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

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

Dendritic cells tip the balance towards induction of regulatory T cells upon priming in experimental autoimmune encephalomyelitis

Counter-balancing regulatory mechanisms, such as the induction of regulatory T cells (Treg), limit the effects of autoimmune attack in neuroinflammation. However, the role of dendritic cells (DCs) as the most powerful antigen-presenting cells, which are intriguing therapeutic targets in this context, is not fully understood. Here, we demonstrate that conditional ablation of DCs during the priming phase of myelin-specific T cells in experimental autoimmune encephalomyelitis (EAE) selectively aborts inducible Treg (iTreg) induction, whereas generation of T helper (Th)1/17\ua0cells is unaltered. DCs facilitate iTreg induction by creating a milieu with high levels of interleukin (IL)-2 due to a strong proliferative response. In the absence of DCs, B220 B cells take over priming of Th17\ua0cells in the place of antigen-presenting cells (APCs), but not the induction of iTreg, thus leading to unregulated, severe autoimmunity

Down-regulation of neuronal L1 cell adhesion molecule expression alleviates inflammatory neuronal injury

In multiple sclerosis (MS), the immune cell attack leads to axonal injury as a major cause for neurological disability. Here, we report a novel role of the cell adhesion molecule L1 in the crosstalk between the immune and nervous systems. L1 was found to be expressed by CNS axons of MS patients and human T cells. In MOG35–55-induced murine experimental neuroinflammation, CD4+ T cells were associated with degenerating axons in the spinal cord, both expressing L1. However, neuronal L1 expression in the spinal cord was reduced, while levels of the transcriptional repressor REST (RE1-Silencing Transcription Factor) were up-regulated. In PLP139–151-induced relapsing–remitting neuroinflammation, L1 expression was low at the peak stage of disease, reached almost normal levels in the remission stage, but decreased again during disease relapse indicating adaptive expression regulation of L1. In vitro, activated CD4+ T cells caused contact-dependent down-regulation of L1, up-regulation of its repressor REST and axonal injury in co-cultured neurons. T cell adhesion to neurons and axonal injury were prevented by an antibody blocking L1 suggesting that down-regulation of L1 ameliorates neuroinflammation. In support of this hypothesis, antibody-mediated blocking of L1 in C57BL/6 mice as well as neuron-specific depletion of L1 in synapsinCre × L1fl/fl mice reduces disease severity and axonal pathology despite unchanged immune cell infiltration of the CNS. Our data suggest that down-regulation of neuronal L1 expression is an adaptive process of neuronal self-defense in response to pro-inflammatory T cells, thereby alleviating immune-mediated axonal injury

Cross-recognition of a myelin peptide by CD8+ T cells in the CNS is not sufficient to promote neuronal damage

Multiple sclerosis (MS) is an inflammatory disease of the CNS thought to be driven by CNS-specific T lymphocytes. Although CD8 T cells are frequently found in multiple sclerosis lesions, their distinct role remains controversial because direct signs of cytotoxicity have not been confirmed in vivo. In the present work, we determined that murine ovalbumin-transgenic (OT-1) CD8 T cells recognize the myelin peptide myelin oligodendrocyte glycoprotein 40–54 (MOG) both in vitro and in vivo. The aim of this study was to investigate whether such cross-recognizing CD8 T cells are capable of inducing CNS damage in vivo. Using intravital two-photon microscopy in the mouse model of multiple sclerosis, we detected antigen recognition motility of the OT-1 CD8 T cells within the CNS leading to a selective enrichment in inflammatory lesions. However, this cross-reactivity of OT-1 CD8 T cells with MOG peptide in the CNS did not result in clinically or subclinically significant damage, which is different from myelin-specific CD4 Th17-mediated autoimmune pathology. Therefore, intravital imaging demonstrates that local myelin recognition by autoreactive CD8 T cells in inflammatory CNS lesions alone is not sufficient to induce disability or increase axonal injury