In multiple sclerosis (MS), pathogenic T effector cells (Teff) are believed to orchestrate immune-mediated destruction of the central nervous system (CNS) myelin sheath. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, CNS infiltration by regulatory T cells (Treg), producing the anti-inflammatory cytokine IL-10, promotes the resolution of disease. Currently, little is understood about how Treg function within the inflamed CNS and on which cells they exert their suppressive function. There is a debate as to whether Treg in MS patients are capable of infiltrating the CNS and if they do, it is unclear whether they are functional. Understanding Treg function in EAE and MS could open up new possibilities for treatment, as Treg could be modulated for immunosuppressive therapy. A key step in the development of EAE (and presumably MS) is the ability of Teff cells to cross the blood brain barrier (BBB) and enter the CNS parenchyma. The hypothesis of this work was that Treg facilitate resolution of the inflamed CNS by preventing entry of the pathogenic T cells into the CNS parenchyma, thus preventing further damage. As such, it is important to understand with which immune cells and CNS resident cells Treg communicate to achieve resolution of disease. The presence of Treg in MS lesions was investigated with double immunohistochemistry (IHC) in frozen post-mortem MS brain tissue. CD4+Foxp3+ Treg were present in a subset of patients and their presence was associated with perivascular retention of CD4+Foxp3- and CD8+Foxp3- T cells. Foxp3+ cells in MS lesions predominantly expressed IL-10, indicating regulatory activity, although low-level production of IL-17, TNF-α, IFN-γ and GM-CSF was observed as well. Generally, analysis of total cytokine expression identified distinct patterns of cytokine production between lesions. Nonetheless, these could not be used to discriminate individual patients. These studies were repeated in C57BL/6 mice in which the Treg population was depleted before onset of EAE to mimic lesions with and without Treg presence, as found in MS patients. An immunofluorescent technique to study up to 5 fluorochromes simultaneously was developed to study antigen presenting cell (APC), Teff and Treg location, spatial relationship and function (as measured by cytokine expression) in the CNS of EAE mice at different stages of disease. Using this technique it was found that CD4+Foxp3- Teff and CD4+Foxp3+ Treg were located within 50-100μm of CD11c+ APC in the CNS of EAE affected mice. CNS Teff and Treg predominantly produced IFN-γ or IL-10, although low levels of IL-17 were detected in Teff and Treg as well. IL-17+ Treg were close to IL-17+ Teff, IFN-γ+ Treg were close to IFN-γ+ Teff, but IL-10+ Treg were not in close proximity to IL-10+ T cells in the CNS during EAE. In conclusion, there is evidence for functional Treg in EAE and MS lesions, supporting the concept of enhancing Treg activity as a clinical intervention. Treg seem to be capable of retaining pathogenic T cells at the blood brain barrier in MS lesions. In addition, studies of cytokine expression in MS lesions indicated that there is no sound basis for patient stratification based on peripheral blood cytokine profile. This thesis advances our understanding of Treg location, function and spatial relationship with other immune cells within the inflamed CNS.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:735613 |
| Date | January 2016 |
| Creators | Zandee, Stephanie Elizabeth Johanna |
| Contributors | Anderton, Stephen ; Williams, Anna |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/25779 |
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