Dissecting Hem-1 functions in B lymphocyte Development and Primary Immunodeficiency Disease

剖析 Hem-1 在 B 淋巴细胞发育和原发性免疫缺陷病中的功能

• 批准号: 10385848
• 负责人: BRIAN M IRITANI
• 金额: $ 19.44万
• 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: 2021-04-06 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10385848
• 关键词: Actins; Adhesions; Affect; Antibody Formation; Antibody Response; Antigens; Autoimmune Diseases; Autoimmunity; B-Cell Development; B-Lymphocytes; Bacterial Infections; Biochemical; Biological Models; CDC42 gene; CRISPR/Cas technology; Cell physiology; Cells; Characteristics; Child; Communities; Community-Acquired Infections; Complex; Cre-LoxP; Cytokine Receptors; Cytoskeleton; Development; Disease; Exhibits; Family; Gene Targeting; Genes; Genetic; Genetic Heterogeneity; Genetic Transcription; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Hematopoietic; Hematopoietic stem cells; Homologous Protein; Human; Humoral Immunities; Immune; Immune response; Immune signaling; Immunity; Immunization; Immunodeficient Mouse; Immunologic Deficiency Syndromes; Immunologic Receptors; Impairment; Individual; Infection; Infectious Skin Diseases; Influenza A virus; Inherited; Integrins; Knowledge; Ligands; Link; Malignant Neoplasms; Mediating; Mission; Modeling; Molecular; Mus; Mutation; Otitis Media; Outcome Study; Patients; Peripheral; Phenotype; Point Mutation; Polysaccharides; Production; Proteins; Public Health; Publishing; Reporting; Research; Respiratory Tract Infections; Role; Signal Pathway; Signal Transduction; Site; Streptococcus pneumoniae; System; T-Cell Receptor; Testing; Time; United States National Institutes of Health; Untranslated RNA; Variant; Vascular Endothelium; Virus Diseases; Wasps; Wiskott-Aldrich Syndrome; cell type; chemokine; congenital immunodeficiency; cytokine; depolymerization; disease-causing mutation; epigenomics; fly; genetic regulatory protein; human model; humanized mouse; innovation; loss of function; loss of function mutation; member; migration; mortality; mouse model; pathogen; polymerization; protein complex; receptor; rho; rho GTP-Binding Proteins; trafficking; transcriptomics

PROJECT SUMMARY Primary immunodeficiency diseases (PID) are group of inherited conditions where components of immune signaling pathways are either missing or are dysfunctional, resulting in severe recurring infections that often progress to autoimmune disease or cancer. Of the greater than 300 PIDs that have been identified so far, the molecular basis of a significant number (~100) of PIDs have yet to be defined. Recently, 9 individuals (4 now deceased) from 4 independent families were identified with PIDs that were linked to mutations in NCKAP1L, which encodes for a conserved hematopoietic cell-specific actin regulatory protein called Hematopoietic protein-1 (Hem-1). Affected children presented with recurring respiratory and skin infections, otitis media, impaired 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 primary mammalian immune cells. Our longterm goal is to overcome this knowledge gap by dissecting the cell-specific cellular and molecular functions of Hem-1 in the development and functions of adaptive and innate immune cells. The objective of this proposal is to target Hem-1 in primary murine and human B lymphocytes in a B cell-specific manner to define the roles of Hem-1 in B cell development, signaling, and protective immunity. Our Specific Aims are to: (1) utilize conditional Cre-LoxP mediated gene targeting in mice to define how B cell specific loss-of-function mutations in Hem-1 alter peripheral B cell development, antibody responses, and protective immunity to Pneumococcus and Influenza A virus, two important community acquired infections; (2) determine the impact of Hem-1 mutations on B cell signaling and transcription; (3) utilize CRISPR/Cas9 gene editing in “humanized mice” to assess the impact of loss-of-function mutations in Hem-1 on the dynamic development of primary human B lymphocytes. To demonstrate feasibility, we have generated mice with a non-coding point mutation in Hem1 (Hem1pt/pt), Hem1 null (Hem1-/-) mice, Hem1floxed (Hem1fl/fl) mice, as well as Hem1 deficient primary human hematopoietic stem cells. Utilizing these innovative mouse and human model systems, we will test our overall hypothesis that B cell specific expression of Hem-1 is essential for the development of marginal zone and B1 B cells, T- independent antibody responses, and protective B cell immunity. These studies are highly significant because they will utilize innovative approaches to define for the first time, the cellular and molecular mechanisms of how non-coding mutations in NCLAP1L disrupt B cell development, signaling, and protective humoral immunity, resulting in PID and potentially autoimmunity. Because of extensive genetic heterogeneity of the 4 human PID families, limited number of patients, and concurrent infections, the disruption of Hem1 in cell-type specific manner is critical for dissecting the mechanisms of how mutations in Hem-1 result in PID and autoimmunity.

项目摘要 原发性免疫缺陷疾病（PID）是一组遗传性疾病，其中免疫成分 信号通路缺失或功能失调，导致严重的反复感染通常 发展为自身免疫性疾病或癌症。到目前为止已识别出的300多个PID中 大量PID的分子基础尚未定义。最近，有9个人（现在有4个人） 已故的4个独立家庭的死者被鉴定出与NCKAP1L突变有关的PI​​D， 该编码为造血细胞特异性肌动蛋白调节蛋白编码称为造血蛋白 蛋白-1（Hem-1）。患有反复呼吸道和皮肤感染的受影响儿童，中耳炎， 对肺炎球菌免疫缺陷的抗体反应受损（B细胞免疫缺陷的特征）， 细胞因子产生和自身免疫性失调。虽然Hem-1的细胞和分子功能 苍蝇和蠕虫中的直系同源物的特征相对较好，有关 HEM-1在原发性哺乳动物免疫细胞中的细胞特异性功能。我们的长期目标是克服这一点 通过剖析HEM-1的细胞特异性细胞和分子功能来开发中知识差距 以及适应性和先天免疫小球的功能。该提议的目的是靶向初级中的Hem-1 以B细胞特异性方式定义HEM-1在B细胞中的作用的鼠和人B淋巴细胞 开发，信号传导和保护免疫力。我们的具体目的是：（1）使用条件Cre-loxP 小鼠中介导的基因靶向，以定义B细胞特异性功能丧失突变如何改变 周围B细胞的发育，抗体反应以及对肺炎球菌和流感A的保护免疫 病毒，两个重要的社区感染； （2）确定HEM-1突变对B细胞的影响 信号和转录； （3）在“人性化小鼠”中利用CRISPR/CAS9基因编辑来评估 HEM-1的功能丧失突变对原代人B淋巴细胞的动态发展。到 证明可行性，我们已经在Hem1（Hem1pt/pt）中产生了具有非编码点突变的小鼠，Hem1 null（hem1 - / - ）小鼠，Hem1floxed（Hem1fl/fl）小鼠以及Hem1缺乏原发性人类造血茎 细胞。利用这些创新的小鼠和人类模型系统，我们将检验我们的总体假设B HEM-1的细胞特异性表达对于边际区和B1 B细胞的发展至关重要 独立的抗体反应，并保护B细胞免疫。这些研究非常重要，因为 他们将首次利用创新的方法来定义，即如何定义的细胞和分子机制 NCLAP1L中的非编码突变破坏了B细胞的发育，信号传导和受保护的体液免疫（们） 导致PID和潜在的自身免疫性。由于4人PID的广泛遗传异质性 家族，患者数量有限和并发感染，细胞类型特异性中HEM1的破坏 方式对于解剖HEM-1中突变如何导致PID和自身免疫的机制至关重要。

项目成果

Metabolism meets immunodeficiency disease.

新陈代谢遇到免疫缺陷疾病。

• DOI: 10.1182/blood.2020008875
• 发表时间: 2021
• 期刊: Blood
• 影响因子: 20.3
• 作者: Iritani,BrianM

Hem-1 regulates protective humoral immunity and limits autoantibody production in a B cell-specific manner.

• DOI: 10.1172/jci.insight.153597
• 发表时间: 2022-05-09
• 期刊: JCI INSIGHT
• 影响因子: 8
• 作者: Avalos, Alan;Tietsort, Jacob T.;Suwankitwat, Nutthakarn;Woods, Jonathan D.;Jackson, Shaun W.;Christodoulou, Alexandra;Morrill, Christopher;Liggitt, H. Denny;Zhu, Chengsong;Li, Quan-Zhen;Bui, Kevin K.;Park, Heon;Iritani, Brian M.
• 通讯作者: Iritani, Brian M.