Transcriptomic analyses of cutaneous immune network in patiens with atopic dermatitis challenged with house dust mite (HDM) patch test. 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）患者原代皮肤细胞的激活状态与转录调控程序，相关样本包括经荧光激活细胞分选（FACS）纯化的朗格汉斯细胞（Langerhans cells），以及全活检组织的单细胞分析；所有受试者均接受了对照或屋尘螨（HDM）斑贴试验，本研究共纳入22例患者，其中nr为应答者，nnr为无应答者。斑贴试验施加48小时后，从对照位点与HDM斑贴试验位点采集6mm活检组织。
整体实验设计：本研究招募了在三级转诊中心接受皮肤科医师诊疗的中重度特应性皮炎患者，其湿疹严重程度评分（Eczema Area and Severity Index，EASI）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例入组患者中11例呈现HDM反应阳性，12例HDM未诱导可见反应，构成“HDM无应答组”；另有4例对HDM与对照斑贴试验均产生刺激性反应，1例出现红斑但未局限于斑贴试验部位，归类为“未明确组”。采集所有斑贴试验位点的6mm活检组织，分别用于流式细胞术或单细胞核糖核酸测序（single cell RNA-seq）分析。对CD207、CD1a、HLA-DR阳性的朗格汉斯细胞（LCs）进行FACS分选，收集至RLT缓冲液中，用于批量核糖核酸测序（bulk RNA-seq）。非朗格汉斯细胞经分选后冻存，用于后续Constellation 10X平台分析。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（Drop-seq技术）。