Amir Boufenzer

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

ABSTRACT 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 &mgr;g/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 SvO2, despite lower norepinephrine requirements. LR12, a TREM-like transcript 1–derived peptide, exhibits salutary properties during septic shock in adult minipigs.

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.

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 &mgr;g/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.

Trem-1 is not crucial in psoriasiform imiquimod-induced skin inflammation in mice.

J er emie Joffre, Estelle Hau, Lynda Zeboudj, Ludivine Laurans, Maxime Battistella, Amir Boufenzer, Ad elede Masson, H el ene Le Buanec, St ephanie Cochaud, Martine Bagot, Armand Bensussan, S ebastien Gibot, Jean-David Bouaziz and Hafid Ait-Oufella Paris Cardiovascular Research Center, INSERM U970 and Universit e Paris-Descartes, Paris, France; Medical Intensive Care Unit, CHU SaintAntoine and Universit e Pierre-et-Marie Curie, Paris, France; INSERM and UMRS 976, Laboratory of Oncodermatology, Immunology and Cutaneous Stem cells, Paris, France; University Paris Diderot, Sorbonne Paris Cit e, Paris, France; Dermatology Department, Saint-Louis Hospital, Paris, France; Pathology Department Saint-Louis Hospital and INSERM UMRS 1165, Paris, France; Inotrem SA, Nancy, France; Inserm UMR_S1116, Facult e de M edecine de Nancy, University of Lorraine, Nancy, France; Medical Intensive Care Unit, Hôpital Central, CHU Nancy, Nancy, France Correspondence: Dr Jean-David Bouaziz, Hôpital Saint Louis, INSERM U976, Equerre Bazin, 1 avenue Claude Vellefaux, 75010 Paris, France, Tel.: +33-1-53-72-20-93, Fax: +33-1-42-49-40-38, e-mail: jean-david.bouaziz@sls.aphp.fr †Have equally contributed to that work and share first authorship. ‡Have equally contributed to that work and share last authorship.

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.

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.

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.

0457 : Targeted endothelial Trem-1 deletion protects mice during septic shock

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

0270: TREM-1 is a therapeutic target during myocardial infarction

Introduction The innate immune system has recently been shown to be important in cardiac response to MI. TREM-1 is an immune-receptor expressed by neutrophils, macrophages, and mature monocytes that acts as an amplifier of the innate immune response triggered by Toll-Like Receptors (TLRs) engagement. We here aim at investigating the mechanisms by which LR12 confers protection. Material and method We used adult male mice TREM-1-/-, Rag1-/- (lacking B and T cells) and mice depleted in neutrophils with anti-Ly6G 1A8. Animals were submitted to a permanent MI and then randomized to receive LR12 (a synthetic TREM-1 inhibitory peptide), LR12 scramble or anti- TREM-1 mAb (as a TREM-1 agonist) ip beginning 1 hour after MI, then daily for 5 days. Results In different types of mice, we first observed that TREM-1 was expressed (except in TREM-1-/-) in myocardial tissue, especially in infarcted areas with an expression peaking at 24h after MI. We next observed that in TREM-1-/- and LR12 treated animals infarct size was reduced. Recruitment of inflammatory cells (neutrophils, Ly6Chimonocytes, B cells) was also reduced, while ‘anti-inflammatory’ Ly6Clow monocytes number was increased. In LR12 treated and TREM-1-/- mice we observed a reduction of leucocytes activation. Using both qRT-PCR and zymography we found that deletion or modulation of TREM-1 was associated with a decreased protease activity in infarcted areas (MMP9/TIMP1). Finally, while activation of TREM-1 through anti- TREM-1 mAb administration decreased survival, LR12 and deletion of TREM-1 conferred protection. The phenomena obtained in TREM-1-/- and LR12 treated was also observed in Rag1-/-and mice depleted in neutrophils. Conclusion TREM-1 plays an important role in mediating inflammatory cells recruitment and activation following myocardial infarction. Its therapeutic modulation achieved through LR12 administration confers protection in mice.

0267: Protective effect of TREM-1 modulation during myocardial infarction in rats

Introduction Myocardial healing after infarction is hampered by reperfusion injury and ventricular remodeling, partly mediated by detrimental inflammatory responses via Toll-like Receptors (TLRs) signaling. We hypothesized that modulation of the Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), an amplifier of the innate inflammatory response, by the use of a small synthetic peptide, namely LR-12, could improve myocardial injury in a rat model of myocardial infarction. Materials and methods Wistar rats underwent either permanent occlusion of the left coronary artery or transient ischemia for 60 minutes followed by reperfusion. They were randomly assigned to receive LR-12 or vehicle for 5 days. Cardiac function and dimensions were assessed at baseline through positron emission tomography (PET) imaging and 6 weeks after infarction by PET and conductance catheter. Results Following permanent ischemia, LR-12 treatment significantly reduced ventricular remodeling, as assessed by a lesser increase of end-diastolic volume measured by PET (273±77 vs 198±95 μL; p=0.007), and improved the systolic function: ESPVR (0.98±0.43 vs 1.45±0.49 mmHg.μL-1, p=0.04) or preload recruitable stroke work PRSW (55±23 vs 80±34 mmHg.μL-1, p=0.03). During transient ischemia, LR-12 infusion just before and for 5 days after reperfusion also improved myocardial contractility assessed by conductance catheter (PRSW: 64±25 vs 96±26 mmHg.μL-1, p=0.02; ESPVR 1.00±0.30 vs 1.61±0.69 mmHg.μL-1, p=0.05). Discussion Activation of immune response after myocardial infarction is a double-edged sword: mandatory for wound healing, but deleterious during reperfusion injury and ventricular remodeling. Inhibition of TREM-1 with LR-12 does not inhibit inflammation but modulates the amplification of the inflammatory response. Conclusion LR-12, by inhibiting TREM-1, improves ventricular remodeling and myocardial contractility in different experimental models of myocardial infarction.