Cut Dahlia Iskandar
Polarization of immune cells in Theiler’s murine encephalomyelitis
Multiple sclerosis (MS), one of the most frequent central nervous system (CNS) diseases in young adults, is a chronic demyelinating disease of unknown etiology and possibly multifactorial causes. Microglia and macrophages play a central role for demyelination in Theiler’s murine encephalomyelitis (TME) virus-infection, a commonly used viral mouse model for the chronic-progressive form of MS. Microglia and CNS-infiltrating macrophages play a central role in the pathogenesis of TME virus-induced demyelination, e.g. as target cells for viral persistence. Similar to other demyelination models, such as experimental autoimmune encephalomyelitis (EAE), they also induce bystander demyelination, delayed-type hypersensitivity and myelin-specific autoimmunity. The current concept of microglia/macrophages plasticity describes different cell populations with distinct and even opposing functions. For instance, M1-type microglia/macrophages exhibit pro-inflammatory properties, while M2-type cells exhibit neuroprotective properties. However, so far, only few reports mention the polarizing effects of TME virus upon microglia/macrophages. Therefore, the aim of the present project was to determine dynamic changes of microglia/macrophage polarization in the spinal cord of susceptible SJL mice during the initiation and progression of TME. Moreover, the relevance of regulatory T cells (Treg) for polarization of immune cells, including microglia/macrophages, was investigated by genetic ablation of Foxp3+ Treg in resistant C57BL/6 mice following TME virus infection.
In the first part of the study, the spinal cord of TME virus-infected SJL mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and chronic demyelinating leukomyelitis was confirmed by immunohistochemistry and histology, respectively. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase, indicative of incomplete remyelination.
A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TMEV-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1-polarization, while mounting M2-polarization of macrophages/microglia together with sustained prominent M1-related gene expression were present during the chronic progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32+ M1- and arginase-1+ M2-type cells associated with progressive demyelination. The study provides a comprehensive database of M1/M2-related gene expression involved in the initiation and progression of demyelination, which supports the hypothesis that the perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.
The second part of the study aimed to gain further insights into the relevance of Treg for disease resistance and antiviral immunity in TME, the kinetics of CNS immune cells and the underlying chemokine and cytokine expression following genetic ablation of Treg in BAC-transgenic Foxp3 reporter mice (DEREG mice) with a C57BL6 background. As determined by RT-qPCR, DEREG mice were resistant to TME virus infection and cleared the virus, regardless of the presence or absence of Treg. Nevertheless, priming of strong effector T cell responses was observed in the periphery following Treg ablation, which subsequently resulted in a transient increase of IFNγ-producing T cells in the brain. Histology, Immunohistochemistry and flow cytometric analysis revealed that this transient increase of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice was not associated with an augmented antiviral response or increased inflammation-mediated tissue damage, respectively. Expression of interleukin-10 in the infected brain was unaltered despite of Treg depletion, which might play a role for dampening the inflammatory damage caused by increased number of effector T cells. Thus, unlike susceptible SJL mice, Treg have only negligible effects on virus-induced pathologies in the CNS of the resistant C57BL/6 mice.
In conclusion, the present findings of the first part support the hypothesis of a dual function of microglia/macrophges with promoting effects upon antiviral immunity and immunopathology, respectively. Modulating the microglia polarization of the spinal cord might represent a prerequisite to stimulate endogenous regeneration and future transplantation approaches. However, since M1-type cells are pivotal for virus elimination, in contrast to primarily autoimmune CNS disorders, reconstitution of immunomodulatory microglia/macrophages might be necessary, rather than simple suppression of M1-responses to establish CNS recovery in this infectious MS model. Hence, in contrast to the therapeutic effect of M2-dominence in primary autoimmune diseases, such as EAE, only a well-orchestrated and timely balanced polarization of macrophages/microglia might have the ability to prevent virus persistence and reduce myelin loss in this infectious MS model.
Results of the second part confirm that resistance in C57BL/6 mice to TME virus infection is largely due to the induction of effective CD4+ and CD8+ T cell responses, which is not significantly influenced by Treg depletion. Sustained expression of interleukin-10, probably by neuroprotective M2-type microglia/macrophages during early infection, might compensate for the lack of Treg and limit the extent of damage caused by an unwanted immune response in the brain.
7. ZUSAMMENFASSUNG
Cut Dahlia Iskandar
Polarisation von Immunzellen bei der Theilerschen murinen Enzephalomyelitis
Bei der Mulitplen Sklerose des Menschen handelt es sich um eine der häufigsten Erkrankungen des zentralen Nervensystems (ZNS) bei jungen Erwachsenen. Die Erkrankung führt zu einer chronisch-entzündlichen Entmarkung, wobei die primäre Ursache bislang ungeklärt ist. Die demyelinisierende murine Theilervirus-Enzephalomyelitis (TME) stellt ein sehr gutes und viral-induziertes Modell für MS dar. Mikroglia und Makrophagen spielen bei der Entmarkung der TME eine wichtige Rolle, insbesondere als Zielzellen während der chronischen Phase, wodurch es zur Viruspersistenz kommt. Außerdem rufen diese Zellen bei MS und TME, wie auch bei anderen Entmarkungskrankheiten (z.B. experimentelle autoimmune Enzephalomyelitis [EAE]) Hypersensitivitätsreaktionen vom verzögerten Typ und eine myelinspezifische Autoimmunität hervor und setzen myelintoxische Substanzen frei (bystander demyelination). Man geht derzeit davon aus, dass die Mikroglia/Makrophagen-Population zwei unterschiedliche, zum Teil gegensätzliche Funktionen aufweisen. Dabei wird zwischen den Mikroglia/Makrophagen vom und M2-Typ unterschieden. M1-Mikroglia/Makrophagen weisen pro-inflammatorische Eigenschaften auf, während die Mikroglia/Makrophagen vom M2-Typ neuroprotektive Eigenschaften zeigen. Bisher gibt es nur wenige Daten zur Mikroglia/Makrophagen-Polarisierung im Verlauf der TME. Aus diesem Grund war es Ziel dieser Studie, die Veränderungen der Mikroglia/Makrophagen-Polarisierung im Rückenmark von virusinfizierten, empfänglichen SJL-Mäusen im Initialstadium der TME und zu späteren Zeitpunkten der progressiven Erkrankung zu untersuchen. Weiterhin wurde die Bedeutung des Einflusses von regulatorischen T Zellen (Treg) auf die Immunzellen, inklusive Mikroglia/Makrophagen, bei der TME untersucht.
Im ersten Teil dieser Studie wurde das Rückenmark von TME-Virus-infizierten SJL-Mäusen mittels Genexpressionsanalyse und Immunfluoreszenz untersucht. Darüber hinaus wurden die Viruspersistenz und die chronisch-demyelinisierende Leukomyelitis mittels Immunhistochemie und Histologie dargestellt. Die elektronenmikroskopische Untersuchung ergab einen kontinuierlichen Myelinverlust, der mit abortiven Reparaturmechanismen bzw.
einer unvollständigen Remyelinisierung während der chronischen Infektionsphase einherging.
Für die Genexpressionsanalyse im Rückenmark von TME-Virus-infizierten SJL-Mäusen wurden 151 Gene ausgewählt, von denen 59 differentiell exprimiert waren. Zu Beginn der virusinduzierten Demyelinisierung wurde eine dominante M1-Polarisierung festgestellt. Die Spätphase der Demyelinisierung war durch eine Zunahme der M2-polarisierten Mikroglia/Makrophagen charakterisiert, die allerdings weiterhin von einer dominierenden M1-Polarisierung begleitet war. Die Ergebnisse der Genexpressionsanalyse wurden durch die Resultate der Immunfluoreszenz verifiziert. Hierbei zeigte sich ein vermehrter Nachweis von CD16/32+ M1- und Arginase-1+ M2-polarisierten Zellen in der chronisch-progressiven Phase.
Diese Studie stellt umfassende Daten zur Expression von M1- und M2-assoziierten Genen
während der Initiation und Progression der TME-Virus-induzierten Demyelinisierung zur Verfügung. Darüber hinaus unterstützen die Ergebnisse die Hypothese, dass die kontinuierliche Interaktion zwischen dem Erreger und Mikroglia/Makrophagen für die persistente Entzündung und Remyelinisierungsstörung bei der TME mitverantwortlich ist.
Im zweiten Teil der Studie sollte die Bedeutung der Treg für Mechanismen der TME-Resistenz und der antiviralen Immunantwort bei C57BL/6-Mäusen näher untersucht werden.
Hierfür wurde die Immunzellinfiltration sowie das zugrunde liegende Chemokin- und Zytokinexpressionsprofil in TME-Virus-infizierten BAC-transgenen Foxp3-Reporter-Mäusen (DEREG-Mäuse) mit einem C57BL/6-Hintergrund untersucht. Unabhängig vom Vorhandensein von Treg wurde mittels RT-qPCR gezeigt, dass DEREG-Mäuse resistent gegenüber der TME-Virus-Infektion sind und das Virus eliminieren. In peripheren lymphatischen Zellen konnte eine deutliche Effektor-T Zellantwort nach Ablation der Treg in infizierten DEREG-Mäusen festgestellt werden. Dies resultierte in einer transienten Zunahme IFNγ-produzierender T-Zellen im Gehirn. Mittels Histologie, Immunhistochemie und Durchflusszytometrie wurde festgestellt, dass dieser transiente Anstieg IFNγ-produzierender T-Zellen bei Treg-depletierten DEREG-Mäusen nicht mit einer gesteigerten antiviralen Immunantwort oder einem deutlicherem Gewebeschaden im Gehirn vergesellschaftet war.
Die Expression von Interleukin-10 war trotz der Treg-Ablation im Gehirn von virusinfizierten Mäusen unverändert, was für einen potentiell neuroprotektiven Effekt des Zytokins bei der TME spricht. Folglich haben Treg in TME-resistenten C57BL/6-Mäusen einen nur sehr geringen Effekt auf die virusinduzierten pathologischen Veränderungen im ZNS, im Gegensatz zu TME-empfänglichen SJL-Mäusen.
Zusammenfassend weisen die Ergebnisse der ersten Studie darauf hin, dass Mikroglia und Makrophagen eine duale Funktion besitzen und sowohl die antivirale Immunantwort als auch die Immunpathologie bei der TME beeinflussen. Die Modulation der Makrophagen/Mikroglia-Polarisation im Rückenmark könnte eine Methode zur Stimulation der endogenen Regeneration und eine adjuvante Therapiestrategie bei zukünftigen Transplantations-Studien darstellen.
Im Gegensatz zu primär autoimmunen Erkrankungen des ZNS (z.B. EAE) spielen bei der TME M1-polarisierte Mikroglia/Makrophagen anscheinend eine große Rolle für die Viruselimination. Aus diesem Grund muss daher die Abfolge der M1- und M2-induzierten Effekte ausbalanciert und zeitlich abgestimmt sein, um die Viruspersistenz zu vermeiden und um den Myelinverlust zu limitieren. Die Ergebnisse des zweiten Teils dieser Studie konnten zeigen, dass die Resistenz der C57BL/6-Mäuse gegenüber der TME weitestgehend auf einer Induktion von CD4+ und CD8+ T Zellen beruht, die nicht signifikant von der Treg-Ablation beeinflusst wird. Eine anhaltende Interleukin-10-Expression, möglicherweise durch Mikroglia/Makrophagen vom M2-Typ während der Frühphase der Infektion, kompensiert hierbei möglicherweise den Treg-Mangel und limitiert den Gewebsschaden.
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