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: 7928446
• 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/7928446
• 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; 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）： 总结本提案是一项修订后的应用，旨在检查细胞内电压门控钠通道NaV1.5对人巨噬细胞的调节。巨噬细胞在细胞内细菌和病毒感染的发病机制中起核心作用，如在结核病（TB）和HIV中所见，并且它们直接介导自身免疫性疾病如多发性硬化症（MS）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，我们发现了两种新的电压门控钠通道变体，NaV1.5和NaV1.6，它们在人巨噬细胞中表达，可能代表新的药理学靶点。在这个修订后的申请中，我们专注于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，并在细胞内表达于晚期内体上。初步数据表明，巨噬细胞NaV1.5可以作为病原体生物传感器，以增强炎症反应，它调节巨噬细胞内体酸化，吞噬作用和炎症信号。 该提议的假设是电压门控钠通道NaV1.5的人巨噬细胞剪接变体具有调节细胞内加工和信号传导的新分子特性。本提案经修订后的具体目标是分析：一）巨噬细胞NaV1.5的电生理特性; II.）NaV1.5调节巨噬细胞加工;和III.）NaV1.5调节细胞内信号传导。这些目标的实验方法将是多学科的，将包括电生理学，显微镜，荧光和生物化学技术。为了表征通道是如何被激活的，在分离的内体中进行单通道记录的新方法将与在过表达NaV1.5的转染细胞中进行全细胞膜片钳记录的更传统的方法相结合。为了评估通道的细胞免疫功能，将使用卡米尔-盖林芽孢杆菌（BCG）作为细胞内分枝杆菌感染的模型，除标准方法外，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。 这些研究代表了内体通道和亚细胞电压门控钠通道的第一个电生理特征之一。由于这项工作将在原代人类细胞和细胞系中进行，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药物。这些钠通道特异性药物可能代表免疫调节的更经济的选择，因为与许多目前基于单克隆抗体的生物治疗相比，它们的成本相对较低。这些研究的一个目标是开发治疗策略，选择性地抑制巨噬细胞NaV1.5，以治疗退伍军人的慢性炎症，如TB，HIV和MS。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性疾病活性的生物标志物，以帮助诊断和优化治疗。 公共卫生关系： 由艾滋病毒/艾滋病和结核病（TB）等感染或多发性硬化症（MS）等自身免疫引起的慢性炎症性疾病对许多退伍军人的健康产生深远影响。大约22，000名感染艾滋病毒的患者通过退伍军人健康管理局（VHA）的设施接受护理，VHA是这一患者群体的单一最大提供者。艾滋病患者仍然容易受到机会性感染，沿着无家可归的退伍军人，他们面临着结核病发展的巨大风险。VHA在历史上也一直是结核病和MS患者的主要医疗保健提供者。巨噬细胞介导的免疫力在这些疾病中的每一种中引起损伤（炎性病变的发展和持续）。这项研究在细胞水平上研究了人类巨噬细胞的独特特征。这些研究需要开发有效的抗巨噬细胞治疗三种严重的人类疾病-艾滋病，结核病和MS。