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.
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