WAVE Regulatory Complex in Primary Immunodeficiency Disease and autoimmunity

原发性免疫缺陷病和自身免疫性疾病中的 WAVE 调节复合体

• 批准号: 10179093
• 负责人: BRIAN M IRITANI
• 金额: $ 58.19万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-10 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10179093
• 关键词: Actins; Affect; Antibody Formation; Antibody Response; Antibody-mediated protection; Antigens; Autoantibodies; Autoimmunity; B-Cell Activation; B-Cell Antigen Receptor; B-Cell Development; B-Lymphocytes; B-cell receptor repertoire sequencing; Bacteremia; Bacterial Infections; Biological Models; Bone Marrow; CRISPR/Cas technology; Cell physiology; Cells; Characteristics; Child; Communities; Complex; Cre-LoxP; Cytokine Receptors; Development; Diffusion; Disease; Exhibits; Family; Flow Cytometry; Gene Expression; Gene Targeting; Genes; Genetic Heterogeneity; Genetic Transcription; Goals; Guanosine Triphosphate Phosphohydrolases; Hematopoietic; Homeostasis; Homing; Homologous Protein; Human; Humoral Immunities; Image; Immune; Immune Tolerance; Immunity; Immunization; Immunoglobulin Class Switching; Immunologic Deficiency Syndromes; Immunologic Receptors; Impairment; Individual; Infection; Infectious Skin Diseases; Inflammatory; Influenza A virus; Intestines; Knowledge; Lead; Ligation; Link; Lymphoid; Mediating; Microscopy; Mission; Modeling; Molecular; Mus; Mutation; Patients; Plasma Cells; Pneumococcal Pneumonia; Point Mutation; Predisposition; Production; Prognosis; Proteins; Public Health; Receptor Signaling; Research; Resolution; Respiratory Tract Infections; Role; Signal Transduction; Streptococcus pneumoniae; System; T-Cell Receptor; T-Independent Antigens; T-Lymphocyte; Technology; Testing; Time; Toll-like receptors; United States National Institutes of Health; Untranslated RNA; Variant; Virus Diseases; autoreactivity; base; cell type; chemokine; congenital immunodeficiency; cytokine; fly; genome wide association study; human disease; human model; humanized mouse; humoral immunity deficiency; improved; innovation; loss of function; loss of function mutation; member; migration; mortality; mouse model; novel therapeutics; pathogen; polymerization; premature; respiratory pathogen; response; rho; stem cells; transcriptome; transcriptome sequencing; translational impact; two-photon

PROJECT SUMMARY Of the greater than 350 Primary Immunodeficiency Diseases (PID) in humans that have been identified to date, the molecular basis of a significant number (~100) of PIDs have yet to be defined. Recently, 9 children (4 now deceased) from 4 independent families were identified with severe PIDs that were linked to mutations in the NCKAP1L gene encoding for Hematopoietic protein-1 (Hem-1), a conserved hematopoietic cell-specific component the WAVE actin regulatory complex (WRC). Affected children presented with severe recurring respiratory and skin infections, failed antibody responses to pneumococcal immunization (characteristic of B cell immunodeficiency), dysregulated cytokine production, and autoimmunity. Although the cellular and molecular functions of Hem-1 orthologues in flies and worms are relatively well characterized, there is a critical knowledge gap regarding the cell specific functions of Hem-1 in the development and functions of primary immune cells. Our longterm goal is to overcome this knowledge gap by dissecting the cell-specific roles of Hem-1 in the development and functions of adaptive and innate immune cells. The objective of this proposal is to disrupt Hem-1 expression in primary murine and human B lymphocytes in a B cell-specific manner to define the roles of Hem-1 in B cell development, protective humoral immunity, and autoimmunity. Our Specific Aims are to utilize inducible B cell specific gene targeting in mice, and CRISPR/Cas9 mediated Hem1 deletion in “humanized mice”, to test our central hypotheses that B cell specific disruption in Hem1 results in: (1) impaired B cell development in part due to reduced homing and retention of developing B cell progenitors in essential lymphoid niches;(2) absent T cell independent antibody responses resulting in crippled protective immunity to influenza A virus and Streptococcus pneumoniae, important community acquired respiratory pathogens; and (3) hyper-responsive B cell signaling and T-bet driven transcriptome, resulting in increased autoantibody production. To demonstrate feasibility, we have generated innovative mouse models to emulate Hem1 PID patients including mice with a non-coding point mutation in Hem1 (Hem1pt/pt), Hem1 null (Hem1-/-) mice, Hem1floxed (Hem1fl/fl) mice, as well as Hem1 deficient ”humanized mice” which contain Hem1 deficient primary human hematopoietic cells. Based on our preliminary results which strongly support our hypotheses, we expect that the results of these studies will be highly significant and will have a high impact because they will define for the first time, the cellular and molecular mechanisms of how loss-of-function variants in NCLAP1L disrupt B cell development, signaling, and protective antibody-mediated immunity resulting in PID and autoimmunity. Because of extensive genetic heterogeneity of the 4 human PID families, limited number of patients, and concurrent infections, the development of these innovative mouse model systems are critical for dissecting the cellular and molecular mechanisms of how mutations in Hem-1 result in PID and autoimmunity, and to provide much needed platforms to develop and test therapies to treat and cure Hem1 deficient children.

项目摘要 迄今已确定的人类中，大于350个原发性免疫缺陷疾病（PID） 大量PID的分子基础尚未定义。最近，有9个孩子（现在有4个 已故的）来自4个独立家庭的严重PID，这些家族与与突变有关的严重PID。 NCKAP1L编码造血蛋白-1（HEM-1）的NCKAP1L基因，一种构型造血细胞特异性 组件波肌动蛋白调节复合物（WRC）。受影响的儿童出现严重的经常性 呼吸道和皮肤感染，对肺炎球菌免疫的抗体反应失败（B的特征 细胞免疫缺陷），细胞因子产生失调和自身免疫性。虽然细胞和 苍蝇和蠕虫中Hem-1直系同源物的分子功能相对较好，有一个关键 关于HEM-1的细胞特异性功能在原发性发展和功能中的知识差距 免疫细胞。我们的长期目标是通过剖析细胞特异性角色来克服这一知识差距 HEM-1在适应性和先天免疫球的发展和功能中。该提议的目的是 以B细胞特异性的方式破坏原代鼠和人B淋巴细胞中的HEM-1表达以定义 HEM-1在B细胞发育，保护体液免疫（和自身免疫）中的作用。我们的具体目标 要利用小鼠中的诱导B细胞特异性基因靶向，CRISPR/CAS9介导的HEM1缺失 “人性化小鼠”，以测试我们的中心假设，即HEM1中B细胞特异性破坏会导致：（1）受损 B细胞发育部分是由于基本生长中的B细胞祖细胞的归巢和保留。 淋巴酸粒;（2）缺乏T细胞独立抗体反应，导致受保护的免疫原子对 影响肺炎的病毒和链球菌，重要的社区获得了呼吸道病原体；和 （3）超响应B细胞信号传导和T-Bet驱动转录组，导致自身抗体增加 生产。为了证明可行性，我们生成了创新的小鼠模型来模拟HEM1 PID 包括Hem1（Hem1pt/pt），Hem1 null（Hem1-/ - ）小鼠的小鼠在内的患者，包括非编码点突变 Hem1floxed（Hem1fl/fl）小鼠，以及含有HEM1缺乏的Hem1缺乏的“人源化小鼠” 原代人造血细胞。根据我们的初步结果，强烈支持我们的假设， 我们希望这些研究的结果将非常重要，并且会产生很大的影响，因为它们 将首次定义的，即功能丧失变体的细胞和分子机制 NCLAP1L破坏B细胞的开发，信号传导和受保护的抗体介导的免疫力，导致PID 和自身免疫。由于四个人PID家族的广泛遗传异质性，数量有限 患者和同时感染，这些创新的小鼠模型系统的发展对于 解剖HEM-1突变如何导致PID和自身免疫性的细胞和分子机制， 并提供急需的平台来开发和测试疗法，以治疗和治愈HEM1缺乏的儿童。