{"id":{"repo_id":"sherbrooke","oai_identifier":"oai:usherbrooke.scholaris.ca:11143/21759"},"canonical_url":"https://search.dev.ndltd.org/etd/sherbrooke/oai:usherbrooke.scholaris.ca:11143/21759","repository":{"repo_id":"sherbrooke","name":"Université de Sherbrooke","base_url":"https://usherbrooke.scholaris.ca/server/oai/request"},"display":{"title":"Early and late events in NET formation","abstract":"Neutrophils are immune cells that are central to host defense against infection and injury. Among other antimicrobial responses, neutrophils are known to produce NETs (for neutrophil extracellular traps) – networks of extruded decondensed chromatin adorned with antimicrobial molecules, which primarily serve to immobilize and inactivate pathogens (bacteria, viruses, yeasts, and fungi). In addition, it has been widely reported that NETs play a role in the progression of various pathologies such as autoimmune, inflammatory and cardiovascular diseases, as well as cancer. Hence the importance of understanding the molecular mechanisms underlying NET generation. In humans, this response spans over about four hours ex vivo, with distinct early (≤ 30 min) and late (90-180 min) signaling events. However, our understanding of the various downstream cellular processes remains incomplete. In this work, we report that histone citrullination (as determined by immunoblot) is an immediate-early event in NET generation (<5 min), that is under the control of the p38, MEK, and TAK1 signaling pathways, which govern the early phase of NET generation. Conversely, chromatin decondensation is a late event (occurring at 3-3.5 h as assessed by confocal microscopy) that is controlled by all signaling pathways known to affect NET formation (p38, MEK, TAK1, Syk, and PI3K), whether they act early or belatedly. Furthermore, we demonstrate (using epifluorescence microscopy and a pharmacological antagonist) that endogenous mediators released by NETing neutrophils feed back on the cells by engaging a common receptor, RAGE, thereby driving NET induction. Other endogenous RAGE ligands similarly participate in the conditioning of adherent neutrophils to quickly form NETs (i.e. within 1h, instead of 4h). Finally, we show that regarding the late-acting PI3K signaling pathway, different PI3K isoforms are mobilized depending on the class of physiological stimuli (e.g. inflammatory cytokines and chemoattractants) employed to trigger NET generation. Collectively, our results shed light on various cellular processes driving the NET response. In particular, the key role of endogenous mediators is a novel finding that offers new therapeutic opportunities for the various pathological conditions in which NETs are involved.","abstract_html":"Neutrophils are immune cells that are central to host defense against infection and injury. Among other antimicrobial responses, neutrophils are known to produce NETs (for neutrophil extracellular traps) – networks of extruded decondensed chromatin adorned with antimicrobial molecules, which primarily serve to immobilize and inactivate pathogens (bacteria, viruses, yeasts, and fungi). In addition, it has been widely reported that NETs play a role in the progression of various pathologies such as autoimmune, inflammatory and cardiovascular diseases, as well as cancer. Hence the importance of understanding the molecular mechanisms underlying NET generation. In humans, this response spans over about four hours ex vivo, with distinct early (≤ 30 min) and late (90-180 min) signaling events. However, our understanding of the various downstream cellular processes remains incomplete. In this work, we report that histone citrullination (as determined by immunoblot) is an immediate-early event in NET generation (&lt;5 min), that is under the control of the p38, MEK, and TAK1 signaling pathways, which govern the early phase of NET generation. Conversely, chromatin decondensation is a late event (occurring at 3-3.5 h as assessed by confocal microscopy) that is controlled by all signaling pathways known to affect NET formation (p38, MEK, TAK1, Syk, and PI3K), whether they act early or belatedly. Furthermore, we demonstrate (using epifluorescence microscopy and a pharmacological antagonist) that endogenous mediators released by NETing neutrophils feed back on the cells by engaging a common receptor, RAGE, thereby driving NET induction. Other endogenous RAGE ligands similarly participate in the conditioning of adherent neutrophils to quickly form NETs (i.e. within 1h, instead of 4h). Finally, we show that regarding the late-acting PI3K signaling pathway, different PI3K isoforms are mobilized depending on the class of physiological stimuli (e.g. inflammatory cytokines and chemoattractants) employed to trigger NET generation. Collectively, our results shed light on various cellular processes driving the NET response. In particular, the key role of endogenous mediators is a novel finding that offers new therapeutic opportunities for the various pathological conditions in which NETs are involved.","abstract_has_math":false,"creators":["De Carvalho Oliveira, Vanessa"],"institution":"Université de Sherbrooke","degree_name":"M. Sc.","degree_level":"Maîtrise","degree_discipline":"Immunologie","degree_department":null,"school":null,"contributors":[],"advisors":["Amrani, Abdelaziz"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022","date_published":"2022","updated_at":"2026-07-27T21:07:45Z","subjects":["Neutrophil extracellular traps","Endogenous mediators","RAGE","PI3K isoforms","NETs","Médiateurs endogènes","Isoformes de PI3K"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/11143/21759","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Amrani, Abdelaziz"]},{"key":"dc:creator","label":"Author","values":["De Carvalho Oliveira, Vanessa"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2024-07-16T15:16:50Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2024-07-16T15:16:50Z"]},{"key":"dc:date.issued","label":"Date","values":["2022"]},{"key":"dc:publisher","label":"Institution","values":["Université de Sherbrooke"]},{"key":"dc:type","label":"Dc Type","values":["Mémoire de maîtrise"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Immunologie"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Maîtrise"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M. 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Among other antimicrobial responses, neutrophils are known to produce NETs (for neutrophil extracellular traps) – networks of extruded decondensed chromatin adorned with antimicrobial molecules, which primarily serve to immobilize and inactivate pathogens (bacteria, viruses, yeasts, and fungi). In addition, it has been widely reported that NETs play a role in the progression of various pathologies such as autoimmune, inflammatory and cardiovascular diseases, as well as cancer. Hence the importance of understanding the molecular mechanisms underlying NET generation. In humans, this response spans over about four hours ex vivo, with distinct early (≤ 30 min) and late (90-180 min) signaling events. However, our understanding of the various downstream cellular processes remains incomplete. In this work, we report that histone citrullination (as determined by immunoblot) is an immediate-early event in NET generation (<5 min), that is under the control of the p38, MEK, and TAK1 signaling pathways, which govern the early phase of NET generation. Conversely, chromatin decondensation is a late event (occurring at 3-3.5 h as assessed by confocal microscopy) that is controlled by all signaling pathways known to affect NET formation (p38, MEK, TAK1, Syk, and PI3K), whether they act early or belatedly. Furthermore, we demonstrate (using epifluorescence microscopy and a pharmacological antagonist) that endogenous mediators released by NETing neutrophils feed back on the cells by engaging a common receptor, RAGE, thereby driving NET induction. Other endogenous RAGE ligands similarly participate in the conditioning of adherent neutrophils to quickly form NETs (i.e. within 1h, instead of 4h). Finally, we show that regarding the late-acting PI3K signaling pathway, different PI3K isoforms are mobilized depending on the class of physiological stimuli (e.g. inflammatory cytokines and chemoattractants) employed to trigger NET generation. Collectively, our results shed light on various cellular processes driving the NET response. In particular, the key role of endogenous mediators is a novel finding that offers new therapeutic opportunities for the various pathological conditions in which NETs are involved.","Les neutrophiles sont des cellules immunitaires essentielles à la défense de l’hôte contre les infections et les blessures. Parmi les diverses réponses antimicrobiennes déployées par les neutrophiles figure la production de NETs (neutrophil extracellular traps), soient des réseaux extracellulaires de chromatine décondensée auxquels sont associées diverses molécules antimicrobiennes. Les NETs servent principalement à immobiliser et à inactiver les agents pathogènes (bactéries, virus, levures et champignons); il a également été rapporté que les NETs participent à la progression de diverses pathologies telles que les maladies autoimmunes, inflammatoires chroniques et cardiovasculaires, et qu’ils favorisent l’établissement de foyers secondaires dans le cas du cancer. D’où l’importance de comprendre les mécanismes moléculaires sous-tendant la génération des NETs. Chez l’humain, cette réponse requiert environ 4h ex vivo, et implique des événements de signalisation précoces (≤ 30 min) et tardifs (90-180 min). Cependant, on connaît mal les divers processus cellulaires en aval de cette signalisation. Dans ce mémoire, nous rapportons que la citrullination de certaines histones (tel que déterminé par immunobuvardage) survient de façon très précoce (<5 min) lors de la génération des NETs, et est sous le contrôle des voies de signalisation p38, MEK, et TAK1, dont on sait qu’elles régissent la phase précoce de la génération des NETs. En comparaison, la décondensation de chromatine est un événement tardif (se produisant vers 3-3.5 h, tel qu’évalué par microscopie confocale) qui est régulé par toutes les voies de signalisation qui contrôlent la formation des NETs (p38, MEK, TAK1, Syk, PI3K), peu importe si elles agissent de façon précoce ou tardive. En outre, nous démontrons (par microscopie d’epifluorescence et à l’aide d’un antagoniste pharmacologique) que les neutrophiles formant des NETs libèrent des médiateurs endogènes qui exercent une rétroaction positive en se liant à un récepteur commun, RAGE, induisant ainsi des NETs. D’autres ligands endogènes de RAGE participent au conditionnement des neutrophiles adhérents permettant la formation rapide des NETs (i.e., dans un délai de 1h, au lieu de 4h). Enfin, nous montrons que pour la voie de signalisation à action tardive PI3K, diverses isoformes de l’enzyme sont mobilisées selon la classe de stimuli physiologiques utilisés (i.e. cytokines inflammatoires ou chimioattractants) pour déclencher la génération des NETs. Collectivement, nos résultats mettent en lumière divers processus cellulaires impliqués dans la génération des NETs. En particulier, le rôle clé des médiateurs endogènes est une avancée qui offre de nouvelles possibilités thérapeutiques pour les diverses conditions pathologiques dans lesquelles les NETs sont impliquées."]},{"key":"dc:title","label":"Title","values":["Early and late events in NET formation"]}]}],"canonical_facts":{"dc:contributor.advisor":["Amrani, Abdelaziz"],"dc:creator":["De Carvalho Oliveira, Vanessa"],"dc:date.accessioned":["2024-07-16T15:16:50Z"],"dc:date.available":["2024-07-16T15:16:50Z"],"dc:date.issued":["2022"],"dc:description.abstract":["Neutrophils are immune cells that are central to host defense against infection and injury. Among other antimicrobial responses, neutrophils are known to produce NETs (for neutrophil extracellular traps) – networks of extruded decondensed chromatin adorned with antimicrobial molecules, which primarily serve to immobilize and inactivate pathogens (bacteria, viruses, yeasts, and fungi). In addition, it has been widely reported that NETs play a role in the progression of various pathologies such as autoimmune, inflammatory and cardiovascular diseases, as well as cancer. Hence the importance of understanding the molecular mechanisms underlying NET generation. In humans, this response spans over about four hours ex vivo, with distinct early (≤ 30 min) and late (90-180 min) signaling events. However, our understanding of the various downstream cellular processes remains incomplete. In this work, we report that histone citrullination (as determined by immunoblot) is an immediate-early event in NET generation (<5 min), that is under the control of the p38, MEK, and TAK1 signaling pathways, which govern the early phase of NET generation. Conversely, chromatin decondensation is a late event (occurring at 3-3.5 h as assessed by confocal microscopy) that is controlled by all signaling pathways known to affect NET formation (p38, MEK, TAK1, Syk, and PI3K), whether they act early or belatedly. Furthermore, we demonstrate (using epifluorescence microscopy and a pharmacological antagonist) that endogenous mediators released by NETing neutrophils feed back on the cells by engaging a common receptor, RAGE, thereby driving NET induction. Other endogenous RAGE ligands similarly participate in the conditioning of adherent neutrophils to quickly form NETs (i.e. within 1h, instead of 4h). Finally, we show that regarding the late-acting PI3K signaling pathway, different PI3K isoforms are mobilized depending on the class of physiological stimuli (e.g. inflammatory cytokines and chemoattractants) employed to trigger NET generation. Collectively, our results shed light on various cellular processes driving the NET response. In particular, the key role of endogenous mediators is a novel finding that offers new therapeutic opportunities for the various pathological conditions in which NETs are involved.","Les neutrophiles sont des cellules immunitaires essentielles à la défense de l’hôte contre les infections et les blessures. Parmi les diverses réponses antimicrobiennes déployées par les neutrophiles figure la production de NETs (neutrophil extracellular traps), soient des réseaux extracellulaires de chromatine décondensée auxquels sont associées diverses molécules antimicrobiennes. Les NETs servent principalement à immobiliser et à inactiver les agents pathogènes (bactéries, virus, levures et champignons); il a également été rapporté que les NETs participent à la progression de diverses pathologies telles que les maladies autoimmunes, inflammatoires chroniques et cardiovasculaires, et qu’ils favorisent l’établissement de foyers secondaires dans le cas du cancer. D’où l’importance de comprendre les mécanismes moléculaires sous-tendant la génération des NETs. Chez l’humain, cette réponse requiert environ 4h ex vivo, et implique des événements de signalisation précoces (≤ 30 min) et tardifs (90-180 min). Cependant, on connaît mal les divers processus cellulaires en aval de cette signalisation. Dans ce mémoire, nous rapportons que la citrullination de certaines histones (tel que déterminé par immunobuvardage) survient de façon très précoce (<5 min) lors de la génération des NETs, et est sous le contrôle des voies de signalisation p38, MEK, et TAK1, dont on sait qu’elles régissent la phase précoce de la génération des NETs. En comparaison, la décondensation de chromatine est un événement tardif (se produisant vers 3-3.5 h, tel qu’évalué par microscopie confocale) qui est régulé par toutes les voies de signalisation qui contrôlent la formation des NETs (p38, MEK, TAK1, Syk, PI3K), peu importe si elles agissent de façon précoce ou tardive. En outre, nous démontrons (par microscopie d’epifluorescence et à l’aide d’un antagoniste pharmacologique) que les neutrophiles formant des NETs libèrent des médiateurs endogènes qui exercent une rétroaction positive en se liant à un récepteur commun, RAGE, induisant ainsi des NETs. D’autres ligands endogènes de RAGE participent au conditionnement des neutrophiles adhérents permettant la formation rapide des NETs (i.e., dans un délai de 1h, au lieu de 4h). Enfin, nous montrons que pour la voie de signalisation à action tardive PI3K, diverses isoformes de l’enzyme sont mobilisées selon la classe de stimuli physiologiques utilisés (i.e. cytokines inflammatoires ou chimioattractants) pour déclencher la génération des NETs. Collectivement, nos résultats mettent en lumière divers processus cellulaires impliqués dans la génération des NETs. En particulier, le rôle clé des médiateurs endogènes est une avancée qui offre de nouvelles possibilités thérapeutiques pour les diverses conditions pathologiques dans lesquelles les NETs sont impliquées."],"dc:identifier.uri":["http://hdl.handle.net/11143/21759"],"dc:language.iso":["en"],"dc:publisher":["Université de Sherbrooke"],"dc:subject":["Neutrophil extracellular traps","Endogenous mediators","RAGE","PI3K isoforms","NETs","Médiateurs endogènes","Isoformes de PI3K"],"dc:title":["Early and late events in NET formation"],"dc:type":["Mémoire de maîtrise"],"thesis:degree_discipline":["Immunologie"],"thesis:degree_level":["Maîtrise"],"thesis:degree_name":["M. Sc."],"thesis:institution_name":["Faculté de médecine et des sciences de la santé"]},"updated_at":"2026-07-27T21:07:45Z"}