The role of cytokine-producing B cells in initiation and regulation of EAE

Multiple sclerosis (MS) is a common disease of the central nervous system with almost 2.5 million patients worldwide. It is characterized by chronic autoimmune inflammation targeting the myelin sheath and resulting in tissue inflammation, demyelination, axonal damage and loss. Traditionally, research efforts in MS and its animal model experimental autoimmune encephalomyelitis (EAE) focused on autoreactive T cells specific for myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). These autoreactive T cells are thought to be primed in the periphery before they enter the CNS where they drive inflammatory processes that lead to lesion formation and tissue damage. However, in the last years, clinical data suggested an additional important role of B cells in MS pathogenesis. Increasing evidence supports the hypothesis that among the B cell effector functions, antigen presentation and cytokine production rather than secretion of antibodies are crucial for disease initiation and progression. Thereby, B cells may promote differentiation of autoreactive T helper (Th) cells into pathogenic subsets such as Th1 and Th17 cells. To test by which mechanisms antigen-specific B cells could cooperate with antigen-specific T cells to induce EAE, B cells isolated from Th mice, which harbor B cells specific for the CNS antigen MOG, were characterized. In this thesis it was demonstrated that Th B cells have an expanded compartment of marginal zone B cells, a B cell subset often associated with different autoimmune diseases. Furthermore, Th B cells produced significantly more pro-inflammatory cytokines such as IL-6 and TNFα, and less anti-inflammatory IL-10 indicating a more pathogenic potential of MOG- specific B cells. To investigate how cytokine production by antigen-specific B cells in the context of antigen-presentation can shape T cell responses in vivo, a novel adoptive co-transfer system was developed, in which activated MOG-specific B cells stimulate MOG-specific CD4+ T cells to induce EAE. Using an in vitro culture system, different cytokines were overexpressed in MOG-specific B cells before adoptive transfer to evaluate the role of these cytokines in initiation of EAE. It could be shown that overexpression of IL-6 and IL-23p19 leads to an accelerated EAE onset mediated by different mechanisms. In contrast, overexpression of anti-inflammatory IL-10 in plasmablasts could contribute to disease regulation in a Th17 adoptive transfer EAE model by increasing the frequency of anti-inflammatory T cells directly in the CNS. Lastly, a novel IL-23p19 reporter mouse was developed to identify the cellular sources of IL-23p19, a cytokine crucial for differentiation and stabilization of pathogenic Th17 cells. It was discovered that activated T cells express and secrete high levels of IL-23p19, but that T cell-derived IL-23p19 is dispensable for EAE. Overall, the role of different B cell-derived cytokines was investigated in this thesis, and it was shown that B cells can contribute to both the initiation but also the regulation of EAE depending on the cytokines they produce. Furthermore, the sources of the cytokine IL-23p19, which is crucial for Th17 differentiation, and its role in T cell biology during EAE were investigated. These findings shed light on the role of different B cell subsets, effector functions, and cytokines during EAE and MS, whose understanding is crucial to develop novel, more specific therapies for MS.Multiple Sklerose (MS) ist eine chronisch-entzündliche Autoimmunerkrankung, bei der die Myelinscheiden im zentralen Nervensystem (ZNS) von Immunzellen angegriffen werden. Dies führt zu lokalen Entzündungen und Demyelinisierung sowie zur Schädigung und zum Verlust von Nervenzellen. Obwohl MS eine häufige Erkrankung ist, von der weltweit fast 2,5 Millionen Menschen betroffen sind, ist die Pathogenese noch nicht abschließend verstanden. Traditionell fokussierte sich die Forschung an MS und seinem Tiermodell, der experimentellen autoimmunen Enzephalomyelitis (EAE), auf autoreaktive T-Zellen, die spezifisch auf Myelin-Antigene wie beispielsweise das Myelin- Oligodendrozyten-Glykoprotein (MOG) reagieren. Man geht davon aus, dass diese autoreaktiven T-Zellen in der Peripherie aktiviert werden und dann ins ZNS einwandern, wo sie Entzündungsreaktionen auslösen, die zu Läsionen und Gewebsschädigung führen. In den letzten Jahren verdichteten sich jedoch die Hinweise auf eine zusätzliche wichtige Beteiligung von B-Zellen an der MS-Pathogenese. Besonders klinische Daten deuten darauf hin, dass B-Zellen zum Krankheitsbeginn und -verlauf beitragen, indem sie Antigene präsentieren und Zytokine sezernieren, die möglicherweise die Differenzierung von pathogenen T-Helfer (Th-)Zellen wie Th1- oder Th17-Zellen fördern. Um zu testen, durch welche Mechanismen antigen-spezifische B-Zellen mit antigen- spezifischen T-Zellen kooperieren und EAE induzieren können, wurden B-Zellen von Th Mäusen, die MOG-spezifische B-Zellen besitzen, isoliert und charakterisiert. In dieser Dissertation konnte gezeigt werden, dass Th B-Zellen einen erhöhten Anteil an Marginalzonen B-Zellen besitzen, eine B-Zell-Population, die im Zusammenhang mit verschiedenen Autoimmunkrankheiten steht. Des Weiteren produzierten die Th B-Zellen deutlich mehr entzündungsfördernde Zytokine wie IL-6 oder TNFα und weniger entzündungshemmendes IL-10, was auf ein pathogenes Potenzial der MOG- spezifischen B-Zellen hindeutet. Um zu untersuchen, ob antigen-spezifische B-Zellen die T-Zellantwort in vivo durch die Produktion von Zytokinen im Kontext der Antigen-Präsentation beeinflussen können, wurde ein neues Ko-Transfermodell entwickelt, in dem aktivierte MOG-spezifische B-Zellen MOG-spezifische CD4+ T-Zellen stimulieren und so EAE induzieren können. In einem in vitro Zellkultursystem wurden vor dem Transfer verschiedene Zytokine in MOG-spezifischen B-Zellen überexprimiert und so die Rolle dieser Zytokine für die Initiierung der Krankheit bewertet. Es konnte gezeigt werden, dass die Überexpression von IL-6 und IL-23p19 zu einem beschleunigten Krankheitsbeginn führt; ein Effekt, der je nach Zytokin durch unterschiedliche Mechanismen vermittelt wird. Im Gegensatz dazu trägt die Überexpression von entzündungshemmendem IL-10 in Plasmablasten zur Krankheitsregulation bei, indem sie den Anteil entzündungshemmender T-Zellen im ZNS erhöht. Außerdem wurde in dieser Dissertation eine neuartige IL-23p19 Reportermaus entwickelt, um die zellulären Quellen des Zytokins IL-23p19 zu untersuchen, das für die Differenzierung und Stabilisierung pathogener Th17-Zellen von entscheidender Bedeutung ist. Es konnte gezeigt werden, dass aktivierte T-Zellen IL-23p19 exprimieren und sezernieren, dass aber das von T-Zellen stammende IL-23p19 unwesentlich für die Entwicklung der EAE ist. Zusammenfassend wurde in dieser Arbeit die Rolle von verschiedenen B-Zell-Zytokinen untersucht und es konnte gezeigt werden, dass B-Zellen, abhängig von den produzierten Zytokinen, sowohl zur Krankheitsauslösung als auch zur -regulation beitragen können. Außerdem wurden die zelluläre Herkunft des Zytokins IL-23p19 sowie seine Rolle in der T-Zellbiologie im Zusammenhang mit Entzündungsprozessen in der EAE untersucht. Diese Erkenntnisse geben Aufschluss über die Rolle von verschiedenen B-Zellpopulationen, Effektor-Funktionen und Zytokinen während der EAE. Dieses Verständnis ist ausschlaggebend für die Entwicklung neuer, gezielterer Therapien für MS

Conversion of Anergic T Cells Into Foxp3−^- IL-10+^+ Regulatory T Cells by a Second Antigen Stimulus In Vivo

T cell anergy is a common mechanism of T cell tolerance. However, although anergic T cells are retained for longer time periods in their hosts, they remain functionally passive. Here, we describe the induction of anergic CD4$^+$ T cells in vivo by intravenous application of high doses of antigen and their subsequent conversion into suppressive Foxp3$^-$ IL-10$^+$ Tr1 cells but not Foxp3$^+$ Tregs. We describe the kinetics of up-regulation of several memory-, anergy- and suppression-related markers such as CD44, CD73, FR4, CD25, CD28, PD-1, Egr-2, Foxp3 and CTLA-4 in this process. The conversion into suppressive Tr1 cells correlates with the transient intracellular CTLA-4 expression and required the restimulation of anergic cells in a short-term time window. Restimulation after longer time periods, when CTLA-4 is down-regulated again retains the anergic state but does not lead to the induction of suppressor function. Our data require further functional investigations but at this stage may suggest a role for anergic T cells as a circulating pool of passive cells that may be re-activated into Tr1 cells upon short-term restimulation with high and systemic doses of antigen. It is tentative to speculate that such a scenario may represent cases of allergen responses in non-allergic individuals

Reduced Gray to White Matter Tissue Intensity Contrast in Schizophrenia

BACKGROUND: While numerous structural magnetic resonance imaging (MRI) studies revealed changes of brain volume or density, cortical thickness and fibre integrity in schizophrenia, the effect of tissue alterations on the contrast properties of neural structures has so far remained mostly unexplored. METHODS: Whole brain high-resolution MRI at 3 Tesla was used to investigate tissue contrast and cortical thickness in patients with schizophrenia and healthy controls. RESULTS: Patients showed significantly decreased gray to white matter contrast in large portions throughout the cortical mantle with preponderance in inferior, middle, superior and medial temporal areas as well as in lateral and medial frontal regions. The extent of these intensity contrast changes exceeded the extent of cortical thinning. Further, contrast changes remained significant after controlling for cortical thickness measurements. CONCLUSIONS: Our findings clearly emphasize the presence of schizophrenia related brain tissue changes that alter the imaging properties of brain structures. Intensity contrast measurements might not only serve as a highly sensitive metric but also as a potential indicator of a distinct pathological process that might be independent from volume or thickness alterations

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Core-shell 3D printed biodegradable calcium phosphate cement—Alginate scaffolds for possible bone regeneration applications

The core/shell 3D printing process using CPC and alginate is intended to create biodegradable scaffolds that have a similar stability to bone tissue and also offer sufficient and continuous antibiotic release. In this way, a patient-specific and patient-friendly process will be established, which should optimally support the human organism in its regeneration. To generate the best possible strength values, the printed scaffolds underwent various post-treatments and were then tested in a material test. The test methods included self-setting, storage in a drying cabinet with a water-saturated atmosphere at 37°C, followed by incubation in PBS, freeze-drying, and coating the samples with alginate. Additionally, a degradation test at pH 7.4 and pH 5 was carried out to test stability under in vitro conditions. It was shown that the untreated and freeze-dried samples failed at a maximum load of 30–700 N, while the remaining scaffolds could withstand a load of at least 2,000 N. At this failure load, most of the test series showed an average deformation of 43.95%. All samples, therefore, remained below the strength of cancellous bone. However, based on a 20% load after surgery, the coated scaffolds represented the best possible alternative, with a Young’s modulus of around 1.71 MPa. We were able to demonstrate that self-setting occurs in core-shell printed CPC/alginate scaffolds after only 1 day, and that mass production is possible. By coating with alginate, the compressive strength could be increased without the need for additional post-treatment. The mechanical strength was sufficient to be available as a scaffold for bone regeneration and additionally as a drug delivery device for future applications and experiments

A scoping review on associations between paratuberculosis and productivity in cattle

Paratuberculosis (PTB), or Johne’s disease, is a disease with worldwide distribution caused by Mycobacterium avium subsp. paratuberculosis (MAP) that leads to chronic enteritis, primarily in ruminants. Even subclinical infection significantly reduces the animals’ performance, and consequences of the disease lead to high economic losses for the cattle industry. To estimate the economic burden of bovine PTB and to evaluate the benefits of a potential control program, accurate estimates of the production effects associated with the disease are required. Therefore, the aim of this scoping review was to provide a comprehensive overview of associations between MAP infection and production parameters in cattle. The studies were collected from three electronic databases. Of the total 1,605 identified studies, 1,432 did not meet the set criteria in the title and abstract screening and a further 106 were excluded during full-text review. Finally, data on 34 different production parameters were extracted from 67 publications. Results show that the magnitude of reported performance losses varies depending on several factors, such as the type of diagnostic test applied, disease status or number of lactations. Studies reported a reduction in milk yield, changes in milk quality (e.g., higher somatic cell count, lower amount of produced milk fat and protein), reduced fertility (e.g., prolonged calving interval and service period, higher abortion rate and calving difficulties), reduced weaning weight, slaughter weight and slaughter value, or a higher risk for mastitis. Results from the studies included in our review show a median decrease of milk yield per infected cow of −452 kg/lactation for raw and −405 kg/lactation for modeled data. Similarly, the amount of produced milk protein fell by a median of −14.41 kg/lactation for modeled data and the amount of produced milk fat by a median of −13.13 kg/lactation. The reviewed studies revealed a prolonged calving interval by around 30 days and a 1.5 to 3 times higher likeliness of culling per lactation in PTB positive animals. Results from this scoping review provide evidence-based inputs for the development of economic models aiming at the estimation of the costs and benefits associated with different disease control scenarios for PTB