一、研究背景和目的

变应性接触性皮炎（allergic contact dermatitis，ACD）是一种常见的湿疹性皮肤病，是在人体致敏后，皮肤再次接触该化学物质引起的一种迟发型超敏反应。典型表现是边界清楚的瘙痒性湿疹样皮疹，可以是急性皮损（水泡、渗出、水肿）或慢性皮损（苔藓化或鳞屑性斑块)。可出现于任何人群，在大多数情况下会持续患者终生。

该病的发病机制主要是化学特异性CD8阳性T细胞快速募集，从而诱导角质形成细胞的凋亡。此外，CD4阳性Th1和Th17通过释放激活角质形成细胞和其他皮肤驻留细胞的促炎细胞因子，进一步引起炎症反应的扩展。但ACD中角质形成细胞的非凋亡死亡方式以及死亡方式之间的相互调控研究尚不明确。

自噬，是真核细胞用于维持营养稳态和控制细胞器质量的主要分解代谢机制。它主要由一组进化上保守的自噬相关基因(autophagy-related gene，ATG）介导，在维护组织和细胞内环境稳定中起着非常关键的生物学作用。同时，自噬在细胞凋亡、铁死亡、调节性坏死等调控性细胞死亡中起到相互连结、相互调控的作用。

本实验在上述研究背景下，旨在明确在ACD中表皮角质形成细胞中的自噬和细胞死亡，并研究自噬关键基因对调控性细胞死亡的调控作用。

二、实验方法

1、于基因表达总库（gene expression omnibus，GEO）数据库中下载变应性接触性皮炎数据集GSE6281，生物信息学分析患者病变组织0h和96h的差异基因。通过基因本体论（gene ontology，GO）分析和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）分析获得差异基因富集后的相关通路。

2、通过CIBERSORT程序包分析ACD患者接触致敏原镍96h皮肤组织样本中免疫细胞比例。通过genecard网站筛选出自噬、凋亡、焦亡、坏死性凋亡相关基因，运用生物信息学分析疾病组和对照组之间自噬及调节性细胞死亡的差异基因。通过spearman相关性分析免疫细胞与前述自噬及调节性细胞死亡差异基因的相关性。

3、通过体外添加肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和干扰素-γ（interferon-γ，IFN-γ）模拟ACD炎症环境，分别运用乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验和蛋白免疫印迹（Western blotting）分析检测角质形成细胞的细胞死亡和自噬。

4、通过背部、右耳涂抹二硝基氯苯（dinitrochlorobenzene，DNCB）致敏及激发的方法制备小鼠变应性接触性皮炎模型。

5、观察角质形成细胞内特异性敲除Atg5的模式鼠（Krt14^cre/+ -Atg5 ^flox/flox，Atg5 cKO）以及其同窝对照鼠 (Krt14^+/+ -Atg5 ^flox/flox)造模后右耳的皮损变化并通过拍照、评分与耳朵厚度测量评价两组炎症程度差异。

6、通过冰冻组织免疫荧光和Western blotting分析检测小鼠耳部ATG5蛋白水平。

7、通过免疫组化染色检测小鼠耳部MPO阳性、F/480阳性、CD4阳性、CD8阳性免疫细胞变化。

8、通过苏木精—伊红染色法（hematoxylin-eosin staining，H&E）检测小鼠耳部组织病理学变化。通过脱氧核糖核苷酸末端转移酶（TdT）介导的缺口末端标记法(terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL)检测小鼠耳部表皮细胞死亡水平。

9、通过冰冻组织免疫荧光和Western blotting分析检测凋亡关键蛋白（Cleaved-Caspase 3、PARP）蛋白水平。

三、实验结果

1、数据集差异基因通路富集分析聚集到凋亡、坏死性凋亡等通路。

2、生物信息学分析显示ACD患者接触致敏原镍96h皮肤组织样本中以CD4阳性T淋巴细胞、CD8阳性T淋巴细胞、M2型巨噬细胞等免疫细胞浸润为主。相关性分析显示自噬及调节性细胞死亡(regulated cell death，RCD)与CD4阳性淋巴细胞、γδT细胞、树突状细胞等密切相关。

3、体外TNF-α和IFN-γ模拟的ACD炎症环境中存在角质形成细胞自噬和细胞死亡。

4、小鼠变应性接触性皮炎模型中角质形成细胞Atg5基因敲除导致炎症表型加重。

5、小鼠变应性接触性皮炎模型中DNCB诱导剂不干扰ATG5表达的变化。

6、小鼠表皮特异性敲除Atg5导致加重的炎症表型与免疫细胞浸润无关。

7、小鼠表皮特异性敲除Atg5导致加重的炎症表型中角质形成细胞总死亡水平增加。

8、小鼠表皮特异性敲除Atg5导致加重的炎症表型中角质形成细胞凋亡水平增加。

四、结论

1、自噬及多种RCD（凋亡、焦亡、程序性坏死）的相关基因在ACD患者中高表达，且与CD4阳性记忆T细胞、γδT等免疫细胞密切相关。自噬及RCD可能参与ACD的免疫病理过程。

2、小鼠角质形成细胞自噬关键基因Atg5敲除后，细胞自噬被抑制，死亡方式转化为凋亡，引起变应性接触性皮炎炎症加重，而炎症变化不依赖于免疫细胞的变化。

Background and objectives

Allergic contact dermatitis (ACD) is a common skin disease of eczematous dermatitis. It is a delayed type hypersensitivity reaction caused by skin re-exposure to the chemical after sensitization. The typical phenomenon is a well-defined itchy eczema-like rash that can be acute (blisters, exudation, edema) or chronic (lichenization or scale). It can appear in any population and in most cases lasts patients’ lifetime.

The pathogenesis of this disease is mainly the rapid recruitment of chemical specific CD8 positive T cells, which induces apoptosis of keratinocytes. In addition, CD4 positive Th1 and Th17 further promote the expansion of the inflammatory response by releasing proinflammatory cytokine that activate keratinocyte and other skin-resident cells. However, the keratinocyte of non-apoptotic death and the regulation of death in ACD remain unclear.

Autophagy is a major catabolic mechanism in eukaryotic cells to maintain nutrient homeostasis and control organelle quality. It is mainly mediated by a group of evolutionarily conserved autophagy-related gene (ATG), which plays a key biological role in the maintenance of tissue and intracellular environmental stability. Meanwhile, autophagy is related to regulate cell death, such as apoptosis, ferroptosis and necroptosis.

In this study, we aim to clarify autophagy and cell death in epidermal keratinocytes and to investigate the role of autophagy key genes in the regulation of regulated cell death in ACD.

Materials and methods

1. GSE6281 data was downloaded from gene expression omnibus database (GEO), and the differential genes were bioinformatics analyzed at 0h and 96h. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis were used to obtain the related pathways after gene enrichment.

2. The proportion of immune cells in skin tissue samples from ACD patients exposed to nickel allergen for 96 hours was analyzed by CIBERSORT package. Genes related to autophagy, apoptosis, pyroptosis and necroptosis were screened by the genecard website. And the differential genes of autophagy and RCD between the disease group and the control group were analyzed by bioinformatics. And Spearman correlation was used to analyze correlation between immune cells and the genes of autophagy and RCD).

3. The inflammatory environment of ACD was simulated by adding the tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in vitro, and the cell death and autophagy of keratinocytes were detected by lactate dehydrogenase (LDH) release assay and Western blotting analysis, respectively.

4. The model of ACD in mice was established by sensitization and provocation of dinitrochlorobenzene (DNCB) on their back and right ears.

5. After building ACD models in Atg5 keratinocyte condition knockout mice (Krt14^cre/+ -Atg5 ^flox/flox, Atg5 cKO) and control mice (Krt14^+/+ -Atg5 ^flox/flox) , the changes of skin lesions in the right ears were evaluated by taking pictures, scoring and measuring ears thickness.

6. The level of ATG5 protein in mice ears was detected by immunofluorescence and western blotting.

7. The changes of MPO positive, F4/80 positive, CD4 positive and CD8 positive immune cells were detected by immunohistochemistry.

8. Hematoxylin-eosin (H&E) staining was used to detect the changes of ear histopathology in mice. The level of mice ears epidermal cell death was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining).

9. The levels of key apoptosis proteins (Cleasved-Caspase 3, PARP) were detected by immunofluorescence and Western blotting.

Results

1. The data of differential gene pathway enrichment analysis clusters to apoptosis, necroptosis and other pathways.

2. Bioinformatics analysis shows that CD4 positive T lymphocytes, CD8 positive T lymphocytes, M2 macrophages and other immune cells are mainly infiltrated in the skin tissue samples of ACD patients exposed to nickel for 96 hours. Correlation analysis shows that autophagy and RCD are closely related to CD4 positive lymphocytes, γδT cells and dendritic cells.

3. Keratinocyte autophagy and cell death are involved in the inflammatory environment of ACD simulated by tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in vitro.

4. Keratinocyte-specific Atg5 deficiency presents aggravation of phenotype in DNCB-induced ACD mice.

5. DNCB motivation does not lead to change of ATG5 expression.

6. Infiltrated immune cells might not contribute to aggravation of phenotype of DNCB-stimulated Atg5 cKO mice.

7. Increase of keratinocyte death is involved in aggravation of phenotype in DNCB-stimulated Atg5 cKO mice.

8. Atg5 deficiency in keratinocytes induces the increase of keratinocyte apoptosis in DNCB-induced ACD.

Conclusions

Autophagy and RCD (apoptosis, pyroptosis, necroptosis) related genes are highly expressed in ACD patients, and are closely related to CD4 positive memory T cells, γδT and other immune cells. Autophagy and RCD may be involved in the immunopathological process of ACD.

Deficiency of Atg5 (a key gene for autophagy) in mouse keratinocytes inhibits autophagy but increases apoptosis, aggravating skin damage in DNCB-induced ACD mice. This inflammatory changes has no relationship with the involvement of immune cells.