Kevin Carrasco1,2, Amir Boufenzer1, Lucie Jolly1,2, Helene Le Cordier3, Guanbo Wang4, Albert JR Heck4, Adelheid Cerwenka5, Emilie Vinolo1, Alexis Nazabal6, Alexandre Kriznik7, Pierre Launay8, Sebastien Gibot2 & Marc Derive1
- INOTREM, Vandoeuvre-les-Nancy, France
- UMR-S 1116, Defaillance cardiovasculaire aigue et chronique, Vandoeuvre-les-Nancy, France
- UMR7365, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), CNRS-Université de Lorraine, Vandoeuvre-les-Nancy, France
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences and Netherlands Proteomics Center, Utrecht University, Utrecht, The Netherlands
- Innate Immunity, German Cancer Research Center (DKFZ), Heidelberg, Germany
- CovalX, Zurich, Switzerland
- Service Commun de Biophysique Interactions Moléculaires (SCBIM), FR3209, Biopôle de l’Université de Lorraine, Vandoeuvre-les-Nancy, France
- Inatherys, Evry Cedex, France
The triggering receptor expressed on myeloid cells-1 (TREM-1) is a receptor expressed on innate immune cells. By promoting the amplification of inflammatory signals that are initially triggered by Toll-like receptors (TLRs), TREM-1 has been characterized as a major player in the pathophysiology of acute and chronic inflammatory diseases, such as septic shock, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. However, the molecular events leading to the activation of TREM-1 in innate immune cells remain unknown. Here, we show that TREM-1 is activated by multimerization and that the levels of intracellular Ca2+ release, reactive oxygen species, and cytokine production correlate with the degree of TREM-1 aggregation. TREM-1 activation on primary human monocytes by LPS required a two-step process consisting of upregulation followed by clustering of TREM-1 at the cell surface, in contrast to primary human neutrophils, where LPS induced a rapid cell membrane reorganization of TREM-1, which confirmed that TREM-1 is regulated differently in primary human neutrophils and monocytes. In addition, we show that the ectodomain of TREM-1 is able to homooligomerize in a concentration-dependent manner, which suggests that the clustering of TREM-1 on the membrane promotes its oligomerization. We further show that the adapter protein DAP12 stabilizes TREM-1 surface expression and multimerization. TREM-1 multimerization at the cell surface is also mediated by its endogenous ligand, a conclusion supported by the ability of the TREM-1 inhibitor LR12 to limit TREM-1 multimerization. These results provide evidence for ligand-induced, receptor-mediated dimerization of TREM-1. Collectively, our findings uncover the mechanisms necessary for TREM-1 activation in monocytes and neutrophils.
CovalX Technology Used (Click each option to learn more)
The recombinant protein hTREM-1-ECD (21-200), a recombinant human TREM-1 (residues Ala21-Arg200) is available commercially and was subjected to crosslinking with the K200 Stabilization Kit. The protein was diluted at 1:4 ratio with the crosslinking buffer and the sample was mixed with 1 μl of the K200 reagent before remaining at room temperature for 3 hours. 1 μl of the final solution was spotted on the MALDI plate and crystallized at room temperature before analysis. The plates were analyzed using a MALDI TOF mass spectrometer modified with a CovalX HM4 detection system. The data was analyzed using the CovalX Complex Tracker software.