Superficial Conjunctival Cells from Dupilumab-treated Atopic Dermatitis Patients with Ocular Adverse Events Display a Transcriptomic Psoriasis Signature

Dupilumab has demonstrated efficacy in the treatment of atopic dermatitis (AD). However, a subset of patients experiences ocular adverse events (OAE), including conjunctivitis and dry eye syndrome, the pathological mechanisms of which are still unknown Study design We conducted a bicentric clinical study between Toulouse and Nantes Hospitals. It is ancillary to the clinical study in Toulouse from 2017 and the multicentric DUPIOEIL study from 2019 in Toulouse and Nantes. This study was conducted in accordance with the declaration of Helsinki. Informed consent was obtained from all participants at M0. Population All patients who needed to be treated with dupilumab were aged over 18 years. Dermatology and ophthalmology examinations were performed on the same day, before treatment introduction. Four months after the beginning of dupilumab treatment (M4) the same ophthalmologist (MC in Toulouse and MW in Nantes) examined the patients again. We excluded patients who could not be consulted before treatment or at M4. The ophthalmological exams concerns the ocular surface status (slit lamp exam, Shirmer test I, Oxford scale and conjunctival impression). Occurrence of OAE was defined as an aggravation of the Oxford scale or development of a conjunctivitis at M4 compared to M0. At M4, we divided the patients in two groups: one group who developed OAE (OAE+) and a control group with patients presenting a comparable ophthalmological exam as before dupilumab treatment (OAE-). Conjunctival cells collection At M0 and M4 examinations, a conjunctival impression was performed in routine examination to analyze ocular surface. The conjunctival impression was collected by the same practitioner (MC in Toulouse and MW in Nantes) for all patients at M0 and M4. After an instillation of anesthetic drop (oxybuprocain, Théa Pharma, Clermont-Ferrand, France), a conjunctival impression device (Eyeprim, Opia technologies SAS, Paris, France) was used to collect cells from temporal superior quadrant at 2 mm from the limbus. The sampling was immediately preserved in liquid nitrogen and then stocked à - 80°C for later evaluation. RNA extraction and isolation Total RNA was directly extracted from the membranes with Qiagen MicroRNA extraction kit according to the manufacturer's instructions (Qiagen S.A.S., Cortaboeuf, France). Reverse transcriptase was performed using the Invitrogen Superscript III VILO kit according to the manufacturer’s recommendations (ThermoFisher Scientific, Villebon-sur-Yvette, France). RNA quantity and quality were determined using QBIT system (Thermofisher Scientific) and Agilent (Agilent, CA). Selected samples had an RNA integrity number above 8 and RNA concentration above 25 ng/µl. Microarray Analysis A full transcriptome microarray analysis was performed using the human Clariom™ S Assay platform (Affymetrix, Thermo Fisher Scientific,Waltham, MA, USA) at the Genotoul Get Biopuces facility (Toulouse, France). Five ng per sample was processed.度普利尤单抗（dupilumab）在特应性皮炎（atopic dermatitis, AD）的治疗中已被证实具有疗效。然而，部分患者会出现眼部不良事件（ocular adverse events, OAE），包括结膜炎与干眼症，其病理机制目前仍未明确。 研究设计 本研究为图卢兹与南特医院开展的双中心临床研究，作为2017年图卢兹地区临床研究及2019年图卢兹、南特多中心DUPIOEIL研究的附属研究进行。本研究遵循《赫尔辛基宣言》开展，所有受试者均于M0时间点签署知情同意书。 研究人群 所有需接受度普利尤单抗治疗的患者均为18岁以上成人。在治疗启动当日，同步完成皮肤科与眼科检查。度普利尤单抗治疗启动4个月后（M4），由同一名眼科医师（图卢兹中心为MC，南特中心为MW）再次对患者进行检查。本研究排除了无法在治疗前或M4时间点接受随访的患者。眼科检查内容涵盖眼表状态评估，包括裂隙灯检查（slit lamp exam）、施墨I试验（Shirmer test I）、牛津评分（Oxford scale）及结膜印迹细胞学检查（conjunctival impression）。眼部不良事件的定义为：与M0时间点相比，M4时间点牛津评分升高或新发结膜炎。在M4时间点，我们将患者分为两组：出现眼部不良事件的OAE+组，以及眼科检查结果与度普利尤单抗治疗前无显著差异的对照组（OAE-组）。 结膜细胞采集 在M0及M4时间点的检查中，常规开展结膜印迹细胞学检查以分析眼表状态。该样本采集工作由同一名医师（图卢兹中心为MC，南特中心为MW）完成。先予以奥布卡因滴眼液（oxybuprocain, Théa Pharma, 法国克莱蒙费朗）表面麻醉，随后使用结膜印迹采集装置（Eyeprim™, Opia technologies SAS, 法国巴黎），在距角膜缘2mm的颞上象限采集细胞。采集完成后立即将样本置于液氮中保存，后续于-80℃冰箱储存以待后续检测。 RNA提取与分离 按照制造商操作规程，使用Qiagen MicroRNA提取试剂盒（Qiagen S.A.S., 法国科尔塔伯夫）直接从印迹膜中提取总RNA。按照制造商推荐方案，使用Invitrogen Superscript III VILO试剂盒（ThermoFisher Scientific, 法国维勒邦叙尔伊维特）进行反转录反应。采用QBIT系统（Thermofisher Scientific）与安捷伦生物分析仪（Agilent, 美国加利福尼亚州）检测RNA的浓度与质量，入选样本的RNA完整性指数（RNA integrity number, RIN）需大于8，且RNA浓度需高于25 ng/µl。 微阵列分析 在法国图卢兹的Genotoul Get Biopuces平台，采用人源Clariom™ S检测试剂盒（Affymetrix, Thermo Fisher Scientific, 美国马萨诸塞州沃尔瑟姆）开展全转录组微阵列分析。每个样本取5 ng RNA进行实验。