Signal Transduction of Paired Inhibitory Receptors of NK Cells and Macrophages

NK 细胞和巨噬细胞配对抑制性受体的信号转导

• 批准号: 9343586
• 负责人: Daniel W. McVicar
• 金额: $ 125.87万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/9343586
• 关键词: Aconitate Hydratase; Alleles; Animal Model; Back; Biochemical; CD94 Antigen; Cancer Patient; Cardiovascular Diseases; Case-Control Studies; Cell surface; Cells; Conserved Sequence; Data; Databases; Development; Dissection; Distal; Endotoxins; Expressed Sequence Tags; Family; Future; Gene Expression; Genes; Genetic; Glycolysis; Goals; Haplotypes; Human; ITAM; Immune; Immune system; Infection Control; Institutes; Interleukin-10; Investigation; Laboratories; Location; Lysine; Malignant Neoplasms; Mediating; Messenger RNA; Metabolic; Metabolic Pathway; Mitochondria; Molecular Analysis; Molecular Biology; Mus; Myeloid Cells; NADH dehydrogenase (ubiquinone); National Human Genome Research Institute; Nitric Oxide; Open Reading Frames; Oxidative Phosphorylation; Pathology; Production; Proteins; RNA; Receptor Signaling; Regulation; Role; Signal Transduction; Signal Transduction Pathway; System; TYROBP gene; Transcript; Transfection; Transmembrane Domain; United States National Institutes of Health; Variant; Work; arm; base; cancer prevention; coronary artery calcification; human disease; interest; killer immunoglobulin-like receptor; mRNA Expression; macrophage; metabolomics; monocyte; mouse model; neutrophil; operation; rare variant; receptor; receptor coupling; receptor function; research study; response; therapeutic development; transcriptome sequencing; tumor; tumor progression

Work has progress in the analysis of multiple DAP12 and/or Immunoreceptor tyrosine-based Activation Motif (ITAM)-coupled receptor systems. Of particular interest is Triggering Receptor Expressed on Myeloid Cells (TREM)-Like Transcript-4 (Treml4). Treml4 is a potential new player in human disease. Previous work on Treml4 has focused on the murine receptor. The syntenic human gene contains an apparent open reading frame predicted to encode a TREM-like protein with a non-canonical leader sequence, conserved V-set Ig domain, short stalk and a hydrophobic domain that is truncated just distal to the lysine residue that would be predicted to facilitate interaction with DAP12. Few ESTs exist within public databases, giving little clue as to the function of TREML4 in humans. We collaborated with Shurjo Sen, of the national Human Genome Research Institute of NIH, who initiated an RNA Seq case-control study of coronary artery calcification (CAC) using a large network of characterized donors known as ClinSeq. This unbiased approach identified multiple genes with apparent association with CAC. The top four immune genes were TREML4, and three KIR loci associated within B-type KIR haplotypes. Working closely with NHGRI, we characterized two SNPs that are associated with mRNA expression for TREML4 in humans, rs2803495 and rs2803496. Using these SNPs we defined three apparent haplotypes, those with a rare variant at rs2803495 but common at rs2803496, those common at rs2803495 but rare at rs2803496, and the majority of donors with the common variants at both locations. We found substantial TREML4 mRNA only in donors who were carriers of at least one copy of a rare allele, either rs2803495 or rs2803496; thus we term these alleles "permissive". Similar to mouse, we found that TREML4 mRNA could be detected in purified monocytes and neutrophils from donors with permissive alleles, the latter having approximately 100-fold higher expression. Molecular analysis of the TREML4 mRNA confirmed a mRNA of 1.8 kb with an intact open reading frame. Transfection experiments showed that when armed with an effective leader sequence, the transmembrane domain was capable of anchoring TREML4 to the cell surface. However, the native TREML4 leader is incapable of effectively targeting the protein to the cell surface, suggesting that TREML4 may regulate neutrophil function as an intracellular or secreted protein or via RNA-mediated mechanisms. Future plans for Treml4 are collaborative studies with the Biesecker lab at NHGRI, the laboratory that initiated the work on coronary artery calcification, and with Dr. Frank Kolodgie at CV Path Institute, a nonprofit pathology based organization that specializes in the pathological analysis of cardiovascular disease and are not detailed here. In addition, the laboratory has begun an extensive investigation into the regulation of metabolic pathways within innate immune cells and its relevance to cancer development and progression. Initial data has demonstrated the existence of an IL-10 driven metabolic rheostat. This regulatory loop involves the role of endogenous IL-10 in the control of both oxidative phosphorylation pathways as well as glycolysis. Biochemical, transcriptional, metabolic, and metabolomics analysis of macrophages where metabolic pathways are disrupted by limited fuel utilization and/or genetic targeting has implicated the regulation of nitric oxide as a key step in regulation. The data suggest that endogenous IL-10 produced in response to the shift to glycolysis associated with endotoxin stimulation acts back on macrophages to regulate nitric oxide production with in turn regulated oxidative phosphorylation via the suppression of Complex I of the electron transport chain, and mitochondrial aconitase. Analysis of transcriptional data from cancer patients via cBioportal, reveals the potential operation of this rheostat in cancers by showing that tumors high in Il10 expression display a statistically significant shift to expression of genes associated with oxidative phosphorylation. The implications of these data in murine models of cancer are underway.

在分析多个DAP 12和/或基于免疫受体酪氨酸的激活基序（ITAM）偶联受体系统方面的工作取得了进展。特别感兴趣的是髓样细胞上表达的触发受体（TREM）样转录物-4（Treml 4）。Treml 4是人类疾病的潜在新参与者。以前关于Treml 4的工作集中在鼠受体上。该同线人类基因含有一个表观开放阅读框，预测该开放阅读框编码具有非典型前导序列、保守的V-组IG结构域、短茎和疏水结构域的TREM样蛋白，该疏水结构域在赖氨酸残基的远端被截短，预测该赖氨酸残基促进与DAP 12的相互作用。在公共数据库中几乎没有EST存在，对于TREML 4在人类中的功能几乎没有线索。我们与美国国立卫生研究院国家人类基因组研究所的Shurjo Sen合作，他使用一个名为ClinSeq的大型特征化供体网络启动了冠状动脉钙化（CAC）的RNA Seq病例对照研究。这种无偏倚的方法鉴定了与CAC明显相关的多个基因。前四位的免疫基因是TREML 4，和B型KIR单倍型内相关的三个KIR基因座。与NHGRI密切合作，我们表征了与人类TREML 4 mRNA表达相关的两个SNP，rs 2803495和rs 2803496。使用这些SNPs，我们定义了三个明显的单倍型，那些在rs 2803495处具有罕见变异但在rs 2803496处常见的单倍型，那些在rs 2803495处常见但在rs 2803496处罕见的单倍型，以及大多数在两个位置具有常见变异的供体。我们发现大量的TREML 4 mRNA仅在供体中，这些供体是至少一个拷贝的罕见等位基因rs 2803495或rs 2803496的携带者;因此我们将这些等位基因称为“允许的”。与小鼠类似，我们发现TREML 4 mRNA可以在来自具有允许等位基因的供体的纯化的单核细胞和中性粒细胞中检测到，后者具有约100倍的高表达。TREML 4 mRNA的分子分析证实了具有完整开放阅读框的1.8 kb的mRNA。转染实验表明，当装备有有效的前导序列时，跨膜结构域能够将TREML 4锚定到细胞表面。然而，天然TREML 4前导序列不能有效地将蛋白质靶向细胞表面，表明TREML 4可以作为细胞内或分泌蛋白或通过RNA介导的机制调节嗜中性粒细胞功能。Treml 4的未来计划是与NHGRI的Biesecker实验室合作研究，该实验室发起了冠状动脉钙化的工作，并与CV Path Institute的Frank Kolodgie博士合作，CV Path Institute是一家非营利性病理学组织，专门从事心血管疾病的病理分析，这里没有详细介绍。此外，该实验室已开始广泛研究先天免疫细胞内代谢途径的调节及其与癌症发展和进展的相关性。初步数据表明存在IL-10驱动的代谢变阻器。该调节环涉及内源性IL-10在氧化磷酸化途径以及糖酵解的控制中的作用。对代谢途径被有限的燃料利用和/或遗传靶向破坏的巨噬细胞的生物化学、转录、代谢和代谢组学分析已经暗示了一氧化氮的调节是调节中的关键步骤。这些数据表明，响应于与内毒素刺激相关的糖酵解转变而产生的内源性IL-10反过来作用于巨噬细胞，以调节一氧化氮的产生，进而通过抑制电子传递链的复合物I和线粒体顺乌头酸酶来调节氧化磷酸化。通过cBioportal分析癌症患者的转录数据，揭示了这种变阻器在癌症中的潜在作用，表明Il 10表达高的肿瘤显示出与氧化磷酸化相关的基因表达的统计学显著变化。这些数据在小鼠癌症模型中的意义正在进行中。