Our study represents a comprehensive investigation of the dual functions of soluble TNF and transmembrane TNF in the pathophysiology of EAE carried out with pharmacological inhibitors. By comparing the effects of XPro1595, a selective soluble TNF inhibitor, and etanercept, a non-selective TNF inhibitor, we directly demonstrate that transmembrane TNF signalling is necessary for functional recovery, axon preservation and, most importantly, remyelination in a murine model of multiple sclerosis. Our findings build upon genetic evidence showing that soluble TNF is acutely proinflammatory, whereas transmembrane TNF is chronically involved in repair, and suggest that pharmacological inhibition of soluble TNF may be effectively translated into a therapy for multiple sclerosis. Furthermore, this work includes a complete investigation of TNF receptor cellular localization in the spinal cord of EAE mice and of patients affected by progressive multiple sclerosis, providing a basis for a better understanding of the role of TNF signalling in the human pathology, as well as its experimental model.
The evidence emerging from our study, in addition to extensive data in the literature, leads us to formulate the hypothesis that selective blockade of soluble TNF, which has the highest affinity for TNFR1, and preservation of signalling mediated by transmembrane TNF, which conversely has the highest affinity for TNFR2, causes a shift in the balance of TNF receptor activation towards TNFR2, and the protective functions associated with transmembrane TNF would likely be attributed to TNFR2-mediated processes (Caminero et al., 2011
). However, mindful that transmembrane TNF is also capable of activating TNFR1, we cannot exclude the possibility that transmembrane TNF-mediated TNFR1 activation could also result, at least in part, in protection.
We show that in MOG-induced EAE, inhibition of soluble TNF significantly improves the clinical outcome when administered after disease onset, hence with a clinically relevant regimen. After overcoming the acute phase of disease, which develops similarly to etanercept-treated and control mice, XPro1595-treated mice recover from paralysis and maintain long-term function. Although it is well documented that activation of TNFR1 signalling is associated with a proinflammatory effect that sustains the initiation phase of EAE and causes demyelination (Korner et al., 1997
; Akassoglou et al., 1998
; Eugster et al., 1999
; Kassiotis et al., 1999
; Arnett et al.
; Kassiotis and Kollias, 2001
; Gimenez et al.
), our data suggest that long-term improvement is not dependent, at least not exclusively, on the ability of XPro1595 to antagonize inflammation by inhibiting this pathway. Indeed, both XPro1595 and etanercept equally attenuate inflammation, as demonstrated by reduced macrophage infiltration and expression of proinflammatory cytokines and chemokines (B and ). Nevertheless, the anti-inflammatory effect of etanercept is not sufficient to stimulate recovery, indicating that it is the protective effect of transmembrane TNF signalling, which is unaffected by XPro1595, to ultimately drive the positive outcome in chronic disease. It is also noteworthy that, except for the reduction in macrophage infiltrates into the cord, treatment with either drug after disease onset did not alter the immune cell profile in spleen and spinal cord, particularly encephalitogenic CD4 and CD8 T cell subsets. It has been recently shown that prophylactic treatment with a TNFR1-selective antagonistic TNF mutant reduces the severity of EAE by suppressing Th1 and Th17 responses, as well as immune cell infiltration into the cord (Nomura et al., 2009
). This is not the case in our experimental paradigm, as we begin administration after disease onset, when activation of the immune response is already underway. Based on our data, we can conclude that delayed therapeutic inhibition of soluble TNF in fully developed disease is independent of immune cell modulation, and is rather associated with a direct effect on the CNS compartment. Indeed, our findings show that XPro1595 treatment increases the number of myelinated axons and the expression of neuronal-specific molecules in EAE spinal cords, while drastically diminishing the number of degenerated axons, suggesting that transmembrane TNF may play a role in axonal sparing. Although we did not assess neuronal survival per se
, the reduction in axonal pathology could be the consequence of the ability of transmembrane TNF to directly or indirectly activate neuroprotective cascades. Based on our hypothesis of an XPro1595-mediated balance shift towards TNFR2 activation, we can speculate that TNFR2-mediated neuroprotection is occurring, while at the same time TNFR1-mediated neurotoxic events are minimized. This concept is supported by published evidence showing that activation of TNFR2 promotes neuronal survival under a variety of pathological conditions, such as ischaemia reperfusion, glutamate- and β-amyloid-induced cytotoxicity (Shen et al., 1997
; Fontaine et al., 2002
; Marchetti et al., 2004
), whereas TNFR1-mediated signalling is associated with neuronal cell death (McCoy et al., 2006
; Wen et al., 2006
; He et al., 2007
; McCoy and Tansey, 2008
; McAlpine et al., 2009
). Our own immunohistochemical data show that TNFR2 is absent in neurons, both in mouse EAE and human multiple sclerosis ( and ), suggesting that transmembrane TNF/TNFR2-mediated neuroprotective effects occur via indirect mechanisms. On the contrary, TNFR1 is highly expressed in neurons ( and ); therefore, by reducing the tone of TNFR1 activation as a consequence of blocking soluble TNF, we may be directly dampening TNFR1-mediated neurotoxicity and pro-apoptotic function. This could be especially important in the human pathology, where administration of a selective soluble TNF inhibitor, by virtue of a limited neuronal TNFR1 activation, could play a role in delaying or reducing neurodegeneration, and hence prevent patient disability. On the other hand, it has also been described that NF-ΚB signalling engaged downstream of TNFR1 activation in neurons is responsible for counteracting TNFR1-mediated pro-apoptotic effect (Kaltschmidt et al., 1999
; Taoufik et al., 2007
), therefore we cannot exclude that transmembrane TNF-mediated activation of TNFR1 may be acting, at least partially, as anti-apoptotic signal preventing neuronal death. This possibility is further corroborated by the findings of Taoufik et al.
(2011) which, in agreement with our study, demonstrate that selective inhibition of soluble TNF by XPro1595 is protective in EAE and such effect is dependent, at least in part, upon maintenance of neuroprotective neuronal NF-ΚB activity.
A crucial finding of our study is the demonstration that transmembrane TNF signalling is essential in preserving myelin integrity and compaction and, most importantly, in promoting remyelination. This, combined with the immunohistochemical evidence on EAE models that TNFR1 and TNFR2 are both expressed on oligodendrocytes and TNFR2 is expressed on oligodendrocyte precursor cells, suggests that TNF plays a direct role in modulating properties and functions of the oligodendrocyte compartment. In our EAE model, and in agreement with previous reports (Arnett et al., 2001
), remyelination is accompanied by a significant increase in the number of NG2+
oligodendrocyte precursor cells (F); therefore, a plausible scenario is that signalling mediated by transmembrane TNF via TNFR2 induces the proliferation and/or survival of oligodendrocyte precursor cells, thereby increasing the pool of cells readily available to remyelinate spared axons. We also found that oligodendrocytes express TNFR1. Hovelmeyer et al. (2005
) have documented that TNFR1-mediated oligodendrocyte apoptosis is a key event in the induction of EAE. More recently, Paintlia et al. (2011
) demonstrated that synergistic action of TNF and IL17 can also cause oligodendrocyte apoptosis via a TNFR1-mediated mechanism. Based on this evidence we can speculate that, by shifting the balance towards a preferential activation of TNFR2, XPro1595 may also contribute to prevent oligodendrocyte apoptosis.
Another possibility in explaining the protective effect of Xpro1595 is that transmembrane TNF is required for establishing immune tolerance. The inhibition by etanercept of transmembrane TNF signalling on specific cell populations, e.g. dendritic cells, which have tolerogenic potential (Fu and Jiang, 2010
), could disrupt cross-talk with effector T cells and prevent the formation of tolerizing T cells, allowing for continued immune attack of oligodendrocyte precursor cells and oligodendrocytes resulting in ongoing demyelination.
Another important finding of our study is that TNFR2 is highly expressed in microglia following EAE (). It has been recently shown by Veroni et al. (2010
) that TNFR2 stimulation in microglia promotes the expression of anti-inflammatory and neuroprotective genes such as granulocyte colony-stimulating factor (GCSF), adrenomedullin and IL-10, suggesting that microglia may contribute to the counter-regulatory response activated in neuropathological conditions. On this basis, we can speculate that microglial TNFR2 activated by transmembrane TNF may be promoting the synthesis of protective molecules participating in the overall positive outcome of XPro1595 treatment. We also found expression of TNFR2 in microglia and macrophages localized within and around multiple sclerosis lesions in the spinal cord, suggesting that similar transmembrane TNF/TNFR2-mediated protective events could also be taking place in the human pathology. To our knowledge, this is the first report of TNFR2 expression in microglia in multiple sclerosis tissue, since thus far microglial TNFR2 was only identified in the brains of patients with AIDS (Sippy et al., 1995
It should also be noted that an interesting pattern of TNFR1 expression was found in spinal cord multiple sclerosis lesions, specifically around and within CNPase+
myelin rings (C). The presence of TNFR1 in oligodendrocyte myelin lamellas wrapped around axons, and possibly at the axon–oligodendrocyte interface, provides strong anatomical evidence of the involvement of TNF signalling in the direct regulation of oligodendrocyte function. Our finding complements previous works showing TNFR1 expression in oligodendrocytes in the brain of patients with multiple sclerosis (Raine et al., 1998
). Similarly to the mouse, this may suggest that TNFR1 activation could lead to oligodendrocyte damage and apoptosis. Therefore, in view of a possible therapeutic application in multiple sclerosis, we can speculate that by shifting the balance towards a preferential TNFR2 activation, XPro1595 administration could result in reduction and/or inhibition of oligodendrocyte apoptosis, hence protection from demyelination.
Lastly, it is worth mentioning that, unlike etanercept, XPro1595 does not directly block lymphotoxin (Zalevsky et al., 2007
), a cytokine belonging to the tumour necrosis factor superfamily and capable of binding to both TNFR1 and TNFR2. Lymphotoxin is secreted by activated T cells (Zipp et al., 1995
) and is expressed in multiple sclerosis plaques (Selmaj et al., 1991
). Although the role of lymphotoxin in EAE is controversial, most studies seem to indicate that inhibition of the lymphotoxin pathway may be protective against EAE (Aktas et al., 2006
). Our work with XPro1595 and etanercept, although it was not specifically directed at investigating the role of lymphotoxin, seems to indicate that lymphotoxin plays a marginal role in EAE, at least in sustaining the chronic phase of the disease. Indeed XPro1595, which does not block lymphotoxin, has a chronic protective effect, while etanercept, which blocks lymphotoxin, does not show protection. We cannot exclude, however, that XPro1595 may indirectly reduce the tone of lymphotoxin signalling by reducing lymphotoxin expression, which could contribute to the protective effect of the compound in EAE.
Collectively, our data suggest that, in the balance between demyelination and remyelination, it is the positive effect of transmembrane TNF in the remyelination process that ultimately accounts for the observed functional recovery and resolution of EAE. This carries important clinical implications for multiple sclerosis therapy. Since human multiple sclerosis is primarily a relapsing–remitting disease characterized by alternating damage and repair phases, the challenge in therapies targeting multiple sclerosis is to block the damage while still allowing repair to take place. Using a pharmacological approach, here we show that soluble TNF is responsible for the damage, whereas transmembrane TNF drives the repair process. XPro1595 maintains the protective properties of TNF and allows remyelination to occur. Therefore, unlike non-selective TNF inhibitors, which are associated with demyelination when administered in human therapy, XPro1595 represents a promising new candidate to be added to the limited repertoire of multiple sclerosis modulating drugs, finally opening the door to the introduction of a TNF inhibitor into multiple sclerosis therapy.