The Genetics, Pathogenesis, and Treatment of Primary Immunodeficiency

原发性免疫缺陷的遗传学、发病机制和治疗

• 批准号: 8555872
• 负责人: Ashish Jain
• 金额: $ 136.88万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555872
• 关键词: Antibodies; Antibody Formation; Antigen-Presenting Cells; Apoptosis; B-Cell Development; B-Lymphocytes; Benign; Binding; C-terminal; CD3 Antigens; CD4 Positive T Lymphocytes; CD40 Ligand; Cells; Chromatin; Code; Common Variable Immunodeficiency; Complex; Custom; DNA; DNA Binding Domain; DNA Resequencing; Defect; Dendritic Cells; Deubiquitinating Enzyme; Deubiquitination; Disease; Disease susceptibility; Ectodermal Dysplasia; Exhibits; Exons; Failure; Gene Expression; Gene Mutation; Genes; Genetic; Genomics; Growth; Histone H3; Hour; IKK alpha; Immune; Immunoglobulin Class Switching; Immunoglobulin Isotypes; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunologic Deficiency Syndromes; In Vitro; Infection; Inherited; Interleukin-12; Introns; Knockout Mice; Laboratories; Length; Lipopolysaccharides; Lymphoid; MAP3K7 gene; MAPK14 gene; MAPK8 gene; Mediating; Microarray Analysis; Missense Mutation; Mitogen-Activated Protein Kinases; Molecular; Molecular Profiling; Mus; Mutation; NF-kappa B; NFKB Signaling Pathway; Nuclear Translocation; Nucleotides; Online Systems; Organ; Pathogenesis; Pathway interactions; Patients; Peripheral; Phenotype; Phosphorylation; Phosphotransferases; Play; Polyubiquitination; Post-Translational Protein Processing; Predisposition; Production; Proteomics; Protocols documentation; Regulation; Regulatory T-Lymphocyte; Role; Sampling; Screening procedure; Signal Transduction; Signaling Molecule; Single Nucleotide Polymorphism; Single-Gene Defect; Site; Susceptibility Gene; Syndrome; T-Cell Development; T-Lymphocyte; TLR4 gene; TNF gene; TNFRSF5 gene; TNFSF5 gene; Thymus Gland; Transcript; Ubiquitin; Ubiquitination; Up-Regulation; Zinc Fingers; activation-induced cytidine deaminase; aurora B kinase; base; cell growth; congenital immunodeficiency; cost effective; cytokine; histone acetyltransferase; inhibitor/antagonist; insight; neutralizing antibody; novel; overexpression; prevent; promoter; protein complex; protein phosphatase inhibitor-2; response; tool; transcription factor; tumor

Employing the candidate genetic approach, we previously identified mutations in the gene encoding NEMO (NF- kB essential modulator), an intracellular signaling constituent of the NF-kB pathway, results in ectodermal dysplasia with an immune deficiency. Mutations in the zinc finger domain of NEMO block CD40 mediated activation of NF- kB and prevent B cells from undergoing class switch recombination (CSR) and APCs from synthesizing NF -kB regulated cytokines such as IL -12 or TNF-a when stimulated with CD40 ligand. The zinc finger domain of NEMO is a site for the covalent attachment of ubiquitin and this step is necessary for the full activation of NF-kappa B. The majority of patients with ectodermal dysplasia with immune deficiency (EDI) have mutations in the zinc finger domain and we have found that such mutations impair the post- translational modification of NEMO by ubiquitin. In subsequent studies we demonstrated that the NEMO zinc finger domain in the regulation of mitogen-activated protein kinase (MAPK) activity. We found that dendritic cells from patients with EDI caused by a C-terminal E391X deletion of the zinc finger of NEMO exhibit impaired MAPK activation in response to lipopolysaccharide (LPS) stimulation. Interestingly, DCs from patients with a C417R missense mutation within the zinc finger domain of NEMO in which ubiquitination of NEMO is preserved are also defective in JNK and ERK activity following LPS stimulation. Our findings indicate that the structural integrity of the NEMO ZF domain is more important than its polyubiquitination for full activation of the MAPK. Furthermore, phosphorylation and polyubiquitination of upstream TAK1 were significantly reduced in the E391X zinc finger deleted patients, indicating that the NEMO zinc finger may play an important role in assembling the proximal signaling complex for MAPK activation. More recently we have studied patients with ectodermal dysplasia with immune deficiency who have normal NEMO coding sequence but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of antigen presenting cells (APCs) from these patients induced normal cytoplasmic activation and nuclear translocation of NF-kappa B. However, cells deficient in-full length NEMO were defective in expression of NF-kappa B regulated cytokines such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-kappa B transcription factors. TLR4-stimulated APCs from these patients were defective in IKK alpha-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-kappa B heterodimers Rel A and cRel to the promoter. Expression of a super active form of IKK alpha E, E restored IL-12 production in NEMO knockdown THP-1 cells following LPS treatment. Our findings indicate that failure to express full-length NEMO is associated with an immune deficiency phenotype, and offer new insights into diminished NF-kappa B signaling in patients with EDI. We found that EDI patient B cells can be characterized by an absence of Ig somatic hypermutation (SHM) and defective class switch recombination (CSR) despite normal induction of activation-induced cytidine deaminase (AID) and Iepsilon-Cepsilon transcripts. We used microarray analysis compared the gene expression profiles of activated B cells from patients with EDI to reference controls to identify candidate factors that may be necessary for CSR and SHM in B cells. NIR (novel INHAT repressor) is a transcriptional co-repressor with inhibitor of histone acetyltransferase (INHAT) activity and has previously been shown to physically interact with and suppress p53 transcriptional activity and function. NIR expression was induced at 6 hours and peaked at 24 hours following CD40 stimulation of control B cells. However, such regulation was absent in patient B cells. We therefore subjected NIR to detailed analysis. Using a proteomic approach, we havve identified the Aurora kinase B as a novel binding partner of NIR. We show that Aurora B, NIR and p53 exist in a protein complex in which Aurora B binds to NIR, thus also indirectly associates with p53. Functionally, overexpression of Aurora B or NIR suppresses p53 transcriptional activity, and depletion of Aurora B or NIR causes p53-dependent apoptosis and cell growth arrest, due to the up-regulation of p21 and Bax. We then demonstrate that Aurora B phosphorylates multiple sites in the p53 DNA-binding domain in vitro, and this phosphorylation probably also occurs in cells. Importantly, the Aurora B-mediated phosphorylation on Ser(269) or Thr(284) significantly compromises p53 transcriptional activity. Taken together, these results provide novel insight into NIR-mediated p53 suppression and also suggest an additional way for p53 regulation. We have recently completed a conditional knockout mouse for NIR and plan to study its function in B cell development. We have also undertaken efforts to identity new disease susceptibility genes for Common Variable Immunodeficiency (CVI). These are heterogeneous disorders characterized by a predisposition to serious infection and impaired or absent neutralizing antibody responses. Although a number of single gene defects have been associated with these immune deficiency disorders, the genetic basis of many cases is not known. To facilitate mutation screening in patients with these syndromes, we have developed a custom 300-kb resequencing array, the Hyper-IgM/CVID chip, which interrogates 1,576 coding exons and intron-exon junction regions from 148 genes implicated in B-cell development and immunoglobulin isotype switching. Genomic DNAs extracted from patients were hybridized to the array using a high-throughput protocol for target sequence amplification, pooling, and hybridization. A Web-based application, SNP Explorer, was developed to directly analyze and visualize the single nucleotide polymorphism (SNP) annotation and for quality filtering. Several mutations in known disease-susceptibility genes such as CD40LG, TNFRSF13B, IKBKG, AICDA, as well as rare nucleotide changes in other genes such as TRAF3IP2, were identified in patient DNA samples and validated by direct sequencing. We conclude that the Hyper-IgM/CVID chip combined with SNP Explorer may provide a cost-effective tool for high-throughput discovery of novel mutations among hundreds of disease-relevant genes in patients with inherited antibody deficiency. We are now subjecting candidate disease susceptibility genes to detailed analysis. Related efforts in the laboratory include making genetically altered mice with targeted mutations in signaling molecules, which regulate NF-kappaB. CYLD is a deubiquitinating enzyme that targets signaling constituents of the NF-kB signaling pathway, including NEMO. Alteration in CYLD have been described in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. In mice deficient deficient in CYLD, we found that regulatory T cells (Tregs) are markedly increased numbers in the peripheral lymphoid organs, but not in the thymus. In vitro stimulation of CYLD-deficient nave T cells with anti-CD3/28 in the presence of TGF-β led to a marked increase in the number of Foxp3-expressing T cells compared with stimulated naive control CD4+ cells. Under endogenous conditions, CYLD formed a complex with Smad7 in which CYLD regulated Smad7 through deubiquitination of K360 and K374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Our results show that CYLD plays a role in regulating TGF-β signaling in T cells and the development of Treg cells through deubiquitination of Smad7.

采用候选遗传方法，我们之前发现编码 NEMO（NF-kB 必需调节剂）的基因发生突变，NEMO 是 NF-kB 通路的细胞内信号传导成分，可导致外胚层发育不良并伴有免疫缺陷。 NEMO 锌指结构域的突变可阻断 CD40 介导的 NF-kB 激活，并阻止 B 细胞进行类别转换重组 (CSR)，并防止 APC 在受到 CD40 配体刺激时合成 NF-kB 调节的细胞因子，例如 IL -12 或 TNF-a。 NEMO 的锌指结构域是泛素共价附着的位点，这一步骤对于 NF-κ B 的完全激活是必要的。大多数患有免疫缺陷 (EDI) 的外胚层发育不良患者的锌指结构域存在突变，我们发现此类突变会损害泛素对 NEMO 的翻译后修饰。 在随后的研究中，我们证明了 NEMO 锌指结构域可以调节丝裂原激活蛋白激酶 (MAPK) 活性。我们发现，由于 NEMO 锌指 C 端 E391X 缺失而导致 EDI 患者的树突状细胞在脂多糖 (LPS) 刺激下表现出 MAPK 激活受损。有趣的是，来自 NEMO 锌指结构域内存在 C417R 错义突变且保留了 NEMO 泛素化的患者的 DC 在 LPS 刺激后的 JNK 和 ERK 活性也存在缺陷。我们的研究结果表明，对于 MAPK 的完全激活，NEMO ZF 结构域的结构完整性比其多泛素化更重要。此外，在 E391X 锌指缺失患者中，上游 TAK1 的磷酸化和多泛素化显着降低，表明 NEMO 锌指可能在组装 MAPK 激活的近端信号复合物中发挥重要作用。 最近，我们研究了患有免疫缺陷的外胚层发育不良的患者，他们具有正常的 NEMO 编码序列，但全长 NEMO 蛋白的表达显着减少。来自这些患者的抗原呈递细胞 (APC) 的 TLR4 刺激诱导了正常的细胞质激活和 NF-κ B 的核转位。然而，全长 NEMO 缺陷的细胞在 NF-κ B 调节的细胞因子（如 IL-12）的表达方面存在缺陷，表明 NF-κ B 转录因子的染色质可及性存在下游缺陷。来自这些患者的 TLR4 刺激的 APC 在 IL-12 启动子处的 IKK α 依赖性 H3 组蛋白磷酸化和启动子上的 NF-κ B 异二聚体 Rel A 和 cRel 募集方面存在缺陷。 LPS 处理后，NEMO 敲低 THP-1 细胞中超活性形式 IKK α E、E 的表达恢复了 IL-12 的产生。 我们的研究结果表明，未能表达全长 NEMO 与免疫缺陷表型相关，并为 EDI 患者中 NF-κ B 信号减弱提供了新的见解。 我们发现，尽管激活诱导胞苷脱氨酶 (AID) 和 Iepsilon-Cepsilon 转录物正常诱导，但 EDI 患者 B 细胞的特点是不存在 Ig 体细胞超突变 (SHM) 和缺陷类转换重组 (CSR)。我们使用微阵列分析将 EDI 患者的激活 B 细胞的基因表达谱与参考对照进行比较，以确定 B 细胞中 CSR 和 SHM 可能必需的候选因子。 NIR（新型 INHAT 阻遏蛋白）是一种转录共阻遏蛋白，具有组蛋白乙酰转移酶 (INHAT) 活性抑制剂，之前已被证明与 p53 转录活性和功能发生物理相互作用并抑制 p53 转录活性和功能。 NIR 表达在 CD40 刺激对照 B 细胞后 6 小时被诱导，并在 24 小时达到峰值。然而，患者 B 细胞中不存在这种调节。 因此，我们对 NIR 进行了详细分析。使用蛋白质组学方法，我们已经确定 Aurora 激酶 B 是 NIR 的新型结合伴侣。我们发现 Aurora B、NIR 和 p53 存在于蛋白质复合物中，其中 Aurora B 与 NIR 结合，因此也间接与 p53 结合。从功能上来说，Aurora B 或 NIR 的过表达会抑制 p53 转录活性，而 Aurora B 或 NIR 的耗竭会由于 p21 和 Bax 的上调而导致 p53 依赖性细胞凋亡和细胞生长停滞。然后我们证明 Aurora B 在体外磷酸化 p53 DNA 结合结构域中的多个位点，并且这种磷酸化也可能发生在细胞中。重要的是，Aurora B 介导的 Ser(269) 或 Thr(284) 磷酸化显着损害 p53 转录活性。总而言之，这些结果为 NIR 介导的 p53 抑制提供了新的见解，并提出了 p53 调节的另一种方法。我们最近完成了 NIR 条件性基因敲除小鼠，并计划研究其在 B 细胞发育中的功能。 我们还努力鉴定常见变异免疫缺陷（CVI）的新疾病易感基因。 这些是异质性疾病，其特征是易于严重感染和中和抗体反应受损或缺失。尽管许多单基因缺陷与这些免疫缺陷性疾病有关，但许多病例的遗传基础尚不清楚。为了便于对患有这些综合征的患者进行突变筛查，我们开发了一种定制的 300 kb 重测序阵列，即 Hyper-IgM/CVID 芯片，它可以询问来自与 B 细胞发育和免疫球蛋白同种型转换有关的 148 个基因的 1,576 个编码外显子和内含子-外显子连接区域。使用高通量方案将从患者中提取的基因组 DNA 与阵列杂交，以进行靶序列扩增、汇集和杂交。开发了基于 Web 的应用程序 SNP Explorer，用于直接分析和可视化单核苷酸多态性 (SNP) 注释并进行质量过滤。在患者 DNA 样本中鉴定出已知疾病易感基因（例如 CD40LG、TNFRSF13B、IKBKG、AICDA）中的几种突变，以及其他基因（例如 TRAF3IP2）中的罕见核苷酸变化，并通过直接测序进行验证。我们的结论是，Hyper-IgM/CVID 芯片与 SNP Explorer 相结合，可以提供一种经济有效的工具，用于高通量发现遗传性抗体缺陷患者的数百个疾病相关基因中的新突变。 我们现在正在对候选疾病易感基因进行详细分析。 实验室的相关工作包括培育具有信号分子靶向突变的基因改造小鼠，这些信号分子可调节 NF-kappaB。 CYLD 是一种去泛素化酶，针对 NF-kB 信号通路（包括 NEMO）的信号成分。 家族性圆柱瘤病患者的 CYLD 发生改变，该病以大量良性附件肿瘤为特征。在CYLD缺陷的小鼠中，我们发现外周淋巴器官中的调节性T细胞（Treg）数量显着增加，但胸腺中却没有。 在 TGF-β 存在的情况下，用抗 CD3/28 体外刺激 CYLD 缺陷的原始 T 细胞，与刺激的原始对照 CD4+ 细胞相比，表达 Foxp3 的 T 细胞数量显着增加。 在内源条件下，CYLD与Smad7形成复合物，其中CYLD通过K360和K374残基的去泛素化来调节Smad7。 此外，Smad7 的这种位点特异性泛素化是 TAK1 和 p38 激酶激活所必需的。 我们的结果表明，CYLD 通过 Smad7 去泛素化在调节 T 细胞中的 TGF-β 信号传导和 Treg 细胞的发育中发挥作用。