Identification of triggering receptor expressed on myeloid cells (TREM)s ligands

TREM (triggering receptor expressed on myeloid cell) proteins are a family of cell receptors expressed on myeloid cells that include both activating and inhibiting receptors, based on their ability to interact with the adaptor protein DAP12 or on the presence of a cytoplasmic domain containing ITIM motifs. TREMs are expressed on cells of myeloid origin that function both in the immune system and in remodelling host tissue: these receptors are expressed on granulocytes, monocytes, macrophages, microglia, dendritic cells, osteoclasts, and platelets. Identified as receptors that ‘trigger’ cellular activation, it is now clear that they are modulators of the cellular response, having both positive and negative functions in regulating the activation and differentiation of myeloid cells. They have been proposed to fall into the group of receptors that set signaling “threshold” for cells. TREM-1 functions as an amplifier of inflammation: it is mainly expressed in neutrophils and monocytes/macrophages, in which microbial stimuli strongly upregulate its expression. TREM-1 triggering with activating antibodies does not results in activation by itself, but synergizes with cellular activation induced by Pattern Recognition Receptor, such as TLRs and NOD receptors, in terms of production of inflammatory cytokines and chemokines and induction of phagocytosis and cellular effector function. Intracellularly, TREM-1 engagement activates the canonical activation cascade of protein kinases described for the ITAM containing adaptor DAP12. In vivo, the soluble form of the receptor, potentially acting as a decoy, has inhibiting functions in acute inflammation models, such as endotoxemia and Cecal Ligation and Puncture (CLP). Multiple functions have been described for TREM-2. TREM-2 is expressed broadly by mononuclear phagocytes, including macrophages, microglia and osteoclast precursors. In these cells TREM-2 inhibits TLR-induced cytokine production and potentiates phagocytosis and mediates an alternative activation of DAP12 signaling that leads to anti-inflammatory effects. TREM-2 is fundamental for proper osteoclast differentiation both in vitro and in vivo. So far, no ligand has been identified for any of the members of the family. Using a soluble form of TREM receptors we identified activated neutrophils as TREM-1 ligand expressing cells. We cloned several genes that were selectively expressed by TREM-1L+ neutrophils, but none of the proteins encoded by those genes interacts with TREM-1, leaving an open question on the nature of TREM-1L. Conversely, upon the identification of a candidate ligand for TREM-2 expressed at cell surface of several tumor cell lines, we have isolated a TREM-2 binding protein, galectin-1, a β-lactose lectin, using a biochemical approach. The interaction was confirmed by immunoprecipitation studies and in vitro assay with immobilized Gal-1. TREM-2 binding with GAL-1 is specific since: i) anti Gal-1 antibodies and ii) a recombinant Gal-1 inhibit binding of a soluble form of TREM-2 (TREM-2/Ig) to ligand positive cells; iii) Gal-1 does not interact with other TREM family members as demonstrated by the in vitro binding assay. We also found that Gal-1 interacts with TREM-2 in a protein-protein manner, not involving its carbohydrate recognition domain. In order to validate a functional role of this interaction, we demonstrated that in an in vitro osteoclastogenesis model recombinant Gal-1 enhanced TRAP+ multinucleated cell formation and increases nuclei/cells ratio. Monocytes from TREM-2 deficient patients are unable to differentiate in vitro into mature osteoclasts and addition of the Gal-1 did not rescue the differentiation. This shows the role of the newly identified TREM-2 ligand, galectin-1, in the process of TREM-2-dependent osteoclastogenesis.