Regulation of TSLP receptor expression and function in eczema in mice and man

描述(申请人提供)：Th2促进细胞因子，胸腺基质淋巴生成素(TSLP)，与慢性炎症性皮肤病特应性皮炎(AD)的发病机制有关。了解提高阿尔茨海默病患者对TSLP反应性的免疫机制有助于开发新的治疗方法，抑制这种疾病背后的炎症级联反应。AD患者对TSLP的反应性是通过一种依赖于变应原连接Fc受体后树突状细胞(DC)上调TSLP受体(TSLPR)能力的过程来增强的。其目的是阐明TSLPR在AD中表达的免疫遗传学机制，并确定该受体与Th2驱动的皮肤炎症反应的功能相关性。人类和小鼠的互补方法将把AD患者细胞的体外研究(AIMS 1和AIMS 2)与AD的活体小鼠模型(AIM 3)结合起来。在目标1中，将测试一系列与树突状细胞表面受体(过敏原和Toll样受体配体)结合的分子上调TSLPR和相关信号成分的能力，这些信号成分通过流式细胞仪和基因沉默技术得到阐明。在DC/T细胞共培养中，将验证在扩增TSLP介导的Th2反应时是否需要上调TSLPR。作为辅助手段，我们将探讨细胞因子对TSLPR的抑制作用，以确定这一过程在AD中是否处于失调状态。为了评估体内相关性，将用流式细胞仪和共聚焦显微镜技术分析皮损皮肤和特应性斑贴试验部位离散DC类型的TSLPR表达。在目的2中，将检测TSLPR基因的遗传变异和该基因的表观遗传变化对TSLPR上调的影响。将使用大量患者的样本对TSLPR基因进行测序，以确认5‘上游区域并识别单核苷酸多态。然后将使用生物信息学和荧光素酶报告分析来鉴定和确认增强TSLPR基因活性的启动子元件和单倍型。将通过染色质免疫沉淀和DNA酶超敏试验评估树突状细胞中TSLPR基因座的染色质重塑。编码微丝蛋白的基因功能丧失突变也将被分型，以探索皮肤屏障中的遗传缺陷与易导致TSLPR上调的因素之间的关系。微丝蛋白是一种对皮肤屏障功能至关重要的蛋白质。在目标3中，将在缺乏朗格汉斯细胞或CD11c树突状细胞的小鼠身上检测表皮和真皮树突状细胞在TSLP驱动的炎症中的作用。朗格汉斯细胞对TSLP的直接反应的要求将在骨髓重建研究中进行测试。为了确定导致TSLP驱动的皮肤Th2型炎症的主要细胞类型(S)，将使用混合嵌合小鼠来限制TSLP对特定细胞类型的反应性。TSLPR基因的条件零等位基因也将产生小鼠，使TSLP无反应性被限制在特定的谱系。

DESCRIPTION (provided by applicant): The Th2-promoting cytokine, thymic stromal lymphopoietin (TSLP), has been implicated in the pathogenesis of the chronic inflammatory skin condition, atopic dermatitis (AD). Understanding immune mechanisms that increase responsiveness to TSLP in AD could aid in developing new treatments that inhibit the inflammatory cascade underlying this disease. Responsiveness to TSLP is enhanced in AD patients through a process that depends upon the capacity for dendritic cells (DCs) to upregulate TSLP receptor (TSLPR) following ligation of Fc receptors by allergen. The objective is to elucidate the immunogenetic mechanisms involved in modulating expression of TSLPR in AD and to determine the functional relevance of this receptor to Th2-driven inflammatory responses in the skin. Complementary approaches in humans and mice will incorporate in vitro studies of cells from AD patients (Aims 1 and 2) with an in vivo mouse model of AD (Aim 3). In Aim 1, an array of molecules that bind surface receptors on DCs (allergens and toll-like receptor ligands) will be tested for their capacity to upregulate TSLPR and the relevant signaling components elucidated using flow cytometry and gene silencing techniques. The requirement for TSLPR upregulation in amplification of TSLP-mediated Th2 responses will be verified in DC/T cell co-cultures. As an adjunct, cytokine-mediated suppression of TSLPR will be explored in order to determine whether this process is dysregulated in AD. To assess in vivo relevance, TSLPR expression will be analyzed on discrete DC types in lesional skin and at atopy patch test sites by flow cytometry and confocal microscopy techniques. In Aim 2, the influence of genetic variants of the TSLPR gene and epigenetic changes at this locus on TSLPR upregulation will be examined. Sequencing of the TSLPR gene will be carried out using samples from a large cohort of patients in order to confirm the 5' upstream region and to identify single nucleotide polymorphisms. Bioinformatics and luciferase reporter assays will then be used to identify and confirm promoter elements and haplotypes that enhance TSLPR gene activity. Chromatin remodeling at the TSLPR locus will be assessed in DCs by chromatin immunoprecipitation and DNase hypersensitivity assays. Loss-of-function mutations in the gene encoding filaggrin, a protein essential to skin barrier function, will also be genotyped to probe the relationship between an inherited defect in the skin barrier and factors predisposing to TSLPR upregulation. In Aim 3, the role of epidermal and dermal DCs in TSLP-driven inflammation will be examined in mice that lack Langerhans cells or CD11c+ DCs. The requirement for Langerhans cells to respond directly to TSLP will be tested in bone marrow reconstitution studies. To identify the principal cell type(s) responsible for TSLP-driven Th2-type inflammation in the skin, mixed chimeric mice will be used to limit TSLP responsiveness to specific cell types. Mice will also be generated with a conditional null allele of the TSLPR gene, allowing TSLP non-responsiveness to be limited to specific lineages.