Humanized mice to study mast cell function

描述(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、癌症转移和牙周病有关。人和啮齿动物的肥大细胞在表型和功能上有重要的差异。毫不奇怪，使用啮齿动物模型开发的治疗肥大细胞介导的疾病的方法，如哮喘，在人类身上缺乏疗效。因此，为了更好地了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至关重要。在这项建议中，主要目标是开发一种人源化的小鼠模型，用于在活体内对人类肥大细胞功能进行遗传操作。高亲和力的IgE受体(Fc？RI)聚集在肥大细胞上，导致C3a的产生，导致人肥大细胞大量脱颗粒，并可能在体内参与IgE介导的变态反应。我们实验室最近的研究发现了新的信号通路，G蛋白偶联受体激酶(GRK5，GRK6)和适配分子β-arrestin 1和Na+/H+交换调节因子(NHERF1和NHEFR2)参与了C3a诱导的肥大细胞脱颗粒。我们还表明，GRK2和GRK3通过激动剂诱导C3aR的磷酸化来减敏C3a诱导的反应。鉴于C3a在体内可能对IgE介导的反应起作用，我们假设肥大细胞中C3aR信号的调节调节了体外和体内的过敏反应。在目标1中，我们将优化在两种表达人肥大细胞生长因子的免疫缺陷小鼠中将人CD34+细胞分化为有功能的人肥大细胞的条件。然后，我们将针对一个有助于(NHERF1)和一个抑制(GRK3)C3a诱导的肥大细胞脱颗粒的蛋白质。慢病毒shRNA将被用来沉默这些蛋白在人脐血来源的CD34+细胞中的表达，并移植到免疫缺陷小鼠中，用于体内发育基因操纵的人肥大细胞。我们将使用这一新方法作为“概念验证”研究，以测试体内对人类肥大细胞的基因操作可用于调节体外支气管收缩的想法。小鼠肥大细胞表达的C3aR与实验性被动皮肤过敏反应(PCA)有关，但与被动全身过敏反应(PSA)无关。在目标2中，我们将利用人源化的小鼠来验证这样的假设，即肺中C3a反应的人肥大细胞的存在有助于PSA中血管肺通透性的增加。我们将NHERF1或GRK3沉默的人CD34+细胞植入免疫缺陷小鼠，并在体内测试它们对PCA和PSA反应的影响。我们相信，在人源化小鼠中对人类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，并最终可能导致开发新的治疗方案。

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.