Signal Transduction of Paired Inhibitory Receptors of NK Cells and Macrophages

在分析多个DAP12和/或免疫受体酪氨酸激活基序(ITAM)偶联受体系统方面取得了进展。特别令人感兴趣的是触发髓样细胞(Trem)样转录本-4(Treml4)上表达的受体。Treml4在人类疾病中是一个潜在的新参与者。之前对Treml4的研究主要集中在小鼠受体上。同线人类基因含有一个明显的开放阅读框，预测编码一个具有非规范前导序列的Trem样蛋白，保守的V-set Ig结构域，短柄和一个疏水结构域，该结构域在赖氨酸残基的远端被截断，预计有助于与DAP12的相互作用。公共数据库中几乎没有EST，几乎没有关于TREML4在人类中功能的线索。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，他利用一个名为ClinSeq的大型捐赠者网络，发起了一项关于冠状动脉钙化(CAC)的RNA序列病例对照研究。这种不偏不倚的方法确定了多个与CAC明显相关的基因。前4位免疫基因为TREML4，3个KIR基因座与B型KIR单倍型相关。与NHGRI密切合作，我们确定了两个与人类TREML4基因表达相关的SNP，rs2803495和rs2803496。利用这些SNP，我们定义了三种明显的单倍型，在rs2803495有罕见变异但在rs2803496有常见的单倍型，在rs2803495有常见的但在rs2803496有罕见的单倍型，以及在两个位置都有共同变异的大多数捐赠者。我们发现，只有携带至少一个稀有等位基因(rs2803495或rs2803496)拷贝的供者体内才有大量的TREML4基因；因此，我们称这些等位基因为“允许的”。与小鼠相似，我们发现在具有允许等位基因的供者的纯化的单核细胞和中性粒细胞中可以检测到TREML4的mRNA，后者的表达水平大约高出100倍。对TREML4mRNA的分子分析证实，TREML4的mRNA为1.8kb，具有完整的开放阅读框架。转染实验表明，当携带有效的前导序列时，跨膜结构域能够将TREML4锚定在细胞表面。然而，天然的TREML4先导不能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的蛋白质或通过RNA介导的机制来调节中性粒细胞的功能。Treml4的未来计划是与NHGRI的Biesecker实验室(发起冠状动脉钙化工作的实验室)以及CV Path Institute的Frank Kolodgie博士进行合作研究，CV Path Institute是一家专门从事心血管疾病病理分析的非营利性组织，此处不作详细介绍。此外，该实验室已经开始对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进行广泛的调查。初步数据表明存在由IL-10驱动的代谢变阻器。这一调节环路涉及内源性IL-10在控制氧化磷酸化途径和糖酵解中的作用。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。巨噬细胞的代谢途径受到燃料利用有限和/或基因靶向的干扰。这些数据表明，内毒素刺激引起的糖酵解反应产生的内源性IL-10作用于巨噬细胞，通过抑制电子传递链复合体I和线粒体乌头酸酶来调节一氧化氮的产生，进而调节氧化磷酸化。通过cBioPortal对癌症患者的转录数据进行分析，揭示了这种变阻器在癌症中的潜在操作，结果表明，IL10高表达的肿瘤表现出统计上显著的向氧化磷酸化相关基因表达的转变。这些数据对小鼠癌症模型的影响正在进行中。

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.