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During sepsis, neutrophil activation induces endothelial cell (EC) dysfunction partly through neutrophil extracellular trap (NET) release. The triggering receptor expressed on myeloid cell-1 (TREM-1) is an orphan immune receptor that amplifies the inflammatory response mediated by Toll-like receptor-4 (TLR4) engagement. Although the key role of TLR4 signaling in NETosis is known, the role of TREM-1 in this process has not yet been investigated. Here, we report that TREM-1 potentiates NET release by human and murine neutrophils and is a component of the NET structure. In contrast, pharmacologic inhibition or genetic ablation of TREM-1 decreased NETosis in vitro and during experimental septic shock in vivo. Moreover, isolated NETs were able to activate ECs and impair vascular reactivity, and these deleterious effects were dampened by TREM-1 inhibition. TREM-1 may, therefore, constitute a new therapeutic target to prevent NETosis and associated endothelial dysfunction.

Background:One of main new avenue of exploration in critical care medicine is the development of personalized medicine approaches to better determine which therapy provides the optimal risk/benefit ratio for subgroups of septic shock patients presenting with similar clinical syndromes. This paper describes the early clinical development of a personalized medicine approach for a novel therapeutic agent targeting the TREM-1 pathway, nangibotide, in the treatment of septic shock.Methods:Soluble TREM-1 (sTREM-1) is a mechanism-based candidate biomarker with the potential to select patients at high risk of death and who may respond best to an anti-TREM-1 strategy. Validation of a potential cut-off value and choice of surrogate endpoint for phase 2b clinical trial are built on data from a retrospective cohort study of 293 septic shock patients and data from a multicentre prospective phase 2a study of 49 septic shock patients treated either by placebo or nangibotide. Logistic regression modelling is combined with receiver operator characteristic analysis to validate the prognostic potential of sTREM-1. In addition, the potential predictive cut-off is defined by modelling the ability of different values to detect treatment effect.Results:sTREM-1 at baseline in the AdrenOSS-1 cohort is strongly associated with 28day mortality with a standardized odds ratio for mortality of 2.86 (95%CI 1.91-4.27, pConclusions: This study improves our understanding of the behaviour of sTREM-1 as a potential mechanism-based biomarker with the potential to offer both prognostic and predictive enrichment in clinical trials of nangibotide. These data inform the design of the ongoing ASTONISH phase 2b study which intends to validate the proposed cut-off value as a predictive biomarker in septic shock.Trial Registration: AdrenOSS-1 study: clinicaltrials.gov: NCT02393781, MOT-C-201: clinicaltrials.gov: NCT03158948

TREM-1 (triggering receptor expressed on myeloid cells-1) is a transmembrane receptor expressed by innate immune cells, including endothelial cells and platelets. TREM-1 is a crucial mediator of septic shock that acts by synergizing with Toll-like receptors (TLRs) to amplify the inflammatory responses to pathogens, thus promoting sepsis-induced immune dysregulation and organ dysfunction. [...]

International audience; BACKGROUND: The use of mesenchymal stem cells (MSCs) is being extensively studied in clinical trials in the setting of various diseases including diabetes, stroke, and progressive multiple sclerosis. The unique immunomodulatory properties of MSCs also point them as a possible therapeutic tool during sepsis and septic shock, a devastating syndrome associated with 30-35% mortality. However, MSCs are not equal regarding their activity, depending on their tissue origin. Here, we aimed at comparing the in vivo properties of MSCs according to their tissue source (bone marrow (BM) versus Wharton's jelly (WJ)) in a murine cecal ligation and puncture (CLP) model of sepsis that mimics a human peritonitis. We hypothesized that MSC properties may vary depending on their tissue source in the setting of sepsis.METHODS: CLP, adult, male, C57BL/6 mice were randomized in 3 groups receiving respectively 0.25 × 106 BM-MSCs, 0.25 × 106 WJ-MSCs, or 150 μL phosphate-buffered saline (PBS) intravenously 24 h after the CLP procedure.RESULTS: We observed that both types of MSCs regulated leukocyte trafficking and reduced organ dysfunction, while only WJ-MSCs were able to improve bacterial clearance and survival.CONCLUSION: This study highlights the importance to determine the most appropriate source of MSCs for a given therapeutic indication and suggests a better profile for WJ-MSCs during sepsis.

Background New evidence has shown the link between inflammation and thrombosis. Triggering receptor expressed on myeloid cells-1 (TREM-1) is an immunoreceptor expressed mostly on neutrophils and monocytes/macrophages. TREM-1 acts as an amplifier of the inflammatory response, and its pharmacological inhibition displays protective effects in various models of inflammatory disorders, in particular by dampening coagulation abnormalities and thrombocytopenia observed during acute inflammation. Objectives We aimed to decipher the role of TREM-1 in fostering thrombin generation. Methods We measured thrombin generation (TG) by the use of calibrated automated thrombography with whole blood, and isolated primary human neutrophils and monocytes upon stimulation with lipopolysaccharide (LPS). Tissue factor (TF) expression was measured by flow cytometry and its activity by ELISA. Phosphatidylserine (PtdSer) exposure was determined by flow cytometry. A dodecapeptide (LR12) was used as a specific inhibitor of TREM-1. Results LPS increased TG, TF expression, and activity, as well as the exposure of PtdSer on the surface of monocytes. LR12 dampened TF activity through the decrease of PtdSer exposure, leading to a reduction of thrombin generation. Conclusions TREM-1 inhibition decreases thrombin generation and could be an interesting target for the development of new inhibitors of leukocyte-associated thrombotic activity.

Aims TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) is an immunoreceptor expressed on neutrophils and monocytes/macrophages whose role is to amplify the inflammatory response driven by Toll-Like Receptors engagement. The pharmacological inhibition of TREM-1 confers protection in several pre-clinical models of acute inflammation. In this study, we aimed to decipher the role of TREM-1 on the endothelium. Methods and results We first showed by qRT-PCR, flow cytometry and confocal microscopy that TREM-1 was expressed in human pulmonary microvascular endothelial cells as well as in mouse vasculature (aorta, mesenteric artery, and pulmonary vessels). TREM-1 expression was upregulated following septic insult. We next observed that TREM-1 engagement impaired mouse vascular reactivity and promoted vascular inflammation. The pharmacological inhibition of TREM-1 (using the synthetic inhibitory peptide LR12) prevented these disorders both in vitro and in vivo. We generated endothelium-conditional Trem-1 ko mice (EndoTREM-1-/-) and submitted them to a caecal ligation and puncture-induced septic shock. As compared with wild-type littermates, targeted endothelial Trem-1 deletion conferred protection during septic shock in modulating inflammatory cells mobilization and activation, in restoring vasoreactivity, and in improving the survival. Conclusion We reported that TREM-1 is expressed and inducible in endothelial cells and plays a direct role in vascular inflammation and dysfunction. The targeted deletion of endothelial Trem-1 conferred protection during septic shock in modulating inflammatory cells mobilization and activation, restoring vasoreactivity, and improving survival. The effect of TREM-1 on vascular tone, while impressive, deserves further investigations including the design of endothelium-specific TREM-1 inhibitors.

SummaryTriggering Receptor Expressed on Myeloid cells-1 (TREM-1) is an immunoreceptor initially known to be expressed on neutrophils and monocytes/macrophages. TREM-1 acts as an amplifier of the inflammatory response during both infectious and aseptic inflammatory diseases. Another member of the TREM family, The Triggering receptor expressed on myeloid cells Like Transcript-1 (TLT-1) is exclusively expressed in platelets and promotes platelet aggregation. As the gene that encodes for TLT-1 is located in the TREM-1 gene cluster, this prompted us to investigate the expression of TREM-1 on platelets. Here we show that TREM-1 is constitutively expressed in α-granules and mobilised at the membrane upon platelet activation. Pharmacologic inhibition of TREM-1 reduces platelet activation as well as platelet aggregation induced by collagen, ADP, and thrombin in human platelets. Aggregation is similarly impaired in platelets from Trem-1−/− mice. In vivo, TREM-1 inhibition decreases thrombus formation in a carotid artery model of thrombosis and protects mice during pulmonary embolism without excessive bleeding. These findings suggest that TREM-1 inhibition could be useful adducts in antiplatelet therapies.Supplementary Material to this article is available online at www.thrombosis-online.com.

Introduction The Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) amplifies the inflammatory response driven by TLR/NLR engagement. In various models of acute inflammation, septic or not, others and we have shown that the genetic deletion or pharmacologic inhibition of Trem-1 protect animals from hyperresponsiveness and death. In this study we demonstrate that a targeted deletion of Trem-1 on endothelial cells confers protection during septic shock in mice. Methods We generated constitutive Trem-1 -/- KO ( Trem-1 -/- ) as well as endothelium-conditional Trem-1 -/- KO (Endo Trem1 -/- ) mice and submitted them to polymicrobial sepsis through CLP. Organs (BM, spleen, lungs, aorta and mesenteric artery) and blood were harvested at different time points and analyzed for cellular content, gene expression, cytokine/chemokine concentrations, and vasoreactivity. Survival was monitored for 1week. Results Trem-1 -/- and Endo Trem1 -/- mice were equal in size, weight and fertility to littermate controls. Moreover, the composition and abundance of immune cells in blood, BM, spleen, and lungs did not differ between groups. Trem-1 deletion altered inflammatory cells mobilization and recruitment in lungs and spleen and favors the accumulation of reparative cells (Ly6Cl ow monocytes and M2 macrophages) in the lungs. This effect was even more pronounced in Endo Trem1 -/- . Consequently, the activation of many inflammatory genes was reduced in the lungs as well as the concentrations of various cyto/ chemokines (MCP-1, VCAM-1, IL6…). Sepsis induced a profound vascular hyporeactivity in WT mice. This phenomenon was absent in the absence of Trem-1 . Interestingly, in Endo Trem1 -/- while vasoconstriction was still slightly impaired, endothelium-dependent vasodilation remained intact. Finally, survival was improved in the Trem-1 -/- group and even more in the Endo Trem1 -/- group. Conclusion The targeted deletion of endothelial Trem-1 confers protection during septic shock in modulating inflammatory cells mobilization and activation and restoring vasoreactivity. The mechanism by which cellular recruitment is reduced is probably linked to a reduction of chemokines production by endothelial cells. The effect of TREM-1 on vascular tone, while impressive, deserves further investigations. The next step should be to design endothelium specific TREM-1 inhibitors. The author hereby declares no conflict of interest

A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), in rat whole blood. Sample treatment was optimized using protein precipitation and solid phase extraction. Chromatographic separation was carried out in a gradient mode using a core-shell C18 column (150 × 4.6 mm, 3.6 µm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the Food and Drugs Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50-500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre-analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.