Characterization of a dendritic cell specific receptor critical for SJS

对 SJS 至关重要的树突状细胞特异性受体的表征

基本信息

批准号：
10219058
负责人：
Xinzhong Dong
金额：
$ 40.94万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-16 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10219058
关键词：
Affect Alleles Allopurinol Animal Model Antigen Presentation Antigen-Presenting Cells Antigens Binding Biological Assay Bulla Calcium Cell Death Cell Surface Receptors Cell physiology Cell surface Cells Characteristics Coculture Techniques Complex Cornea Cytotoxic T-Lymphocytes Data Dendritic Cells Development Dose Drug Exposure Epidermis Excision Exposure to Eye Eyelid structure Family Flow Cytometry G-Protein-Coupled Receptors Genetic Goals Hand Hemorrhage Homologous Gene Hospitalization Human Image Immune Immune response Immune system Immunohistochemistry Inflammation Mediators Inflammatory Knock-out Knockout Mice Knowledge Lead Lesion Life Major Histocompatibility Complex Mediating Mediator of activation protein Molecular Molecular Target Mucous Membrane Mus Mutation Necrosis Open Reading Frames Oral Oral Ingestion Pathway interactions Patients Pharmaceutical Preparations Pharmacotherapy Phenotype Play Process Reaction Recovery Research Reverse Transcriptase Polymerase Chain Reaction Role Site Spleen Stevens-Johnson Syndrome Symptoms Syndrome T-Cell Activation T-Cell Proliferation T-Lymphocyte Tissues adaptive immune response adverse drug reaction cellular targeting conjunctiva cytokine cytotoxic experimental study foot genetic approach in vivo keratinocyte lamotrigine lymph nodes medication safety member mouse model mutant novel novel therapeutics prevent receptor receptor expression response

项目摘要

Project Summary The goal of our research is to understand the cellular and molecular mechanisms behind the development of Stevens-Johnson syndrome. Adverse drug reactions (ADRs) are a serious drug safety concern that can be hard to predict and treat. Stevens-Johnson syndrome (SJS) is one of the most serious and life- threatening ADRs characterized by keratinocyte cell death and mucosal breakdown; patients often form lesions on their palms of hands and soles of their feet and blistering and necrosis of their eyelids, conjunctiva and cornea. The exact cellular and molecular mechanisms behind drugs triggering SJS is unknown. Using a Ca2+ imaging assay, we found certain members of the Mrgpr GPCR family are activated by multiple known SJS- causing drugs. To examine the immune effects of these SJS drugs, we developed a novel mouse treatment where daily oral ingestion of a SJS-causing drug resulted in WT mice forming mucosal secretion in their eyes and blister bleeding in their hindpaws, similar to symptoms seen in patients suffering from SJS. We generated a mouse line where the specific Mrgpr's open reading frame was deleted and replaced with GFP and this genetic removal prevented formation of the SJS-like phenotype. Strikingly, heterozygous Mrgpr +/GFP mice, mice retaining 1 functional copy of the certain Mrgpr allele, developed the SJS-like phenotype, implying an important role for this Mrgpr in drug-caused SJS. Further experiments revealed the Mrgpr was expressed in a subset of dendritic cells, cells known to play an important role in antigen-presentation and immune response initiation. In this proposal, we will use molecular, cellular, genetic approaches and a potential novel in vivo animal model to uncover the role of the Mrgpr-expressing dendritic cells in drug-activated SJS. Aim I will focus on establishing our novel drug-induced SJS mouse model. We will examine what immune changes occur as mice are dosed with specific SJS-causing drugs and then we will determine what role the specific Mrgprs play in these immune responses. In Aim II, we will examine the specific characteristics of Mrgpr-expressing dendritic cells. We will find what type of dendritic cell expresses the Mrgprs and the role they play in the immune system. We will then examine how the cells change upon exposure to SJS-causing drugs and how these changes cause a cytotoxic immune response and SJS phenotype. We will also confirm the pivotal role these Mrgpr-expressing cells play in the development of SJS by rescuing the phenotype in the Mrgpr KO mouse. In Aim III, we will continue examining the immune cascade triggered by the SJS drugs. Dendritic cells are known for their important role of presenting antigens to T cells, which activate and in turn cause an immune response. We will determine what T cells are being activated by the Mrgpr-expressing dendritic cells and what cellular effects and mediators are released by these activated T cells. We will establish the necessity of T cells in continuing the immune response that is initiated and maintained by activation of the Mrgpr-expressing dendritic cell. Data collected from this project will help reveal some of the cellular and molecular reasons behind certain drugs causing SJS; these new cellular and molecular targets could lead to new treatments and drug therapies for SJS and adverse drug reactions.

项目概要我们研究的目标是了解背后的细胞和分子机制史蒂文斯-约翰逊综合征的发展。药物不良反应（ADR）是一个严重的药物安全问题这可能很难预测和治疗。史蒂文斯-约翰逊综合征 (SJS) 是最严重且致命的疾病之一以角质形成细胞死亡和粘膜破坏为特征的威胁性不良反应；患者常形成病变手掌、脚底、眼睑、结膜和眼睑起泡、坏死角膜。药物引发 SJS 的确切细胞和分子机制尚不清楚。使用 Ca2+ 成像分析中，我们发现 Mrgpr GPCR 家族的某些成员被多种已知的 SJS-激活引起毒品。为了检查这些 SJS 药物的免疫效果，我们开发了一种新型小鼠治疗方法每天口服引起 SJS 的药物会导致 WT 小鼠在眼睛中形成粘膜分泌物后爪出现水泡出血，与 SJS 患者的症状相似。我们生成了删除特定 Mrgpr 开放阅读框并用 GFP 替换的小鼠品系基因去除阻止了 SJS 样表型的形成。引人注目的是，杂合 Mrgpr +/GFP 小鼠，保留某些 Mrgpr 等位基因的 1 个功能性拷贝的小鼠，形成了 SJS 样表型，这意味着该 Mrgpr 在药物引起的 SJS 中发挥着重要作用。进一步的实验表明 Mrgpr 表达于树突状细胞的子集，已知在抗原呈递和免疫反应中发挥重要作用的细胞引发。在这个提案中，我们将使用分子、细胞、遗传方法和一种潜在的体内新颖方法动物模型揭示了表达 Mrgpr 的树突状细胞在药物激活的 SJS 中的作用。我会专注的目标建立我们的新型药物诱导 SJS 小鼠模型。我们将检查发生的免疫变化给小鼠注射特定的引起 SJS 的药物，然后我们将确定特定的 Mrgprs 所起的作用在这些免疫反应中。在目标 II 中，我们将研究 Mrgpr 表达的具体特征树突状细胞。我们将发现什么类型的树突状细胞表达 Mrgprs 以及它们在免疫系统。然后我们将检查细胞在接触引起 SJS 的药物后如何变化以及如何变化。这些变化会引起细胞毒性免疫反应和 SJS 表型。我们还将确认关键作用这些表达 Mrgpr 的细胞通过拯救 Mrgpr KO 中的表型来参与 SJS 的发展老鼠。在目标 III 中，我们将继续检查 SJS 药物引发的免疫级联反应。树突状细胞因其向 T 细胞呈递抗原的重要作用而闻名，T 细胞激活并进而引起免疫回复。我们将确定哪些 T 细胞被表达 Mrgpr 的树突状细胞激活，以及哪些 T 细胞被激活。这些激活的 T 细胞释放细胞效应和介质。我们将确定T细胞的必要性继续由表达 Mrgpr 的激活启动和维持的免疫反应树突状细胞。该项目收集的数据将有助于揭示一些细胞和分子原因某些导致 SJS 的药物背后的原因；这些新的细胞和分子靶标可能会带来新的治疗方法和 SJS 的药物治疗和药物不良反应。