Transcriptomic analyses of cutaneous immune network in patiens with atopic dermatitis challenged with house dust mite (HDM) patch test

Our study investigates the activation and transcriptional programming of primary skin cells (FACS-purified Langerhans cells and single cell analysis of the whole biopsy) from patients with atopic dermatitis (n=22, nr = rensponding, nnr=nonresponding), exposed to a control or HDM patch test. 6 mm biopsies were taken from control and HDM patch test site 48h post application. Overall design: Patients with moderate to severe atopic dermatitis eczema severity scores (EASI) median = 17.70, IQR:10.20 – 30.90, max = 51.40) under the care of a dermatologist in a tertiary referral centre were recruited to the study. 48h post application of a patch test to buttock skin, 11 out of the 28 patients were HDM-reactive, while In 12 patients, HDM did not induce a visible response, creating “HDM-non-reactive” group. 4 patients reacted to both HDM and control patch test "irritant", and one showed redness which was not patch test localised "undefined". 6 mm biopsies were taken from all patch test sites, and processed for flow cytometry or single cell RNA-seq analysis. CD207, CD1a, HLA-DR+ Langerhans cells (LCs)were FACS-sorted into RLT buffer and processed for bulk RNA-seq. Non-LCs cells were sorted and cryopreserved for further analysis with Constellation 10X. Freshly dissociated whole skin biopsies from 6 biopsies were suspended in RNAse-out buffer and processed on ice to the co-encapsulation of single cells with genetically-encoded beads (Drop-seq).

本研究探究了特应性皮炎（atopic dermatitis, AD）患者原代皮肤细胞的激活状态与转录调控程序，实验样本包含经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化的朗格汉斯细胞（Langerhans cells, LCs）以及全皮肤活检组织的单细胞分析样本；受试者接受对照斑贴或屋尘螨（House Dust Mite, HDM）斑贴试验干预。本研究标注总样本量n=22，其中nr为应答者（responding）、nnr为无应答者（nonresponding）。
斑贴试验施加48小时后，从对照斑贴与HDM斑贴试验部位采集6mm直径皮肤活检组织。
整体实验设计：
本研究招募了在三级转诊中心皮肤科就诊的中重度特应性皮炎患者，其湿疹面积和严重程度指数（Eczema Area and Severity Index, EASI）的中位数为17.70，四分位距为10.20–30.90，最大值为51.40。
在臀部皮肤施加斑贴试验48小时后，28名入组患者中11名呈现HDM阳性反应，12名患者未出现HDM诱导的可见反应，由此构建"HDM-non-reactive"组；另有4名患者对HDM与对照斑贴均呈现刺激性反应，1名患者出现的红斑未局限于斑贴试验部位，归类为"undefined"（不确定）组。
从所有斑贴试验部位采集6mm直径活检组织，分别用于流式细胞术或单细胞RNA测序（single cell RNA-seq, scRNA-seq）分析。
对CD207、CD1a、HLA-DR阳性的朗格汉斯细胞进行FACS分选，将其置于RLT缓冲液中，随后进行批量RNA测序（bulk RNA-seq）。
对非朗格汉斯细胞进行分选并冷冻保存，后续通过Constellation 10X平台开展进一步分析。
取6份新鲜解离的全皮肤活检组织，悬浮于RNA酶抑制剂（RNAse-out）缓冲液中，在冰上进行处理，实现单细胞与基因编码微球的共包裹（即Drop-seq技术）。