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

小鼠和人湿疹中 TSLP 受体表达和功能的调节

• 批准号: 8106840
• 负责人: Judith A Woodfolk
• 金额: $ 58.16万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8106840
• 关键词: Alleles; Allergens; Animal Model; Antigen-Presenting Cells; Atopic Dermatitis; Automobile Driving; Binding; Bioinformatics; Biological Assay; Blood; Bone Marrow; CD4 Positive T Lymphocytes; Cells; Chronic; Clinical; Coculture Techniques; Collaborations; Confocal Microscopy; Cutaneous; DNA Sequence; Defect; Dendritic Cells; Deoxyribonucleases; Dependence; Dermal; Disease; Eczema; Elements; Epigenetic Process; Fc Receptor; Flow Cytometry; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Variation; Genotype; Haplotypes; Human; Hypersensitivity; ITGAX gene; Immune; Immunogenetics; In Vitro; Individual; Inflammation; Inflammatory; Inflammatory Response; Inherited; Langerhans cell; Ligands; Ligation; Luciferases; Mediating; Molecular; Molecular Profiling; Molecular Target; Mus; Mutation; Patch Tests; Pathogenesis; Pathway interactions; Patients; Phenotype; Play; Population; Predisposing Factor; Predisposition; Process; Promoter Regions; Proteins; Receptor Gene; Regulation; Reporter; Reporting; Research Institute; Role; Sampling; Severity of illness; Signal Transduction; Single Nucleotide Polymorphism; Site; Skin; Small Interfering RNA; Stimulus; Surface; System; T-Lymphocyte; TSLP gene; Techniques; Testing; Th2 Cells; Toll-like receptors; Universities; Up-Regulation; Virginia; atopy; base; cell type; chromatin immunoprecipitation; chromatin remodeling; cohort; cytokine; filaggrin; genetic variant; human TSLP protein; in vivo; insight; interdisciplinary approach; loss of function mutation; man; mast cell; mouse model; promoter; receptor; receptor expression; receptor function; receptor upregulation; reconstitution; research study; response

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. PUBLIC HEALTH RELEVANCE: The cytokine, TSLP, has been implicated in driving Th2 responses that underlie the chronic inflammatory skin condition, atopic dermatitis. Nothing is known about how TSLP receptor may modulate responses to TSLP. The proposed studies, which investigate TSLP receptor expression and function in mice and man could yield new insight into why some individuals are susceptible to the effects of TSLP while others are not. Such studies could identify new molecular targets for treatment of this debilitating disease.

描述（由申请人提供）：Th 2促进细胞因子，胸腺基质淋巴细胞生成素（TSLP），与慢性炎症性皮肤病，特应性皮炎（AD）的发病机制有关。了解增加AD患者对TSLP反应性的免疫机制有助于开发抑制这种疾病潜在炎症级联反应的新治疗方法。AD患者对TSLP的反应性通过依赖于树突状细胞（DC）在通过过敏原连接Fc受体后上调TSLP受体（TSLPR）的能力的过程而增强。目的是阐明参与调节TSLPR在AD中的表达的免疫遗传学机制，并确定该受体与Th 2驱动的皮肤炎症反应的功能相关性。在人类和小鼠中的互补方法将结合来自AD患者的细胞的体外研究（目的1和2）与AD的体内小鼠模型（目的3）。在目标1中，将测试结合DC上的表面受体（变应原和toll样受体配体）的一系列分子上调TSLPR的能力，并使用流式细胞术和基因沉默技术阐明相关信号传导组分。将在DC/T细胞共培养物中验证TSLP介导的Th 2应答的扩增中TSLPR上调的要求。作为一种辅助手段，将探索马槟榔碱介导的TSLPR抑制，以确定该过程是否在AD中失调。为了评估体内相关性，将通过流式细胞术和共聚焦显微镜技术在病变皮肤和特应性斑贴试验部位的离散DC类型上分析TSLPR表达。在目的2中，将检查TSLPR基因的遗传变体和该基因座的表观遗传变化对TSLPR上调的影响。TSLPR基因的测序将使用来自大量患者的样本进行，以确认5'上游区域并鉴定单核苷酸多态性。然后将使用生物信息学和荧光素酶报告基因测定来鉴定和确认增强TSLPR基因活性的启动子元件和单倍型。通过染色质免疫沉淀和DNA酶超敏性测定在DC中评估TSLPR基因座处的染色质重塑。编码聚丝蛋白（一种对皮肤屏障功能至关重要的蛋白质）的基因中的功能缺失突变也将进行基因分型，以探索皮肤屏障中的遗传缺陷与诱发TSLPR上调的因素之间的关系。在目标3中，将在缺乏朗格汉斯细胞或CD 11 c + DC的小鼠中检查表皮和真皮DC在TSLP驱动的炎症中的作用。将在骨髓重建研究中检测朗格汉斯细胞对TSLP直接应答的要求。为了鉴定负责皮肤中TSLP驱动的Th 2型炎症的主要细胞类型，将使用混合嵌合小鼠来限制TSLP对特定细胞类型的响应性。还将产生具有TSLPR基因的条件无效等位基因的小鼠，从而允许将TSLP无应答性限于特定谱系。 公共卫生相关性：细胞因子TSLP与驱动Th 2应答有关，Th 2应答是慢性炎症性皮肤病特应性皮炎的基础。关于TSLP受体如何调节对TSLP的反应尚不清楚。这项研究调查了小鼠和人类中TSLP受体的表达和功能，可能会对为什么有些人容易受到TSLP的影响而另一些人则不然产生新的见解。这些研究可以确定治疗这种使人衰弱的疾病的新分子靶点。