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.

项目摘要我们研究的目的是了解背后的细胞和分子机制史蒂文斯 - 约翰逊综合症的发展。不良药物反应（ADR）是严重的药物安全问题这可能很难预测和治疗。史蒂文斯 - 约翰逊综合症（SJS）是最严重和生活的一种 - 威胁性ADR，其特征是角质形成细胞死亡和粘膜崩溃；患者经常形成病变在他们的手掌和脚底，眼睑，结膜和坏死的手掌上角膜。触发SJS的药物背后的确切细胞和分子机制尚不清楚。使用Ca2+ 成像测定法，我们发现MRGPR GPCR家族的某些成员被多个已知的SJS-激活引起毒品。为了检查这些SJS药物的免疫作用，我们开发了一种新型的小鼠治疗每天口服引起SJS的药物会导致WT小鼠在他们眼中形成粘膜分泌并在后爪中出血，类似于患有SJS的患者的症状。我们生成了在其中删除了特定MRGPR的开放式阅读框并用GFP替换的鼠标系遗传去除阻止了SJS样表型的形成。令人惊讶的是，杂合MRGPR +/GFP小鼠，保留某些MRGPR等位基因的1个功能副本的小鼠开发了类似SJS的表型，这意味着该MRGPR在引起药物引起的SJS中的重要作用。进一步的实验表明，MRGPR在A中表达树突状细胞的子集，即已知在抗原表征和免疫反应中起重要作用的细胞引发。在此提案中，我们将使用分子，细胞，遗传方法和潜在的新型体内新颖动物模型揭示了表达MRGPR的树突状细胞在药物激活的SJ中的作用。目的我将集中精力建立我们的新型药物诱导的SJS小鼠模型。我们将检查哪些免疫变化发生小鼠用特定的SJS引起药物剂量，然后我们将确定特定的MRGPR扮演的角色在这些免疫反应中。在AIM II中，我们将检查MRGPR表达的特定特征树突状细胞。我们会发现哪种类型的树突细胞表达了MRGPR及其在免疫系统。然后，我们将研究细胞在暴露于SJS引起SJS的药物时如何变化，以及如何这些变化会导致细胞毒性免疫反应和SJS表型。我们还将确认关键角色这些表达MRGPR的细胞通过在MRGPR KO中拯救表型来发挥SJS的发展老鼠。在AIM III中，我们将继续研究SJS药物触发的免疫级联反应。树突状细胞以将抗原呈现给T细胞的重要作用而闻名，T细胞激活并导致免疫回复。我们将确定由表达MRGPR的树突细胞激活哪些T细胞以及什么这些活化的T细胞释放了细胞效应和介体。我们将确定T细胞的必要性在继续通过表达MRGPR的激活开始和维持的免疫反应时树突状细胞。从该项目收集的数据将有助于揭示一些细胞和分子原因引起SJ的某些药物的背后；这些新的细胞和分子靶标可能会导致新的治疗方法 SJS和不良药物反应的药物疗法。