Potentiation of NETs release is novel characteristic of TREM-1 activation and the pharmacological inhibition of TREM-1 could prevent from the deleterious consequences of NETs release in sepsis.

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.

TREM-1 orchestrates angiotensin II-induced monocyte trafficking and promotes experimental abdominal aortic aneurysm.

The triggering receptor expressed on myeloid cells 1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of angiotensin II-induced (AngII-induced) AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalized with macrophages. Trem1 gene deletion (Apoe-/-Trem1-/-), as well as TREM-1 pharmacological blockade with LR-12 peptide, limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2, and Mmp9 mRNA expression, and led to a decreased macrophage content due to a reduction of Ly6Chi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6Chi monocyte aorta infiltration after AngII infusion through CD62L upregulation and promoted proinflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6Chi monocytes through AngII receptor type I (AT1R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared with patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in humans.

Comparison of sTREM-1 and associated periodontal and bacterial factors before/after periodontal therapy, and impact of psychosocial factors.

AIM: The immune receptor triggering receptor expressed on myeloid cell-1 (TREM-1) is responsible for an amplification of the immuno-inflammatory response in inflammatory diseases. Its role in the aetiopathogenesis of periodontitis is underexplored. The aim of this case-control and before-after study was to determine the evolution of soluble form of TREM-1 (sTREM-1) concentrations after scaling and root planing (SRP), and its prognostic value and evaluate associated microbial, periodontal and psychosocial factors. METHODS: Gingival crevicular fluid was collected in two pathological sites (periodontal pocket depth (PPD) ≥ 5 mm) and one healthy site (PPD ≤ 3 mm) from thirty periodontitis patients (before/after SRP), and in one healthy site from thirty controls (patients without periodontal disease). Each patient filled-in stress/anxiety self-assessment questionnaires and provided a saliva sample. Diseased patients were followed for a total of 13-15 weeks in initial periodontal treatment. sTREM-1 and salivary cortisol levels were determined by ELISA and periodontopathogens by PCR. RESULTS: Before SRP, higher crevicular sTREM-1 levels were positively associated with some increased clinical parameters (Plaque Index, tooth mobility, bleeding on probing, p < .05) and inversely with Aggregatibacter actinomycetemcomitans abundance (p = .03). No correlation with psychological factors nor cortisol was found with salivary sTREM-1 concentrations. After SRP, crevicular sTREM-1 levels decreased (p < .001) and were not linked to a PPD decrease but remained higher in pathological than in healthy sites (p < .001). Higher concentrations were also found out in unimproved sites (no change or increase in PPD) compared to improved ones (p = .02). Higher sTREM-1 levels were associated with Porphyromonas gingivalis, Treponema denticola and Campylobacter rectus in pathological sites after SRP (p < .05). CONCLUSION: Crevicular sTREM-1 level decreased after SRP but did not appear to be a site outcome predictive factor of periodontal healing and remained an inflammatory parameter.

Inhibition of triggering receptor expressed on myeloid cells-1 impairs thrombin generation.

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.

Triggering Receptor Expressed on Myeloid Cells-1 Inhibitor Targeted to Endothelium Decreases Cell Activation.

TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) is an immunoreceptor expressed on neutrophils, monocytes/macrophages, and endothelial cells. It amplifies the inflammatory response driven by Toll-Like Receptors (TLR) engagement. The pharmacological inhibition of TREM-1 confers protection in several pre-clinical models of acute inflammation. In this study, we aimed to investigate the role of TREM-1 in endothelial cells using a sneaking ligand construct (SLC) inhibiting TREM-1 in the endothelium. The SLC was made of 3 modules: an E-selectin targeting domain, a Pseudomonas aeruginosa exotoxin a translocation domain, and a 7 aa peptide (LSKSLVF) that contains the interaction site between TREM-1 and its adaptor protein DAP-12. SLC peptide was effectively picked up by endothelial cells following LPS stimulation. It decreased LPS induced TREM-1 up-regulation and cell activation, neutrophils extravasation, and improved median survival time during experimental peritonitis in mice. We reported that a targeted endothelial TREM-1 inhibition is able to dampen cell activation and to confer protection during septic shock in mice. The use of such cell-specific, ligand- independent TREM-1 inhibitors deserve further investigations during acute or chronic inflammatory disorders.

Bone marrow vs Wharton's jelly mesenchymal stem cells in experimental sepsis: a comparative study.

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.

TREM-1 multimerization is essential for its activation on monocytes and neutrophils.

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.

Clinical-grade mesenchymal stem cells derived from umbilical cord improve septic shock in pigs.

BACKGROUND: Septic shock is the leading cause of death in intensive care units. The pathophysiological complexity of this syndrome contributes to an absence of specific treatment. Several preclinical studies in murine models of septic shock have shown improvements to organ injury and survival after administration of mesenchymal stem cells (MSCs). To better mimic a clinical approach in humans, we investigated the impact of randomized controlled double-blind administration of clinical-grade umbilical cord-derived MSCs to a relevant pig model of septic shock. METHODS: Septic shock was induced by fecal peritonitis in 12 male domestic pigs. Animals were resuscitated by an experienced intensivist including fluid administration and vasopressors. Four hours after the induction of peritonitis, pigs were randomized to receive intravenous injection of thawed umbilical cord-derived MSCs (UCMSC) (1 × 106 UCMSCs/kg diluted in 75 mL hydroxyethyl starch (HES), (n = 6) or placebo (HES alone, n = 6). Researchers were double-blinded to the treatment administered. Hemodynamic parameters were continuously recorded. Gas exchange, acid-base status, organ function, and plasma cytokine concentrations were assessed at regular intervals until 24 h after the onset of peritonitis when animals were sacrificed under anesthesia. RESULTS: Peritonitis induced profound hypotension, hyperlactatemia, and multiple organ failure. These disorders were significantly attenuated when animals were treated with UCMSCs. In particular, cardiovascular failure was attenuated, as attested by a better mean arterial pressure and reduced lactatemia, despite lower norepinephrine requirements. As such, UCMSCs improved survival in this very severe model (60% survival vs. 0% at 24 h). CONCLUSION: UCMSCs administration is beneficial in this pig model of polymicrobial septic shock.

Targeted endothelial gene deletion of triggering receptor expressed on myeloid cells-1 protects mice during septic shock.

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.

Triggering Receptor Expressed on Myeloid cells-1: a new player in platelet aggregation.

Triggering 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.

Genetic and Pharmacological Inhibition of TREM-1 Limits the Development of Experimental Atherosclerosis.

BACKGROUND: Innate immune responses activated through myeloid cells contribute to the initiation, progression, and complications of atherosclerosis in experimental models. However, the critical upstream pathways that link innate immune activation to foam cell formation are still poorly identified. OBJECTIVES: This study sought to investigate the hypothesis that activation of the triggering receptor expressed on myeloid cells (TREM-1) plays a determinant role in macrophage atherogenic responses. METHODS: After genetically invalidating Trem-1 in chimeric Ldlr-/-Trem-1-/- mice and double knockout ApoE-/-Trem-1-/- mice, we pharmacologically inhibited Trem-1 using LR12 peptide. RESULTS: Ldlr-/- mice reconstituted with bone marrow deficient for Trem-1 (Trem-1-/-) showed a strong reduction of atherosclerotic plaque size in both the aortic sinus and the thoracoabdominal aorta, and were less inflammatory compared to plaques of Trem-1+/+ chimeric mice. Genetic invalidation of Trem-1 led to alteration of monocyte recruitment into atherosclerotic lesions and inhibited toll-like receptor 4 (TLR 4)-initiated proinflammatory macrophage responses. We identified a critical role for Trem-1 in the upregulation of cluster of differentiation 36 (CD36), thereby promoting the formation of inflammatory foam cells. Genetic invalidation of Trem-1 in ApoE-/-/Trem-1-/- mice or pharmacological blockade of Trem-1 in ApoE-/- mice using LR-12 peptide also significantly reduced the development of atherosclerosis throughout the vascular tree, and lessened plaque inflammation. TREM-1 was expressed in human atherosclerotic lesions, mainly in lipid-rich areas with significantly higher levels of expression in atheromatous than in fibrous plaques. CONCLUSIONS: We identified TREM-1 as a major upstream proatherogenic receptor. We propose that TREM-1 activation orchestrates monocyte/macrophage proinflammatory responses and foam cell formation through coordinated and combined activation of CD36 and TLR4. Blockade of TREM-1 signaling may constitute an attractive novel and double-hit approach for the treatment of atherosclerosis.

TREM-1 Mediates Inflammatory Injury and Cardiac Remodeling Following Myocardial Infarction.

RATIONALE: Optimal outcome after myocardial infarction (MI) depends on a coordinated healing response in which both debris removal and repair of the myocardial extracellular matrix play a major role. However, adverse remodeling and excessive inflammation can promote heart failure, positioning leucocytes as central protagonists and potential therapeutic targets in tissue repair and wound healing after MI. OBJECTIVE: In this study, we examined the role of triggering receptor expressed on myeloid cells-1(TREM-1) in orchestrating the inflammatory response that follows MI. TREM-1, expressed by neutrophils and mature monocytes, is an amplifier of the innate immune response. METHODS AND RESULTS: After infarction, TREM-1 expression is upregulated in ischemic myocardium in mice and humans. Trem-1 genetic invalidation or pharmacological inhibition using a synthetic peptide (LR12) dampens myocardial inflammation, limits neutrophils recruitment and monocyte chemoattractant protein-1 production, thus reducing classical monocytes mobilization to the heart. It also improves left ventricular function and survival in mice (n=20-22 per group). During both permanent and transient myocardial ischemia, Trem-1 blockade also ameliorates cardiac function and limits ventricular remodeling as assessed by fluorodeoxyglucose-positron emission tomographic imaging and conductance catheter studies (n=9-18 per group). The soluble form of TREM-1 (sTREM-1), a marker of TREM-1 activation, is detectable in the plasma of patients having an acute MI (n=1015), and its concentration is an independent predictor of death. CONCLUSIONS: These data suggest that TREM-1 could constitute a new therapeutic target during acute MI.

Pharmacological inhibition of the triggering receptor expressed on myeloid cells-1 limits reperfusion injury in a porcine model of myocardial infarction.

AIMS: Limitation of ischemia/reperfusion injury is a major therapeutic target after acute myocardial infarction (AMI). Toll-like receptors are implicated in the inflammatory response that occurs during reperfusion. The triggering receptor expressed on myeloid cells (TREM)-1 acts as an amplifier of the immune response triggered by toll-like receptor engagement. We hypothesized that administration of a TREM-1 inhibitory peptide (LR12) could limit reperfusion injury in a porcine model of AMI. METHODS AND RESULTS: AMI was induced in 15 adult minipigs by a closed-chest coronary artery occlusion-reperfusion technique. Animals were randomized to receive LR12 or vehicle before reperfusion (LR12 n = 7, vehicle n = 8), and were monitored during 18 h. AMI altered hemodynamics and cardiac function, as illustrated by a drop of mean arterial pressure, cardiac index, cardiac power index, ejection fraction, and real-time pressure-volume loop-derived parameters. TREM-1 inhibition by LR12 significantly improved these dysfunctions (P < 0.03) and limited infarct size, as assessed by lower creatine phosphokinase and troponin I concentrations (P < 0.005). Pulmonary, renal, and hepatic impairments occurred after AMI and were attenuated by LR12 administration as assessed by a better PaO2 to FiO2 ratio, a less positive fluid balance, and lower liver enzymes levels (P < 0.05). CONCLUSION: Inhibition of the TREM-1 pathway by a synthetic peptide limited myocardial reperfusion injury in a clinically relevant porcine model of AMI.

Attenuation of responses to endotoxin by the triggering receptor expressed on myeloid cells-1 inhibitor LR12 in nonhuman primate.

BACKGROUND: The triggering receptor expressed on myeloid cells-1 is an immunoreceptor that amplifies the inflammatory response mediated by toll-like receptors engagement. Triggering receptor expressed on myeloid cells-1 inhibitory peptides such LR12 have been shown to prevent hyperresponsiveness and death in several experimental models of septic shock. METHODS: Twelve adult male Cynomolgus (Macaca fascicularis) monkeys exposed to an intravenous bolus of endotoxin (10 µg/kg) were randomized to receive LR12 or placebo (n = 6 per group) as an initial intravenous bolus followed by an 8-h continuous intravenous infusion. An additional group of four only received vehicle infusion. Vital signs were monitored for 8 h. Blood was sampled at H0, 1, 2, 4, and 8 for analysis of clinical chemistries, leukocyte count, coagulation parameters, and cytokine plasma concentration. RESULTS: LR12 showed no effect on heart rate and body temperature. By contrast to the placebo group, which experienced a 25 to 40% blood pressure decrease after endotoxin administration, LR12-treated monkeys remained normotensive. Endotoxin induced leukopenia at 2 h (mean leukocyte count, 7.62 g/l vs. 21.1 at H0), which was attenuated by LR12. LR12 also attenuated cytokine production. CONCLUSIONS: The triggering receptor expressed on myeloid cells-1 inhibitor LR12 is able to mitigate endotoxin-associated clinical and biological alterations, with no obvious side effects. This study paves the way for future phases Ia and Ib trials in humans.

Effects of a TREM-like transcript 1-derived peptide during hypodynamic septic shock in pigs.

The objective of this study was to determine the effects of a TREM (triggering receptor expressed on myeloid cells 1)-like transcript 1-derived peptide (LR12) administration during septic shock in pigs. Two hours after induction of a fecal peritonitis, anesthetized and mechanically ventilated adult male minipigs were randomized to receive LR12 (n = 6) or its vehicle alone (normal saline, n = 5). Two animals were operated and instrumented without the induction of peritonitis and served as controls (sham). Resuscitation was achieved using hydroxyethyl starch (up to 20 mL/kg) and norepinephrine infusion (up to 10 µg/kg per minute). Hemodynamic parameters were continuously recorded. Gas exchange, acid-base status, organ function, and plasma cytokines concentrations were evaluated at regular intervals until 24 h after the onset of peritonitis when animals were killed under anesthesia. Peritonitis induced profound hypotension, myocardial dysfunction, lactic acidosis, coagulation abnormalities, and multiple organ failure. These disorders were largely attenuated by LR12. In particular, cardiovascular failure was dampened as attested by a better mean arterial pressure, cardiac index, cardiac power index, and S(v)O(2), despite lower norepinephrine requirements. LR12, a TREM-like transcript 1-derived peptide, exhibits salutary properties during septic shock in adult minipigs.