The Formation of Melanocyte Apoptotic Bodies in Vitiligo and the Relocation of Vitiligo Autoantigens under Oxidative Stress.

Jun Tian,Bin Jiao,Tao Wang,Jiaxi Chen,Kai Li,Chunying Li,Yue Zhang,Xiuli Yi,Yaojun Wang,Ming Ding,Zhe Jian,Gang Wang,Ling Liu,Qiang Li,Jiaoni Chi,Ye Huang,Tianwen Gao,Chengfeng Zhang

doi:10.1155/2021/7617839

Abstract

Background Oxidative stress has a vital role in the early stages of vitiligo. Autoantigens released from apoptotic melanocytes (MC) under oxidative stress are involved in the presentation and recognition of antigens. However, the transport of autoantigens to the cell surface and their release to the extracellular environment are still unclear. Apoptotic bodies (ABs) have always been considered as a key source of immunomodulators and autoantigens. Yet, the role of ABs in the immune mechanism of vitiligo is still unknown. Purpose To explore whether MC's autoantigens translocate into ABs during oxidative stress-induced apoptosis and study the molecular mechanisms underlying autoantigen migration and AB formation. Methods PIG3V (an immortalized human vitiligo melanocyte cell line) were treated with H2O2, and ABs were separated. Transmission electron microscopy, flow cytometry, Western blot, mass spectrometry, and other methods were used to determine the relocation of specific antigens in PIG3V cells to ABs. After pretreatment with specific inhibitors (Rho kinase (Y-27632), myosin light chain kinase (MLCK, ML-9), pan-caspase (zVAD-FMK), and JNK (SP600125)), the pathway of autoantigen translocation into ABs and the formation of apoptotic bodies were determined. Results When treated with 0.8 mM H2O2, ABs were released from these cells. Autoantigens such as tyrosinase-related protein 1 (TYRP-1) and cleavage nuclear membrane antigen Lamin A/C (Asp230) were concentrated in ABs. The expression of autoantigens and the formation of ABs increased in a time- and dose-dependent manner after treatment with H2O2, while the application of specific inhibitors inhibited the formation of apoptotic bodies, i.e., the expression of antigens. Conclusion Vitiligo autoantigens translocate into ABs in the process of apoptosis induced by oxidative stress. The cytoskeletal protein activation pathway and the JNK-related apoptosis pathway are involved in the transport of autoantigens and the formation of ABs. ABs may be the key bridge between MC cell apoptosis and cellular immunity.

Highlights

Oxidative stress has a critical role in the activation of autoimmunity related to vitiligo [1]
The morphological characteristics of apoptotic bodies were analyzed by transmission electron microscopy (TEM), and the functions of apoptotic bodies were analyzed by flow cytometry
PS is highly expressed on the membrane surface of Apoptotic bodies (ABs) as a key feature of ABs [8, 9], and to further characterize the quality of the collected ABs, we stained them with annexin V and analyzed them by flow cytometry [10], and the results suggested that the results showed that more than 90% of the examined particles were positive for annexin V (Figure 1(c)), suggesting that the majority of the particles we collected were from ABs

Summary

Introduction

Oxidative stress has a critical role in the activation of autoimmunity related to vitiligo [1]. Autoantigens released from apoptotic melanocytes (MC) under oxidative stress are involved in the presentation and recognition of antigens. To explore whether MC’s autoantigens translocate into ABs during oxidative stress-induced apoptosis and study the molecular mechanisms underlying autoantigen migration and AB formation. Transmission electron microscopy, flow cytometry, Western blot, mass spectrometry, and other methods were used to determine the relocation of specific antigens in PIG3V cells to ABs. After pretreatment with specific inhibitors (Rho kinase (Y-27632), myosin light chain kinase (MLCK, ML-9), pan-caspase (zVAD-FMK), and JNK (SP600125)), the pathway of autoantigen translocation into ABs and the formation of apoptotic bodies were determined. The cytoskeletal protein activation pathway and the JNK-related apoptosis pathway are involved in the transport of autoantigens and the formation of ABs. ABs may be the key bridge between MC cell apoptosis and cellular immunity

Methods

Results

Discussion

Conclusion