Multiple Sclerosis (MS) is a chronic, inflammatory disease of the central nervous system. MS is caused by the immune-mediated destruction of myelin and oligodendrocytes, resulting in demyelination and neurodegeneration. The microRNA miR-145-5p has been demonstrated to be upregulated in MS lesions. Our lab has previously shown that dysregulation of miR-145-5p can interfere with oligodendrocyte differentiation in mice and that knockout of miR-145-5p protects mice from experimental autoimmune encephalomyelitis (EAE), a model for MS. The objective of this study is to determine if inhibition of miR-145-5p with an antisense oligonucleotide (ASO) is sufficient to protect mice from EAE. Female mice were induced with EAE and then treated with a control or miR-145 ASO at the onset of disease. We evaluated disease progression by monitoring clinical severity, and evaluating molecular and structural characteristics of EAE by RT-qPCR, histology, immunohistochemistry and electron microscopy. We have shown that the miR-145 ASO reduced miR-145-5p expression in the lumbar spinal cord, spleen and thymus following EAE induction. Treatment with the miR-145-5p ASO resulted in improved clinical severity of EAE, reduced neuroinflammation and increased myelination. Inhibition of miR-145-5p may represent a novel treatment for MS.

One of the major focuses for our lab involves examining the immunosuppressive properties of various agents and receptors in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). This dissertation encompasses an investigation of cannabidiol in the EAE model, the endocannabinoid CB1 receptor in the EAE model, staphylococcal superantigens (SAgs) as immunosuppressive agents, and various aspects of the EAE model. The first chapter covers the existing literature pertinent to these studies, the second and third chapters cover the material, methods, and results from the studies, and the fourth chapter is a discussion of how those results fit into the existing body of literature. A fifth chapter has also been included which covers two additional studies designed to develop alternative EAE models for our lab; however, both studies turned out differently than expected. One of the most interesting developments from this final chapter was the discovery of an age dependent difference in the memory T cell response of older mice, which allows for more robust disease to be induced when cells from 6 month old mice are used in the passive EAE (P-EAE) model as opposed to mice 10 weeks of age.

No description available.

No description available.

No description available.

Multiple sclerosis (MS) is a demyelinating neurodegenerative disease that typically has a relapsing-remitting pattern of progression superimposed on a gradual worsening of disease symptoms. Experimental autoimmune encephalomyelitis (EAE) is a model of MS where animals develop relapses, demyelination and accumulate neurological deficits. Studies using the EAE model have provided evidence that cannabinoids are beneficial in reducing disease symptoms and may impact long term neurodegeneration, but side-effects of exogenous cannabinoid receptor agonists may limit their potential as therapeutic agents for MS. Targeting enzymes involved in degradation of endocannabinoids such as the anandamide-degrading enzyme fatty acid amide hydrolase (FAAH) may be an attractive alternative strategy. Using experimental allergic encephalomyelitis (EAE) as a mouse model of MS, two complementary approaches were used to assess FAAH as a potential therapeutic target. The FAAH deficient (ABH.FAAH-/-) developed similar paralytic relapsing disease of similar severity of disease compared to the wild-type, but showed a poorer recovery following the acute phase. However, following a relapsing-remitting disease course, the FAAH deficient mice showed a substantial improvement in clinical score, improved motor control, and lost less neurofilament compared to wild-type mice. These findings indicate that fatty acid amides may be neuroprotective in EAE. Secondly, a selective FAAH inhibitor (PF-3845; 10 mg/kg) was used to treat mice during the relapse phase of the disease course. Treatment with PF-3845 caused an elevation of anandamide in the CNS. This treatment resulted in a small reduction in neurofilament loss, but no reduction in clinical score or improvement in motor control was observed compared to the vehicle treated group. To investigate at a cellular level how FAAH might affect disease progression in the EAE model, immunohistochemistry was used to analyse FAAH expression in the CNS. Employing novel antibodies to FAAH in combination with neuronal and glial cell markers, it was found that, in addition to previously reported neuronal expression of FAAH, FAAH is highly expressed 3 in oligodendrocytes, but not in other glial cell types. Thus, genetic deletion or pharmacological inhibition of FAAH may affect both neuronal activity and oligodendroglial function (e.g. myelination). The role of FAAH in oligodendrocytes was investigated in vitro. An oligodendrocyte precursor cell (OPC) monoculture was used to monitor differentiation, and a co-culture comprising neurons and OPCs was used to monitor myelination. During the differentiation of OPCs, FAAH expression was detected in the entire oligodendroglia lineage, but with high expression only in mature myelin basic protein (MBP) expressing cells. Treatment with the FAAH inhibitor PF-3845 (0.1 μM to 1 μM) increased differentiation of OPCs into mature oligodendrocytes. However, the same treatment of co-cultures had no effect on the myelination of neurites. In conclusion, this study has: i) obtained evidence that genetic deletion of FAAH is neuroprotective in a mouse model of MS and ii) provided new insights on FAAH expression in the CNS. Further investigation of FAAH, in particular its role(s) in oligodendrocytes, will be required to fully unlock the therapeutic potential of FAAH inhibition in the treatment of MS.

Self-reactive CD4+ helper T cells (Th) are key causal agents in the pathogenesis of many autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a CD4+T cell model of the demyelinating autoimmune disease multiple sclerosis (MS). It has been shown that EAE is caused by CD4+ T-cells that produce pro-inflammatory cytokines IFN-γ (Th1) and IL-17 (Th17). As such, understanding how these Th cells are generated and controlled is essential. There is debate as to whether Th1 and Th17 cells act independently in EAE or if there is plasticity between these two subtypes, and whether the capacity to switch from Th1 to Th17 confers pathogenic capacity. T-bet was first described as the master transcription factor for Th1 cells, and is thought to have a critical role in EAE even though IFN-γ, the Th1 archetypal cytokine, has been shown to be redundant. More recent work has shown that T-bet is expressed in multiple immune cell types, and it remains unclear in what cells the expression of T-bet is required for EAE. Considerable efforts have been put into understanding the role of T-bet in EAE pathogenesis, with a view to modulate cells expressing T-bet for therapy. The hypothesis of this work was that T-bet has multifaceted roles in EAE, in initiating and directing an immune response in innate antigen presenting cells such as dendritic cells (DC) as well as programming pathogenic effector CD4+ T cell (Teff) response to antigen. T-bet-/- mice were studied using different models of EAE to dissect the role of T-bet in disease pathogenesis. Active immunisation of C57BL/6 mice with the immunodominant peptide from myelin oligodendrocyte glycoprotein (MOG35-55) showed that T-bet-/- mice developed EAE with an IL-17 dominated profile and critically, T-bet-/- mice were able to produce GM-CSF which has recently been described as a key cytokine for EAE. T-bet-/- cells were not able to transfer EAE in a model of passive transfer EAE, where CD4+ T cells were polarised towards a Th1 profile in vitro. Illustrating that T-bet is required in CD4+ T cells for Th1 mediated EAE. DC driven EAE showed that T-bet-/- DC were able to activate CD4+ T cells in vitro and cause EAE upon co-transfer into host mice with transgenic CD4+ T cells. Thus, it has been shown that T-bet is not required in EAE. This work represents a step further towards understanding the disease mechanisms involved in EAE and suggests T-bet is not an appropriate therapeutic target for the treatment of MS.

Regulatory T cells (Treg) play a crucial role in controlling immune homeostasis. Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Treg. While several mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain unknown. The Treg pool consists of highly diverse subpopulations, indicating that there is a potential to optimise Treg-targeted therapies if disease-relevant mechanisms can be established. Microarray data from our lab suggests a marked upregulation of the integrin αv as well as the IL-33 receptor ST2 in Treg retrieved from the inflamed central nervous system (CNS) during experimental autoimmune encephalomyelitis compared to peripheral lymphoid organs. These two molecules were further investigated within this PhD project with the aim to understand their role in Treg function during chronic inflammatory disease. αvβ integrins have been reported to be needed for effector T cell migration to inflamed sites through binding of extracellular matrix components and are involved in TGF-β activation by a variety of cell types. Conditional knockout mice lacking the integrin αv specifically in Foxpγ+ Treg were generated to address the role of αv integrins on regulatory T cells in inflammatory disease. αv-/- Treg showed a deficiency in activating latent TGF-β, but were able to suppress responder T cell proliferation in vitro as well as in vivo. αv-/- Treg were also able to migrate to the inflamed CNS during EAE and resolve disease. However, αv-/- Treg were detected at significantly lower numbers and proportions in the inflamed gut during a curative T cell transfer model of colitis; this led to a quantitative impairment in the ability of αv-/- Treg to cure colitis when compared to wild-type (WT) Treg. Whether this is a deficit in migration, survival, proliferation, or Foxp3 stability, remains to be investigated. IL-33 acts as an alarmin and is best studied as a cytokine released upon tissue damage that induces a potent type 2 immune response by acting on a multitude of immune cells. Expression of the IL-33 receptor ST2 on Treg has recently been associated with positive metabolic parameters in visceral adipose tissue, protection from gut inflammation, and tissue-restorative function in other inflamed tissues such as injured muscle or lung. This project showed that in steady state, ST2+ Treg expressed high levels of several markers which have been associated with potent regulatory function. When stimulated in vitro, ST2+ Treg showed a better survival and expansion rate compared to their ST2- counterparts, even more so in the presence of IL-33. T-bet deficiency in Treg resulted in an increased ST2+ Treg pool, and T-bet-associated cytokine IFN-γ was found to antagonise IL-33-induced expansion of the ST2+ Treg pool in a T-bet-independent manner. When ST2+ and ST2- Treg were tested for their respective suppressive capacity in vivo, ST2+ Treg were able to suppress responder T cell expansion despite being found only at low numbers in secondary lymphoid organs compared to ST2- Treg. However, in a curative model of T cell transfer colitis, ST2+ Treg were less capable of controlling the ongoing immune response than ST2- Treg. A possible explanation for the superiority of ST2- Treg in this setting can be found in the fact that injected ST2- Treg acquired a distribution of ST2 expression reminiscent of WT Treg over the course of disease. On the other hand, an increased starting pool of ST2+ Treg as occurs in T-bet-/- Treg significantly enhanced the capacity of Treg to control colitis compared to WT Treg. In conclusion, both ST2- and ST2+ Treg are likely to have a distinct, non-redundant role in suppressing T cell activation in secondary lymphoid organs and controlling ongoing inflammation in peripheral tissue, respectively.

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.

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell mediated autoimmune disease of the central nervous system and shares many characteristics with multiple sclerosis (MS). Induction of EAE is mediated by myelin reactive CD4+ T helper (Th) cells, particularly Th1 and Th17 cells, which can be provoked by the immunization with myelin derived protein (or peptide) and Toll-like receptor (TLR) stimulus (eg, complete Freund¡s adjuvant, CFA). If given an injection of soluble peptide before immunization, mice do not develop EAE (they are tolerant). This approach has been widely applied, evoking tolerance in primary responses (i.e., in naive T cells). Therefore the first hypothesis of this thesis is that peptide induced protection from EAE is a result from T cell deletion or / and anergy. As MS patients have ongoing disease and over 85% of MS patients develop a relapsing-remitting course, memory T cells are key targets when considering peptide-induced tolerance as a therapeutic strategy. Thus, a model for ¡memory EAE¡ was established to test a second hypothesis that the myelin reactive memory T cells can be controlled by the administration of soluble peptide. Here, adoptive transfer of T cells from T cell receptor transgenic mice (2D2) recognizing myelin oligodendrocyte glycoprotein 35-55 (pMOG) was used to investigate the pMOG-reactive memory responses. Soluble pMOG administration could induce a transient expansion of 2D2 T cells followed by their loss through apoptosis. A model using double immunization was established by immunizing mice first with pMOG together with unmethylated CpG oligonucleotide (CpG) as an adjuvant, and subsequently immunizing with pMOG in CFA. This produced EAE with early onset and high incidence compared to mice which received pMOG/CFA only. Cells from mice that received the double immunization protocol produced high levels of IFN-γ, suggesting that memory T cell responses have been triggered in the mice. Administration of soluble peptide before secondary immunization could ameliorate EAE, indicating that memory T cells are susceptible to tolerance induction. pMOG-reactive memory T cells were further assessed by isolating CD4+ CD25- CD44high CD62Llow cells from pMOG-experienced 2D2 mice. These cells showed early and high production of IFN-γ, and early but transient production of IL-2, compared with naive population. These data provide basic information relevant to translating peptide-induced T cell tolerance from mice to humans.