PURPOSE: In the mouse, the macrophage adhesion G protein-coupled receptor (ad-GPCR) molecule, F4/80, is required for the development of regulatory T cells in two models of tolerance, the eye and gut. Since F4/80 is not expressed in humans, the purpose of this research is to determine the human analog of F4/80. F4/80 belongs to a novel family of Epidermal growth factor-seven transmembrane (EGF-TM7) molecules, which include the EGF module-containing mucin-like hormone receptor (EMR) molecules. In the human, EMR1 has sequential homology with F4/80 and EMR2 has shown immune suppressing function in tumor cells. Thus, we investigate the possible suppressor role of the EMR family in human ocular tolerance. METHODS: Human peripheral blood mononuclear cells (huPBMC) were treated with porcine TGFβ2 and LPS or an antigenic stimulant for at least six hours to generate tolerogenic antigen presenting cells (APC). Cells were characterized by flow cytometric analysis for expression of CD14, CD40, PDL1, ILT3, and EMR2. Later, T regulatory cells were generated by incubating tolerogenic APCs with autologous huPBMC for five to seven days. Post culture, the T cells were stained and characterized for expression of CD4, CD25, and FoxP3. RESULTS: Post treatment of huPBMC with TGFβ2 and antigen, the resulting tolerogenic APCs expressed PDL1, ILT3, and EMR2. CD40 remained unchanged and CD14 was constitutively expressed. Post five to seven day culture, tolerogenic APCs treated with TGFβ2 increased the CD4+ CD25+ FoxP3+ lymphocyte populations. CONCLUSIONS: The upregulation of EMR2 on human tolerogenic APCs suggests that EMR2 may have a role in inducing tolerance in humans. Much like its mouse counterpart, F4/80, EMR2 is an adhesion molecule that may facilitate the induction of naïve T lymphocytes to regulatory T lymphocytes. Once the F4/80 analog is established for humans, novel therapies may be developed to interfere or encourage signaling in the treatment of tumors or immune inflammatory diseases, respectively.

MiRNAs control skin homeostasis through post-transcriptional gene repression by binding to their target mRNAs. However, their role in regulation of apoptosis and hair loss in alopecia areata (AA) is largely unknown, which became the aim of this study. In AA mouse model (C3H/HeJ), global miRNA profiling revealed 22 miRNAs with significant changes in their expression in AA affected skin. Amongst these miRNAs, miR-486-5p was dramatically decreased in alopecic skin in both humans and mice, in striking contrast to its prominent expression in the hair follicle (HF) epithelium of healthy anagen skin. Moreover, the expression of both pri-miR-486 and miR-486 is down-regulated in the human anagen HFs and keratinocytes treated with IFN-g, one of the key factors contributing to the immune privilege (IP) collapse in HFs. Intradermal delivery of miR-486-5p mimic into mouse skin affected by AA prevented premature entrance of HFs into catagen phase and reduced the numbers of CD4+ and CD8+ lymphocytes in the peri- and intra-follicular skin compartments. Consistently, subcutaneous administration of miR-486-5p inhibitor delayed anagen progression associated with a higher number of intrafollicular NKG2D+ cells in C3H/HeJ mice. Silencing of miR-486-5p in human anagen HFs ex vivo caused premature catagen development and led to suppression of IP by up-regulating HLA class 1, IRF1, ICAM1 and CADM1 expression of which CADM1 was confirmed to be a direct target of miR-486- 5p. Transcriptome profiling of primary human epidermal keratinocytes overexpressing miR-486-5p revealed damping the signalling pathways associated with inflammatory chemokines, cytokines and interleukins. Taken together, these data suggest that miR-486-5p plays a protective role in the pathogenesis of AA by maintaining anagen phase and preventing the IP collapse. / National Alopecia Areata Foundation

Bei der vorliegenden Arbeit handelt es sich um experimentelle Untersuchungen zu funktionellen Aspekten der neurovaskulären Einheit im Tiermodell. Mittels Licht,- Fluoreszenz- und der Elektronenmikroskopie sowie diverser immunhistochemischer Nachweisverfahren konnten wir verschiedene Populationen der neurovaskulären Einheit näher charakterisieren. So konnten wir nachweisen, dass im Hirnparenchym eine Population CD11c-positiver, dendritischer Zellen existiert, welche im gesunden Gehirn hauptsächlich an Prädilektionsstellen für Entmarkungsherde im Rahmen der Multiplen Sklerose vorkommt. Weiterhin zeigten wir im Tiermodell, dass die über Diphterietoxin vermittelte Oligodendrozytendepletion mit einer Demyelinisierung der Axone im Gehirn einhergeht, wobei die Freisetzung und Drainage der Antigene in zervikale Lymphknoten keine gegen das Gehirn gerichtete Autoimmunität auslöst. Ebenso untersuchten wir den Beitrag endothelialer Tight junctions zur Bluthirnschrankenstörung im Modell der fokalen Ischämie an der Ratte. Hierbei waren wir in der Lage nachzuweisen, dass entgegengesetzt zur herrschenden Lehrmeinung diese nicht verantwortlich für die erhöhte Gefäßpermeabilität im Rahmen des Schlaganfalls im Tiermodell zu sein scheint. Vielmehr konnten wir mit Hilfe der Elektronenmikroskopie einen neuen Mechanismus aufzeigen. Diese Ergebnisse liefern neue Erkenntnisse bezüglich der Interaktion der verschiedenen Populationen der neurovaskulären Einheit und können somit zur Entwicklung neuer Modelle verschiedener Pathologien des Zentralnervensystems beitragen.

Bei der vorliegenden Arbeit handelt es sich um experimentelle Untersuchungen zu funktionellen Aspekten der neurovaskulären Einheit im Tiermodell. Mittels Licht,- Fluoreszenz- und der Elektronenmikroskopie sowie diverser immunhistochemischer Nachweisverfahren konnten wir verschiedene Populationen der neurovaskulären Einheit näher charakterisieren. So konnten wir nachweisen, dass im Hirnparenchym eine Population CD11c-positiver, dendritischer Zellen existiert, welche im gesunden Gehirn hauptsächlich an Prädilektionsstellen für Entmarkungsherde im Rahmen der Multiplen Sklerose vorkommt. Weiterhin zeigten wir im Tiermodell, dass die über Diphterietoxin vermittelte Oligodendrozytendepletion mit einer Demyelinisierung der Axone im Gehirn einhergeht, wobei die Freisetzung und Drainage der Antigene in zervikale Lymphknoten keine gegen das Gehirn gerichtete Autoimmunität auslöst. Ebenso untersuchten wir den Beitrag endothelialer Tight junctions zur Bluthirnschrankenstörung im Modell der fokalen Ischämie an der Ratte. Hierbei waren wir in der Lage nachzuweisen, dass entgegengesetzt zur herrschenden Lehrmeinung diese nicht verantwortlich für die erhöhte Gefäßpermeabilität im Rahmen des Schlaganfalls im Tiermodell zu sein scheint. Vielmehr konnten wir mit Hilfe der Elektronenmikroskopie einen neuen Mechanismus aufzeigen. Diese Ergebnisse liefern neue Erkenntnisse bezüglich der Interaktion der verschiedenen Populationen der neurovaskulären Einheit und können somit zur Entwicklung neuer Modelle verschiedener Pathologien des Zentralnervensystems beitragen.

Mouse embryonic stem cells (ESCs) are capable of differentiating into any somatic cell type and are known to display fragile immune privilege in vivo and in vitro. The extent to which the depletion of essential amino acids (EAAs) by ESCs contributes to this phenomenon was investigated. ESCs were found to express various enzymes capable of catabolising EAAs within the culture medium. In particular, depletion of threonine, valine and lysine was found to have significant impact on T cell proliferation and differentiation, biasing their polarisation towards a FoxP3⁺ T regulatory (T_reg) phenotype. Supplementing ESC conditioned medium with these three EAAs alone rescued normal T cell proliferation, whereas artificially limiting their availability was sufficient to induce T_reg cell differentiation to a level equivalent to general EAA depletion. The pattern of EAA catabolism by mouse ESC was shared by induced pluripotent stem cells, while mouse melanoma cell lines and human ESCs displayed distinct patterns of EAA depletion. The cytosolic branched chain aminotransferase enzyme, Bcat1, catalyses the first step of branched chain amino acid catabolism (isoleucine, leucine and valine), and is highly expressed by both mouse and human ESCs. The contribution of this enzyme to the establishment of acquired immune privilege by ESC-derived tissues was, therefore, investigated. ESC lines were derived from mice lacking Bcat1 activity and were characterised. Bcat1^−/− ESC lines displayed no difference to their wildtype counterparts (Bcat1^LoxP) in terms of in vitro proliferation and their capacity to form teratomas in vivo. Furthermore, the loss of Bcat1 function had little impact on the inhibition of T cell proliferation in culture, ability to induce T_reg cell commitment or their ability to prevent rejection by T cell receptor transgenic recipients, suggesting the minimal contribution of Bcat1 to the depletion of EAAs by ESCs. In conclusion, EAA depletion by mouse ESC may provide a mechanistic explanation for the previously described immune-suppressive capacity of ESC.

Myelin-assoziierte Epitope können Ziel destruktiver T-Zell Antworten während autoimmuner Erkrankungen wie der Multiplen Sklerose oder der experimentellen autoimmunen Enzephalomyelitis werden. Dagegen reagieren selbstspezifische T-Zellen nach axonaler Degeneration nicht mit destruktiver Autoimmunität, obwohl die entsprechenden Epitope durch Myelin-phagozytierende Mikroglia präsentiert werden. Im Modell der entorhinalen Kortexläsion von Ratte und Mause zeigten wir, daß Autoimmunität nach solchen Läsionen durch die Expression des Todesliganden CD95L (FasL, Apo1L) auf Astrozyten verhindert wird, da hochaktivierte T-Zellen durch CD95L apoptotisch eliminiert werden. Myelin-phagozytierende Mikroglia reguliert MHC-II und B7-2 hoch, nicht aber das kostimulatorische B7-1 Moleküle, das mit Autoimmunität im Gehirn assoziiert ist. In Zonen retrograder Degeneration, wo Axone am Sproutingprozess beteiligt sind, zeigen Mikrogliazellen bis mindestens 90 Tage nach Läsion einen MHC-II und B7-2 positiven Immunphänotyp. Trotz Anwesenheit von CD4/B7-2 positiven a/b T-Zellen, behält Mikroglia ihre ramifizierte, ruhende Morphologie. Im Gegensatz zu autoimmunen Erkrankungen im Gehirn, erfolgt die Antigenpräsentation nach axonaler Läsion durch Mikroglia also nicht über das B7-1 Molekül. Dies kann der Grund für das Ausbleiben destruktiver Autoimmunität nach axonaler Schädigung sein. / Myelin-associated epitopes are targets of destructive T cell responses during autoimmune diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). On the other hand, autoimmune T cells do not respond in a destructive way to mechanically-induced axonal degeneration despite myelin phagocytosis and presentation by local microglia. Using entorhinal cortex lesion, a model of axonal degeneration and reactive sprouting, we showed that autoimmunity in the brain is prevented by the expression of the death Ligand CD95L expressed on astrocytes lading to apoptosis of highly activated T cells. Moreover, myelin phagocytosing microglia upregulate MHC-II and B7-2, but lack expression of B7-1, a costimulatory molecule related to destructive immunity. In zones of retrograde axonal degeneration, where axons undergo secondary damage and later contribute to the sprouting response, MHC-II/B7-2 positive microglia are still found at 90 days post lesion. These cells exhibit the ramified morphology of resting microglia in the presence of CD4/B7-2 positive a/b T cells. Thus, in contrast to autoimmune brain disease, axonal degeneration is lacking a signal to induce B7-1 on microglial cells and the recruited T cells do not induce microglial activation. Differences in B7-phenotype of local antigen-presenting cells might provide an explanation for the important finding that autoimmune T cells elicit protective rather than destructive effects following axonal degeneration in the CNS.

The mammalian pregnancy comprises a challenge to the maternal immune system since the fetus is semi-allogeneic and could thus be rejected. Pregnancy success is associated with the placenta that is not only essential for oxygen supply, nourishment and pregnancy hormones but also plays a role in the protection of the fetus against maternal immunologic attack. The aim of the current studies was to elucidate the role of human placenta as an immunomodulatory organ with a special focus on placental exosomes as vehicles for establishment of maternal tolerance to the fetus. We discovered that the syncytiotrophoblast in human normal pregnancy constitutively produces and secretes exosomes. Exosomes are 30-100 nanometer-sized membrane vesicles of endosomal origin that convey intercellular communication. Exosomes are produced and released through the endosomal compartment and reflect the type and the activation state of the cells that produce and secrete them. They carry cytosolic and membrane-bound proteins and nucleic acids and can influence and re-program recipient cells. Depending on their interactions with cells of the immune system they can be divided into immunostimulatory or immunosuppressive. We developed methods for isolation and culture of trophoblast and placental explants from human normal first trimester pregnancy and isolated exosomes from the culture supernatants.  These exosomes were characterized biochemically and functionally regarding mechanisms with potential importance in the establishment of maternal tolerance towards the fetus. The following aspects were studied: 1) exosomal modulation of the NKG2D receptor-ligand system, a major cytotoxic pathway for NK- and cytotoxic T cells and thus potentially dangerous to the fetus; 2) placental exosome-mediated apoptosis of activated immune effector cells; and 3) Foxp3-expressing T regulatory cells in human pregnant uterine mucosa, the decidua. Using immuno electron microscopy we show that human early syncytiotrophoblast constitutively expresses the stress-inducible NKG2D ligands MICA/B and ULBP1-5, and the apoptosis inducing molecules FasL and TRAIL. While MICA/B were expressed both on the cell surface and intracellularly on the limiting membrane of multivesicular bodies (MVB) and on exosomes, the ULBP1-5, FasL and TRAIL  were solely  processed through the MVB of the endosomal compartment and secreted on exosomes. The NKG2D ligand-expressing placental exosomes were able to internalize the cognate receptor from the cell surface of activated NK- and T cells thus down regulating their cytotoxic function. In our studies of apoptosis we found that placental exosomes carry the proapoptotic ligands FasL and TRAIL in their active form as a hexameric complex of two homotrimeric molecules, required for triggering of the apoptotic signaling pathways. This finding was supported by the ability of isolated placental FasL/TRAIL expressing exosomes to induce apoptosis in activated peripheral blood mononuclear cells (PBMC) and Jurkat T cells. Additionally, we studied Foxp3-expressing T regulatory (Treg) cells in paired human decidual and blood samples from pregnant women compared to non-pregnant controls. The CD4+CD25+Foxp3+ Treg cells were 10 fold enriched in the decidual mucosa compared to peripheral blood of pregnant women and non-pregnant controls. We discovered a pool of Foxp3-expressing, CD4+CD25- cells in human decidua, a phenotype consistent with naïve/precursor Foxp3+ Treg cells. These results suggest local enrichment of Treg cells in decidua of normal pregnancy. Furthermore, we have results indicating that the exosomes, isolated from placental explant cultures, carry PD-L1 and TGFβ on their surface, molecules known to promote induction of Treg cells. Taken together, our results provide evidence that placental exosomes are immunosuppressive and underline their role in the maternal immune modulation during pregnancy. The constitutive production and secretion of immunosuppressive placental exosomes create a protective exosomal gradient in the blood surrounding the feto-placental unit. This “cloud of immunosuppressive exosomes” conveys immunologic privilege to the developing fetus and thus contributes to the solution of the immunological challenge of mammalian pregnancy.

Die vorliegende Arbeit befaßt sich mit T-Helferzellen und ihren Interaktionen mit Gewebszellen, wie sie im gesunden Organismus und in Autoimmunerkrankungen auftreten. Es werden Fragen der Toleranzinduktion durch orale Gabe von Antigenen, speziell der oralen Verabreichung von Collagen II bei Patienten mit rheumatoider Arthritis diskutiert. Eine Immundeviation als Mittel, inflammatorische Th1-Zellantworten in anti-inflammatorische Th2-Zellantworten zu verwandeln, kann durch Eingriffe in die T-Zell-Signaltransduktion erreicht werden. Es werden neue Ansätze zu Mechanismen diskutiert, die das Immunprivileg des Zentralnervensystems gewährleisten. Die hirnresidenten Immunzellen, zu denen Mikrogliazellen und Astrozyten zählen, besitzen Eigenschaften, die eine Entzündung unwahrscheinlich machen. Sie müssen aktiviert werden, um Antigene präsentieren zu können. In organtypischen entorhinal-hippocampalen Schnittkulturen konnte gezeigt werden, dass Mikrogliazellen durch Th1-Zellen aktiviert, von Th2-Zellen hingegen deaktiviert werden. Die Möglichkeit, dass die Costimulation über CD80 oder CD86 differentielle Effekte auf den Charakter der Immunantwort hat, wird diskutiert. Der Einfluß von pro-inflammatorischen Zytokinen auf Mikrogliaaktivierung und den Erhalt von Nervenfasern wurde ebenfalls in Hirnschnittkulturen untersucht. Astrozyten sind wesentlicher Bestandteil der Blut-Hirn-Schranke. Diese kann jedoch von aktivierten T-Zellen überwunden werden. In dieser Arbeit wird gezeigt, dass Astrozyten über eine Expression von CD95L in aktivierten T-Zellen Apoptose induzieren können. Davon sind jedoch nicht alle T-Zellen betroffen. Andererseits wird eine T-Zellproliferation unterdrückt, indem T-Zellen unter Astrozyteneinfluß verstärkt CTLA-4 exprimieren, was einen Zellzyklusarrest zur Folge hat. Darüber hinaus ist eine verstärkte Produktion von Nervenwachstumfaktor (NGF; nerve growth factor) nach antigenspezifischer Interaktion von Astrozyten mit Th1- und Th2-Zellen als zusätzliches Mittel, eine Neuroinflammation einzudämmen, anzusehen. Die Arbeit stellt diese Ergebnisse in fünf Kapiteln dar, welche gleichzeitig eine Einführung in die als Anlagen enthaltenen zehn Publikationen geben. / This thesis deals with T helper cells and their interactions with tissue cells as they occur in the healthy organism and in autoimmune diseases. Questions of tolerance induction by oral application of antigens are discussed especially oral treatment with type II collagen in patients with rheumatoid arthritis. In order to transform inflammatory Th1 responses into anti-inflammatory Th2 responses, immune deviation can be reached by interference with T-cell signal transduction. New approaches towards the different ways that the immune privilege of the central nervous system is maintained are discussed. The resident immune cells, i.e. microglia and astrocytes possess properties that make inflammation unlikely. They have to be activated in order to present antigens. It has been shown in organotypic entorhinal-hippocampal slice cultures that Th1 cells activate whereas Th2 cells deactivate microglial cells. The possibility is discussed as to whether costimulation via CD80 or CD86 differentially influences the character of the immune response. The influence of pro-inflammatory cytokines on microglial activation and preservation of nerve fibers has also been studied in brain slice cultures. Astrocytes are an essential part of the blood-brain barrier, which can be crossed by activated T cells. The thesis shows that astrocytes can induce apoptosis in activated T cells via expression of CD95L. However, not all T cells are affected. T cell proliferation is suppressed by increased CTLA-4 expression in T cells under the influence of astrocytes, resulting in a cell cycle arrest. An additional mechanism of confining neuroinflammation is increased production of the nerve growth factor (NGF) following antigen-specific interaction of astrocytes and Th1 and Th2 cells, respectively. These results are presented in five chapters that also introduce the ten attached publications.

Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...

Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...