Immune responses in demyelinating diseases

One proposed etiology of MS is cell-mediated autoimmunity against CNS components, due to a failure of negative selection of autoreactive lymphocytes in the thymus and breakdown of peripheral immunological tolerance (Oleszak et al., 2004; Sospedra and Martin, 2005). Innate and adaptive immune responses, including T-cells, B-cells and macrophages contribute to disease progression in MS (Figure 1-4).

The most important target of autoaggressive immune cells in MS is the myelin sheath (Dal Canto et al., 1995). CD4⁺ T-cells directed against MBP have been detected in the cerebrospinal fluid (CSF) of MS patients (Burns et al., 1983). In addition, CD8⁺ T-cells are involved in myelin damage (Sospedra and Martin, 2005). They can be observed in the CSF and appear to persist for months or years in MS patients (Huseby et al., 2001). CD8⁺ T-cells are supposed to be a central reservoir of memory T-cells which could be mobilized during acute attacks (Lassmann et al., 2007).

20 Chapter 1 - Introduction

Figure 1-4. Pathogenesis of multiple sclerosis.

B- and T-cells are primed in the periphery, mainly in lymph nodes and spleen. This T-cell activation leads to receptor expressions that attract immune cells to the CNS. Inside the CNS T-cells are reactivated by myelin fragments presented by antigen presenting cells (macrophages, microglia and astrocytes). The reactivation induces cytokine and chemokine release that attract more pro-inflammatory cells to the CNS. Infiltrating immune cells and activated microglia are able to cause direct damage to oligodendrocytes due to their cytotoxic products such as reactive oxygen (Baranzini et al., 1999; Baranzini, 2004). Ag: antigen, IFN-γ: interferon-gamma, IL: interleukin, MHC:

major histocompatibility complex, NO^-: nitric oxide, O2: oxygen,TCR: T-cell receptor, TNF-α: tumor necrosis factor alpha.

The observation that CD4⁺ and CD8⁺ T-cells use different effector mechanisms within the CNS has important implications for the understanding of the pathogenesis of MS (Hartung et al., 2005). Usually T-cells directed against host proteins are eliminated by Fas-mediated apoptosis. Nonetheless, few autoimmune cells may escape this selection process. T-cells that recognize myelin can be observed in the peripheral circulation of MS patients, as well as in healthy humans without neurological disorders

Chapter 1 - Introduction 21

(Pette et al., 1990). Therefore, additional factors have to play a role in the fate of these autoimmune cells and the induction of clinical symptoms. Peripheral immunological tolerance and autoaggressive lymphocytes are controlled by regulatory T-cells.

Different, yet undetermined stimuli cause a dysregulation of this regulatory cell population (von Herrath and Harrison, 2003) and a subsequent failure of peripheral tolerance mechanisms of the immune system in MS. A promising strategy for new therapies is the modulation of peripheral tolerance pathways (Hartung et al., 2005).

Activated autoreactive T-cells release cytokines that are important for the migration and homing of cells to the target site and for the initiation of inflammation, including Th1 and the Th2 immune responses. In MS, acute relapses are associated with a dominance of the Th1 immune response (Steinman, 2000). Treatments that restore the immune balance or promote a Th2 phenotype represent potential strategies to prevent relapses (Hartung et al., 2005). Th17-cells are also involved in MS, playing an important role in the induction of the disease (Aranami and Yamamura, 2008). It has been observed that IL-17 mRNA expression is increased in acute CNS lesions and in the CSF of MS patients during relapses (Lock et al., 2002; Matusevicius et al., 1999). It is supposed that Th17-cells are attracted to the CNS or that this cell population is expanded in the CNS. In addition, CD8⁺ T-cells, astrocytes and oligodendrocytes produce IL-17. The release of neurotoxic substances leads to demyelination (Tzartos et al., 2008). Furthermore, Th17-cells initiate astrocytic IL-6 production in MS (Graber et al., 2008). It has been shown that the regulatory cytokine TGF-β in conjunction with IL-6 activates the Th17 cell lineage (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006). Therefore, one could speculate that an excessive IL-6 production might break the immunological tolerance and perpetuates an autocrine loop in which the Th17-cell lineage is expanded, which induces more IL-6 with disastrous consequences in MS patients (Graber et al., 2008). Natural Tregs expressing FOXP3 represent a lineage that maintains central immunological tolerance (Sakaguchi, 2006). Studies have demonstrated that MS patients have either decreased or equal numbers of circulating Tregs compared to healthy individuals (Khoury et al., 2000; Viglietta et al., 2004). In addition, it has been shown, that the suppressive activity of Tregs of MS patients is reduced in vitro (Kumar et al., 2006). Furthermore, decreased levels of

22 Chapter 1 - Introduction

FOXP3 have been observed in natural Tregs of affected patients, indicative of an impaired immune regulatory mechanism (Huan et al., 2005). However, since other studies have not observed any differences of the FOXP3 expression between healthy individuals and MS patients, the exact role of Treg remains to be determined (Viglietta et al., 2004).

The detection of activated B-cells and elevated immunoglobulin titers in the CSF of MS patients indicates a local production of myelin-specific antibodies (Baranzini et al., 1999). These antibodies can be observed as oligoclonal bands by electrophoresis and represent an important diagnostic feature of MS. Unlike T-cells, which appear to be activated in an episodic fashion, B-cells are permanently activated. Therefore, antibodies can be identified in the CSF of MS patients even when the disease is clinically silent (Cepok et al., 2005a). Interestingly, the presence of activated B-cells correlates with the local immunoglobulin production as well as with the inflammatory disease activity. Autoreactive myelin-specific B-cells are generated spontaneously during the process of genetic rearrangement of precursor B-cells and are retained in the lymph nodes and spleen. Upon activation, these B-cells undergo clonal expansion in peripheral lymphoid organs and migrate to the CNS. So far, the factors that trigger the activation and clonal expansion of B-cells in the periphery are largely unknown (Cepok et al., 2005a).

Autoimmune mechanisms such as epitope spreading and molecular mimicry appear to be involved in MS (Sospedra and Martin, 2005). Epitope spreading causes priming of lymphocytes with self-antigens during demyelination, resulting in the generation of myelin-reactive T-cells (Katz-Levy et al., 2000). In MS patients, a closure of the blood brain barrier (BBB) is supposed to isolate the CNS from peripheral lymphoid organs and favors local antigen presentation, as well as plasma cells formation and antibody production within the brain. This process of compartmentalization might be responsible for prolonged neuroinflammation and possibly therapy failure in some MS patients (Lassmann et al., 2007). So far, the onset and dynamics of CNS compartmentalization in MS remain undetermined (Meinl et al., 2008; Serafini et al., 2004).

Chapter 1 - Introduction 23

1.4.2 Theiler’s murine encephalomyelitis

The TMEV-induced demyelination is associated with a delayed-type hypersensitivity reaction (Dal Canto et al., 2000). Demyelinated lesions in the white matter of the spinal cord of susceptible SJL/J-mice are characterized by a large number of T-cells, including virus-specific CD4⁺ and CD8⁺ T-cells as well as B-cells and macrophages (Gerhauser et al., 2007b; Oleszak et al., 1995). Immune responses during TME in the CNS have been described as protective, but they also contribute to the immune-mediated demyelination process. Resistant C57BL/6-mice eliminate the virus from the CNS gray matter after the acute phase, using specific humoral and cellular immunity (Monteyne et al., 1997), as well as by apoptosis of infected cells (Tsunoda, 2008).

Activated Natural Killer (NK)-cells are crucial for viral elimination during the early disease phase in TMEV-resistant C57BL/6-mice (Welsh et al., 2004). In addition, T-cells, especially CD8⁺ cytotoxic T-cells are involved in the TMEV-clearance (Tsunoda and Fujinami, 1996). However, CD4⁺ and CD8⁺ lymphocytes also contribute to myelin damage in advanced lesions of susceptible SJL/J-mice (Oleszak et al., 2004). These immune cells destroy virus-infected cells but also uninfected oligodendrocytes. The failure to clear the virus observed in susceptible SJL/J-mice leads to viral persistence, mostly in macrophages and/or glial cells (Tsunoda and Fujinami, 1996). The mechanisms involved in impaired viral clearance are related to an inhibition of CD8⁺ cytotoxic T-cells, due to an increased CNS production of TGF-β, produced by infiltrating immune cells (Oleszak et al., 2004). The inadequate antiviral immune response in SJL/J-mice leads to a subsequent delayed-type hypersensitivity, and autoimmune processes in the spinal cord white matter during the chronic phase (Lipton, 1975; Monteyne, 1999; Tsunoda and Fujinami, 1996; Tsunoda, 2008). Th 17-cells have been found to promote chronic demyelinating disease in TMEV-infected mice. IL-17 up-regulates antiapoptotic molecules and, consequently, increases persistent infection by enhancing the survival of virus-infected cells and blocking target cell destruction by cytotoxic T-cells (Hou et al., 2009).

Ongoing mechanisms in the CNS of susceptible SJL/J-mice include epitope spreading (McMahon et al., 2005) and the development of apoptosis resistance of myelin-specific

24 Chapter 1 - Introduction

lymphocytes during the chronic disease phase (Oleszak et al., 2003; Drescher et al., 1997; Njenga et al., 2004). In TMEV, macrophages and microglia are responsible for presenting viral- and autoantigens to T-cells (Zheng et al., 2001) as well as for the production of toxic factors, reactive oxygen species (Oleszak et al., 2004) and matrix-metalloproteinases (Ulrich et al., 2006; Liuzzi et al., 1995; Katz-Levy et al., 2000).