Sepsis is a clinical syndrome characterized by the systemic activation of inflammatory and coagulation pathways in response to microbial infection of normally sterile parts of the body. Despite considerable advances in our understanding of sepsis pathophysiology, sepsis remains the leading cause of death in non-coronary intensive care units (ICU) with a global disease burden between 15 and 19 million cases per year (Dellinger et al., 2008). Severe sepsis, defined as sepsis associated with organ dysfunction is associated with mortality rates of 33% to 45%. The incidence of severe sepsis continues to increase by 1.5% per annum due to the aging population, a rise in the prevalence of comorbidities, and the wider use of immunosuppressive agents and invasive procedures (Angus et al., 2001). Over the past several decades, many potential treatments for sepsis have shown early promise, yet have failed to improve survival in over 100 Phase II and Phase III clinical trials (Marshall, 2014) suggesting that some fundamental knowledge is lacking in our understanding of sepsis pathophysiology. Emerging studies on cell-free DNA (cfDNA), DNA released extracellularly into the circulation, demonstrate that cfDNA is a crucial link between inflammation and coagulation . In various conditions characterized by excessive inflammatory responses or aberrant prothrombotic responses, cfDNA has been implicated in exacerbating disease pathology (Atamaniuk, Kopecky, Skoupy, Säemann, & Weichhart, 2012; Fuchs, Brill, & Wagner, 2012; Swystun, Mukherjee, & Liaw, 2011). In clinical sepsis, levels of cfDNA upon admission into the ICU have strong prognostic value in predicting mortality (Dwivedi et al., 2012; Saukkonen et al., 2008). However, it is unclear whether these increases in cfDNA are an epiphenomenon during sepsis progression, or whether cfDNA actively plays a role in sepsis pathophysiology. In this work, in vivo studies were conducted to characterize the role of cfDNA in sepsis, the effects of DNase administration, and the potential mechanism by which cfDNA is released during experimental sepsis. In addition, mortality studies were conducted to identify surrogate markers of death to promote the design of humane and ethical animal studies in conducting sepsis research. Polymicrobial sepsis was induced via a surgical procedure whereby the cecum is exteriorized, ligated and punctured twice to introduce a continuous source of microorganisms, a model termed cecal ligation and puncture (CLP). In our CLP sepsis model, levels of cfDNA increased in a time-dependent manner. These increases accompanied an early pro-inflammatory response marked by increased pro-inflammatory IL-6, a transient increase in anti-inflammatory IL-10, and elevated lung myeloperoxidase (MPO) activity. Septic mice with elevated cfDNA levels also had high bacterial loads in the lungs, blood, and peritoneal cavity fluid. Organ damage was also observed in mice following CLP surgery versus mice subjected to the non-septic sham control surgery marked by increased levels of creatinine and alanine aminotransferase (ALT) indicative of kidney and liver injury, respectively. Histological analyses further confirmed lung and kidney damage following CLP surgery. Changes in coagulation were also observed in septic mice as mice subjected to CLP had sustained increases in thrombin-antithrombin (TAT) complexes. In addition, plasma from CLP-operated mice had increased thrombin generation (i.e. increased endogenous thromin potential, increased peak thrombin, decreased time to peak, and decreased lag time) mediated by FXIIa and enhanced by platelets. Following CLP-induced sepsis, elevations in cfDNA levels accompanied pro-inflammatory and pro-coagulant responses. The effects of in vivo DNase treatment in septic mice were time-dependent. Early DNase treatment when cfDNA levels were low resulted in an exaggerated pro-inflammatory response marked by increased plasma IL-6 levels and increased lung damage. In contrast, delayed DNase treatment at time-points when cfDNA levels were elevated suppressed inflammation characterized by an increase in anti-inflammatory IL-10 and reductions in cfDNA, IL-6, lung MPO, and ALT activity. Furthermore, delayed DNase administration resulted in decreased bacterial load in the lungs, blood, and peritoneal cavity fluid. Delayed DNase treatment also resulted in blunted pro-coagulant responses as levels of TAT complexes were suppressed and thrombin generation from septic mouse plasma was normalized. Moreover, DNase treatment when cfDNA levels were elevated increased survival in CLP-operated mice by 80% and reduced lung and liver damage. These findings suggest that administration of DNase when cfDNA levels are elevated may reduce pro-inflammatory and pro-coagulant responses and that delayed DNase treatment may infer protection in the CLP model of sepsis. One mechanism by which cfDNA is released is via the formation of neutrophil extracellular traps (NETs). Upon inflammatory stimulation, some neutrophils release chromatin material and antimicrobial proteins (i.e. neutrophil elastase, MPO, and histones) in an active process termed NETosis. Although NETs ensnare bacteria and exert antimicrobial properties, NETs may also exert harmful effects on the host by activating inflammation and coagulation. While some in vitro evidence suggest that neutrophils are the main source of cfDNA released following inflammatory stimulation, others have reported that neutrophils are not the main source of circulating cfDNA following septic challenge. To determine whether NETs contribute to cfDNA released during CLP sepsis, genetically modified mice that are incapable of forming NETs, PAD4-/- mice, were used. Levels of cfDNA in PAD-/- mice were significantly lower than cfDNA levels in C57Bl/6 mice following CLP surgery, suggesting that NETs were a source of cfDNA in our model. Levels of IL-6, MPO, and bacterial load in the lungs, blood, and peritoneal cavity were significantly reduced, indicating that NETs exert pro-inflammatory effects in CLP sepsis. Thrombin generation was also suppressed in PAD4-/- mice which suggests that NETs contribute to thrombin generation following CLP sepsis. NETs contribute to increases in circulating cfDNA and may exacerbate pathology by driving pro-inflammatory and pro-coagulant responses in CLP-induced sepsis. Appreciating the implications of conducting research using animals, it is pertinent that researchers ensure the highest ethical standards and design animal studies in the most humane, yet scientifically rigorous manner. Using mortality studies, we validated the utility of physiological and phenotypic markers to assess disease severity and predict death in murine sepsis. Temperature via a rectal probe monitor and sepsis scoring systems which assess components such as orbital tightening, level of consciousness, and activity were effective surrogate markers of death. These tools offer a non-invasive assessment of disease progression which do not artificially exacerbate sepsis pathology and immediate information regarding any changes in the health status. Surrogate markers of death also provide reliable monitoring to meet increasing standards of ethical, humane animal research and a feasible and cost-efficient means to obtain vital signs in small rodents. We have proposed a scoring system which can be used for assessing disease severity, endpoint monitoring, and predicting death to obviate inhumane methods of using death as an endpoint in sepsis studies. In summary, cfDNA levels are elevated in CLP-induced sepsis and these elevations accompany pro-inflammatory and pro-coagulant responses. NETosis may be a mechanism by which cfDNA is released and NETs may drive inflammation and coagulation in CLP sepsis. Delayed DNase administration may suppress inflammation and coagulation and may be protective in polymicrobial sepsis. In future animal sepsis studies, surrogate markers of death and a sepsis scoring system can be used in place of death as an endpoint to raise the standards in conducting ethical, humane sepsis research. / Thesis / Doctor of Philosophy (PhD)

Transaldolase-Mangel (TALDO) ist ein extrem seltener, angeborener Stoffwechseldefekt, von dem weltweit nur 34 Fälle bekannt sind. Der Defekt geht auf den Verlust des Enzyms Transaldolase 1 aus dem nicht-oxidativen Pentosephosphat-Weg (nicht-oxPPW) zurück und äußert sich in einem weiten Spektrum klinischer Symptome. Die schwerwiegendsten Folgen sind Leber- und Nierenmangelfunktionen, die zum sehr frühen Tod führen können. Desweiteren leiden 15 % der Patienten an wiederkehrenden Infektionen. Neutrophile Granulozyten (Neutrophile) sind die häufigsten weißen Blutkörperchen im Menschen und essentiell für die angeborene Immunantwort gegen Infektionserreger. Ich habe hier funktionale Aspekte von TALDO-Neutrophilen untersucht. Der oxidative Pentosephosphat-Weg (oxPPW) stellt das Reduktionsäquivalent NADPH bereit, welches indirekt für die Entstehung von reactive oxygen species (ROS)-abhängigen Neutrophil Extracellular Traps (NETs) verantwortlich ist. Der Beitrag des nicht-oxPPW zur ROS-abhängigen NET-Bildung ist bislang nicht bekannt. In dieser Arbeit konnte ich für Neutrophile aus drei TALDO-Patienten eine jeweils komplett abwesende Entstehung ROS-abhängiger NETs und einen deutlich verringerten oxidativen Burst nach PMA-Stimulation zeigen. Um diese Beobachtungen in einem unabhängigen Modelsystem zu bestätigen, habe ich mit Hilfe des CRISPR-Cas9-Systems, ‚knock-out‘ Mutanten von Transaldolase 1 und dessen Partnerenzym Transketolase in der Neutrophil-ähnlichen Zelllinie PLB-985 hergestellt. Die dergestalt genetisch manipulierten Zellen waren nicht mehr zu PMA-induziertem Zelltod in der Lage. Dies ist somit der erste genetische Beweis für die Abhängigkeit des oxidativen Burst und der Bildung von NETs vom nicht-oxPPW. Diese Erkenntnis trägt zum einen zum mechanistischen Verständnis der NET-Entstehung bei und liefert zum anderen eine potentielle Erklärung für einige der bei TALDO beobachteten Symptome. Desweiteren wurden einige der metabolischen Erfordernisse für die Bildung von NETs mit Hilfe von Inhibitoren untersucht. Die erhaltenen Erkenntnisse zeigen, dass das initiale Maximum des oxidativen Bursts für NET-Bildung unerheblich ist und vielmehr die ROS-Generierung nach ca. 50 Minuten entscheidende Bedeutung für diese hat. / Transdaldolase 1-deficiency (TALDO) is a rare genetic disease with only 34 described cases globally. Transaldolase 1 is part of the non-oxidative pentose phosphate pathway (PPP) and its deficiency results in many clinical symptoms including kidney and liver failure, which can lead to early child-mortality. Some of these patients suffer from recurrent infections, for example in the respiratory tract. Neutrophils are the most abundant white blood cells and essential for the innate immune defence against bacterial and fungal pathogens. The PPP generates reduced NADPH that is crucial for the generation of superoxide by the NADPH oxidase NOX2. In turn, NOX2 is essential for neutrophil extracellular trap (NET) formation. NETs occur through the neutrophil-specific cell death netosis and consist of chromatin decorated with granular proteins. Here I report that neutrophils of three TALDO patients did not make NETs. Deletion of transaldolase 1, and its partner enzyme transketolase, in the neutrophil-like PLB-985 cell line reduced ROS generation and cell death. This confirms that transaldolase 1 is required for NET formation. We present, to the best of our knowledge, the first genetic evidence that the non-oxidative PPP is required for ROS generation and NET formation. Furthermore, some of the metabolic requirements for NET formation were assessed. The obtained data indicate that the initial peak of the oxidative burst is irrelevant for NET formation but the ROS generation after 50 minutes on the contrary has crucial significance.

Les granulocytes de faible densité (LDGs) sont un sous-type de neutrophiles mis en évidence initialement dans le sang de patients atteints de différentes maladies telles que le lupus érythémateux systémique ou le psoriasis. Cependant, des études rapportent également leur présence chez des individus sains. On connait mal à ce jour les caractéristiques des LDGs, notamment en ce qui a trait à leur profil inflammatoire. De plus, leur biogenèse demeure toujours mal connue. Mes travaux de maîtrise visaient à comparer les propriétés des LDGs à celles des neutrophiles de densité normale (NDNs). Pour ce faire, 8 chevaux atteints d’asthme équin sévère et 11 chevaux sains bien caractérisés ont été sélectionnés pour l’étude et sur lesquels des isolations de NDNs ainsi que des LDGs ont été réalisées. La morphologie des neutrophiles a ensuite été évaluée par microscopie optique. Le contenu en myéloperoxidase, un composant des granules primaires azurophiles des neutrophiles, et la présence de récepteurs du N-formylméthionine-leucyl-phénylalanine (fMLP-R) ont été évalués par cytométrie de flux et immunofluorescence, respectivement. Enfin, la capacité fonctionnelle de ces cellules à produire spontanément des pièges extracellulaires des neutrophiles (NETs) a été étudiée in vitro par microscopie confocale. Les résultats démontrent que le nombre de LDGs est augmenté dans le sang des chevaux asthmatiques lors d'exacerbation de la maladie. De plus, ces cellules présentent une morphologie différente puisqu’elles sont de taille plus petite et contiennent plus de fMLP-R que les NDNs. Le contenu en myéloperoxidase est cependant similaire dans les deux populations de neutrophiles. Enfin, les LDGs produisent plus de NETs, et sont plus sensibles aux stimuli activateurs que les NDNs. Ces caractéristiques sont similaires dans les 2 groupes de chevaux suggérant ainsi que ce sont des propriétés intrinsèques des LDGs et qu’ils représentent une population cellulaire préactivée et qui de plus, est majoritairement mature. Cette étude caractérise et compare pour la première fois les LDGs chez des animaux sains et ceux retrouvés chez des animaux atteints d’une maladie inflammatoire chronique. / Low-density granulocytes (LDGs) are a subset of neutrophils first described in the bloodstream upon pathological conditions. However, several studies also reported the presence of these cells in the blood of healthy patients. Whether LDGs characteristics, especially their enhanced pro-inflammatory profile, are specific to this subset of neutrophils and not related to disease states is unknown. Thus, we sought to compare the properties of LDGs to those of autologous normal-density neutrophils (NDNs), in both health and disease. We studied 8 horses with severe equine asthma and 11 healthy animals. Neutrophil morphology was studied using optical microscopy, and content in myeloperoxidase and N-formylmethionine-leucyl-phenylalanine receptors (fMLP-R) evaluated using flow cytometry and immunofluorescence, respectively. Confocal microscopy was used to determine their functional capacity to spontaneously release neutrophil extracellular traps (NETs) stimulating with phorbol-12-myristate-13-acetate (PMA). LDGs were smaller and contained more fMLP-R than NDNs, but myeloperoxidase content was similar in both populations of neutrophils. They also had an increased capacity to produce NETs, and were more sensitive to activation stimuli. These characteristics were similar in both healthy and diseased horses, suggesting that these are intrinsic properties of LDGs. Furthermore, these results suggest that LDGs represent a population of primed and predominantly mature cells. Our study is the first to characterize LDGs in health, and to compare their characteristics with those of animals with a naturally occurring disease.

One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.

No description available.

Type 1 diabetes (T1D) is an autoimmune disease with multifactorial aetiology that involves an attack of self-reactive cytotoxic CD8 lymphocytes on insulin-producing beta cells in the pancreas. In the T1D pathophysiology, both innate and adaptive immunity mechanisms cooperate in the development of inflammation leading to autoimmune destruction. Autoreactive T lymphocytes are the canonical destructors of the beta cells, and B cells produce autoantibodies; the innate immunity cells are considered the initiators of the pathological autoimmune reaction by promoting T and B cell activation. Here, we provide evidence of both innate and adaptive immunity cell types dysregulation in patients with T1D, and that these changes occur before the onset of the disease. The changes in T regulatory lymphocytes (Tregs) and B cell subpopulations occur already in asymptomatic T1D first-degree relatives. During the first year after the onset of the disease, there is a gradual decrease in the neutrophil numbers in the periphery, which probably infiltrate the pancreas. We have focused more closely on the innate immunity dysregulation and its contribution to T1D pathogenesis. Initially, we describe that neutrophil products called neutrophil extracellular traps (NETs) are able to induce IFNγ-producing T cells through...

Les neutrophiles peuvent synthétiser et relâcher l’angiopoïétine 1 (Ang1), un facteur de croissance cytosolique impliqué dans l’angiogenèse, capable d’induire plusieurs activités pro-inflammatoires chez les neutrophiles. Ces derniers induisent la synthèse et la relâche de neutrophil extracellular traps (NETs), des filaments d’ADN nucléaires décondensés transportant des protéines telles que l’élastase, la myélopéroxydase (MPO), la protéinase (PR3) et la calprotectine (S100A8/S100A9) qui contribuent à la réponse immunitaire innée afin de combattre les pathogènes (i.e. bactéries). Les NETs ont des effets pro-inflammatoires, pro-thrombotiques, jouant un rôle dans la dysfonction endothéliale, et ont récemment été retrouvés dans l’insuffisance cardiaque (IC) et le diabète de type 2 (T2DM). Le but de cette étude est de déterminer la synthèse et la relâche d’Ang1 chez des patients T2DM et insuffisants cardiaques avec fraction d’éjection préservée (HFpEF) (stables ou en décompensation aigue (ADHFpEF)) avec ou sans T2DM en comparant à des volontaires sains (VS) comme groupe témoin. Nos résultats démontrent qu’en absence de traitement (PBS) et avec un traitement avec LPS, les niveaux de NETs augmentent chez les patients ADHFpEF + T2DM comparé aux VS. Nous observons également que le LPS, le PMA ou A23187 augmentent la synthèse de l’Ang1 (de 150 à 250%) chez des VS et cet effet est amplifié chez les patients T2DM et IC. L’Ang1 est relâchée à 100% par les neutrophiles de tous les groupes à l’étude et ne se lient pas sur les NETs, comparé à la calprotectine. Notre étude suggère que les patients en insuffisance cardiaque aigue (ADHFpEF + T2DM) synthétisent et relâchent plus de NETs et que l’exocytose de l’Ang1 est indépendante de la synthèse et la relâche de NETs. / Neutrophils can induce the synthesis and release of angiopoietin 1 (Ang1), a cytosolic growth factor involved in angiogenesis capable of inducing several neutrophil-driven pro-inflammatory activities. Neutrophils also synthesize and release neutrophil extracellular traps (NETs) composed of decondensed nuclear DNA filaments carrying proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), proteinase 3 (PR3) and calprotectin (S100A8/S100A9) which contribute to the innate immune response to combat pathogens (e.g., bacteria). NETs are also implicated in various pathological conditions through pro-inflammatory, pro-thrombotic properties, leading to endothelial dysfunction, and have recently been found in heart failure (HF) and type 2 diabetes (T2DM). The purpose of this study is to determine the synthesis and release of Ang1 in patients with T2DM and HF with preserved ejection fraction (HFpEF) (stable or acute decompensated (ADHFpEF)) with or without T2DM, compared to healthy volunteers (HV) as control group. Our results show that in absence of agonist (PBS) and after LPS treatment, NETs levels are increased in ADHFpEF + T2DM patients compared to HV. We also observed that LPS, PMA or A23187 treatments increase the synthesis of Ang1 (from 150 to 250%) in HV and this effect is amplified in T2DM and HF patients. Ang1 is completely released (100%) by neutrophils isolated from all cohorts and does not bind to NETs compared to calprotectin. Our study suggests that severely ill patients with decompensated heart failure (ADHFpEF + T2DM) synthesize and release more NETs and that Ang1 exocytosis is independent from NETs synthesis.

Neutrophile sind die häufigsten weißen Blutkörperchen im menschlichen Blut. Sie bilden die erste Verteidigungslinie und töten eindringende Krankheitserreger ab. Neutrophile extrazelluläre Fallen (NETs) sind netzartige Strukturen, die aus dekondensiertem Chromatin bestehen und mit zytotoxischen Proteinen dekoriert sind. NETs können Mikroben in vitro und in vivo einfangen und abtöten, sind aber auch für verschiedene Krankheiten verantwortlich. Frühere Studien haben eine spezifische Gruppe von 20-50 Neutrophilenproteinen identifiziert, die an NETs gebunden sind und von denen einige eine mikrobizide Wirkung haben. Wie diese Proteine an die NETs binden, wie sie interagieren und wie die Bindung ihre antimikrobielle Aktivität beeinflusst, ist noch nicht bekannt. In dieser Dissertation habe ich die Verteilung von acht neutrophilen Proteinen und Nukleosomen auf NETs mit Hilfe der Superauflösungsmikroskopie untersucht. Es wurden drei unabhängige Techniken mit Auflösungen von mehr als 90 nm verwendet. Die Nukleosomen bildeten auf den NETs periodische Cluster mit deutlich größeren Abständen im Vergleich zum kondensierten Chromatin. Drei NET-Proteine waren ebenfalls in periodischen Clustern auf den NETs lokalisiert und zwei von ihnen waren stark mit Nukleosomen kolokalisiert. Alle anderen analysierten Proteine zeigten keine Muster der Bindung an NETs. Zusammengenommen zeigen diese Ergebnisse, dass die Bindung von Proteinen an NETs zumindest teilweise spezifisch ist und teilweise durch Wechselwirkungen mit Nukleosomen vermittelt wird. Die erfolgreiche Einführung der superauflösenden Mikroskopie für schwierige NET-Proben in Kombination mit einem vorgeschlagenen rekonstituierten NET-System eröffnet neue Möglichkeiten für das Verständnis der molekularen Mechanismen der NET-Bildung und der Protein-Protein-Interaktion bei der NET-vermittelten Abtötung. / Neutrophils are the most abundant human white blood cell in circulation. They are the first line of defense and kill invading pathogens. Neutrophil Extracellular Traps (NETs) are weblike structures composed of decondensed chromatin decorated with cytotoxic proteins. NETs can trap and kill microbes in vitro and in vivo, but also mediate several diseases. Previous studies identified a specific set of 20-50 neutrophil proteins bound to NETs, several with microbicidal activity. It remains unknown how these proteins bind to NETs, how they interact and how binding influences their anti-microbial activity. In this dissertation, I studied the distribution of eight neutrophil proteins and nucleosomes on NETs using super-resolution microscopy. Three independent techniques with resolutions larger than 90nm were used. Nucleosomes formed periodic clusters on NETs, with significantly larger spacing compared to condensed chromatin. Three NET proteins also localized in periodic clusters on NETs and two of them strongly co-localized with nucleosomes. All other proteins analyzed showed no patterns binding to NETs. Taken together, these findings demonstrate that, at least some, protein binding to NETs is specific and in part mediated by interactions with nucleosomes. The successful introduction of super-resolution microscopy to the challenging NET samples in combination with a proposed reconstituted NET system opens new possibilities to understand the molecular mechanisms behind NET formation and protein-protein interaction in NET mediated killing.