Human Macrophage Sodium Channels: Novel Targets for Inflammatory Diseases

人巨噬细胞钠通道：炎症性疾病的新靶点

• 批准号: 8196324
• 负责人: MICHAEL D CARRITHERS
• 金额: --
• 依托单位: WM S. MIDDLETON MEMORIAL VETERANS HOSP
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196324
• 关键词: AIDS Dementia Complex; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Acute; Antigens; Apoptosis; Apoptotic; Arrhythmia; Attenuated; Autoimmune Diseases; Autoimmunity; Autophagocytosis; Autophagosome; Bacillus (bacterium); Bacterial Infections; Biochemical; Biological Markers; Biosensor; Brain; Calcium; Calcium Signaling; Carbamazepine; Cardiac; Caring; Cell Death; Cell Death Signaling Process; Cell Line; Cell physiology; Cell surface; Cells; Characteristics; Chronic; Clinical; Clinical Treatment; Clone Cells; Communicable Diseases; Coupled; Data; Dependence; Development; Diagnosis; Diagnostic Procedure; Disease; Endosomes; Environment; Epilepsy; Extracellular Space; Genus Mycobacterium; Goals; HIV; HIV Envelope Protein gp120; Health; Health Personnel; Homelessness; Human; Immune; Immunity; Infection; Inflammation; Inflammatory; Inflammatory Response; Injury; Interferon Type II; Laboratories; Lead; Lesion; Life; Ligands; Link; Lipopolysaccharides; MHC Class II Genes; Macrophage Activation; Measures; Mediating; Membrane; Methods; Microscopic; Microscopy; Modeling; Modification; Molecular; Molecular Weight; Monitor; Monoclonal Antibodies; Multiple Sclerosis; Mus; Mycobacterium Infections; Mycobacterium bovis; Neurologic; Opportunistic Infections; Pathogenesis; Pathway interactions; Patients; Phagocytosis; Pharmaceutical Preparations; Phenytoin; Phosphorylation; Physiological; Play; Post-Translational Protein Processing; Process; Property; Proteins; Provider; Quality of life; RNA Splicing; Regulation; Relapse; Relative (related person); Research; Risk; Rodent; Role; Safety; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sodium Channel; Sodium Channel Blockers; Spinal Cord Lesions; Techniques; Therapeutic; Tissues; Translating; Tuberculosis; United States; Variant; Veterans; Virus Diseases; Work; antigen processing; base; channel blockers; chronic pain; computerized data processing; cost; cost effective; cytotoxicity; design; disability; health administration; human disease; humanized monoclonal antibodies; immune function; immunoregulation; improved; in vivo; innate immune function; killings; late endosome; macrophage; novel; novel diagnostics; patch clamp; pathogen; patient population; prevent; public health relevance; research study; response; small hairpin RNA; voltage; young adult

DESCRIPTION (provided by applicant): Summary This proposal is a revised application that examines regulation of human macrophages by the intracellular voltage-gated sodium channel, NaV1.5. Macrophages play a central role in the pathogenesis of intracellular bacterial and viral infections as seen in tuberculosis (TB) and HIV, and they directly mediate tissue injury in autoimmune diseases such as multiple sclerosis (MS). Studies examining unique features of human macrophages at the cellular level are required to develop specific anti-macrophage therapies for human disease. Recently, we identified two novel variants of voltage-gated sodium channels, NaV1.5 and NaV1.6, that are expressed in human macrophages and may represent novel pharmacologic targets. In this revised application, we focus on the role of the NaV1.5 channel in human macrophage function. Macrophage NaV1.5 is unique to human macrophages and is expressed intracellularly on the late endosome. Preliminary data suggest that macrophage NaV1.5 may act as a pathogen biosensor to enhance inflammatory responses and that it regulates macrophage endosomal acidification, phagocytosis, and inflammatory signaling. The hypothesis of this proposal is that the human macrophage splice variant of the voltage- gated sodium channel NaV1.5 has novel molecular properties that regulate intracellular processing and signaling. The revised Specific Aims of this proposal are to analyze: I.) The electrophysiologic properties of macrophage NaV1.5; II.) NaV1.5 regulation of macrophage processing; and III.) NaV1.5 regulation of intracellular signaling. The experimental approach to these aims will be multi-disciplinary and will include electrophysiologic, microscopic, fluorometric, and biochemical techniques. To characterize how the channel is activated, novel methods of single channel recordings in isolated endosomes will be combined with more traditional methods of whole cell patch clamp recordings in transfected cells that over-express NaV1.5. To assess the cellular immune function of the channel, Bacillus Camille-Guerin (BCG) will be used as a model of intracellular mycobacteria infection, and NaV1.5-dependent endosomal processing and intracellular signaling will be assessed by live cell microscopy in addition to standard approaches. These studies represent one of the first electrophysiologic characterizations of endosomal channels and subcellular voltage-gated sodium channels. Since this work will be performed in primary human cells and cell lines, these studies are a unique opportunity to characterize novel properties of human macrophage function and to translate these findings to developing anti-macrophage therapeutics and new diagnostic techniques. Voltage-gated sodium channels are attractive pharmacologic targets because their function can be blocked by low molecular weight, orally active drugs that are currently used clinically to treat epilepsy. Related novel agents that specifically modulate macrophage sodium channels also could be developed. These sodium channel-specific agents may represent a more economical choice for immune modulation because of their relatively low cost as compared with many current monoclonal antibody-based biologic treatments. One goal of these studies is to develop therapeutic strategies to inhibit macrophage NaV1.5 selectively to treat chronic inflammatory conditions such as TB, HIV, and MS in veterans. A second goal is to determine whether or not expression and activity of these macrophage proteins can serve as a biomarker of inflammatory disease activity to aid diagnosis and permit optimization of treatment. PUBLIC HEALTH RELEVANCE: Relevance Statement Chronic inflammatory diseases due to infections such as HIV/AIDS and tuberculosis (TB) or to autoimmunity such as multiple sclerosis (MS) have a profound impact on the health of many veterans. About 22,000 patients infected with HIV receive their care through Veterans Health Administration (VHA) facilities, and the VHA is the single largest provider to this patient population. Patients with AIDS remain susceptible to opportunistic infections and, along with homeless veterans, are at great risk for the development of TB. The VHA historically also has been a major health care provider for patients with TB and MS. Macrophage-mediated immunity causes damage (development and persistence of inflammatory lesions) in each of these diseases. The research proposed examines unique features of human macrophages at the cellular level. Such studies are needed to develop effective anti-macrophage treatments for three serious human diseases -- HIV, TB, and MS.

描述（由申请人提供）： 摘要 该提案是一项修订后的申请，旨在检查细胞内电压门控钠通道 NaV1.5 对人类巨噬细胞的调节。巨噬细胞在结核病 (TB) 和 HIV 等细胞内细菌和病毒感染的发病机制中发挥着核心作用，并且它们直接介导多发性硬化症 (MS) 等自身免疫性疾病中的组织损伤。需要在细胞水平上研究人类巨噬细胞的独特特征，以开发针对人类疾病的特异性抗巨噬细胞疗法。最近，我们发现了电压门控钠通道的两种新变体 NaV1.5 和 NaV1.6，它们在人类巨噬细胞中表达，可能代表新的药理学靶点。在这个修订后的申请中，我们重点关注 NaV1.5 通道在人类巨噬细胞功能中的作用。巨噬细胞NaV1.5是人类巨噬细胞所特有的，在细胞内的晚期内体上表达。初步数据表明，巨噬细胞 NaV1.5 可能作为病原体生物传感器来增强炎症反应，并调节巨噬细胞内体酸化、吞噬作用和炎症信号传导。 该提议的假设是电压门控钠通道 NaV1.5 的人类巨噬细胞剪接变体具有调节细胞内处理和信号传导的新分子特性。该提案修订后的具体目标是分析： I.) 巨噬细胞 NaV1.5 的电生理特性； II.) NaV1.5巨噬细胞加工的调节； III.) NaV1.5 细胞内信号传导的调节。实现这些目标的实验方法将是多学科的，包括电生理学、显微镜、荧光和生化技术。为了表征通道是如何激活的，在分离的内体中进行单通道记录的新方法将与在过度表达 NaV1.5 的转染细胞中进行全细胞膜片钳记录的更传统方法相结合。为了评估通道的细胞免疫功能，将使用卡米尔-格林芽孢杆菌（BCG）作为细胞内分枝杆菌感染的模型，除了标准方法外，还将通过活细胞显微镜评估NaV1.5依赖性内体加工和细胞内信号传导接近。 这些研究代表了内体通道和亚细胞电压门控钠通道的最早的电生理学特征之一。由于这项工作将在原代人类细胞和细胞系中进行，因此这些研究是表征人类巨噬细胞功能的新特性并将这些发现转化为开发抗巨噬细胞疗法和新诊断技术的独特机会。电压门控钠通道是有吸引力的药理学靶点，因为它们的功能可以被目前临床上用于治疗癫痫的低分子量口服活性药物阻断。还可以开发特异性调节巨噬细胞钠通道的相关新型药物。这些钠通道特异性药物可能代表免疫调节更经济的选择，因为与许多当前基于单克隆抗体的生物治疗相比，它们的成本相对较低。这些研究的目标之一是开发选择性抑制巨噬细胞 NaV1.5 的治疗策略，以治疗退伍军人的慢性炎症性疾病，如结核病、艾滋病毒和多发性硬化症。第二个目标是确定这些巨噬细胞蛋白的表达和活性是否可以作为炎症疾病活动的生物标志物，以帮助诊断并优化治疗。 公共卫生相关性： 相关性声明 HIV/AIDS 和结核病 (TB) 等感染或多发性硬化症 (MS) 等自身免疫引起的慢性炎症对许多退伍军人的健康产生深远影响。大约 22,000 名艾滋病毒感染者通过退伍军人健康管理局 (VHA) 机构接受护理，VHA 是该患者群体最大的单一提供者。艾滋病患者仍然容易受到机会性感染，并且与无家可归的退伍军人一样，面临着罹患结核病的巨大风险。 VHA 历来也是结核病和多发性硬化症患者的主要医疗保健提供者。巨噬细胞介导的免疫会导致这些疾病的损害（炎症病变的发展和持续）。该研究提议在细胞水平上检查人类巨噬细胞的独特特征。需要进行此类研究来开发针对三种严重人类疾病（艾滋病毒、结核病和多发性硬化症）的有效抗巨噬细胞疗法。