Characterization of a dendritic cell specific receptor critical for SJS

项目摘要 我们研究的目标是了解细胞和分子机制背后的 Stevens-Johnson综合征药物不良反应（ADR）是一个严重的药物安全问题 Stevens-Johnson综合征（SJS）是一种最严重的， 以角质形成细胞死亡和粘膜破坏为特征的威胁性ADR;患者通常形成病变 手掌和脚底，眼睑、结膜和 角膜药物触发SJS的确切细胞和分子机制尚不清楚。使用Ca 2 + 通过成像分析，我们发现Mrgpr GPCR家族的某些成员被多种已知的SJS激活， 导致毒品。为了检查这些SJS药物的免疫作用，我们开发了一种新型小鼠治疗方法 其中每天口服引起SJS的药物导致WT小鼠在其眼睛中形成粘膜分泌物 和水泡出血在他们的后爪，类似的症状，看到病人患有SJS。我们产生 一个小鼠系，其中特定的Mrgpr开放阅读框被删除并被GFP取代， 遗传去除阻止了SJS样表型的形成。引人注目的是，杂合子Mrgpr +/GFP小鼠， 保留1个特定Mrgpr等位基因功能拷贝的小鼠，发展出SJS样表型，这意味着 Mrgpr在药物引起的SJS中的重要作用。进一步的实验表明，Mrgpr在一个细胞中表达。 树突状细胞亚群，已知在抗原呈递和免疫应答中发挥重要作用的细胞 入会仪式在本提案中，我们将使用分子、细胞、遗传方法和潜在的体内新方法 动物模型来揭示表达Mrgpr的树突状细胞在药物激活的SJS中的作用。我会集中精力 建立我们的新型药物诱导的SJS小鼠模型。我们将研究免疫系统的变化， 给小鼠服用特定的引起SJS的药物，然后我们将确定特定的Mrs. in these immune免疫responses反应.在目的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哪种类型的树突状细胞表达Mrs 19，以及它们在免疫系统中的作用。 免疫系统然后，我们将研究细胞在暴露于引起SJS的药物后如何变化，以及 这些变化引起细胞毒性免疫应答和SJS表型。我们还将确认 这些表达Mrgpr的细胞通过挽救Mrgpr KO中的表型在SJS的发展中发挥作用 老鼠.在目标III中，我们将继续研究SJS药物引发的免疫级联反应。树突状细胞 以其向T细胞呈递抗原的重要作用而闻名，T细胞激活并反过来引起免疫应答。 反应我们将确定表达Mrgpr的树突状细胞激活了哪些T细胞， 细胞效应和介质由这些活化的T细胞释放。我们将建立T细胞的必要性 在持续免疫应答中，所述免疫应答是通过激活表达Mrgpr的 树突状细胞从这个项目收集的数据将有助于揭示一些细胞和分子的原因 这些新的细胞和分子靶点可能会导致新的治疗方法， SJS的药物治疗和药物不良反应。

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.