El concepto de teoría de Suppes

1.4.1 T cells

MS pathology is suggested to be initiated by infiltration of the CNS by encephalitogenic T cells, followed by B cell recruitment and involvement. T helper cells (Th cells) are characterised by expression of CD4 molecules and are functionally distinct from cytotoxic T cells which express CD8 molecules. The association of MS with particular MHC II haplotypes suggests that antigen presentation to CD4+ T cells is a key event in pathogenesis (Gourraud et al., 2012). Polarisation of CD4+ Th cells into different functional subsets defined by the secretion of signature effector cytokines, following antigen recognition and activation of naïve T cells, is influenced by a number of factors, particularly the local cytokine environment (Dittel, 2008; Kaiko et al., 2008).

Th type 1 (Th1) cells typically secrete pro-inflammatory cytokines (IFNγ, TNF and LTα) and are important for macrophage activation and cell-mediated immunity, while secretion of interleukin (IL)-4, and IL5 is characteristic of Th type 2 (Th2) cells, which are potent helpers of

the humoral response and antibody production by B cells (Dittel, 2008; Zhu et al., 2010). Historically, the Th1 subset has been thought to be predominantly responsible for pathology, since MS-like phenotypes can be induced in naïve mice following adoptive transfer of Th1 but not Th2 cells (Khoruts et al., 1995). Also, administration of IFNγ, a Th1 effector cytokine, to RRMS patients induced disease exacerbations (Panitch et al., 1987). However the focus on the role of the Th1 subset has been shown to be overly simplistic, since other subsets have been shown to contribute to pathogenesis in animal models of MS. Investigation into the role of the Th17 subset, characterised by the expression of IL17A and IL22, has shown that adoptive transfer of MOG-specific Th17 cells in naïve mice induces meningeal TLO formation, in addition to an MS-like phenotype (Peters et al., 2011). However, while CD4+ cells are a component of perivascular cuffs and have been isolated from MS lesions, they are outnumbered by CD8+ cells (Babbe et al., 2000; Skulina et al., 2004). Depletion of CD4+ T cells in MS patients also failed to produce an improvement in the number of lesions detected by MRI, suggesting that the view of CD4+ T cells as the main pathogenic protagonists in MS too narrow (Van Oosten et al., 1997). Further investigation has revealed the important contributions of cytotoxic T cells and B cells to MS pathology (Van Oosten et al., 1997; Archelos et al., 2000; Bar-Or et al., 2010; Mars et al., 2011).

CD8+ cytotoxic T cells induce the death of cells expressing their cognate antigen on MHC I molecules through a variety of mechanisms, including release of lytic enzymes and Fas ligand- receptor binding (Mars et al., 2011). CD8+ cells also secrete pro-inflammatory cytokines including IFNγ, TNF and LTα, and thereby recruit and activate macrophages and microglia. Interestingly, CD8+ T cells from SPMS patients show co-stimulation independent secretion of LTα compared to cells isolated from RRMS patients, which is postulated to be a critical stage in the progression of MS from the earlier RRMS stage to SPMS, due to the cytotoxic effect of secreted LTα (Buckle et al., 2003). Clonal expansions of CD8+ T cells have been observed in active lesions (Babbe et al., 2000), CSF (Jacobsen et al., 2002) and serum (Skulina et al., 2004), and the number of CD8+ T cells correlates with a marker of axonal damage in MS lesions, suggestive of an active pathogenic role (Bitsch et al., 2000; Kuhlmann et al., 2002).

1.4.2 B cells

B cells form a critical component of the humoral immune response and may be activated in either a T cell dependent or independent manner. Naïve B cells which encounter their cognate antigen in the secondary lymphoid organs mature into memory B cells, which have undergone

somatic hypermutation and isotype class switch to increase the affinity of the antibodies they secrete on stimulation. Naïve and memory B cells both express MHC II molecules, enabling antigen presentation function to T cells, while memory B cells may terminally differentiate into plasma cells, enabling increased secretion of antibodies (Archelos et al., 2000).

Increasing evidence suggests that B cells play a crucial and pathogenic role in MS, since CSF B cell counts correlate with cortical inflammation in RRMS and CIS patients detected by MRI (Kuenz et al., 2008), and the presence of meningeal aggregates of proliferating B cells in F+SPMS cases is associated with more severe pathology (Villar et al., 2003; Serafini et al., 2004; Magliozzi et al., 2007). Expansion of B cell clones specific for myelin antigens has been observed in the CSF (Lovato et al., 2011), and the persistence of intrathecal autoantibodies is indicative of continual production, since the half-life of serum Ig is less than 3 weeks and is assumed to be similar for CSF (Archelos et al., 2000). Extensive investigation of Ig in serum and CSF from MS patients has revealed a range of autoantibody specificities, and no unifying autoantibody common to all MS cases has yet been identified (Moller et al., 1989; Reindl et al., 1999; Archelos et al., 2000; Haase et al., 2001; Nylander and Hafler, 2012). This suggests that either a range of autoantigens may be important in MS pathogenesis, or that initial autoreactivity to a single antigen may be followed by epitope spreading to involve additional epitopes or antigens, as has been shown in animal models of MS (Tuohy et al., 1999; Miller et al., 2007; Kuerten et al., 2012). Prominent antibody deposition has been observed in a subset of active WMLs (Lucchinetti et al., 2000; Lassmann et al., 2001), and the presence of oligoclonal CSF bands is associated with conversion from RRMS to SPMS and a significantly increased EDSS score (Villar et al., 2003), indicating a role for autoantibodies in pathology and clinical progression (Archelos et al., 2000).

Autoantibodies may opsonise myelin, resulting in macrophage-mediated demyelination, or may activate the complement cascade, leading to the formation of the membrane attack complex (MAC) and deposition of complement fragments and complexes on the myelin sheath. Complement fragments act as chemoattractants for macrophages and lymphocytes, resulting in release of cytokines, reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), which causes local inflammation and break down of the BBB. Deposition of MAC (C5b-9) on the myelin sheath opens pores in the membrane, disrupting the ionic balance and facilitating a calcium influx that disrupts impulse propagation and activates myelin-integral proteases which degrade the myelin sheath (Hartung et al., 1995; Hartung and Rieckmann, 1997). The C9neo antigen signifies formation of the terminal lytic complement

complex and has been observed co-deposited with Ig in active lesions, in phagocytic macrophages and on oligodendrocytes (Storch et al., 1998a). Antibodies have also been detected between the clathrin-coated pits of phagocytic macrophages and myelin debris (Prineas and Graham, 1981).

In addition to direct pathogenic effects such as autoantibody production, B cells may also play a significant role as APCs, by facilitating activation of T cells and their recruitment to the CNS. Depletion of B cells in the CSF by Rituximab treatment resulted in significant reduction of CSF T cells in RRMS patients (Genain et al., 1999; Cross et al., 2006) and T cell infiltration is blunted in B cell deficient mice during the onset of the in vivo model of MS, experimental autoimmune encephalomyelitis (EAE) (Pierson et al., 2013). B cells express MHC II and reactivate CD4+ T cells infiltrating the CNS, and may preferentially reactivate Th1 T cells (Pierson et al., 2013). A higher proportion of Th1 T cells in the CNS may result in increased levels of pro-inflammatory cytokines in the CSF and creation of an inflammatory milieu that is suggested to drive pathology in MS (Reynolds et al., 2011). B cells themselves may also contribute to this pro-inflammatory environment through secretion of pleiotropic cytokines including LTα and TNF (Bar-Or et al., 2010).

Although autoantibodies and complement components cannot cross the BBB, activated T and B cells can extravasate across the intact BBB into the Virchow-Robin space. Here, presentation of autoantigen on MHC II molecules results in activation of B and T cells resulting in cytokine release and intrathecal production of autoantibodies, leading to a compartmentalisation of the immune response behind the BBB. Formation of TLOs may then result in sites of persistent autoantigen presentation, autoreactive cell activation and intrathecal autoantibody production.

1.4.3 Microglia and macrophages

Microglia are the most numerous cell of myeloid origin in the CNS, and are highly specialised to carry out a variety of diverse functions including neuroprotection and repair. However, they can also initiate neurotoxic responses, and are implicated in contributing to neurodegeneration (Correale, 2014). “Resting” microglia actively and continually survey the local environment, and have a ramified morphology, characterised by fine, highly branched and motile processes with a relatively small cell soma (Nimmerjahn et al., 2005). When activated, microglia adopt a more amoeboid morphology, typified by short, thickened processes with a more rounded cell

soma, and show increased proliferation, motility, expression of pro- and anti-inflammatory mediators and antigen presenting molecules (Bogie et al., 2014). In SPMS, microglial activation is not only associated with demyelination and lesions, but also with NAGM and NAWM, indicative of a global diffuse inflammatory response within the CNS (Kutzelnigg et al., 2005).

Although considerable infiltration of phagocytic macrophages is observed in newly formed GMLs in early MS (Lucchinetti et al., 2011; Popescu and Lucchinetti, 2012), few infiltrating macrophages are observed in GMLs from progressive cases (Bo et al., 2003a). Cells with the morphology of activated microglia are observed to account for over 90% of phagocytic cells in chronic active GMLs (Peterson et al., 2001). The role of microglia in GML formation and development is unclear, as many studies have focused on WMLs, and it is uncertain to what extent, if any, the pathogenesis of WMLs and GMLs differs. Phagocytosis of myelin by microglia is thought to be a key contributing process to demyelination. Evidence of several mechanisms of demyelination have been detected in active WMLs from biopsies and autopsy material from MS cases of short duration (Lucchinetti et al., 2000), but it is suggested that the dominant mechanism in established MS WMLs becomes antibody- and complement-mediated phagocytosis (Breij et al., 2008). Microglial expression of the complement receptor CR3 allows activation of the complement cascade and phagocytosis, following binding with complement components deposited on myelin sheaths (Smith, 2001). However, the role of complement in GML pathogenesis is controversial, since significant deposition of complement fragments was not observed in purely cortical lesions (Brink et al., 2005). Microglia are known to phagocytose myelin via expression of Fc receptors, the rate of which is increased in vitro by the addition of antibodies specific for myelin components, suggesting a dependence on autoantibodies in vivo (Smith, 2001). Activation of microglia by infiltrating autoreactive T cells has also been shown to increase phagocytosis of myelin by microglia (Nielsen et al., 2009). In addition, Fc receptor- mediated phagocytosis leads to ROS production (Ueyama et al., 2004), resulting in the release of glutamate and excitotoxic damage, to which oligodendrocytes are highly susceptible (Mcdonald et al., 1998; Werner et al., 2001; Barger et al., 2007; Sierra et al., 2013). Secretion of cytokines and cytotoxic inflammatory mediators by activated microglia may also contribute to pathology and oligodendrocyte loss, since microglial activation by TNF in vitro induced production of nitric oxide, TNF and ROS (Kuno et al., 2005).

In addition to possible roles in the pathogenesis of MS, microglia and macrophages are also important for regenerative processes, and microglial expression of neurotrophic factors may

contribute to neuroprotection (Batchelor et al., 1999). The varied functions and phenotypes of microglia and macrophages can be characterised by the M1 (pro-inflammatory) – M2 (anti- inflammatory) paradigm, where the classical inflammatory functions, including secretion of pro- inflammatory cytokines and antigen presentation, are associated with polarization to the M1 phenotype, while the M2 phenotype is associated with regenerative functions and the secretion of anti-inflammatory cytokines and growth factors (Bogie et al., 2014). During remyelination, microglia and macrophages switch from the M1 to the M2 phenotype, which has been shown to drive oligodendrocyte differentiation and is crucial for efficient remyelination (Miron et al., 2013). In addition, microglia may promote remyelination through phagocytosis of cellular and myelin debris, which inhibit axonal regeneration and OPC maturation (Robinson and Miller, 1999; Wang et al., 2002; Kotter et al., 2006; Plemel et al., 2013).