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Humanized mice to study mast cell function

研究肥大细胞功能的人源化小鼠

基本信息

批准号：
8643443
负责人：
Hydar Ali
金额：
$ 20万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-15 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8643443
关键词：
ADRBK1 gene ADRBK2 gene Acute Affinity Agonist Allergens Allergic Allergic Disease Allergic rhinitis Anaphylatoxins Anaphylaxis Animal Model Antigen-Antibody Complex Antigens Asthma Atherosclerosis Autoimmune Diseases Bronchoalveolar Lavage Fluid Bronchoconstriction Bulla CD34 gene Cell Degranulation Cell physiology Cell surface Cells Cleaved cell Collaborations Complement 3a Complement 5a Complement Activation Complement Membrane Attack Complex Complex Data Development Disease Experimental Models G protein coupled receptor kinase G-Protein-Coupled Receptors GRK5 gene GRK6 gene Generations Genetic Growth Factor Host Defense Human IgE IgE Receptors Immune Immune system Inflammation Inflammatory Inflammatory Response Laboratories Lead Lung Lytic Mediating Mediator of activation protein Methods Mouse Strains Mus Natural Immunity Neoplasm Metastasis Passive Cutaneous Anaphylaxis Pathogenesis Pathway interactions Pattern Recognition Systems Periodontal Diseases Phosphorylation Plasma Play Predisposition Protein Tyrosine Kinase Proteins Reaction Receptor Gene Receptor Signaling Regulation Relative (related person)Research Personnel Rheumatoid Arthritis Rodent Rodent Model Role Signal Pathway Signal Transduction Single Nucleotide Polymorphism Skin Slice Surface Antigens Testing Therapeutic Tryptase Umbilical Cord Blood Work airway inflammation allergic response arrestin 1 base complement C3 precursor complement system genetic manipulation in vivo in vivo Model mast cell microbial mouse development mouse model novel novel strategies novel therapeutic intervention pathogen progenitor protein expression public health relevance pulmonary vascular permeability receptor response small hairpin RNA therapy development

项目摘要

DESCRIPTION (provided by applicant): Mast cells are multifunctional immune cells that play an important role in host defense but also promote asthma, allergic rhinitis, rheumatoid arthritis, atherosclerosis, skin-blistering diseases, cancer metastasis and are implicated in periodontal disease. There are important phenotypic and functional differences between human and rodent mast cells. Not surprisingly, therapeutic approaches developed using rodent models for mast cell-mediated disease such as asthma lack efficacy in humans. Thus, to better understand the mechanisms of mast cell-mediated diseases, it is critical to utilize in vivo models that are relevant to humans. In this proposal, the major objective is to develop a humanized mouse model for the genetic manipulation of human mast cell function in vivo. Aggregation of high affinity IgE receptors (Fc¿RI) on mast cells leads to the generation of C3a, which causes substantial degranulation in human mast cells and likely contributes to IgE-mediated allergic responses in vivo. Recent studies performed in our laboratory have identified novel signaling pathways via which G protein coupled receptor kinases (GRK5, GRK6) and adapter molecules ¿-arrestin 1 and Na+/H+ exchange regulatory factors (NHERF1 and NHEFR2) contribute to C3a-induced mast cell degranulation. We have also shown that GRK2 and GRK3 desensitize C3a-induced responses via the agonist-induced phosphorylation of C3aR. Given that C3a likely contributes to IgE-mediated responses in vivo, we hypothesize that modulation of C3aR signaling in mast cells regulates allergic responses ex vivo and in vivo. In aim #1, we will optimize conditions for the differentiation of human CD34+ cells into functional human mast cells in two strains of immune-deficient mice expressing growth factors for human mast cells. We will then target one protein that contributes to (NHERF1) and one that inhibits (GRK3) C3a-induced mast cell degranulation. Lentiviral shRNA will be used to silence the expression of these proteins in human cord blood- derived CD34+ cells and engrafted into immune deficient mice for the development of genetically manipulated human mast cells in vivo. We will use this novel approach as a "proof of concept" study to test the idea that genetic manipulation of human mast cells in vivo can be used to modulate bronchoconstriction ex vivo. C3aR expressed in mouse mast cells contribute to experimental passive cutaneous anaphylaxis (PCA) but not passive systemic anaphylaxis (PSA). In aim #2, we will utilize humanized mice to test the hypothesis that the presence of C3a-responsive human mast cells in the lung contributes to increased vascular pulmonary permeability in PSA. We will engraft NHERF1 or GRK3-silenced human CD34+ cells into immune deficient mice and test their impact on PCA and PSA reactions in vivo. We believe that genetic manipulation of human mast cells in humanized mice will provide a better understanding of their role in allergic and non-allergic diseases and may eventually lead to the development of novel treatment options.

描述（由申请人提供）：肥大细胞是多功能免疫细胞，在宿主防御中发挥重要作用，但也促进哮喘、过敏性鼻炎、类风湿性关节炎、动脉粥样硬化、皮肤起泡疾病、癌症转移，并与牙周病有关。人和啮齿类动物肥大细胞之间存在重要的表型和功能差异。毫不奇怪，使用啮齿动物模型开发的肥大细胞介导的疾病（如哮喘）的治疗方法在人类中缺乏疗效。因此，为了更好地理解肥大细胞介导的疾病的机制，利用与人类相关的体内模型是至关重要的。在这项提案中，主要目标是开发一种人源化小鼠模型，用于体内人类肥大细胞功能的遗传操作。肥大细胞上高亲和力IgE受体（Fc <$RI）的聚集导致C3 a的产生，这导致人肥大细胞中的大量脱粒，并可能有助于体内IgE介导的过敏反应。最近在我们实验室进行的研究已经确定了新的信号转导途径，通过这些途径，G蛋白偶联受体激酶（GRK 5，GRK 6）和接头分子<$-arrestin 1和Na+/H+交换调节因子（NHERF 1和NHEFR 2）有助于C3 a诱导的肥大细胞脱粒。我们还发现GRK 2和GRK 3通过激动剂诱导的C3 aR磷酸化使C3 a诱导的反应脱敏。鉴于C3 a可能有助于体内IgE介导的反应，我们假设肥大细胞中C3 aR信号转导的调节调节离体和体内过敏反应。在目标#1中，我们将在表达人肥大细胞生长因子的两种免疫缺陷小鼠品系中优化人CD 34+细胞分化为功能性人肥大细胞的条件。然后，我们将靶向一种蛋白质，有助于（NHERF 1）和一个抑制（GRK 3）C3 a诱导的肥大细胞脱粒。慢病毒shRNA将用于沉默这些蛋白在人脐带血来源的CD 34+细胞中的表达，并植入免疫缺陷小鼠中用于体内遗传操作的人肥大细胞的发育。我们将使用这种新的方法作为“概念验证”研究，以测试体内人类肥大细胞的遗传操作可用于调节离体支气管收缩的想法。C3 aR在小鼠肥大细胞中的表达有助于实验性被动皮肤过敏反应（PCA），但不有助于被动全身过敏反应（PSA）。在目标#2中，我们将利用人源化小鼠来测试肺中C3 a应答性人肥大细胞的存在有助于PSA中血管肺渗透性增加的假设。我们将NHERF 1或GRK 3沉默的人CD 34+细胞移植到免疫缺陷小鼠中，并测试它们对体内PCA和PSA反应的影响。我们相信，在人源化小鼠中对人肥大细胞进行遗传操作将更好地了解它们在过敏性和非过敏性疾病中的作用，并可能最终导致开发新的治疗方案。