Cell Type-Specific Roles of NF-κB Linking Inflammation and Thrombosis

The transcription factor NF-κB is a central mediator of inflammation with multiple links to thrombotic processes. In this review, we focus on the role of NF-κB signaling in cell types within the vasculature and the circulation that are involved in thrombo-inflammatory processes. All these cells express NF-κB, which mediates important functions in cellular interactions, cell survival and differentiation, as well as expression of cytokines, chemokines, and coagulation factors. Even platelets, as anucleated cells, contain NF-κB family members and their corresponding signaling molecules, which are involved in platelet activation, as well as secondary feedback circuits. The response of endothelial cells to inflammation and NF-κB activation is characterized by the induction of adhesion molecules promoting binding and transmigration of leukocytes, while simultaneously increasing their thrombogenic potential. Paracrine signaling from endothelial cells activates NF-κB in vascular smooth muscle cells and causes a phenotypic switch to a “synthetic” state associated with a decrease in contractile proteins. Monocytes react to inflammatory situations with enforced expression of tissue factor and after differentiation to macrophages with altered polarization. Neutrophils respond with an extension of their life span—and upon full activation they can expel their DNA thereby forming so-called neutrophil extracellular traps (NETs), which exert antibacterial functions, but also induce a strong coagulatory response. This may cause formation of microthrombi that are important for the immobilization of pathogens, a process designated as immunothrombosis. However, deregulation of the complex cellular links between inflammation and thrombosis by unrestrained NET formation or the loss of the endothelial layer due to mechanical rupture or erosion can result in rapid activation and aggregation of platelets and the manifestation of thrombo-inflammatory diseases. Sepsis is an important example of such a disorder caused by a dysregulated host response to infection finally leading to severe coagulopathies. NF-κB is critically involved in these pathophysiological processes as it induces both inflammatory and thrombotic responses

Insights in GM-CSF Differentiated Monocyte Derived Cells during Autoimmune Inflammation and Fibrin-Deposition

Granulocyte macrophage colony stimulating factor (GM-CSF) wurde anfangs als Wachstumsfaktor für myeloische Vorläuferzellen entdeckt, gilt aber heute als einer der wichtigsten Faktoren in der Pathologie von Autoimmunerkrankungen wie Multiple Sklerose (MS) und rheumatoide Arthritis (RA). Patienten, die von solchen Krankheiten betroffen sind, weisen erhöhte Konzentrationen von GM-CSF im Blutplasma auf, was die Mobilisierung von Neutrophilen sowie von inflammatorischen Monozyten fördert. Aufgrund der GM-CSF-gesteuerten Differenzierung werden Monozyten darauf vorbereitet entscheidende Merkmale zu entwickeln um die Entzündung, eine effiziente Antigenpräsentation und Gewebeschäden zu verursachen. Mit dieser Arbeit möchte ich Einblicke in GM-CSF-differenzierte inflammatorische Monozyten geben und deren Rolle in einer autoimmunen neurologischen Entzündungsreaktion beleuchten. Wie bereits gezeigt wurde, werden Reifung und Polarisierung myeloider Zellen durch die Signalachse PI3K-AKT-mTOR entscheidend reguliert. Durch die konditionierte Gen-Deletion von PTEN wurde eine hyperaktive unkontrollierte PI3K-Signalübertragung in myeloiden Zellen implementiert, wodurch Auswirkungen in verschiedene Krankheitsszenarien wie Autoimmunneuroinflammation, Lungenfibrose und Tumorimmunüberwachung beobachtet wurden. Zusätzlich zeigen wir, dass der Polarisationsphänotyp sowie die Effizienz in Antigen Präsentation von GM-CSF differenzierten Antigen-präsentierenden Zellen (APCs) durch die PI3K-regulatorischen Untereinheit p85 reguliert wird. Da das PI3K AKT-mTOR-Signal als inhibitorischer Mechanismus für die Aktivierung myeloider Zellen fungiert, konnten wir aufzeigen, dass in p85 defizienten APCs eine erhöhte Expression von inflammatorischen Zytokinen vorliegt und APCs dazu tendieren Antigenpräsentations-assoziierte Rezeptoren zu exprimieren. Der proinflammatorische Phänotyp von GM-CSF definierten APCs führte zu einer milderen Pathogenese für ein experimentelles Autoimmun-Enzephalomyelitis-Modell (EAE). Anhand dieser Publikationen, formulieren wir die Hypothese worin die PI3K-PTEN-Achse in APCs eine entscheidende Rolle einnimmt um die Zytokin-vermittelte Aktivierung von autoreaktiven Effektor-T-Zellen auszugleichen. Weiterführend untersuchten wir die GM-CSF-gesteuerte Differenzierung von Monozyten auf die Expression von extrinsischen Gerinnungsfaktoren und deren Konsequenzen für die Makrophagenpolarisation. Während der progressiven EAE wird die Integrität der Blut-Hirn-Schranke (BBB) gestört und es kommt zum Plasmaaustritt in das neuronale Gewebe des Gehirns. Folglich wird Fibrin gebildet und löst eine Entzündung aus. Da Makrophagen und differenzierte Monozyten (MdCs) signifikante Mengen an Tissue Factor (TF) exprimieren, besteht die Möglichkeit dass diese Zellen zur Fibrinbildung beitragen. Hierzu fragen wir, ob myeloider TF und andere Gerinnungsfaktoren die durch MdCs vermittelte Entzündung beeinflussen. Das GM-CSF-getriebene Priming von Monozyten verursachte einen gerinnungsfördernden Phänotyp in ausdifferenzierten MdCs. Diese Zellen zeigten eine erhöhte Kapazität zur Thrombinerzeugung und könnten daher als potente Initiatoren für die extrinsische Koagulation und die Fibrinbildung gesehen werden. Andererseits wurde festgestellt dass IFN--Signalübertragung in MdCs durch Expression von myeloischen extrinsischen Gerinnungsfaktoren signifikant erhöht wurde. Aktuelle Studien identifizierten eine IFN--vermittelte Genexpressionssignatur auf MdCs, die für die Initiierung der Entzündungsauflösung entscheidend ist. Dazu passend identifizierten wir myeloiden TF die Entzündung in akuten Lungenverletzungen durch Hemmung der IL-6-Expression einzuschränken. Wir schließen daraus, dass die myeloide Expression von extrinsischen Gerinnungsfaktoren die Fibrinablagerung fördern könnte, um Entzündungsprozesse zu unterstützen, aber zusätzlich die Polarisation von GM-CSF-differenzierten MdCs durch IFN--Induktion in Richtung eines entzündungshemmenden Zustands moduliert.Granulocyte macrophage colony stimulating factor (GM-CSF) was originally discovered as a growth factor for myeloid progenitor cells but it is now considered one of the most important mediators for the pathology of autoimmune related diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Patients affected from such diseases exhibit an increased plasma level of GM-CSF, which is understood to drive the mobilization of neutrophils and inflammatory monocytes. Due to GM-CSF driven differentiation, highly plastic monocytes are primed to develop critical features promoting inflammation, efficient antigen presentation and tissue damage. In this thesis, I aimed to provide insights into GM-CSF differentiated inflammatory monocytes in context of autoimmune neuro-inflammation. As demonstrated previously by our group and others, maturation and polarization of myeloid cells are critically regulated by the signaling axis PI3K-AKT-mTOR. Through conditional deletion of PTEN, a hyperactive uncontrolled PI3K signaling was implemented in myeloid cells and resulted in substantial effects in different disease scenarios such as autoimmune neuro-inflammation, lung fibrosis and tumor immune surveillance. Here, we demonstrate that conditional deletion of the PI3K regulatory subunit p85 in GM-CSF derived antigen presenting cells (APCs) alters their polarization phenotype as well as their potential to present antigen to autoreactive T cells. Since PI3K-AKT-mTOR signaling functions as an inhibitory regulator of myeloid cell activation, we showed that in GM-CSF derived APCs deficient of p85 show elevated expression of inflammatory cytokines and are more prone to express antigen-presentation associated receptors. Intriguingly, observed pro-inflammatory alterations in the behavior of GM-CSF derived APCs resulted in an impaired pathogenesis in an experimental autoimmune encephalomyelitis model (EAE). We propose a critical role for PI3K in APCs in order to balance cytokine mediated activation of autoreactive effector T cells. Secondly, we examined GM-CSF driven differentiation emphasizing expression of myeloid extrinsic coagulation factors and their consequences on macrophage polarization. During progressive EAE, the blood brain barrier (BBB) is disrupted and increases leakage of plasma into the neuronal tissue of the brain. Consequently, fibrin is deposited and thrives inflammation. As macrophages and monocyte derived cells (MdCs) are known to express significant levels of tissue factor (TF), these cells might contribute to fibrin deposition. Here, we addressed the question, if myeloid TF and other coagulation factors influence MdCs mediated inflammation. Interestingly, GM-CSF driven priming and differentiation of monocytes revealed a pro-coagulatory phenotype in matured MdCs. These cells exhibited highly increased capacity for thrombin generation and therefore might be considered as potent initiators for extrinsic coagulation and ultimately fibrin deposition. On the other side significantly increased expression of myeloid extrinsic coagulation factors was revealed to emphasize interferon (IFN-) signaling in MdCs. Recent studies identified a IFN- mediated gene expression signature on MdCs, which is critical for initiation of inflammation resolution. In line, we identified myeloid TF to restrict inflammation within an acute lung injury model by inhibiting Interleukin (IL) -6 expression. We conclude that myeloid expression of extrinsic coagulation factors might promote fibrin deposition to support inflammatory processes, but additionally modulates polarization of GM-CSF primed MdCs towards an anti-inflammatory state via IFN- induction.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2019(VLID)512112

Molecular Immunology / PI3K activity in dendritic cells exerts paradoxical effects during autoimmune inflammation

The peripheral activation of autoreactive T cells and subsequent central nervous system (CNS) immune cell infiltration are key events relevant for experimental autoimmune encephalomyelitis (EAE), a commonly employed multiple sclerosis (MS) model, influenced by TH1 and TH17 mediated immunity. The phosphoinositide-3-kinase (PI3K)-AKT kinase pathway modulates outcome during EAE, with direct actions of PI3K on adaptive immunity implicated in deleterious and effects on antigen presenting cells involved in beneficial responses during EAE. Here, by genetically deleting the regulatory subunit of Class Ia PI3K, p85, in selective myeloid cells, we aimed to resolve the impact of PI3K in EAE. While genetically deleting PI3K in LysM expressing cells exerted unremarkable effects, attenuating PI3K function in CD11c dendritic cells (DCs), promoted secretion of pathogenic EAE promoting cytokines, particularly skewing TH1 and TH17 immunity, while notably, improving health in EAE. Neutralizing IFN- activity using blocking antibodies revealed a prolonged TH1 response was critical for the decreased disease of these animals. Thus, PI3K-AKT signaling in DCs acts in a paradoxical manner. While attenuating EAE associated TH1 and TH17 responses, it impairs health during autoimmune inflammation.(VLID)494477