IL-1β, IL-6, and tumour
necrosis factor (TNF)-α, significantly reduced MITF expression in melanogenesis [72]. A recent
study showed significantly increased serum levels of CCL20 and Th17 cells in patients with
vitiligo [73]. CCL20 is a chemokine produced by Th17 cells, which causes CD8+ T cells from
the systemic circulation to infiltrate peripheral tissues [25]; thus, this chemokine may contribute
to melanocyte destruction.
The exosomal pathway is necessary for the regulation of CD8+ T, Treg and Th17 cells in
both pathological and physiological conditions. CD8+ T cells are cytotoxic cells that are

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responsible for destroying cancer cells and infected cells. Immunosuppressive effects of
exosomes on CD8+ T cells have been frequently described in the tumour microenvironment.

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Exosomes derived from malignant cells or immune cells can be taken up by tumour-infiltrating
CD8+ T cells and regulate the function and antitumour effect of normal CD8+ T cells [74].
Tumour-derived exosomes cause a significant loss of CD27/CD28, which leads to the induction

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of CD8+ T cell suppressors [75]. The Gal-1 protein in tumour-derived exosomes has been
demonstrated to induce CD8+ T cell suppressors, but suppression of CD8+ T cells has not been
demonstrated [75]. However, RNA from exosomes also induces the activity of CD8+ T cell
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suppressors [75]. In the cancer microenvironment, the lncRNA profiles of exosomes derived
from exhausted and non-exhausted CD8+ T cells are different [74]. In addition, exosomes
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secreted from exhausted CD8+ cells can be taken up by normal CD8+ T cells and impair their
physiological functions [74].
Furthermore, exosomes contribute significantly to the immunoregulation of Treg cells
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and Th17 cells. Human umbilical cord MSC-derived exosomes increase the proportion of
CD4+CD25+Foxp3+ Treg cells and reduce the proportion of CD4+IL17A+ T cells (Th17 cells)
[76]. They also inhibit the proliferation of CD4+ and CD8+ T cells and increase the proportion of
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CD4+CD25+FoxP3+ Treg cells [76]. Thus, the immunomodulatory effect of exosomes on T cells
was discovered in vitro based on increased and reduced proportions of Treg and Th17 cells,
respectively [76]. Treg cells decrease Th1 cell proliferation and IFN-γ secretion by transferring
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let-7d-containing exosomes to Th1 cells in vivo [77]. In addition, exosomes derived from
multiple sclerosis (MS) can selectively inhibit IFN-γ−IL-17A−Foxp3+CD4+ Treg cells through
exosomal let-7i and consequently suppress insulin-like growth factor 1 receptor and transforming
growth factor beta receptor 1; thus, exosomes may contribute to the underlying mechanisms by
which Treg cell homeostasis is disrupted in MS [78]. A study showed that MSC-derived
exosomes have the capacity to decrease Th17/Treg imbalance in aplastic anaemia through
sphingosine kinase isoenzyme 1 (SphK1)-mediated exosomal sphingosine 1-phosphate
enrichment (S1P) in both in vivo and in vitro experiments [79]. In conclusion, exosomes are
important in regulating several types of immune responses; hence, exosomes may be associated
with disruption of autoimmune tolerance in vitiligo.

4. Interaction of Melanocytes and Keratinocytes via Exosomes

Melanocytes are responsible for melanin synthesis in organelles called melanosomes and
distribute melanin to the epidermis by transporting melanosomes to adjacent keratinocytes for
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skin pigmentation [80]. The mechanism of melanosome transfer has not been fully explored, and
studies have proposed that melanosomes are transferred to keratinocytes from melanocytes by
means of plasma membrane fusion of both cells or phagocytosis of secretory melanosomes or
melanosome-containing dendrite tips from melanocytes [81-82]. In addition, the role of vesicle
shedding in this mechanism has been demonstrated, showing that melanosome-containing
vesicles discharged from melanocytes are phagocytosed by keratinocytes [81-82]. Furthermore,
the transport of melanosomes is regulated by the small GTPase Rab27a [83], and a role of Rab27
in exosome secretion has been reported [37]; therefore, melanosome transport may be related to
exosomes. Hence, intercellular contact is not required, and an interaction may occur through
intercellular mediator vesicles, which may contribute to the mechanism of melanosome transfer.
In addition to melanosome transfer, an interaction between melanocytes and keratinocytes via
exosomes has also been discovered recently.
Both melanocytes and keratinocytes can secrete exosomes, and the secreted exosomes are

of
taken up by the other cell type; this communication induces intercellular activities. First, cultured
supernatants of keratinocytes were found to contain exosomal protein markers such as TSG101

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[84], Alix [85] and CD63 [85-86]. The expression of miRNAs in exosomes is not affected by
RNase intervention in keratinocyte supernatants, as they are encapsulated in the phospholipid
bilayer of exosomes [86]. Transfer of exosomes from keratinocytes to melanocytes has been

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demonstrated recently [84-85]. Cicero’s study used CD63-GFP to label MVBs in keratinocytes
to track the movement of exosomes [85]. CD63-positive compartments were initially detected
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around the nucleus of keratinocytes, and then their movement towards the plasma membrane of
keratinocytes was observed during cultivation with melanocytes. This process led to significantly
increased levels of exosomes in melanocytes after 24 hours of cultivation [85]. Therefore,
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exosomes from keratinocytes are transferred to melanocytes through exocytosis. In addition,

exosomes derived from keratinocytes carry miR-330-5p, which causes a significant increase in
miR-330-5p expression within melanocytes, as detected by fluorescence in situ hybridization
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[84]. This suggests that nucleic acids from keratinocytes are transported to melanocytes via
exosomes [84].
Research on exosomes that are derived from melanocytes and taken up by keratinocytes
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is rarely reported. Because the differences between exosomes and other extracellular vesicles
(EVs) are not well defined or fully established, research on the extracellular vesicles secreted by
melanocytes has been included in this review. UVA radiation is essential for the secretion of EVs
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by human melanocytes, with an underlying mechanism involving activation of lysosome

exocytosis [87]. Under UVA exposure, EV protein markers (flotillin-1), exosomal protein
markers (CD63), and lysosomal-associated membrane proteins (LAMP) are detected in
melanocyte conditioned medium, whereby LAMP-1 is specifically detected on the plasma
membrane of melanocytes [87]. However, no melanosomes are found in the medium [87]. This
suggests that UVA radiation causes melanocyte plasma membrane damage via lysosomes,
followed by EV release but not melanosome release [87]. Coculture of keratinocytes and
melanocytes in conditioned medium showed uptake of GFP-LAMP-1-containing vesicles and
melanosomes by keratinocytes 2 hours and 24 hours after incubation, respectively. Although
melanosomes were transferred successfully, there was no effect on tyrosinase activity [87].

5. Exosomes and Melanogenesis

Skin pigmentation relies on the interaction between keratinocytes and melanocytes in the
epidermis [85], and their communication via exosomes has been investigated. Thus, exosomes
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are closely related to melanogenesis and are involved in the pathogenesis of pigmentation-related
disease. Exosomal miRNAs are involved in melanogenesis through the MAPK signalling
pathway, the Ras signalling pathway, the Rap1 signalling pathway, the cAMP signalling
pathway, endocytosis, axon guidance and the Wnt signalling pathway [88]. Coculture of
melanocytes from human foreskin and exosomes derived from keratinocytes promotes
melanocyte proliferation and increases tyrosinase activity in melanogenesis [88]. Moreover,
exosomes containing miR-330-5p derived from murine keratinocytes are transferred to
melanocytes and induce the inhibition of melanogenesis [84]. These exosomes significantly
decrease tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein-2
(TYRP2) and MITF protein expression in melanocytes, consequently decreasing melanin content
[84]. In addition, the release of miR-675 from human keratinocytes via exosomes has an
inhibitory effect on melanogenesis [86] by reducing the expression of tyrosinase, TYRP1 and
TYRP2 in melanocytes [86]. Phosphorylation of cAMP-responsive element-binding protein

of
(CREB), extracellular signal-regulated kinase (ERK), apoptosis signal-regulating kinase (AKT),
and nuclear factor kappa B is reduced by an miR-675 mimic. Biopsied skin of C57BL/6J mice

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injected with the miR-675 3p mimic showed reduced mRNA expression of MITF, tyrosinase,
TYRP1 and TYRP2 [86]. In addition, miR-675 expression in keratinocytes decreased with H19
knockdown, suggesting that the miR-675 signalling pathway in melanogenesis is regulated by
H19 RNA [86].
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Ultraviolet radiation is an important extrinsic factor in the regulation of melanogenesis
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[89], and a recent study found changes in the biological activity of exosomes during
melanogenesis after UV exposure. Melanocytes treated with exosomes derived from
keratinocytes exposed to UVB radiation showed significantly increased melanin content
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compared with those treated with exosomes keratinocytes that were not exposed to UVB
radiation [85]. Further investigation found that UVB radiation does not affect the number of
MVBs in keratinocytes but alters their biological activity in melanogenesis [85]. Tyrosinase
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activity does not change with normal keratinocyte-derived exosomes from low phototype skin
(Caucasian) unless keratinocytes are exposed to UVB radiation, whereas normal keratinocyte-
derived exosomes from high phototype skin (Black) significantly increase tyrosinase activity in
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Caucasian melanocytes without UVB exposure [85]. Thus, exosomes secreted by keratinocytes
from different phototypes and after UVB irradiation control melanocyte pigmentation to
different extents via different mechanisms [85]. Furthermore, exosomes exposed to UVB
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radiation increase MITF-M and tyrosinase expression in melanocytes. A breakthrough in this

field revealed the role of hsa-miR-3196 from UVB-treated exosomes from Caucasians in
melanogenesis [85].
Melanocyte survival is an important factor in sustaining melanogenesis to prevent DNA
damage under UV radiation and maintain skin pigmentation [90]. The underlying mechanism by
which melanocytes prevent cytotoxicity after repetitive UV radiation is related to exosomes that
carry fibronectin [90]. Melanocytes significantly increase the release of exosome or EV markers
(CD8, heat shock protein 90) and fibronectin after UVB radiation, and fibronectin is contained
within exosomes [90]. Fibronectin-containing vesicles reduce apoptotic bodies, TUNEL-positive
cell death and senescence of melanocytes after UVB radiation, thus preventing cell apoptosis
[90]. Hence, fibronectin-containing vesicles derived from melanocytes play a significant role in
preventing the cytotoxicity of UVB radiation to maintain normal melanogenesis [90].
Effects of exosomes on the melanogenesis pathway have been proposed, and exosomes
may play a role in the pathogenesis of pigmentation disease, but the mechanism is poorly
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understood. Research has discovered differences in exosomal nucleic acids between healthy
populations and active vitiligo patients. The similarity of exosomal RNA between active vitiligo
patients and healthy populations is 27.07%, while the expression of specific factors among total
RNA in active vitiligo patients is 45.73% [91]. Among the total miRNAs present in both
populations, there were 22 upregulated miRNAs and 12 downregulated miRNAs in serum
exosomes from patients with vitiligo [91]. In melasma patients, the fibronectin signal around
melanocytes in the basement membrane increases, indicating prolonged survival of myelocytes
as a result of hyperpigmentation [90]. Fibronectin is known to be contained within exosomes
[90]; thus, hyperpigmentation of melasma is associated with exosomes.

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Fig. 2. MiRNAs from keratinocytes are sorted into ILVs forming MVBs and are secreted
through exocytosis.
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The miRNAs containing exosomes are taken up by melanocytes followed by the transfer of
miRNAs. Furthermore, exosomal miRNAs are involved in the PI3K/Akt, MAPK and cAMP
signalling pathways to regulate transcription of MITF in melanogenesis. Exosomal miR-675
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inhibits the phosphorylation of CREB, ERK, AKT signalling molecules and then causes
inhibition of TYR, TYRP1 and TYRP2 expression. Exosomal miR-330-5p directly inhibits
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the expression of MITF protein and leads to inhibition of TYR, TYRP1 and TYRP2
expression; PI3K/Akt= phosphatidylinositol 3-kinase/protein kinase B, MAPK= mitogen-
activated protein kinase, cAMP= cyclic adenosine monophosphate.

6. Conclusion
Exosomes are nanosized EVs that carry proteins, lipids and nucleic acids [1] and serve as
mediators of intercellular communication by delivering signalling molecules to target cells [5].
Recent studies have implicated exosomes in several physiological and pathological conditions,
including skin homeostasis [30] and skin diseases [35]. Although the role of exosomes has been
described in several skin diseases, studies of this topic in vitiligo are limited. Vitiligo is a
depigmentation skin disease that is characterized by the appearance of disfiguring white patches
[16] and caused by progressive autoimmune destruction of epidermal melanocytes [15].
Autoimmune intolerance has been proposed to play a role in the development of vitiligo via
CD8+, Treg and Th17 cell imbalance [23,68] in adaptive immunity. In the present review, the
role of exosomes in immunity and melanogenesis has been revealed. Exosomes derived under
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physiological conditions can regulate the activation of CD8+ cells [64], the differentiation of
Th17 cells [67] and the proliferation of Treg cells [67] in immunity. However, exosomes derived
under pathological conditions can influence CD8+ [74], Treg and Th17 cell [76] balance in the
disease microenvironment. This suggests a role of exosomes in the underlying mechanism of
autoimmune dysregulation that leads to melanocyte destruction in vitiligo. In addition, exosomes
serve as mediators of communication between keratinocytes and melanocytes in the
melanogenesis pathway and may also be involved in melanosome transport. Exosomes regulate
melanocyte survival [90] and the protein expression of enzymes such as TYR, TYRP1, TYRP2
and MITF in melanogenesis; thus, melanin production is associated with exosomes [84, 86].
Exosomes carry signalling molecules from their cells of origin and reflect the
characteristics of those cells. Thus, signalling molecules from exosomes involved in pathological
processes can be detected in body fluids and help to discover signalling pathways related to the
onset of disease. A recent study proposed changes in exosomal RNA expression in vitiligo

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patients compared to the heathy population, suggesting that RNA in exosomes has the potential
to reflect the pathogenesis of vitiligo [91]. Based on the current findings, we suggest the

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potential application of exosomes as diagnostic biomarkers and therapeutics for vitiligo. They
can be utilized as potential diagnostic biomarkers to assess severity, monitor treatment efficacy,
determine disease subtype and evaluate prognosis for vitiligo in the future. Exosomal

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immunotherapy interventions may become an innovative therapeutic tool for the treatment of
vitiligo that inhibits autoimmune intolerance to prevent melanocyte destruction and regulates
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melanogenesis to promote repigmentation. They may be developed as drug carriers for the
transportation of signalling molecules to depigmentation sites. In conclusion, further studies are
required to develop new diagnostic biomarkers and design efficient treatments for vitiligo and to
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determine how we can manipulate this process to overcome the shortcomings of vitiligo
treatment.
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6.1 Take Home Messages

 Vitiligo is a depigmentation skin disease caused by progressive destruction of autologous
epidermal melanocytes caused by autoimmune intolerance.
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 Exosomes involved in immune regulation, melanocyte–keratinocyte interactions, and

melanogenesis.
 Understanding of the role of exosomes in immune regulation and melanogenesis may
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help to elucidate the pathogenesis of vitiligo and lead to the development of potential
diagnostic markers and therapeutic options.

Disclosure

The authors declare no conflicts of interest.

Funding sources

Supported by the National Natural Science Foundation of China (81972932), the Shanghai
Municipal Health Committee Traditional Chinese Medicine Technology Promotion Project
(zyjx-2017005), the Three-Year Action Plan to Further Speed Up the Development of
Traditional Chinese Medicine in Shanghai, Construction and Cultivation Project of Dominant
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Diseases of Traditional Chinese Medicine (ZY(2018-2020)-ZYBZ-10), and the High-Level

University Summit Project (Huimin Zhang, Summit Plateau Team).

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