In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

<p>Abstract</p> <p>Background</p> <p>Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.</p> <p>Methods</p> <p>We applied <it>ex vivo </it>and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.</p> <p>Results</p> <p>By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.</p> <p>Conclusions</p> <p>The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.</p

Absence of system xc⁻ on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis

Background: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System x(c)- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system x(c)-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT(-/-)) mice and irradiated mice reconstituted in xCT(-/-) bone marrow (BM), to their proper wild type (xCT(+/+)) controls. Results: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT(+/+) mice, xCT(-/-) mice were equally susceptible to EAE, whereas mice transplanted with xCT(-/-) BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system x(c)- on immune cells invading the CNS participates to EAE. Since a total loss of system x(c)- had no net beneficial effects, these results have important implications for targeting system x(c)- for treatment of MS

Die Rolle von Th17-Zellen bei der Initiierung und Chronifizierung von autoimmuner Demyelinisierung des Zentralen Nervensystems

T cells are critical for the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The invasion of encephalitogenic T cells through the blood-brain barrier (BBB) is considered as the initiatory event of the autoimmune pathology. However, antigen- specificity seems not to be essential for the transmigration step as non-CNS- specific T cells are also capable of entering the CNS without promoting any CNS pathology. It has recently been shown that Th17 cells are critically involved in the initiation of EAE. Their differentiation in vitro is dependent on the presence of the pro inflammatory cytokines IL-6 and IL-23 in the context of TGF-β. But there is a still a controversial debate around how far EAE and MS are rather Th1 or Th17 mediated diseases. In the first part of this thesis, the T cell differentiation requirements for a stable induction of EAE were investigated. The focus of experimental investigations was activation status, rounds of restimulation and differentiation (Th1 vs. Th17 cells). Myelin oligodendrocyte glycoprotein (MOG)-specific T cells were completely primed in vitro to generate “pure” Th1 or Th17 cells arising from naïve T cells. Results show that the in vitro generated MOG specific Th17 cells are very capable of inducing EAE in wild type and lymphopenic recipients. Adoptive transfer of differentially stimulated IL-17 producing CD4+ T cells into C57BL/6 mice wild type recipients led to severe, non-remitting clinical EAE resulting in death in a small number of animals, whereas the lymphopenic Rag1-/- recipient mice presented with a fulminant and severely progressive EAE resulting in death in all animals transferred with the MOG-specific Th17 cells. High numbers of the MOG-specific Th17 cells were found in the CNS of the Rag1-/- and C57BL/6 mice. It was observed, that the lymphocytes re- isolated from the CNS of diseased animals showed a T cell lineage shift as they showed also increased IFN-γ production. Titration of MOG-specific Th17 cells showed that even low cell numbers could induce severe disease in the Rag1-/- recipient mice. Only repetitive in vitro stimulation ensured disease induction. Utilising magnet resonance imaging (MRI) blood-brain barrier (BBB) breakdown detected by contrast-agent (Gd-DTPA) enhancement was observed only in the Rag1-/- mice and was correlated to lymphocyte recruitment to the CNS using immunohistochemistry. Two-photon laser scanning microscopy (TPLSM) visualised the potential of the MOG-specific CD4+ Th17 cells to promote neurodegeneration by directly contacting neurons. A comparison of the disease inducing potential of MOG-specific Th17 and Th1 in chronic neuroinflammation was made showing that also Th1 cells were able to induce EAE in Rag1-/- mice. The disease course was very mild when compared to adoptive transfer EAE induced with 2d2 Th17 cells. Additionally, alternative ways to induce adoptive transfer EAE were explored in the context of the investigation of the role of kinin receptor B1 (Bdkrb1). Here, encephalitogenic Bdkrb1 deficient and wild type T cells were transferred into Rag1-/- recipient mice. The transfer of these encephalitogenic T cells led to increased CD4+ T cell numbers in the CNS of mice transferred with encephalitogenic Bdkrb1-/- T cells. The proportion of CD4+ IL-17-secreting T cells was greater in the sick mice that received Bdkrb1-/- T cells, whereas the proportion of IFN-γ-producing T cells was comparable in mice that got Bdkrb1-/- T cells and wild type T cells, respectively. CD4+ Th17 cells treated with Bdkrb1 modulators or vehicle before allowing them to infiltrate into syngeneic hippocampal slice cultures showed a reduced infiltrative behaviour upon Bdkrb1 activation in TPLSM. In the second part, it was investigated if non-CNS-specific T cells that are able to enter the CNS may contribute to pathological processes such as induction of new clinical episodes in a bystander way. Background of this hypothesis is the epidemiological evidence that MS patients which suffer from trivial systemic infection, e.g. of the upper respiratory tracts, are highly prone to consecutively develop a relapse of the CNS disease. It could be demonstrated that OVA-specific Th17 cells are able to induce relapses in chronically sick mice when compared to vehicle control. They are not able to do so in healthy wild type and lymphopenic mice. The OVA-specific Th17 cells show no cross- reactivity with myelin-antigens and therefore seem to contribute to neuropathological processes in the bystander way. OVA specific Th17 cells have also been shown to directly contact neurons and induce damage much similar to MOG-specific Th17 cells. Taken together, these results indicate that repetitive restimulation of MOG-specific Th17 cells is necessary to yield a highly encephalitogenic Th17 subset, which is a potent inducer of EAE in an adoptive transfer model and that also non-CNS-specific Th17 cells can induce relapses in animals with pre-existing CNS pathology and that the kallikrein- kinin system is involved in the regulation of CNS inflammation, limiting encephalitogenic T lymphocyte infiltration into the CNS.T-Zellen spielen eine entscheidende Rolle in der Pathogenese der Multiplen Sklerose (MS) und ihres Tiermodells, der Experimentellen Autoimmunen Enzephalomyelitis (EAE). Die Invasion von enzephalitogenen T-Zellen über die Blut-Hirn-Schranke wird als Anfangspunkt für die autoimmune Pathologie betrachtet. Antigenspezifität scheint für diesen Prozess keine Rolle zu spielen, da sowohl Zellen die spezifisch wie auch unspezifisch für Antigene im Zentralen Nervensystem (ZNS) sind, die Blut-Hirn-Schranke passieren können. Kürzlich, konnte gezeigt werden, dass Th17-Zellen an der Initiierung von EAE beteiligt sind. Ihre Differenzierung in vitro ist abhängig von den proinflammatorischen Zytokinen IL-6 und IL 23 im Zusammenhang mit TGF-β. Im Moment gibt es eine kontroverse Debatte darüber, ob MS und EAE Th1- oder Th17-Zellvermittelte Krankheiten sind. Im ersten Teil der vorliegenden Dissertation wurde untersucht, welche Voraussetzungen in der Differenzierung von T-Zellen für die Induktion von EAE nötig sind. Dabei wurde ein Schwerpunkt auf den Aktivierungsstatus der Zellen, die Anzahl der Restimulationen und den Differenzierungsstatus (Th1- vs. Th17-Zellen) gelegt. Aus naiven Myelin Oligodendrozyten Glykoprotein (MOG)-spezifischen T-Zellen wurden in vitro “reine” Th1- oder Th17- Zellen generiert. Die Ergebnisse zeigen, dass adoptiver Transfer von unterschiedlich oft stimulierten IL 17 produzierenden CD4+ T-Zellen zu einem schweren Krankheitsverlauf sowohl in C57BL/6 Wildtyp Mäusen, als auch in Rag1-/- Mäusen führten. MOG-spezifische Th17-Zellen wurden im ZNS der Rag1-/- und C57BL/6 Mäuse wiedergefunden und zeichneten sich hier durch einen Phänotypwechsel aus, da sie eine erhöhte Produktion von IFN-γ aufzeigten. Eine Titration der Zellen zeigte, dass bereits geringe Zellzahlen in der Lage sind EAE in Rag1-/-Mäusen auszulösen. Mithilfe von Magnetresonanztomographie konnte die zellverursachte Zerstörung der Blut-Hirn- Schranke in Rag1-/- Mäusen beobachtet werden und mit der Rekrutierung von Lymphozyten immunohistologisch korreliert werden. Zwei-Photonen Mikroskopie wurde verwendet um das Potential der MOG-spezifischen CD4+ Th17-Zellen zur Neurodegeneration durch direkten Kontakt mit Neuronen zu visualisieren. Ein Vergleich MOG-spezifischer Th1- und Th17-Zellen in chronischer Neuroinflammation veranschaulichte, dass Th1-Zellen zwar fähig sind EAE in Rag1-/- Mäusen zu induzieren, dass aber in sehr viel abgeschwächterer Form als Th17-Zellen. Zusätzlich wurde die Rolle des Kininrezeptor B1 (Bdkrb1) untersucht. Hierfür wurde eine klassische passive EAE induziert. Bdkrb1-defiziente T-Zellen wurden in Rag1-/- Mäuse transferiert und mit Wildtyp T-Zellen verglichen. Es konnte eine erhöhte Anzahl CD4+ T-Zellen im ZNS der Tiere, die die Bdkrb1-defizienten T-Zellen erhalten haben, nachgewiesen werden. Der Anteil IL 17 produzierender Zellen in dieser CD4+ Fraktion war ebenfalls erhöht. CD4+ T-Zellen, die mit Modulatoren für Bdkrb1 behandelt wurden, zeigten ein weniger invasives Verhalten in hippocampalen Gehirnschnitten im Zwei-Photonen Mikroskop. Im zweiten Teil der Arbeit wurde das Verhalten ZNS-unspezifischer T-Zellen untersucht. Dabei sollte gezeigt werden, ob diese ZNS-unspezifischen T-Zellen in der Lage sind ins ZNS zu gelangen und dort zerstörerische Prozesse anzuregen. Basierend auf dem Hintergrund, dass in MS Patienten, die an einer gewöhnlichen Infektion leiden, Schübe beobachtet werden können, sollte das Verhalten der ZNS-unspezifischen T-Zellen untersucht werden. Es konnte gezeigt werden, dass ZNS-unspezifische T-Zellen in der Lage sind Schübe in chronisch kranken Mäusen zu induzieren. Die T-Zellen waren nicht in der Lage EAE in gesunden Tieren auszulösen und zeigten keine Kreuzreaktivität zu ZNS Antigenen auf, was darauf hinweist, dass sie eine sekundäre Rolle in der Zerstörungskaskade der EAE haben, obwohl aufgezeigt werden konnte, dass sie durchaus direkt auch Neuronen angreifen können. Zusammengenommen zeigen die Ergebnisse auf, dass wiederholte Stimulation MOG spezifischer Th17-Zellen essentiell ist um eine hochenzephalitogene Zellpopulation zu schaffen, die EAE sehr effizient induzieren kann. ZNS-unspezifische T-Zellen sind in der Lage Schübe in chronisch kranken Tieren zu induzieren und der Kininrezeptor B1 scheint eine Rolle in der Regulation von entzündlichen Prozessen im ZNS zu spielen, indem er die Transmigration von T-Zellen ins ZNS verhindert

Electromagnetic flowmeter with capacitive electrodes

Jedním z nových přístupů, jak měřit průtok kapalin s nízkou elektrickou vodicostí, je použití elektromagnetického průtokoměru s kapacitními elektrodami. Tato metoda rovněž umožňuje měřit průtok kapalin s velkým počtem pevných částic a kapalin se sklonem k utvoření usazenin. Diplomová práce se zabývá popisem teoretického modelu a sestavením funkčních modelů průtokoměru, na kterých byla provedena měření ověřující jejich funkčnost. Účelem diplomové práce však není vyřešení všech problémů souvisejících s uvedením do sériové výroby

The phenomenon of hate crimes against the LGBT community in Poland. Discussing selected issues.

Praca dotyczy problemu przestępstw z nienawiści wobec społeczności LGBT w Polsce. Autor analizuje zjawisko hate crime i hate speech na podstawie poszczególnych, krajowych regulacji prawnych - prawa konstytucyjnego, karnego, wykroczeń oraz prawa cywilnego. Ponadto, przywołuje najważniejsze przepisy prawa międzynarodowego i orzeczenia, które dotyczą sytuacji osób LGBT. Na podstawie wybranych badań, podjęto próbę ukazania skali przemocy wobec mniejszości seksualnych. Zaprezentowano sylwetkę sprawcy i profil ofiary hate crimes. Zjawisko wytłumaczono na podstawie wielu definicji odnoszących się do przestępstw z nienawiści i mowy nienawiści, w celu ukazania złożoności zjawiska. Zdefiniowano podstawowe pojęcia: homoseksualność, orientacja seksualna, homofobia. Z racji interdyscyplinarnego charakteru zagadnienia, w pracy tej korzystano z dorobku nie tylko osiągnięć kryminologii, ale też psychologii, psychiatrii, politologii czy socjologii.The master’s thesis discusses the problem of hate crimes against the LGBT community in Poland. The author analysis the phenomenon of hate crime and hate speech on the basis of polish legal regulations - constitutional law, criminal law and civil law. In addition, selected issues of international law are presented. The author shows the scale of violence against sexual minorities based on selected studies.The profile of victim's hate crimes and offender are presented. The phenomenon is explained on the basis of various definitions referring to hate crimes and hate speech to show the complexity of the phenomenon. Due to the interdisciplinary character, science of criminology, psychology, psychiatry, political science and sociology are used in this master’s thesis

Phenotype of antigen unexperienced T(H) cells in the inflamed central nervous system in experimental autoimmune encephalomyelitis

Multiple sclerosis is a chronic, disseminated inflammation of the central nervous system which is thought to be driven by autoimmune T cells. Genetic association studies in multiple sclerosis and a large number of studies in the animal model of the disease support a role for effector/memory T helper cells. However, the mechanisms underlying relapses, remission and chronic progression in multiple sclerosis or the animal model experimental autoimmune encephalomyelitis, are not clear. In particular, there is only scarce information on the role of central nervous system-invading naive T helper cells in these processes. By applying two-photon laser scanning microscopy we could show in vivo that antigen unexperienced T helper cells migrated into the deep parenchyma of the inflamed central nervous system in experimental autoimmune encephalomyelitis, independent of their antigen specificity. Using flow cytometric analyses of central nervous system-derived lymphocytes we found that only antigen-specific, formerly naive T helper cells became activated during inflammation of the central nervous system encountering their corresponding antigen

The role of CD8(+) T cells and their local interaction with CD4(+) T cells in myelin oligodendrocyte glycoprotein(35-55)-induced experimental autoimmune encephalomyelitis

T cells have an essential role in the induction of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Although for CD4(+) T cells it is well established that they contribute to the disease, less is known about the role of CD8(+) T cells. Our aim was to determine the individual contribution of CD4(+) and CD8(+) T cells in myelin oligodendrocyte glycoprotein (MOG)35-55-induced EAE. We investigated MOG35-55-activated CD8(+) T cells to clarify their potential to induce or attenuate EAE. We monitored the behavior of CD8(+) T cells and their interaction with CD4(+) T cells directly at the site of inflammation in the CNS using intravital imaging of the brainstem of EAE-affected living anesthetized mice. We found that mice without CD4(+) T cells did not develop relevant clinical signs of disease, although CD8(+) T cells were present in the CNS of these mice. These CD8(+) T cells displayed reduced motility compared with those in the presence of CD4(+) T cells. In mice that harbored CD4(+) and CD8(+) T cells, we saw a similar extent of clinical signs of EAE as in mice with only CD4(+) T cells. Furthermore, the dynamic motility and viability of CD4(+) T cells were not disturbed by CD8(+) T cells in the lesions of these mice. Therefore, we conclude that in MOG35-55-induced EAE, CD8(+) T cell accumulation in the CNS represents instead an epiphenomenon with no impact on clinical disease or on the effects of CD4(+) T cells, the latter being the true inducers of the disease

Functional characteristics of Th1, Th17, and ex-Th17 cells in EAE revealed by intravital two-photon microscopy

Background!#!T helper (Th) 17 cells are a highly plastic subset of T cells, which in the context of neuroinflammation, are able to acquire pathogenic features originally attributed to Th1 cells (resulting in so called ex-Th17 cells). Thus, a strict separation between the two T cell subsets in the context of experimental autoimmune encephalomyelitis (EAE) is difficult. High variability in culture and EAE induction protocols contributed to previous conflicting results concerning the differential contribution of Th1 and Th17 cells in EAE. Here, we systematically evaluate the role of different T cell differentiation and transfer protocols for EAE disease development and investigate the functional dynamics of encephalitogenic T cells directly within the inflamed central nervous system (CNS) tissue.!##!Methods!#!We compiled the currently used EAE induction protocols reported in literature and investigated the influence of the different Th1 and Th17 differentiation protocols as well as EAE induction protocols on the EAE disease course. Moreover, we assessed the cytokine profile and functional dynamics of both encephalitogenic Th1 and Th17 cells in the inflamed CNS using flow cytometry and intravital two-photon laser scanning microscopy. Lastly, we used astrocyte culture and adoptive transfer EAE to evaluate the impact of Th1 and Th17 cells on astrocyte adhesion molecule expression in vitro and in vivo.!##!Results!#!We show that EAE courses are highly dependent on in vitro differentiation and transfer protocols. Moreover, using genetically encoded reporter mice (B6.IL17A-EGFP.acRFP x 2d2/2d2.RFP), we show that the motility of interferon (IFN)γ-producing ex-Th17 cells more closely resembles Th1 cells than Th17 cells in transfer EAE. Mechanistically, IFNγ-producing Th1 cells selectively induce the expression of cellular adhesion molecules I-CAM1 while Th1 as well as ex-Th17 induce V-CAM1 on astrocytes.!##!Conclusions!#!The behavior of ex-Th17 cells in EAE lesions in vivo resembles Th1 rather than Th17 cells, underlining that their change in cytokine production is associated with functional phenotype alterations of these cells

Flow cytometric analysis of T cell/monocyte ratio in clinically isolated syndrome identifies patients at risk of rapid disease progression

BACKGROUND: Multiple sclerosis is a chronic inflammatory central nervous system disease diagnosed by clinical presentation and characteristic magnetic resonance imaging findings. The role of cerebrospinal fluid (CSF) analysis has been emphasized in particular in the context of differential diagnosis in patients with a first episode suggestive of multiple sclerosis. OBJECTIVE: We investigated here the potential additional value of analysis of CSF cellularity by fluorescence activated cell sorting (FACS) in the setting of a routine diagnostic work-up in our inpatient clinic. METHODS: CSF cells from back-up samples from patients with suspected chronic inflammatory central nervous system disorder were analyzed by FACS and correlated with clinical data, magnetic resonance imaging findings and oligoclonal band status. RESULTS: We found distinct changes of T cell/monocyte (CD4/CD14) and B cell/monocyte (CD20/CD14) ratios between clinically isolated syndrome (CIS)/multiple sclerosis and other neurologic diseases or other inflammatory neurologic diseases. In particular, patients with a rapid transition from CIS to multiple sclerosis had an elevated CD4/CD14 ratio. A subgroup analysis showed diagnostic value of CD4/CD8 ratio in the differential diagnosis of CIS/multiple sclerosis to neurosarcoidosis. CONCLUSION: The diagnostic and prognostic accuracy of autoimmune neuroinflammatory diseases can be improved by FACS analysis of CSF cells

Role of Sortilin in Models of Autoimmune Neuroinflammation

The proneurotrophin receptor sortilin is a protein with dual functions, being involved in intracellular protein transport, as well as cellular signal transduction. The relevance of the receptor for various neuronal disorders, such as dementia, seizures, and brain injury, is well established. In contrast, little is known about the role of sortilin in immune cells and inflammatory diseases. The aim of our study was to elucidate the distribution of sortilin in different immune cell types in mice and humans and to analyze its function in autoimmune CNS inflammation. Sortilin was expressed most profoundly in murine and human macrophages and dendritic cells and to a much lesser extent in B and T cells. In dendritic cells, sortilin had an impact on Ag processing. Accordingly, sortilin was highly expressed by infiltrated perivascular myeloid cells, mainly in vessel cuffs, in the CNS of patients suffering from multiple sclerosis, the most common inflammatory autoimmune disease of the CNS. Yet, sortilin gene-targeted mice (Sort1(-/-)) and chimeras deficient in sortilin in the immune system were as susceptible as wild-type littermates to T cell-dependent experimental autoimmune encephalomyelitis. Considering our results and recent data from other investigators, we conclude that the proneurotrophin receptor sortilin plays a role in innate, rather than in adaptive, immune processes and, thus, not in autoimmune neuroinflammation