Céline Delierneux

Biological Effects of Cardiac Magnetic Resonance on Human Blood Cells

Background—Cardiac magnetic resonance (CMR) is increasingly used for the diagnosis and management of cardiac diseases. Recent studies have reported immediate post-CMR DNA double-strand breaks in T lymphocytes. We sought to evaluate CMR-induced DNA damage in lymphocytes, alterations of blood cells, and their temporal persistence. Methods and Results—In 20 prospectively enrolled healthy men (31.4±7.9 years), blood was drawn before and after (1–2 hours, 2 days, 1 month, and 1 year) unenhanced 1.5T CMR. Blood cell counts, cell death, and activation status of lymphocytes, monocytes, neutrophils, and platelets were evaluated. The first 2-hour post-CMR were characterized by a small increase of lymphocyte B and neutrophil counts and a transient drop of total lymphocytes because of a decrease in natural killer cells. Among blood cells, only neutrophils and monocytes displayed slight and transient activation. DNA double-strand breaks in lymphocytes were quantified through flow cytometric analysis of H2AX phosphorylation (&ggr;-H2AX). &ggr;-H2AX intensity in T lymphocytes did not change early after CMR but increased significantly at day 2 ⩽1 month before returning to baseline levels of 1-year post-CMR. Conclusions—Unenhanced CMR is associated with minor but significant immediate blood cell alterations or activations figuring inflammatory response, as well as DNA damage in T lymphocytes observed from day 2 until the first month but disappearing at 1-year follow-up. Although further studies are required to definitely state whether CMR can be used safely, our findings already call for caution when it comes to repeat this examination within a month.

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice

Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs.

ATP-gated P2X1 ion channels protect against endotoxemia by dampening neutrophil activation

Summary.  Background:  In sepsis, extracellular ATP, secreted by activated platelets and leukocytes, may contribute to the crosstalk between hemostasis and inflammation. Previously, we showed that, in addition to their role in platelet activation, ATP‐gated P2X1 ion channels are involved in promoting neutrophil chemotaxis.

ATP-gated P2X1 ion channels protect against endotoxemia by dampening neutrophil activation.

Summary.  Background:  In sepsis, extracellular ATP, secreted by activated platelets and leukocytes, may contribute to the crosstalk between hemostasis and inflammation. Previously, we showed that, in addition to their role in platelet activation, ATP‐gated P2X1 ion channels are involved in promoting neutrophil chemotaxis.

Tissue factor induced by epithelial-mesenchymal transition triggers a pro-coagulant state that drives metastasis of circulating tumor cells.

Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT-TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270-82. ©2016 AACR.

Overexpression of CD39 in Mouse Airways Promotes Bacteria-Induced Inflammation

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8+ T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X1 or P2X7 receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.

Purinergic control of inflammation and thrombosis: Role of P2X1 receptors

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both in vitro and in vivo. In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1−/ − mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1−/ − mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation.

Rasa3 controls megakaryocyte Rap1 activation, integrin signaling and differentiation into proplatelet.

Rasa3 is a GTPase activating protein of the GAP1 family which targets Ras and Rap1. Ubiquitous Rasa3 catalytic inactivation in mouse results in early embryonic lethality. Here, we show that Rasa3 catalytic inactivation in mouse hematopoietic cells results in a lethal syndrome characterized by severe defects during megakaryopoiesis, thrombocytopenia and a predisposition to develop preleukemia. The main objective of this study was to define the cellular and the molecular mechanisms of terminal megakaryopoiesis alterations. We found that Rasa3 catalytic inactivation altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet forming megakaryocytes. These megakaryocyte alterations were associated with an increased active Rap1 level and a constitutive integrin activation. Thus, these mice presented a severe thrombocytopenia, bleeding and anemia associated with an increased percentage of megakaryocytes in the bone marrow, bone marrow fibrosis, extramedular hematopoiesis, splenomegaly and premature death. Altogether, our results indicate that Rasa3 catalytic activity controls Rap1 activation and integrin signaling during megakaryocyte differentiation in mouse.

Dusp3 deletion in mice promotes experimental lung tumour metastasis in a macrophage dependent manner

Vaccinia-H1 Related (VHR) dual-specificity phosphatase, or DUSP3, plays an important role in cell cycle regulation and its expression is altered in several human cancers. In mouse model, DUSP3 deletion prevents neo-angiogenesis and b-FGF-induced microvessel outgrowth. Considering the importance of angiogenesis in metastasis formation, our study aimed to investigate the role of DUSP3 in tumour cell dissemination. Using a Lewis Lung carcinoma (LLC) experimental metastasis model, we observed that DUSP3-/- mice developed larger lung metastases than littermate controls. DUSP3-/- bone marrow transfer to lethally irradiated DUSP3+/+ mice was sufficient to transfer the phenotype to DUSP3+/+ mice, indicating that hematopoietic cells compartment was involved in the increased tumour cell dissemination to lung tissues. Interestingly, we found a higher percentage of tumour-promoting Ly6Cint macrophages in DUSP3-/- LLC-bearing lung homogenates that was at least partially due to a better recruitment of these cells. This was confirmed by 1) the presence of higher number of the Ly6Bhi macrophages in DUSP3-/- lung homogenates and by 2) the better migration of DUSP3-/- bone marrow sorted monocytes, peritoneal macrophages and bone marrow derived macrophages (BMDMs), compared to DUSP3+/+ monocytes, macrophages and BMDMs, in response to LLC-conditioned medium. Our study demonstrates that DUSP3 phosphatase plays a key role in metastatic growth through a mechanism involving the recruitment of macrophages towards LLC-bearing lungs.

Targeting of C-type lectin-like receptor 2 or P2Y12 for the prevention of platelet activation by immunotherapeutic CpG oligodeoxynucleotides

Essentials CpG oligodeoxynucleotide (ODN) immuotherapeutics cause undesired platelet activating effects. It is crucial to understand the mechanisms of these effects to identify protective strategies. CpG ODN‐induced platelet activation depends on C‐type lectin‐like receptor 2 (CLEC‐2) and P2Y12. Targeting CLEC‐2 or P2Y12 fully prevents CpG ODN‐induced platelet activation and thrombosis.