Skin depigmentation that occurs over time as a result of cutaneous melanocytes is a characteristic of vitiligo, where the classification of vitiligo is divided into segmental vitiligo (SV) and non-segmental vitiligo (NSV), as well as mixed and unclassified vitiligo. ¹ Prevalence of the disease is 0.5–2% of the worldwide population. ² In Indonesia, 187 of the 2700 patients who visited the outpatient Dermatology and Venereology clinic of Kepanjen Hospital in South Malang between 2015 and 2019 had vitiligo, amounting to a prevalence of 7%. The peak occurrence of vitiligo is usually between the first and third decade of life and it has a psychological impact on the sufferer, although vitiligo is non-life threatening. ^{3 , 4} The diagnosis of vitiligo can be made from clinical examination and additional laboratory tests are usually needed only for confirmation. ⁵

The pathogenesis of SV, including neuropeptide, somatic mosaicism, and microvascular skin horning, which eventually cause melanocyte damage, is better known than that of NSV. ^{6 – 8} Otherwise, NSV pathogenesis is still multifactorial and polygenic. The dominant factors in the NSV pathogenesis are oxidative stress and autoimmunity. ^{6 , 9 , 10} However, the mechanisms involved in the onset and progression of vitiligo remain unclear. Several previous studies have linked endoplasmic reticulum (ER) stress, which has a relationship with oxidative and immune stress in the pathogenesis of vitiligo. ¹¹ Oxidative stress disrupts the cellular redox potential that extends to the ER, so that protein will fail to fold and its accumulation will activate a response called the unfolded protein response (UPR). ^{11 – 13}

Binding immunoglobulin protein (BiP), an ER chaperone in the accumulation of unfolded protein, preferentially binds to unfolded protein peptides and releases the ER stress sensor to enable its spontaneous dimerization and activation. ^{14 , 15} Inducible heat shock protein 70 (HSP70i) can also be secreted from the stressed live cells as a chaperone. Intracellular HSP70i will bind native protein in response to stress, to prevent misfolding dan cellular apoptosis. Some experimental animal models have confirmed the action and overexpression of HSP70i in progressive depigmentation. ^{16 – 18} Thus, the presence of UPR is related to the extent and severity of vitiligo because it will eventually cause damage to and destruction of melanocyte cells. ^{6 , 9 , 10 , 19} The progressive depigmentation of NSV and its unpredictable disease course makes it an important subject for research. Therefore, this study aims to compare BiP and HSP70i associated with melanocyte cell death in SV and NSV patients.

This research has been declared to be ethically feasible by the Health Research Ethics Committee, Faculty of Medicine, Brawijaya University, dated July 28 ^th, 2021, with approval number 218/EC/KEPK-S3/07/2021.

The ER in eukaryotic cells has an important role in synthesis, maturing protein, lipid metabolism, and intracellular calcium homeostasis. ^{1 , 12} Physiologically, the ER ensures that proteins are folded, always oxidized around the folds, and filled with calcium. Subtoxic levels of ER-stress-induced UPR in the skin are required for normal cellular function, including differentiation. ¹⁶ Melanocyte are more susceptible to oxidative stress in patients with vitiligo than in healthy individuals and melanocytes release reactive oxygen species (ROS) as a respond to stress. ²⁰ The over-production of ROS and it's accumulation triggers protein oxidation and fragmentation, lipid peroxidation, and DNA damage, which compromises cellular function. ^{10 , 21} In addition, ROS also interfere with the work of the folding machinery of the ER, resulting in UPR. ¹⁰ This situation leads to chronic and continual ER stress that induces UPR, which has a deleterious effect on cells, and the UPR in turn becomes mechanism of a cell death. ¹⁵ The accumulation of UPR also disturbs transcription, translation, and mRNA translation in protein synthesis. ^{22 – 24}

The ER stress response or the so-called UPR has been widely recognized in autoimmune and inflammatory diseases, where the UPR contributes to the course of autoimmune disease through the formation of antigens during the degradation of misfolded proteins, secreting neo-antigens by apoptotic cells or impairing immune tolerance. ^{4 , 25} The UPR can also be activated through an increase in unfolded protein that occurs as a result of the production of large amounts of protein during melanin synthesis. ¹⁰ Intracellular oxidative emphasise that outcomes from outside shows vulnerability to chemicals and harsh environmental factors, besides failure of the intracellular systems to overcome stress, and induces targeted melanocytic destruction. ²⁶ Production of pro-inflammatory cytokines and signals that activate the innate immune system result from the ROS- and UPR-mediated death of keratinocytes and melanocytes. Then, pattern recognition receptors (PRR) and nucleic acid receptors through danger-associated molecular patterns (DAMP) recognize host-derived self-DNA/RNA from damaged cells. ²⁷

The three ER transmembrane sensor proteins control UPR, namely inositol-requiring enzyme 1 alpha (IRE1a), double-stranded RNA-dependent protein kinase-like ER kinase (PERK) and activating transcription factor (ATF) 6. ¹⁶ All three sensors are primarily bound with BiP, which helps to maintain their inactive state, in non-stress conditions. The interplay between these three major arms of the UPR signaling pathways determines whether stressed cells survive or die. ¹¹ The protein is known as a binding immunoglobulin protein because it is found in the ER and is bound to secreted Ig heavy chains. ²⁸ The UPR pathway is activated when the calcium concentration in the ER is reduced, which causes a rapid buildup of unfolded/misfolded proteins and the promotion of dissociated BiP from three ER transmembrane sensor proteins: IRE1a, PERK, and ATF6. ¹⁶ In addition, BiP/GRP78 levels are very high in various human cancers. ²⁹

In response to cellular stress and UPR activation, melanocytes secrete HSP70i, as many other cells do. ²⁷ HSP70i binds native proteins to prevent misfolding and cellular apoptosis. In experimental animal models, the role of HSP70i overexpression in gradual depigmentation has been demonstrated. ^{24 , 30 , 31} The effector T cells are attracted to the dendritic cells by increasing signaling, which is induced by HSP70i. ²⁷ Under stress, DAMPs that convey danger signals cause the innate immune system to become more activated by way of pattern recognition receptors (PRR) that include TLRs, RIG-I similar receptors, and NLRs. By producing cytokines, chemokines, and antigen-presenting molecules, this mechanism has the potential to both negatively destroy melanocytes and spark adaptive immunity. ¹⁸ Furthermore, it activates dendritic cells, and the activated DAMP and CD8 ⁺ T cells stimulate the immune response to again destroy melanocytes. ^{27 , 32 , 33} Melanocyte-specific, cytotoxic CD8+ T lymphocytes are crucial for the melanocyte’s eventual targeted demise. ^{34 – 36} It has been found that the severity of vitiligo disease is related to the intensity of the CD8+ reaction. ^{25 , 37 , 38}

In our study, HSP70i and BiP expression were found to be relatively higher in NSV than in SV, indicating a higher response to oxidative stress that can interfere with transcription, translation, and translation of mRNA in protein synthesis, which is related to the extent and severity of vitiligo in NSV. Thus, dissimilar to SV, melanocyte destruction in NSV is mediated over diverse, intricate, and interconnected mechanisms. ²⁶

Our study showed that expression using HSP70i was higher than with BiP, both in SV and NSV. Stressed vitiligo melanocytes secrete HSP70i that induce dendritic cells to increase the expression of CD80 and CD86, that stimulate immune responses to melanocytes. In a previous study using a mouse model, adding HSP70i alone could worsen vitiligo, perhaps causing over activation of dendritic cells in the skin. Meanwhile, in a mouse model lacking HSP70i expression, there was no depigmentation in the experimental study, which indicates the role of HSP70i in vitiligo. These dendritic cells can be stimulated to express more coactivation markers like CD80 and CD86, which will amplify the immune response against melanocytes. ⁴ As a result, HSP70i, which is overexpressed in lesional vitiligo epidermis compared to normal skin, might cause an autoimmune reaction in response to environmental stressors such oxidative stresses. ^{4 , 35} Thus, HSP70i is preferred because it can serve as a link between the innate and adaptive immune systems. ²⁷

The existence of this UPR is seen as crucial because if it is not immediately addressed, it will disrupt homeostasis and protein, lipid, and DNA synthesis, which will then activate the apoptotic signaling pathway, resulting in melanocyte apoptosis. Furthermore, the apoptosis produces neo antigens that will stimulate the immune system. ^{14 , 16} Melanocyte apoptosis is a programmed cell death mechanism involving endonuclease enzymes, characterized by shriveled melanocyte cells that have the characteristic pattern of a step ladder and eventually become fragmented and release apoptosis bodies. ³⁸ Precisely, CD8 ⁺ T lymphocytes play a variety of roles in mediating melanocyte death. ³⁹ The majority of melanocyte death is caused by immune cells breaching the self-tolerance as a result of melanocyte apoptosis, antigen exposure, and an inflammatory microenvironment. ¹⁸ In our investigation, the SV group had a relatively lower expression of melanocyte cell apoptosis than the NSV group. Although both SV and NSV exhibit melanocyte damage, it is unknown whether the loss of melanocytes in SV is brought on by an autoimmune reaction or a cellular defect that is already present. ¹⁸ However, our study is consistent with other studies showing an elevated anti-melanocyte antibody titer in NSV patients. This can be used as an important basis in the vitiligo pathogenesis. ⁴⁰

In conclusion, the appearance of UPR and oxidative stress affect the progression of vitiligo, especially in NSV. Both HSP70i and BiP expression can be UPR markers, although HSP70i is superior in expressing UPR, compared with BiP, because it represents a link between the adaptive and innate immune systems; thus, HSP70i is recommended for use in future studies. The existence of this UPR is crucial because it can activate the apoptotic signaling pathway resulting in melanocyte apoptosis. Additional studies are necessary to investigate if UPR can be used as a new target in the treatment of vitiligo.

Written informed consent for publication of their clinical details and clinical images was obtained from the patients.