Frédéric Massin

Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock.

ABSTRACT The triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as tumor necrosis factor alpha and interleukin-1β, and improved survival. The protective effect of these peptides on arterial pressure could partly be explained by a decreased production of nitric oxide. These data suggest that in vivo modulation of TREM-1 might be a suitable therapeutic tool for the treatment of sepsis.

TREM‐1 promotes survival during septic shock in mice

Triggering receptor expressed on myeloid (TREM)‐1 is integral to the inflammatory response occurring during septic shock, although its precise function has yet to be determined. Here we show that in vivo silencing of TREM‐1 using siRNA duplexes in a fecal peritonitis mouse model resulted in a blunted inflammatory response and increased mortality. This was associated with impaired bacterial clearance related to marked inhibition of the neutrophil oxidative burst. By contrast, TREM‐1‐silenced mice were highly resistant to a lethal endotoxin challenge, while partial silencing of TREM‐1 in the bacterial peritonitis model produced a significant survival benefit. These data highlight the crucial role of the TREM‐1 pathway in mounting an adequate inflammatory and cytotoxic response to polymicrobial sepsis, and both the therapeutic promise and potential risks of its modulation.

Elevated synovial expression of triggering receptor expressed on myeloid cells 1 in patients with septic arthritis or rheumatoid arthritis

Objective: To determine whether synovial expression of triggering receptor expressed on myeloid cells 1 (TREM-1) is upregulated in patients with distinct types of inflammatory or non-inflammatory arthritis. Methods: Synovial fluid (SF) samples were analysed for levels of soluble TREM-1 (sTREM; n  =  132), tumour necrosis factor alpha (TNFα, n  =  78) and leucocyte TREM-1 messenger RNA (n  =  48). Synovial tissue from four rheumatoid arthritis (RA) patients, two patients with Crohn’s-associated arthritis, one patient with ankylosing spondylitis and one patient with osteoarthritis were examined for TREM-1 expression by immunohistology, and three of the RA samples were also analysed by Western blotting. Results: Synovial fluid sTREM-1 levels in septic arthritis and RA were similar to each other and were each greater than those in gouty arthritis, non-septic/non-RA inflammatory arthritis and non-inflammatory arthritis. Synovial fluid TNFα and sTREM-1 levels correlated with each other, and sTREM-1 and leucocyte TREM-1 mRNA levels each correlated with SF leucocyte counts. TREM-1 in RA was expressed in situ in synovial tissue by cells of myelomonocytic lineage but was not detectably expressed in control osteoarthritis synovial tissue. Conclusions: Synovial TREM-1 expression is increased in septic arthritis and RA. In patients with acute inflammatory arthritis, elevated SF sTREM-1 levels may point the clinician to a diagnosis of septic arthritis or RA. In RA patients, targeting TREM-1 may have therapeutic benefits by reducing local proinflammatory cytokine and chemokine release.

Modulation of the Triggering Receptor Expressed on Myeloid Cells–1 Pathway during Pneumonia in Rats

BACKGROUND Triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell-surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. The aim of the present study was to investigate the effect of the modulation of the TREM-1 pathway during experimental pneumonia in rats. METHODS Adult male Wistar rats were intratracheally inoculated with Pseudomonas aeruginosa (PAO1 strain) and randomly treated or not treated with an analogue synthetic peptide derived from the extracellular moiety of TREM-1 (LP17). RESULTS P. aeruginosa induced a severe pneumonia associated with signs of severe sepsis within the first 24 h. In septic rats, LP17 improved hemodynamic status, attenuated the development of lactic acidosis and hypoxemia, modulated lung and systemic inflammatory responses and coagulation activation, reduced lung histological damage, and improved survival. CONCLUSIONS The modulation of the TREM-1 pathway by the use of such synthetic peptides as LP17 appears beneficial during P. aeruginosa pneumonia in rats in attenuating lung and systemic inflammatory responses.

Growth arrest-specific protein 6 plasma concentrations during septic shock

IntroductionThe product of growth arrest-specific gene 6 (Gas6) is a vitamin K dependent protein that is secreted by leucocytes and endothelial cells in response to injury and participates in cell survival, proliferation, migration and adhesion. Our purpose was to investigate plasma Gas6 concentration and its relation to organ dysfunction in patients with septic shock.MethodsForty-five patients with septic shock admitted to a medical adult intensive care unit were enrolled. Plasma Gas6 concentration was determined using enzyme-linked immunosorbent assay at days 1, 3, 7 and 14.ResultsThe median (interquartile range) Gas6 concentration was 51 (5 to 95) pg/ml at admission. A positive correlation (Spearman rank-order coefficient [rs] = 0.37, P = 0.01) was found between Gas6 level and Sepsis-related Organ Failure Assessment score. Patients requiring renal support had higher Gas6 concentration that those without need for haemofiltration (76.5 [52 to 164] pg/ml versus 10.5 [1.5 to 80.5] pg/ml; P = 0.04). Moreover, there was a positive correlation between Gas6 and aspartate transaminase (rs = 0.42, P = 0.006) and between Gas6 and prothrombin time (rs = 0.45, P = 0.02). Although there was a progressive decline in Gas6 concentration in survivors (analysis of variance, P = 0.01), nonsurvivors exhibited persistently elevated Gas6. However, the two populations diverged only after day 7 (P = 0.04).ConclusionPlasma concentrations of Gas6 correlate with disease severity, especially with renal and hepatic dysfunction, in septic shock.

The relationship between CD4+CD25+CD127- regulatory T cells and inflammatory response and outcome during shock states.

IntroductionAlthough regulatory T lymphocytes (Tregs) have a pivotal role in preventing autoimmune diseases and limiting chronic inflammatory conditions, they may also block beneficial immune responses by preventing sterilizing immunity to certain pathogens.MethodsTo determine whether naturally occurring Treg cells have a role in inflammatory response and outcome during shock state we conducted an observational study in two adult ICUs from a university hospital. Within 12 hours of admission, peripheral whole blood was collected for the measurement of cytokines and determination of lymphocyte count. Sampling was repeated at day three, five and seven. Furthermore, an experimental septic shock was induced in adult Balb/c mice through caecal ligation and puncture.ResultsForty-three patients suffering from shock (26 septic, 17 non septic), and 7 healthy volunteers were included. The percentage of Tregs increased as early as 3 days after the onset of shock, while their absolute number remained lower than in healthy volunteers. A similar pattern of Tregs kinetics was found in infected and non infected patients. Though there was an inverse correlation between severity scores and Tregs percentage, the time course of Tregs was similar between survivors and non survivors. No relation between Tregs and cytokine concentration was found. In septic mice, although there was a rapid increase in Treg cells subset among splenocytes, antibody-induced depletion of Tregs before the onset of sepsis did not alter survival.ConclusionsThese data argue against a determinant role of Tregs in inflammatory response and outcome during shock states.

Soluble Trem-like Transcript-1 Regulates Leukocyte Activation and Controls Microbial Sepsis

The triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role during the onset of sepsis by amplifying the host immune response. The TREM-like transcript-1 (TLT-1) belongs to the TREM family, is selectively expressed on activated platelets, and is known to facilitate platelet aggregation through binding to fibrinogen. In this study, we show that a soluble form of TLT-1 is implicated in the regulation of inflammation during sepsis by dampening leukocyte activation and modulating platelet-neutrophil crosstalk. A 17-aa sequence of the TLT-1 extracellular domain (LR17) is responsible for this activity through competition with the TREM-1 ligand. Whereas early or late LR17 treatment of septic mice improves survival, treml-1−/− animals are highly susceptible to polymicrobial infection. The present findings identify platelet-derived soluble TLT-1 as a potent endogenous regulator of sepsis-associated inflammation and open new therapeutic perspectives. We anticipate soluble TLT-1 to be important in regulating leukocyte activation during other noninfectious inflammatory disorders.

Follicular Proinflammatory Cytokines and Chemokines as Markers of IVF Success

Cytokines are key modulators of the immune system and also contribute to regulation of the ovarian cycle. In this study, Bender MedSystems FlowCytomix technology was used to analyze follicular cytokines (proinflammatory: IL-1β, IL-6, IL-18, IFN-γ, IFN-α, TNF-α, IL-12, and IL-23;, and anti-inflammatory: G-CSF), chemokines (MIP-1α, MIP-1β, MCP-1, RANTES, and IL-8), and other biomarkers (sAPO-1/Fas, CD44(v6)) in 153 women undergoing in vitro fertilization (IVF). Cytokine origin was studied by mRNA analysis of granulosa cells. Higher follicular MIP-1α and CD44(v6) were found to correlate with polycystic ovary syndrome, IL-23, INF-γ, and TNF-α with endometriosis, higher CD44(v6) but lower IL-β and INF-α correlated with tubal factor infertility, and lower levels of IL-18 and CD44(v6) characterized unexplained infertility. IL-12 positively correlated with oocyte fertilization and embryo development, while increased IL-18, IL-8, and MIP-1β were associated with successful IVF-induced pregnancy.

Effects of the Trem-1 pathway modulation during mesenteric ischemia-reperfusion in rats

Objectives: The triggering receptor expressed on myeloid cells (TREM)-1, a receptor expressed on the surface of neutrophils and monocytes/macrophages, synergizes with the Toll-like receptors in amplifying the inflammatory response mediated by microbial components. Because the pathogenesis of ischemia-reperfusion-induced gastrointestinal tissue injury and multiple organ failure implies leukocyte activation and bacterial translocation, we hypothesized that the TREM-1 pathway modulation would prove beneficial in this setting. Design: Animal study. Setting: Research laboratory. Subjects: Adult male Wistar rats (250–300 g). Interventions: Rats were subjected to intestinal ischemia-reperfusion induced by occlusion of the superior mesenteric artery during 60 mins and reperfused for 180 mins. At the time of reperfusion, animals were administered with LP17 (a synthetic TREM-1 inhibitor), a control peptide, or a vehicle (normal saline). Plasma concentrations of tumor necrosis factor-&agr;, interleukin-6, and soluble TREM-1 were measured by enzyme-linked immunosorbent assay. Hepatic activation of the transcriptional factor nuclear factor-&kgr;B was assessed by electrophoretic mobility shift assay. Hepatic oxidant-antioxidant balance was estimated by measurement of lipid peroxidation and catalase activity. Ileal mucosal permeability was estimated by fluorescein dextran-4 clearance and bacterial translocation by mesenteric lymph nodes culture. Measurements and Main Results: Ischemia-reperfusion was associated with cardiovascular collapse, lactic acidosis, and systemic and hepatic inflammatory response that were partly prevented by LP17 administration. Liver lipid peroxidation and catalase depletion were attenuated by LP17. Ischemia-reperfusion induced a marked increase in ileal mucosal permeability and an associated bacterial translocation that was also prevented by TREM-1 modulation. LP17 delayed mortality. Conclusions: The modulation of the TREM-1 pathway by the means of a synthetic peptide may be useful during acute mesenteric ischemia.

Development of a new method for identification and quantification in cerebrospinal fluid of malignant cells from breast carcinoma leptomeningeal metastasis

BackgroundThe diagnosis of leptomeningeal metastasis (LM) in patients with solid tumors remains difficult. The usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI of the entire neuraxis lack both specificity and sensitivity. The Veridex CellSearch® technology has been designed for the detection of circulating tumor cells (CTC) in blood from cancer patients and validated for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer. Our aim was to adapt this technology for the detection and the enumeration of tumor cells in the CSF of breast cancer patients presenting with LM.MethodsOn the occasion of a randomized phase III study evaluating the role of the intrathecal treatment in LM from breast cancer (DEPOSEIN, EudraCT N°: 2010-023134-23), the CellSearch® technology was adapted to direct enrichment, enumeration and visualization of tumor cells in 5 mL CSF samples, collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling.ResultsSixteen CSF of 8 patients with primary breast cancer presenting with LM were studied. EpCAM+/cytokeratin + cells with typical morphology could be observed and enumerated sequentially with reproducible results in low or elevated numbers in 8 patients.ConclusionThis methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of cancer patients with LM, especially to appreciate the efficacy of chemotherapy.