TREM-1 Mediates Inflammatory Injury and Cardiac Remodeling Following Myocardial InfarctionNovelty and Significance
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.
Acute myocardial ischemia induces an intense activation of the immune system leading to cytokines and chemokines production1,2 and to the recruitment of neutrophils and mononuclear cells in the infarcted area.3,4 Early proinflammatory signals are crucial in mediating the response to injury, regulating clearance of dead cardiac myocytes and initiating the cellular events necessary for wound healing. However, optimal healing requires activation of inhibitory mechanisms that suppress cytokine and chemokine synthesis and mediate resolution of the inflammatory infiltrate.5
Therefore, limiting the inflammatory response amplification seems to be important for containment of injury and optimal infarct healing. Triggering receptor expressed on myeloid cells-1 (TREM-1) is an immune-receptor expressed by neutrophils, macrophages, and mature monocytes that acts as an amplifier of the innate immune response.6 It has been shown that blockade of TREM-1 activation by short inhibitory peptides or fusion protein protected from hyper-responsiveness and death in various models of severe infections.7–12 Whether targeting the TREM-1–mediated immune response would be beneficial in acute myocardial infarction (MI) is still unknown.
In this study, we examined the role of TREM-1 in orchestrating the inflammatory response that follows MI. We show that Trem-1 genetic invalidation or pharmacological inhibition dampens myocardial inflammation, limits leukocytes recruitment, and improves heart function. Moreover, the soluble form of TREM-1 (sTREM-1) is found in the plasma of patients having an acute MI, and its concentration is an independent predictor of death.
Trem-1, Rag-1 knockout adult male C57BL/6 (6–8 weeks) and wild-type littermates, as well as adult male Wistar rats (Charles River, Lyon, France), were used. Trem-1−/− mice have recently been described in detail by Weber et al.11 Experiments were approved by our institutional Animal Care and Use Committee.
MI was induced in mice by permanent coronary ligation as described previously.10 In brief, mice were anesthetized with isoflurane (2%/2L O2/min), intubated, and ventilated with an Inspira Advanced Safety Single Animal Pressure/Volume Controlled Ventilator (Harvard Apparatus). The chest wall was shaved and a left thoracotomy was performed in the fourth left intercostal space. The left ventricle (LV) was visualized and the left coronary artery was permanently ligated with monofilament nylon 8–0 sutures (Ethicon) at the site of its emergence from under the left atrium. The chest wall was closed with 7–0 nylon sutures, and the skin was closed with 6-0 nylon sutures. A similar procedure was performed for permanent coronary artery ligation in rats. Ischemia–reperfusion model was achieved in rats through a transient (1 hour) coronary artery ligation. The sham-operated control mice underwent the same intervention except that the ligature was left untied.
TREM-1 Inhibitory Peptide
LR12 (LQEEDAGEYGCM) and LR12-scramble (which is the inactive control peptide) were chemically synthesized (Pepscan Presto BV, Lelystad, The Netherlands) as COOH terminally amidated peptides. The correct peptides were obtained with >99% yields and were homogeneous after preparative purification, as confirmed by mass spectrometry and analytic reversed-phase high–performance liquid chromatography. These peptides were free of endotoxin.
Animals were blindly randomized 2 hours after coronary ligation to receive 5 mg/kg LR12 or LR12-scramble peptides intraperitoneally once a day for 5 days.
LVs were sectioned into transverse slices from apex to base and were embedded in optimal cutting temperature compound (Tissue-Tek) for immunohistochemistry. We stained 5-μm thick sections with a rat monoclonal antibody (mAb) to myeloperoxidase, CD68, or CD163 (Serotec), or a goat polyclonal antibody to TREM-1 (catalog number AF1278, R&D Systems). We identified polymorphonuclear cells as MPO+ cells with a typical multinucleated morphology, and monocytes and macrophages as CD68+/CD163+ mononucleated cells. We detected matrix metalloproteinase 9 (MMP9) with a goat antibody to mouse MMP9 (catalog number AF911, R&D Systems). Cardiac healing after MI was assessed at day 1, 14, and 42. Hearts were excised, rinsed in phosphate-buffered saline, and frozen in liquid nitrogen. Hearts were cut by a cryostat (CM 3050S; Leica) into 7-μm thick sections. Masson’s Trichrome and Sirius Red stainings were performed for infarct size and myocardial fibrosis evaluation.
To prepare single-cell suspensions from infarcted tissues, hearts were harvested; minced with fine scissors; placed into a cocktail of collagenase I, collagenase XI, DNase I, and hyaluronidase (Sigma-Aldrich); and shaken at 37°C for 1 hour. Cells were then triturated and centrifuged (15 minutes, 500g, 4°C).
Spleens were removed, triturated in Hank's Balanced Salt Solution (HBSS) at 4°C with the end of a 3-mL syringe, and filtered through 70-μm nylon filters (BD). The cell suspension was centrifuged at 300g for 10 minutes at 4°C. Red blood cells were lysed (Red Blood Cells Lysis solution; Miltenyi), and the splenocytes were washed with HBSS and resuspended in HBSS supplemented with 0.2% (wt/vol) bovine serum albumin. Peripheral blood was drawn via cardiac puncture with citrate solution as anticoagulant, and red blood cells were lysed. Finally, bone marrow single-cell suspensions were obtained from femurs after flushing them with 1-mL HBSS, filtration through 70-μm nylon filters, and centrifugation at 300g for 10 minutes at 4°C. Total viable cell numbers were determined from aliquots using a hemacytometer with Trypan blue (BioRad).
Cell suspensions were incubated in a cocktail of mAbs against CD4+ or CD8+ T cells (CD4- or CD8-APC, CD3ε-PE, CD45-FITC), B cells (CD19-PE, CD45-FITC), granulocytes (CD11b-PB, CD45-PerCP, Ly-6G-PE), and monocyte subsets (Ly-6C-FITC, F4/80-APC), all antibodies from Miltenyi Biotech. Reported cell numbers were calculated as the product of total living cells (total viable leukocytes per mL) and percentage of cells within selected gate, and reported per mg of tissue (heart), per organ (femur and spleen), or per mL (blood). Data were acquired on FACScalibur cytometer (BD).
Protein Phosphorylation Analysis
Isolated myocardial cells were lysed in PhosphoSafe Extraction Reagent (Novagen; Merck Biosciences, Nottingham, United Kingdom) and centrifuged for 5 minutes at 16 000g at 4°C to collect the supernatant. Protein concentration was determined (BCA Protein Assay Kit, Pierce; ThermoScientific, Brebières, France). Lysates were then analyzed by Western blot (Criterion XT Bis-Tris Gel, 4%–12%; BioRad, Marnes-la-Coquette, France and PVDF membrane; Millipore, Saint-Quentin en Yvelines, France) revealed with antiphospho-p38, antipERK1/2, antipGSK3β, anti-iNOS and the corresponding secondary antibody conjugated to horseradish peroxidase (Cell Signaling; Ozyme, Saint-Quentin en Yvelines, France), and SuperSignal West Femto Substrate (Pierce; ThermoScientific). Nonphosphorylated forms or tubulin (Cell Signaling) were used for normalization. Acquisition and quantitative signal density analyses were performed by a LAS-4000 imager (FSVT, Courbevoie, France) and Multi-Gauge software (LifeScience Fujifilm, France).
Quantitative Reverse Transcriptase-Polymerase Chain Reaction
Total RNAs were extracted from myocardium (infarcted or remote areas) using RNeasy Plus Mini Kit (Qiagen, Courtaboeuf, France) and quantified with NanoDrop (ThermoScientific) before being retrotranscripted using the iScript cDNA synthesis kit (BioRad) and quantified by quantitative polymerase chain reaction (PCR) using Qiagen available probes (Quantitect Primers) for Trem-1, Il-6, Tnf-α, Timp1, Mmp9, and ActB. ActB serves as housekeeping gene. Alternatively, total RNAs were retrotranscripted with RT° First Strand Kit (SABiosciences, Tebu-bio, Le Perray-en-Yvelines, France) for PCR arrays (Mouse Innate Immune/Endothelial Cells RT° Profiler PCR Arrays; SABiosciences). All PCRs were performed in a MyiQ Thermal Cycler and quantified by iQ5 software (Qiagen). PCR array results were analyzed using PCR Array Data Analysis Software (SABiosciences) and normalized with 5 housekeeping genes.
Cytokine concentration measurements were performed using enzyme-linked immunosorbent assay (mouse Quantikine ELISA Kits; R&D Systems) and cytokine panel assays (Proteome Profiler Mouse Cytokine Array Kit; R&D Systems).
In-gel zymography and gelatinase activity assay were performed as previously described.13
Transthoracic echocardiography was performed in anesthetized mice (isoflurane inhalation) 14 and 42 days after surgery using an echocardiograph (ACUSON S3000; Siemens AG, Erlangen, Germany) with a 14 MHz linear transduced. The investigator (P.B.) was blinded to the group assignment. Two-dimensional parasternal long-axis views of the LV were obtained for guided M-mode measurements of the LV internal diameter at end diastole and end systole, interventricular septal wall thickness, and posterior thickness. Percentage fractional shortening was calculated as follows: percentage fractional shortening=[(LV diameter at end diastole–LV diameter at end systole)/LV diameter at end diastole]×100.
Fluorodeoxyglucose-Positron Emission Tomographic Imaging
After a metabolic premedication by acipimox (50 mg/kg), ≈70 MBq of 18F-fluorodeoxyglucose was injected intravenously under a short anesthesia (1.5%–2.5% of isoflurane inhalation). Sixty minutes later, a 20-minute PET recording was started under continuous anesthesia by isoflurane, using a dedicated small animal PET system (Inveon, Siemens, Knoxville, TN). The animals were connected to a standard ECG monitor and images were subsequently reconstructed in 16 cardiac intervals, providing a temporal resolution of 11 to 15 ms for common heart rate values. The axial spatial resolution was <1.5 mm. The extent of necrotic myocardium was determined as the percentage of LV segments showing <50% of fluorodeoxyglucose uptake using the dedicated QPS software on a 17-segment division of LV. LV ejection fraction, as well as end-systolic and end-diastolic volumes, was determined using the fully automatic QGS software. In these conditions, a precise determination of the actual cavity volumes is provided above the level of 100 μL corresponding to the lower limit for the LV end-systolic volume in adult rats.
Conductance Catheter Studies
Rats were anesthetized with isoflurane and a 2F high-fidelity micromanometer catheter (SPR-407; Millar Institute, Houston, TX) was inserted into the LV via the right carotid artery. The Millar catheter was connected to a Harvard Data Acquisition System interfaced with a PC with the AcqKnowledge III software (ACQ 3.2).
The population and methods of the French registry of Acute ST-segment–elevation and non–ST-segment–elevation MI (FAST-MI) have been described in detail in previous publications.14,15 Briefly, all patients aged ≥18 years were included in the registry if they had elevated serum markers of myocardial necrosis higher than twice the upper limit of normal for creatine kinase, creatine kinase-MB, or elevated troponins, and either symptoms compatible with acute MI or electrocardiographic changes on at least 2 contiguous leads with pathological Q waves or persisting ST-elevation or depression. The time from symptom onset to intensive care unit admission had to be <48 hours. Patients were managed according to usual practice; treatment was not affected by participation in the registry. Of the 374 centers in France that treated patients with acute MI at that time, 223 (60%) participated in the registry. Among these, 100 centers recruited 1015 patients who contributed to a serum bank. Each patient provided written informed consent. More than 99% of patients were white. The follow-up was collected through contacts with the patients’ physicians, the patients themselves or their family, and registry offices of their birthplace. The 2-year follow-up was >98% complete. The outcome events were assessed blinded to the results of sTREM-1 measurements. The study was reviewed by the Committee for the Protection of Human Subjects in Biomedical Research of Saint Antoine University Hospital, and the data file was declared to the Commission Nationale Informatique et Libertés. Plasma concentrations of sTREM-1 were determined in duplicate by enzyme-linked immunosorbent assay (RnD Systems).
All data, unless indicated, were normally distributed and then are presented as mean±SD, and statistical significance of differences between groups was analyzed using Student t test or Kruskal–Wallis test. Kaplan–Meier survival curves were analyzed using the log-rank test. A P value <0.05 was deemed significant.
An outcome event was defined as all-cause death or nonfatal MI during the 2-year follow-up period. The primary end point, a composite of all-cause death and nonfatal MI defined as the episode index at inclusion, was adjudicated by a committee whose members were unaware of patients’ medications, and blood measurements. Continuous variables are described as mean±SD and categorical variables as frequencies and percentages. Survival curves according to sTREM-1 tertiles are estimated using the Kaplan–Meier estimator. We used a multivariable Cox proportional hazards model to assess the independent prognostic value of variables with the primary end point during the 2-year follow-up period. The multivariable model comprised sex, age, previous or current smoking, body mass index, family history of coronary disease, history of hypertension, acute MI, heart failure, renal failure, diabetes mellitus, heart rate at admission, Killip class, left ventricular ejection fraction, hospital management (including reperfusion therapy, statins, β-blockers, clopidogrel, diuretics, digitalis, and heparin), and log C-reactive protein levels. The results are expressed as hazard ratios for Cox models with 95% confidence intervals. All statistical tests were 2-sided and performed using the SAS software version 9.1.
Trem-1 Inhibition Improves Heart Function After MI in Mice
In mice, permanent left ventricular ischemia induced Trem-1 expression in the infarcted area (Figure 1A). This expression was transient and observed mainly on infiltrating neutrophils. Trem-1 was also expressed in the human ischemic myocardium (Figure 1B).
We monitored Trem-1−/− mice and wild-type littermates by echocardiography 2 and 6 weeks after left coronary artery ligation (Figure 1C). Trem-1 genetic invalidation led to an improvement of left ventricular fractional shortening (P<0.05). Although there were no differences at day 1, infarct size was significantly reduced by day 14 in Trem-1−/− mice and this decrease was still apparent at 6 weeks (Figure 1D). Moreover, cardiac fibrosis, as well as the number of apoptotic cells (though we were not able to distinguish which cell type underwent apoptosis), was reduced in Trem-1−/− post ischemic myocardium (Figure 1E). Survival was higher in animals than in Trem-1+/+ (91% versus 64%, P=0.008; Figure 1F). Cardiac rupture was found to be the cause of death in 62.5% and 25% of wild-type and Trem-1−/− mice, respectively (P<0.02).
Trem-1 Genetic Invalidation Thus Improved Heart Function and Protected From Death After MI
To confirm these findings, we used 2 complementary approaches. TREM-1 activation can be limited by the use of an inhibitory peptide (LR12) that we previously reported to have protective effects during septic shock.8–10 On the opposite, TREM-1 can be engaged by an agonistic mAb (Trem-1 mAb).6 Although mAb administration decreased survival, LR12 treatment was associated with a reduced death rate, not different to the one observed in Trem-1−/− (Figure 1F). Moreover, LR12 administration also improved cardiac function, reduced infarct size, apoptosis, and fibrosis (Figure 1C–1E).
Trem-1 Controls Leukocyte Recruitment and Activation in Infarcted Myocardium
Because TREM-1 is an amplifier of the innate immune system, we investigated whether its modulation may regulate the inflammatory response.
Among the 168 genes involved in innate immunity we examined, the expression of 156 was altered in the myocardium after coronary artery ligation, mostly at 24 hours after MI. LR12 administration opposed to MI-induced gene activation (Online Table I). Notably, the expression of Tnf-α, Il6, and Trem-1 was reduced, and the plasma concentrations of the related proteins were decreased (Figure 2A) in the LR12-treated group. Administration of LR12 also reduced p38-MAPK and ERK1/2 phosphorylation, decreased the expression of iNOS, and increased the phosphorylation of GSK-3β (Online Figure I).
At 24 hours after MI, Mmp9 mRNA expression increased in infarcted areas (Figure 2B). LR12 treatment was associated with increased Timp-1 and reduced Mmp9 expressions.
We next examined whether the modulation of inflammation may be explained by a reduction of the number of inflammatory infiltrating cells in the infarcted heart.
Using flow cytometry, we analyzed the infiltration of leucocytes within myocardium after coronary ligation. The number of monocytes and macrophages was significantly reduced in Trem-1−/− and LR12-treated mice when compared with Trem-1+/+ (Figure 2C). Trem-1 deletion or pharmacological inhibition almost completely abrogated infiltration of infarcted myocardium by inflammatory Ly-6Chigh monocytes, whereas had few effects on Ly-6Clow recruitment (Figure 2C). Infiltration of neutrophils began early (6 hours) in control mice and was also blocked by LR12 treatment, as well as in Trem-1−/− animals (Figure 2D). This phenomenon has been previously shown in the injured lung.12
The effect of Trem-1 deletion on neutrophils’ migration was also observed in vitro: neutrophils purified from bone marrow were allowed to transmigrate through a porous filter on stimulation with plasma obtained from MI animals or ischemic heart lysates. Neutrophils from Trem-1−/− had a reduced migration compared with wild-type cells. Lipopolysaccharide-induced neutrophil activation was also decreased in the absence of Trem-1 (Online Figure II).
Although not expressed by lymphocytes, TREM-1 influenced B- and T-cell mobilization: B- and CD8+-lymphocyte infiltration was reduced, whereas CD4+-cells recruitment was unchanged in LR12-treated and knockout mice (Online Figure III).
TREM-1 modulation thus limits leukocytes recruitment to the ischemic myocardium.
Trem-1 Regulates Leukocytes Mobilization From Remote Compartments
After coronary ligation in control animals, as previously reported,16 we observed a decrease of bone marrow– and spleen-derived monocytes number at 24 hours followed by a pronounced increase of circulating monocytes number at 72 hours (Figure 3A). Interestingly, Trem-1 genetic deletion or pharmacological modulation induced an accumulation of monocytes in the spleen and in the bone marrow at 24 hours and a reduction in the blood at H72 suggesting that blocking Trem-1 reduced monocytes mobilization.
Peripheral blood neutrophil count was increased 6 hours after MI. This neutrophilia was not observed in Trem-1−/− or LR12-treated mice (Figure 3B). Number of circulating B lymphocytes increased by 72 hours after MI, whereas CD4+ and CD8+ lymphocytes count did not change significantly. Blocking Trem-1 prevented in part from this B-lymphocytes kinetics patterns (Online Figure IV).
To explain the defect of leukocytes mobilization induced by Trem-1 inhibition, we next focused on chemokines. Monocyte chemoattractant protein 1 (MCP-1 or Ccl2), Cx3cl1 (or Fractalkine), and Mcp-3 (or Ccl7) are important chemokines involved in the recruitment of, respectively, Ly-6Chigh, Ly-6Clow monocytes, and lymphocytes to inflammatory sites. Plasma and heart concentrations of Ccl-2 increased early (6 hours), whereas plasma levels of Ccl-7 and Cx3cl1 increased later (24 hours) after MI. Ccl-2 and Ccl-7 levels were reduced, whereas Cx3cl1 concentration did not change in Trem-1−/− and LR12-treated mice (Figure 3C). Ccl-2 production by myocardial neutrophils was decreased in Trem-1−/− and LR12-treated mice (Figure 3D).
After MI, neutrophils infiltrate the ischemic myocardium several hours before monocytes (Figure 2C and 2D). We therefore hypothesized that neutrophils may be responsible for Ccl-2 production that will subsequently attract inflammatory monocytes to the heart. We achieved to efficiently deplete neutrophils in mice by the administration of 1A8 antibody (Figure 4A). In neutrophil-depleted animals, plasma concentration of Ccl-2 was decreased and barely detectable in the ischemic myocardium (Figure 4b). These data suggest that neutrophils are an important cellular source of Ccl-2 in the heart, although we cannot completely exclude a release by another cell type.
Consequently, we observed that neutrophils depletion associated with a reduction of monocytes infiltration in the heart (Figure 4C). Administration of LR12- to 1A8-treated mice did not yield to additional effects on cellular infiltration (not shown), although further reduced the cardiac inflammatory response (Figure 4D).
Finally, to rule out an action of LR12 on lymphocytes, which was highly improbable because of an absence of Trem-1 expression on these cells, we performed experiments using Rag-1−/− animals. In the absence of lymphocytes, LR12 was still able to prevent myocardial neutrophil infiltration and activation (not shown).
Taken together, these data show that after MI, Trem-1 inhibition decreases neutrophil recruitment and activation, which in turn yields to a reduced monocyte mobilization from remote compartment.
Trem-1 Modulation Improves Cardiac Function After MI in Rats
To further extend functional analyses, we moved to a rat model of MI. Within 1 hour after permanent coronary artery ligation, rats were imaged by positron emission tomography with 18F-fluorodeoxyglucose to determine infarct extension and ventricular volumes and then blindly randomized to receive daily intraperitoneal injection for 5 days of LR12 or the control peptide (LR12scr). Six weeks after MI, rats were again subjected to fluorodeoxyglucose-positron emission tomographic imaging (Online Figure V) and invasive hemodynamics studies using a left ventricular conductance catheter. The 2 groups of animals were similar at baseline. Six weeks after MI, a severe left ventricular dilation had occurred in the control animals: this pathological ventricular remodeling was partly prevented by LR12 (Table). Invasive hemodynamics data were available for 12 of 18 LR12scr and 12 of 17 LR12-treated animals because of ventricular arrhythmias in the remaining rats. Main load-independent systolic and diastolic myocardial function parameters were improved by LR12 treatment (Online Figure VI). Similar results were obtained in an ischemia–reperfusion model (Online Figure VII).
TREM-1 modulation thus prevented from ventricular remodeling and systolic–diastolic dysfunction after MI in rats.
Plasma Concentration of Soluble TREM-1 in Patients With Acute Coronary Disease
TREM-1 activation may be assessed through the measurement of its soluble form (sTREM-1) in plasma. We addressed the relevance of our experimental findings to the human disease by evaluating the relationship between circulating sTREM-1 plasma concentrations and survival in a cohort of 1015 patients enrolled in the prospective, multicenter Fast-MI (NCT00673036).14,15 Of the 1015 patients enrolled, 154 patients (15%) died during the 2-year follow-up period. We found that increased concentrations of sTREM-1 at the time of admission in patients with acute MI was related to higher risk of death after 2 years of follow-up, even after adjustment for several multivariable risk factors, including age, sex, body mass index, diabetes mellitus, smoking status, hypertension, previous history of MI, stroke, chronic heart or renal failure, left ventricular ejection fraction, hospital management including reperfusion therapy, recommended drug therapies including statins, and C-reactive protein. The adjusted hazard ratio of death associated with an increase of 1 pg/mL of sTREM-1 was 2.22 (95% confidence interval, 1.69–2.93; P<0.0001), 2 0.29 (95% confidence interval, 1.71–3.06; P<0.0001) with creatine kinase added in the model, and 2.18 (95% confidence interval, 1.61–2.94; P<0.0001) with troponin I.
There were no correlations between sTREM-1 and creatine kinase (R=0.018; P=0.57) or between sTREM-1 and troponin I (R=0.012; P=0.72).
The probability of outcome events as a function of the baseline tertile levels of sTREM-1 is presented in Figure 5. After adjustment with the same variables as above, sTREM-1 plasma concentration remained an independent correlate of the risk of death at 2 years (hazard ratio adjusted at 1.65 [0.87–3.10] and 3.11 [1.72–6.64] for tertile 2 and tertile 3, respectively, compared with tertile 1, chosen as a reference, P<0.0001).
Considerable work has been performed to decipher cellular or molecular targets that may be addressed in MI clinical trials, though with disappointing results.17,18,19 Here, we used several complementary animal models, as well as patients’ samples, to demonstrate the role of TREM-1 in orchestrating the inflammatory response triggered by MI. Genetic invalidation or pharmacological blockade of Trem-1 inhibits the recruitment of inflammatory cells to the infarcted myocardium and their activation. This translates into a reduction of infarct size and an improvement of cardiac function and survival. Using a nationwide cohort of patients admitted for an acute MI, we observed that soluble TREM-1 plasma concentration (a marker of TREM-1 activation) at admission is a strong predictive factor of death during a 2-year follow-up.
A crucial determinant of infarct healing and scar formation resides into the delicate balance between the type and amount of recruited leukocytes.3 Rapidly after ischemia, neutrophils accumulate in the injured myocardium and such an extravasation, when excessive, is thought to be deleterious. Trem-1 inhibition almost completely abrogated myocardial infiltration with neutrophils. Such a phenomenon has recently been described during pneumonia in which Trem-1 inhibition blocked transepithelial migration of neutrophils.12 Shortly after neutrophils, inflammatory Ly-6Chigh monocytes are recruited that if left unchecked may cause infarct expansion and ventricular remodeling. This inflammatory subset is progressively replaced by reparative Ly-6Clow monocytes that promote inflammation resolution and extracellular matrix reorganization.2 The spleen is an important reservoir for monocytes during MI.16 We here observed that Trem-1 is important in mediating not only splenic monocytes exit but also mobilization from the bone marrow and myocardial infiltration. Moreover, although Trem-1 inhibition decreases Ccl-2 production and thus prevents from the accumulation of Ly-6Chigh cells, it does not compromise Ly-6Clow monocytes recruitment triggered by Cx3cl1. Lymphocytes are also present, though in low numbers, in the infarcted area and rapidly proliferate after MI. The exact role of lymphocyte subsets is not clear but recent evidences suggest a deleterious effect of B cells,4 whereas CD4+ T cells probably facilitate wound healing and transition from Ly-6Chigh to Ly-6Clow monocytes.20 Again, Trem-1 seems important in mediating lymphocyte recruitment, especially cytotoxic CD8+ and B cells. By contrast, Trem-1 inhibition does not alter CD4+ recruitment. Although the precise mechanisms remain to be elucidated, all these data suggest that Trem-1 plays an important role in the recruitment of leukocytes, both quantitatively and qualitatively.
Trem-1 is an amplifier of the immune response during various inflammatory diseases.6 We show that the same holds true after MI: Trem-1 inhibition dampens inflammatory gene and protein activation and decreases cytokine/chemokine production. Inhibition of MMPs reduces the risk of fatal cardiac rupture and ventricular remodeling after MI. Myeloid cells, especially neutrophils, are a major source of Mmp9 in the infarcted myocardium.13 Trem-1 inhibition, in decreasing myeloid cells infiltration, reduces Mmp9 expression and activity, whereas improves Timp1 expression. This yields to an overall MMP activity reduction in the injured myocardium and thus may prevent from remodeling.
Besides improving immediate survival, the aims of the treatment of MI are to avoid its major late-onset consequence, the development of chronic heart failure. We therefore studied the consequences of Trem-1 modulation after MI in rats using 2 complementary techniques: microTEP imaging and invasive hemodynamic study. Six weeks after the onset of a permanent myocardial ischemia, left ventricular function remains compromised and important ventricular remodeling had occurred. A short treatment (for the first 5 days) of animals with a Trem-1 inhibitor opposes to this ventricular dilation and improves systolic and diastolic ventricular functions. We also confirmed this heart function improvement during myocardial ischemia–reperfusion.
Considering that inhibition of Trem-1 only occurs during the first 5 days after MI (the LR12 half-life is short),8 we thus speculate that the initial modulation of the inflammatory response must be responsible for this late-onset ventricular function improvement.
Questions often arise about the translation of animal findings into human pathology.5 To decipher whether TREM-1 could also play a role in humans, we measured its soluble form in the plasma of patients having acute MI. Using a nationwide cohort of patients admitted for an acute MI, we observed that soluble TREM-1 plasma concentration at admission is linked to the risk of death during the 2 years of follow-up. Importantly, sTREM-1 concentration remains predictive even after adjustment for all parameters known to impact outcome including therapeutic management, such as reperfusion therapy and recommended medical drugs. TREM-1 thus also seems to be an important mediator during MI in patients.
In conclusion, our results indicate that TREM-1 has a major role in controlling inflammatory cells recruitment and activation after MI. TREM-1 modulation is able to prevent from ventricular dysfunction. Finally, TREM-1 activation is an independent outcome predictor of death in patients after an acute MI. The ability to manipulate TREM-1 activity to achieve therapeutic effects seems promising and needs to be further explored with the goal to finally reduce the epidemic development of postinfarction chronic heart failure.
Fast-MI is a registry if the French Society of Cardiology supported by unrestricted grants from Pfizer and Servier and a research grant from the French Caisse Nationale d’Assurance Maladie. We thank Ariel Cohen (Hôpital St Antoine, Paris), Karl Isaaz (CHU St Etienne, Department of Cardiology), and the physicians who cared for the patients at the participating institutions, Elodie Drouet, Salma Kotti, and the Clinical Research Assistant team of Unité de Recherche Clinique de l’Est Parisien (Assistance Publique–Hôpitaux de Paris and UPMC Paris 06), Benoît Pace, Vincent Bataille and Geneviève Mulak (French Society of Cardiology) for their assistance in designing the electronic case-record form and data management during the follow-up.
Sources of Funding
This work was supported by INSERM, l’Agence Nationale de la Recherche, and la Fondation pour la Recherche Médicale.
M. Derive and S. Gibot are cofounders of INOTREM, a company developing TREM-1 inhibitors. A. Boufenzer, M. Derive, S. Gibot, T. Simon, N. Danchin, and H. Ait-Oufella applied a patent on the measurement of plasma sTREM-1 concentration to predict outcome after acute myocardial infarction. The other authors report no conflicts.
↵In February 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.9 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.305628/-/DC1.
- Nonstandard Abbreviations and Acronyms
- left ventricle
- monoclonal antibody
- myocardial infarction
- matrix metalloproteinase 9
- soluble triggering receptor expressed on myeloid cells-1
- Received November 12, 2014.
- Revision received April 2, 2015.
- Accepted April 3, 2015.
- © 2015 American Heart Association, Inc.
- Nahrendorf M,
- Swirski FK,
- Aikawa E,
- Stangenberg L,
- Wurdinger T,
- Figueiredo JL,
- Libby P,
- Weissleder R,
- Pittet MJ.
- Swirski FK,
- Nahrendorf M.
- Gibot S,
- Kolopp-Sarda MN,
- Béné MC,
- Bollaert PE,
- Lozniewski A,
- Mory F,
- Levy B,
- Faure GC.
- Derive M,
- Bouazza Y,
- Sennoun N,
- Marchionni S,
- Quigley L,
- Washington V,
- Massin F,
- Max JP,
- Ford J,
- Alauzet C,
- Levy B,
- McVicar DW,
- Gibot S.
- Simon T,
- Verstuyft C,
- Mary-Krause M,
- Quteineh L,
- Drouet E,
- Méneveau N,
- Steg PG,
- Ferrières J,
- Danchin N,
- Becquemont L
- Lim P,
- Moutereau S,
- Simon T,
- Gallet R,
- Probst V,
- Ferrieres J,
- Gueret P,
- Danchin N.
- Swirski FK,
- Nahrendorf M,
- Etzrodt M,
- Wildgruber M,
- Cortez-Retamozo V,
- Panizzi P,
- Figueiredo JL,
- Kohler RH,
- Chudnovskiy A,
- Waterman P,
- Aikawa E,
- Mempel TR,
- Libby P,
- Weissleder R,
- Pittet MJ.
- Faxon DP,
- Gibbons RJ,
- Chronos NA,
- Gurbel PA,
- Sheehan F
- Armstrong PW,
- Granger CB,
- Adams PX,
- Hamm C,
- Holmes D Jr.,
- O’Neill WW,
- Todaro TG,
- Vahanian A,
- Van de Werf F.
- Hofmann U,
- Beyersdorf N,
- Weirather J,
- Podolskaya A,
- Bauersachs J,
- Ertl G,
- Kerkau T,
- Frantz S.
Novelty and Significance
What Is Known?
The triggering receptor expressed on myeloid cells (TREM)-1 is an amplifier of the innate immune response induced through toll-like receptors activation by microbial structures and endogenous danger signals.
Trem-1 inhibition has been shown beneficial in various experimental models of severe infections.
Myocardial infarction (MI) triggers an intense inflammatory reaction whose timely resolution is crucial to minimize cardiac dysfunction.
What New Information Does This Article Contribute?
Trem-1 inhibition and genetic invalidation improve heart function after myocardial infarction in mice and rats.
Trem-1 inhibition limits activation and recruitment of neutrophils and inflammatory monocytes to the infarct zone.
In humans, TREM-1 activation after MI is a predictor of mortality.
The innate immune response triggered by the death of cardiac cells during infarction is ambivalent as it is required for wound healing, but detrimental, if not timely restrained. To date, complete inhibition of the immune response by anti-inflammatory therapies has failed to improve prognosis after MI. Therefore, modulating the inflammatory response amplification could be a promising approach. TREM-1 is an amplifier of innate immune response, and we hypothesized that its inhibition could be beneficial in an experimental model of MI. We found that TREM-1 inhibition improved heart function after MI and decreased inflammatory cells recruitment and activation into the heart. We also found that infiltrated neutrophils released monocyte chemoattractant protein-1/Ccl-2, which in turn recruit inflammatory monocytes. Finally, TREM-1 activation is a strong predictor of death in patients after MI. These findings suggest a crucial role for TREM-1 activation after MI.