Impact de l'inflammation parodontale sur l'axe splénique cardiaque : étude des Neutrophils Extracellular Traps (NETs) et des microvésicules spléniques comme médiateurs des réponses procoagulantes et inflammatoires

P.gingivalis (PG) induit la parodontite et est associée au risque cardiovasculaire. Les microvésicules (MV) sont des effecteurs pro-inflammatoires et coagulants libérés des membranes plasmiques. L’effet à distance de PG sur le cœur et la rate de souris est détecté par immuno-marquage, mesure de ROS, et émission de MVs splénocytaires (SMVs). Les lésions gingivales par fils (LIG) imbibés ou non de PG (LIG-PG) sont comparées au gavage (gvg) et à l'injection intrapéritonéale (IP). LIG et LIG-PG réduisent les granulocytes, doublent les monocytes et les neutrophiles spléniques, sans réponse adaptative. IP triple les monocytes, multiplie par 10 les neutrophiles, réduit par 2 lymphocytes T ou B. L'inhibiteur de PAD-4 réduit les ROS, suggérant une Nétose. In vitro, les SMVs de LIG-PG, IP et gvg induisent la sénescence endothéliale. Dans le cœur, VCAM, Gasdermin-D, IL-6 et TNF- inflammatoires sont surexprimés pour IP et LIG-PG.En conclusion, PG induit l’activation splénique et cardiaque à distance, l'émission de SMVs pro-coagulantes et -sénescentes favorisant le risque cardiovasculaire.Periodontitis, mainly induced by P.gingivalis (PG) is associated with cardiovascular risk. Microvesicles (MVs) are pro-inflammatory and -coagulant effectors shed from plasma membranes. We evaluated the remote inflammatory impact of PG on heart and spleen tissue by immunostaining, ROS formation, and on endothelial senescence. In mice, ligature and/or PG gum injury by threads (LIG) soaked or not with PG (LP,LP 4w) were compared to oral gavage (gvg) and intraperitoneal injection (IP). LIG and LIG-PG reduced spleen granulocytes, doubled monocytes and neutrophils with no adaptative responses in 2-4 weeks. IP tripled monocytes with 10 folds neutrophil rise and half T or B lymphocytes. PAD-4 inhibitor decreased ROS , pointing at Netosis contribution. Splenocytes MVs (SMVs) raised in IP. In vitro, LIG-PG, IP and gvg SMVs induced coronary endothelial senescence. In heart extracts, pro-inflammatory VCAM, Gasdermin-D, IL-6, and TNF- were only up-regulated in IP and LIG-PG. Anti-inflammatory IL-10 raised in IP.Altogether, PG induces remote spleen and heart activation, the shedding of pro-coagulant and -senescent SMVs possibly favoring cardiovascular risk

Impact de l'inflammation parodontale sur l'axe splénique cardiaque : étude des Neutrophils Extracellular Traps (NETs) et des microvésicules spléniques comme médiateurs des réponses procoagulantes et inflammatoires

P.gingivalis (PG) induit la parodontite et est associée au risque cardiovasculaire. Les microvésicules (MV) sont des effecteurs pro-inflammatoires et coagulants libérés des membranes plasmiques. L’effet à distance de PG sur le cœur et la rate de souris est détecté par immuno-marquage, mesure de ROS, et émission de MVs splénocytaires (SMVs). Les lésions gingivales par fils (LIG) imbibés ou non de PG (LIG-PG) sont comparées au gavage (gvg) et à l'injection intrapéritonéale (IP). LIG et LIG-PG réduisent les granulocytes, doublent les monocytes et les neutrophiles spléniques, sans réponse adaptative. IP triple les monocytes, multiplie par 10 les neutrophiles, réduit par 2 lymphocytes T ou B. L'inhibiteur de PAD-4 réduit les ROS, suggérant une Nétose. In vitro, les SMVs de LIG-PG, IP et gvg induisent la sénescence endothéliale. Dans le cœur, VCAM, Gasdermin-D, IL-6 et TNF- inflammatoires sont surexprimés pour IP et LIG-PG.En conclusion, PG induit l’activation splénique et cardiaque à distance, l'émission de SMVs pro-coagulantes et -sénescentes favorisant le risque cardiovasculaire.Periodontitis, mainly induced by P.gingivalis (PG) is associated with cardiovascular risk. Microvesicles (MVs) are pro-inflammatory and -coagulant effectors shed from plasma membranes. We evaluated the remote inflammatory impact of PG on heart and spleen tissue by immunostaining, ROS formation, and on endothelial senescence. In mice, ligature and/or PG gum injury by threads (LIG) soaked or not with PG (LP,LP 4w) were compared to oral gavage (gvg) and intraperitoneal injection (IP). LIG and LIG-PG reduced spleen granulocytes, doubled monocytes and neutrophils with no adaptative responses in 2-4 weeks. IP tripled monocytes with 10 folds neutrophil rise and half T or B lymphocytes. PAD-4 inhibitor decreased ROS , pointing at Netosis contribution. Splenocytes MVs (SMVs) raised in IP. In vitro, LIG-PG, IP and gvg SMVs induced coronary endothelial senescence. In heart extracts, pro-inflammatory VCAM, Gasdermin-D, IL-6, and TNF- were only up-regulated in IP and LIG-PG. Anti-inflammatory IL-10 raised in IP.Altogether, PG induces remote spleen and heart activation, the shedding of pro-coagulant and -senescent SMVs possibly favoring cardiovascular risk

Evaluation of EV Storage Buffer for Efficient Preservation of Engineered Extracellular Vesicles

Extracellular vesicles (EVs), detectable in all bodily fluids, mediate intercellular communication by transporting molecules between cells. The capacity of EVs to transport molecules between distant organs has drawn interest for clinical applications in diagnostics and therapeutics. Although EVs hold potential for nucleic-acid-based and other molecular therapeutics, the lack of standardized technologies, including isolation, characterization, and storage, leaves many challenges for clinical applications, potentially resulting in misinterpretation of crucial findings. Previously, several groups demonstrated the problems of commonly used storage methods that distort EV integrity. This work aims to evaluate the process to optimize the storage conditions of EVs and then characterize them according to the experimental conditions and the models used previously. Our study reports a highly efficient EV storage condition, focusing on EV capacity to protect their molecular cargo from biological, chemical, and mechanical damage. Compared with commonly used EV storage conditions, our EV storage buffer leads to less size and particle number variation at both 4 °C and −80 °C, enhancing the ability to protect EVs while maintaining targeting functionality

Porphyromonas gingivalis triggers the shedding of inflammatory endothelial microvesicles that act as autocrine effectors of endothelial dysfunction

International audienceA link between periodontitis and atherothrombosis has been highlighted. The aim of this study was to determine the influence of Porphyromonas gingivalis on endothelial microvesicles (EMVPg) shedding and their contribution to endothelial inflammation. Endothelial cells (EC) were infected with P. gingivalis (MOI = 100) for 24 h. EMVPg were isolated and their concentration was evaluated by prothrombinase assay. EMVPg were significantly increased in comparison with EMVCtrl shedded by unstimulated cells. While EMVCtrl from untreated EC had no effect, whereas, the proportion of apoptotic EC was increased by 30 nM EMVPg and viability was decreased down to 25%, a value elicited by P. gingivalis alone. Moreover, high concentration of EMVPg (30 nM) induced a pro-inflammatory and pro-oxidative cell response including up-regulation of TNF-α, IL-6 and IL-8 as well as an altered expression of iNOS and eNOS at both mRNA and protein level. An increase of VCAM-1 and ICAM-1 mRNA expression (4.5 folds and 3 folds respectively (p < 0.05 vs untreated) was also observed after EMVPg (30 nM) stimulation whereas P. gingivalis infection was less effective, suggesting a specific triggering by EMVPg. Kinasome analysis demonstrated the specific effect induced by EMVPg on main pro-inflammatory pathways including JNK/AKT and STAT. EMVPg are effective pro-inflammatory effectors that may have detrimental effect on vascular homeostasis and should be considered as potential autocrine and paracrine effectors involved in the link between periodontitis and atherothrombosis

Ageing enhances the shedding of splenocyte microvesicles with endothelial pro-senescent effect that is prevented by a short-term intake of omega-3 PUFA EPA:DHA 6:1

International audienceAgeing enhances the shedding of splenocyte microvesicles with endothelial pro-senescent effect that is prevented by a short-term intake of omega-3 PUFA EPA:DHA 6:1