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.

对多个 DAP12 和/或免疫受体酪氨酸激活基序 (ITAM) 偶联受体系统的分析工作取得了进展。特别令人感兴趣的是在骨髓细胞上表达的触发受体 (TREM)-样 Transcript-4 (Treml4)。 Treml4 是人类疾病中潜在的新参与者。之前关于 Treml4 的工作主要集中在小鼠受体上。同线性人类基因包含一个明显的开放阅读框，预计编码具有非规范前导序列的 TREM 样蛋白、保守的 V 型 Ig 结构域、短茎和在赖氨酸残基远端截短的疏水结构域，预计该结构有助于与 DAP12 相互作用。公共数据库中很少有 EST，因此无法提供有关 TREML4 在人类中的功能的线索。我们与 NIH 国家人类基因组研究所的 Shurjo Sen 合作，他利用名为 ClinSeq 的大型特征性供体网络发起了一项冠状动脉钙化 (CAC) 的 RNA Seq 病例对照研究。这种公正的方法鉴定了多个与 CAC 明显相关的基因。前四个免疫基因是 TREML4，以及 B 型 KIR 单倍型中相关的三个 KIR 位点。我们与 NHGRI 密切合作，鉴定了与人类 TREML4 mRNA 表达相关的两个 SNP，rs2803495 和 rs2803496。使用这些 SNP，我们定义了三种明显的单倍型，即在 rs2803495 处具有罕见变异但在 rs2803496 处常见的单倍型，在 rs2803495 处常见但在 rs2803496 处罕见的单倍型，以及大多数供体在两个位置都具有常见变异的单倍型。我们仅在至少携带一个罕见等位基因（rs2803495 或 rs2803496）拷贝的捐赠者中发现大量 TREML4 mRNA；因此，我们将这些等位基因称为“宽容的”。与小鼠类似，我们发现可以在来自具有许可等位基因的供体的纯化单核细胞和中性粒细胞中检测到 TREML4 mRNA，后者的表达量高出大约 100 倍。 TREML4 mRNA 的分子分析证实了 1.8 kb 的 mRNA 具有完整的开放阅读框。转染实验表明，当配备有效的前导序列时，跨膜结构域能够将 TREML4 锚定到细胞表面。然而，天然 TREML4 前导序列无法有效地将蛋白质靶向细胞表面，这表明 TREML4 可能作为细胞内或分泌蛋白质或通过 RNA 介导的机制调节中性粒细胞功能。 Treml4 的未来计划是与 NHGRI 的 Biesecker 实验室（该实验室发起了冠状动脉钙化工作）以及 CV Path Institute 的 Frank Kolodgie 博士进行合作研究，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，此处不再详细介绍。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生和进展的相关性。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 在氧化磷酸化途径和糖酵解控制中的作用。对巨噬细胞的生化、转录、代谢和代谢组学分析表明，一氧化氮的调节是调节的关键步骤，其中代谢途径因有限的燃料利用和/或遗传靶向而受到干扰。数据表明，响应与内毒素刺激相关的糖酵解转变而产生的内源性 IL-10 作用于巨噬细胞，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 对癌症患者的转录数据进行分析，通过显示高 Il10 表达的肿瘤显示出与氧化磷酸化相关的基因表达发生统计学上显着的转变，揭示了这种变阻器在癌症中的潜在作用。这些数据对小鼠癌症模型的影响正在进行中。