High levels of serum soluble TWEAK are associated with neuroinflammation during multiple sclerosis

Multiple sclerosis (MS) is a chronic central nervous system (CNS) disease that is characterized by demyelination, inflammation and degenerative processes leading to irreversible disability [1]. Assuming the common hypothesis that degenerative processes are mainly secondary to focal and diffuse inflammation in MS, biomarkers of ongoing inflammation are needed, particularly in the perspective of therapeutics [2]. While a dozen anti-inflammatory drugs are available to clinicians, treatment decisions remain uncertain because they are based on the concept of escalation therapy. Patients commonly start with a less effective but also a less risky drug, and escalades to a more effective drug only if inflammation remains despite treatment. Thus, the depiction of inflammation is fundamental especially considering that there is an optimal therapeutic window in MS that occurs in the first years after disease onset, where the treatment may limit irreversible disability [3, 4]. The occurrence of clinical relapses and the observation of an inflammatory activity measured by CNS magnetic resonance imaging (MRI) are commonly used to depict ongoing inflammation during MS [5]. Nevertheless, the sensitivity of relapses to detect the inflammatory processes is very low [6]. Additionally, the CNS MRI explorations cannot be as frequent as would be required [7], due to MRI accessibility and cost, time of the exam, patient comfort and potentially deleterious effect of gadolinium accumulation [8]. A blood biomarker of ongoing inflammation would be eagerly beneficial in daily MS care.

MS is associated with communication disorders between the CNS and the immune system. These disorders involve specially cytokines. Among them, the cytokine TWEAK (TNFSF12) is a transmembrane protein that belongs to the TNF (tumor necrosis factor) ligand family and whose transcripts have been found in many tissues including the brain [9]. TWEAK can be released and function as a soluble cytokine [10]. Its main receptor is Fn14, a fibroblast growth factor inducible 14 kDa protein [11]. The main sources of TWEAK protein are monocytes/macrophage family cells including microglia [12]. Based on experimental MS mouse models, TWEAK has pro-inflammatory effects during CNS inflammation. Interestingly blocking the TWEAK/Fn14 pathway during immune cell recruitment across the blood brain barrier (BBB) was protective in the same model [13, 14]. Increased permeability of the BBB is an early and critical event in the development and evolution of MS. It has been established that soluble TWEAK modulated the expression of proteins that are involved in inflammation and opening of the BBB [15]. Besides, Serafini et al. described TWEAK and Fn14 up-regulation in post-mortem MS brain sections [9]. Furthermore, this increase was related to the degree of inflammation and demyelination. The absence of TWEAK/Fn14 expression in healthy brain reinforces the idea that TWEAK/Fn 14 pathway could play a role in MS pathogeny. Additionally, a membrane TWEAK expression has been described on circulating and potentially further CNS infiltrating monocytes of MS patients and not on control monocytes suggesting that TWEAK is involved in the diapedesis of monocytes during neuroinflammation [12]. Of interest, monocytes/macrophages are known to be particularly associated with tissue injury in acute MS lesions [16‐18].

Based upon these previous data, the aim of the present study was to evaluate whether serum soluble TWEAK could be a reliable biomarker of neuroinflammation in MS patients. We first determined TWEAK serum levels during a longitudinal- follow-up of MS patients started at the onset of the disease. Then we studied the relationship between TWEAK serum levels and MS course/MS inflammatory activity. Finally, we investigated the prognostic value of TWEAK serum levels for i) clinical disability and ii) CNS tissue integrity using advanced MRI techniques (magnetization transfer imaging).

The present study brought three main findings: (i) TWEAK serum levels are increased in MS patients; (ii) this increase is related to disease activity (relapses and MRI) and (iii) no relation was evidenced between soluble TWEAK and short term brain alteration of tissue structure, suggesting that TWEAK is mainly a biomarker of inflammation in MS.

Thus, for the first time, an increase of TWEAK serum levels was shown in a homogenous population of early MS patients. All the blood samples were prospectively assessed, using the same procedure and the same ELISA kit purchased from Bender Medsystems for which the team had several years of expertise [22]. A large sample of healthy controls was also assessed to define normal biological value limits. Moreover, in the present study, we took care to control confounding factors such as systemic inflammation or peripheral monocyte proliferation independent of MS. These precautions lead to demonstrate for the first time an increase of TWEAK serum levels under inflammatory conditions in early MS, suggesting the use of TWEAK as a biomarker of inflammation in daily MS care. Nonetheless, this result was expected based on previous findings. Indeed, TWEAK exerts pleiotropic effects including apoptosis [10], angiogenesis [23, 24] but also inflammation in different cell types [15, 25‐29]. In MS brain, TWEAK expression is up regulated compared to controls, particularly in tissue samples with higher inflammatory processes [9]. Macrophages, microglia and astrocytes were identified as the major sources of TWEAK in MS brain [9]. Furthermore, it was previously shown that TWEAK is expressed at the cell surface of circulating monocytes only in MS patients compared to non-MS CNS inflammatory disease controls or to patients with other neurological diseases, especially in patients included just after their first MS relapse [12]. Interestingly, none of the patients displayed TWEAK expression at the surface of lymphocytes. In experimental autoimmune encephalomyelitis (EAE), an increased severity was observed in transgenic mice overexpressing soluble TWEAK [13]. Moreover, anti-TWEAK monoclonal neutralizing antibodies have an anti-inflammatory effect in EAE and reduce disease severity [14]. All these studies strongly suggest that in MS, there is a chronic TWEAK stimulation linked to monocyte related inflammatory processes, which are particularly relevant because associated to neuro-axonal loss in MS [30, 31]. One may suppose that the increased soluble TWEAK serum levels observed in the present study are mainly due to the cleavage of membrane TWEAK from blood monocytes.

Furthermore, an originality of the present study is its longitudinal design for patients’ assessment. Evolution of TWEAK serum levels was assessed over 1 year and patients were prospectively followed over 3 years. At the group level, TWEAK serum levels decreased during follow-up but remained higher than controls. It is notable that TWEAK serum levels do not share the same kinetics at individual levels and that these levels can increase, notably when a relapse occurs. This result suggests that a TWEAK increase may be concomitant with exacerbation of inflammatory processes, and that TWEAK can be regularly measured in patient serum to advise the clinician for the best moment to perform a new MRI. Nevertheless, the kinetics and half-life of TWEAK in the serum have to be further assessed precisely to determine the optimal frequency of blood tests for monitoring MS inflammation.

The second main finding of the study is the association of high TWEAK levels with disease activity. Disease activity assessment is of primary importance in daily patient’s care, as showed, among others, by the re-examination of MS disease phenotypes [5]. In the present study, TWEAK serum levels are increased in patients presenting a relapse or a gadolinium enhanced lesion on MRI. As reported above, TWEAK expression may reflect inflammatory processes related to monocytes. Thus, an increase of TWEAK serum levels concomitant with activity in MS was expected. Moreover, a set of data strongly suggests that TWEAK may play a role in blood brain barrier (BBB) permeability modulation [15, 32‐34]. The BBB is a dynamic structure composed by endothelial cells, basal lamina, astrocytes end-feet processes, pericytes and neurons. Its main function is to regulate the passage of cellular and molecular components into the CNS. BBB integrity is impaired during MS, particularly in gadolinium contrast enhanced lesions [35, 36]. It was previously showed that TWEAK has a direct effect on the interaction between astrocytes and the basal lamina, leading to changes in BBB permeability [37]. This alteration of BBB permeability seems to be induced by the pro-inflammatory effects of TWEAK by promoting secretion of cytokines [15], activating NF-KB pathways [38] and inducing the passage of inflammatory cells, particularly monocytes [39], through the BBB involving an increased expression of MMP-9 [15, 33, 38].

Nevertheless, some discrepancy between TWEAK serum levels and activity as measured in the present study can be explained by the fact that neither relapses nor gadolinium enhancement reflect all inflammatory process in MS. In fact, relapses occur only when clinically eloquent CNS regions are affected by inflammation (phenomenon 5 to 10 times less frequent than new T2 lesions occurrence on CNS MRI) [6]. Moreover, gadolinium enhancement is known to be a transient phenomenon, lasting less than 15 days [7]. Finally, TWEAK expression was showed to be particularly increased at the edges of chronic active white matter lesions [9] that are not enhanced by gadolinium [40‐42]. Thus, an elevated TWEAK serum level can reflect subtle and diffuse inflammatory processes. In this regard, a future study assessing the potential value of TWEAK as a biomarker of inflammation in progressive forms of MS would be of interest, considering that these forms are known to be more affected by diffuse inflammation (microglial activation, chronic active lesions and meningeal inflammation) [43].

The last main finding of our study is that TWEAK serum levels were not related to tissue integrity at baseline and at 1 year of follow up. In the present study, tissue integrity was assessed by MTI, an MRI advanced technique that estimated the physical and chemical interactions between the free water proton pool and the protons close to macromolecules called the bound pool. This technique is widely used in imaging study in MS for two main reasons. The first is that guidelines for good quality and reproducibility of the technique were proposed in a white paper several years ago by the magnetic resonance in multiple sclerosis community (MAGNIMS) together with a group of North American experts, allowing its large use in imaging studies [44]. The second reason is that histological studies have showed in MS a robust correlation between MTR and demyelination and axonal loss [45]. The absence of correlation between TWEAK serum levels and short-term tissue damage assessed by MTI suggests that serum TWEAK up-regulation is not directly associated with demyelination and/or axonal loss.

Nevertheless, these preliminary findings need to be confirmed in large groups of MS patients including all phenotypes with a longitudinal assessment of controls. More importantly, kinetics and half-life of TWEAK in the serum should be assessed precisely and a potential correlation between TWEAK serum levels and ongoing inflammatory processes under anti-inflammatory treatment needs to be more precisely assessed. These conditions are needed for a clinical use of TWEAK as a biomarker of inflammation in clinical routine.

TWEAK level in the serum of MS patients is a promising biomarker of ongoing inflammatory processes in the CNS. It is a simple and reproducible serum test that could be proposed as an accurate marker of ongoing inflammation, contributing to the follow-up and the care of MS patients. Thus, TWEAK is a promising serum marker of the best window to perform brain MRI, optimizing disease control in patients.