Transcriptome Response to Autoantibodies in Human Primary Epidermis Keratinocyte Model for Pemphgus Vulgaris

Pemphigus vulgaris (PV) is an autoimmune disease caused by autoantibodies (AAbs) targeting Desmoglein 1 (DSG1) or Desmoglein 3 (DSG3) on keratinocytes, leading to disrupted cell-cell adhesion and epidermal blistering. To investigate the early signaling events triggered by AAb binding, we examined transcriptomic responses in a human primary epidermis keratinocyte (HPEK) model for PV. After incubating the single-chain variable fragment (scFv) PX43, which targets DSG1 and DSG3, and human IgG as control for 5h, 10h and 24h, differentially expressed genes (DEGs) and regulated pathways was analyzed using DESeq2 and pathway enrichment analysis. Few significantly differentially expressed DEGs (under 10) were identified of PX43 compared to hIgG at all three time points (5h, 10h and 24h) with adjusted pvalue below 0.05 and |log2FC| >1. Upregulated inflammatory related pathways including TNFa signaling were identified only at 24h. Correlation analysis of the log2 transformed transcripts per million (TPM) shows the very high positive correlation of PX43 and hIgG at all time points (spearman correlation above 0.9) These findings indicate that AAbs targeting DSG1 and DSG3 do not drive broad transcriptomic or proteomic changes at the HPEK model. Overall design: Human skin normal human epidermal keratinocytes from three healthy human donors were grown in 2D until 85% confluenent. Calcium concentration was increased to establish cell-cell adhesion and differentiation. Cells were then incubated with DSG-targeting PX43 (DSG1 and DSG3) antibodies and human IgG as controls, with transcriptome data collected by RNA-seq at 5, 10, and 24, 48 hours post-stimulation.

寻常型天疱疮（Pemphigus vulgaris, PV）是一种由靶向角质形成细胞表面桥粒芯糖蛋白1（Desmoglein 1, DSG1）或桥粒芯糖蛋白3（Desmoglein 3, DSG3）的自身抗体（autoantibodies, AAbs）引发的自身免疫病，可导致细胞间黏附受损及表皮水疱形成。为探究自身抗体结合所触发的早期信号事件，本研究针对PV构建了人原代表皮角质形成细胞（human primary epidermis keratinocyte, HPEK）模型，并检测其转录组应答情况。将靶向DSG1与DSG3的单链可变片段（single-chain variable fragment, scFv）PX43，以及作为对照的人IgG分别与细胞孵育5、10、24小时后，本研究采用DESeq2与通路富集分析，对差异表达基因（differentially expressed genes, DEGs）及调控通路进行了分析。在三个时间点（5h、10h、24h），与hIgG对照相比，PX43处理组仅鉴定得到不足10个校正后P值小于0.05且|log₂FC|>1的显著差异表达基因。仅在24小时时，可鉴定得到上调的炎症相关通路，包括TNF-α信号通路。对经log₂转换的每百万转录本（transcripts per million, TPM）进行相关性分析显示，PX43组与hIgG组在所有时间点均呈现极高的正相关（斯皮尔曼相关系数大于0.9）。上述研究结果表明，靶向DSG1与DSG3的自身抗体在HPEK模型中不会引发广泛的转录组或蛋白质组变化。总体实验设计：从3名健康人体供体分离得到的正常人皮肤表皮角质形成细胞以二维培养方式扩增至85%汇合度。随后提高钙浓度以建立细胞间黏附并诱导细胞分化。将细胞分别与靶向DSG的PX43（靶向DSG1与DSG3）抗体及人IgG对照孵育，并在刺激后5、10、24及48小时通过RNA测序（RNA-seq）获取转录组数据。