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Growth differentiation factor 15 is increased in stable MS

Objective To assess whether serum concentrations of the anti-inflammatory cytokine growth differentiation factor 15 (GDF-15) differ in patients with highly active multiple sclerosis (MS) vs patients with stable MS and healthy controls (HCs). Methods GDF-15 concentrations were measured by ELISA in serum and CSF in a cross-sectional cohort of patients with MS, patients with other inflammatory neurologic diseases (OIND), patients with noninflammatory neurologic diseases (NIND), and healthy controls (HC). Serum GDF-15 concentrations were measured in a longitudinally sampled cohort of clinically and radiologically well-characterized patients with MS and corresponding controls. Results Cross-sectionally measured median serum GDF-15 concentrations were significantly higher in patients with OIND (n = 42) (600 pg/mL, interquartile range [IQR] = 320–907 pg/mL) compared with HCs (n = 29) (325 pg/mL, IQR = 275–419 pg/mL; p = 0.0007), patients with NIND (n = 46) (304 pg/mL, IQR = 245–493 pg/mL; p = 0.0002), or relapsing MS (n = 42) (356 pg/mL, IQR = 246–460 pg/mL; p = 0.0002). CSF and serum concentrations of GDF-15 were correlated (r = 0.41, 95% CI = 0.25–0.56, p < 0.0001). In a longitudinally sampled cohort of patients with MS (n = 48), deeply phenotyped with quantitative clinical and MRI assessments, mean GDF-15 concentrations were significantly higher in patients with a stable disease course (405 pg/mL, SD = 202) than in patients with intermittent MRI activity (333 pg/mL, SD = 116; p = 0.02). Conclusions Serum GDF-15 concentrations are increased in patients with MS with a stable disease course. These data suggest that GDF-15 may serve as a biomarker for disease stability in MS.

MS is a chronic inflammatory demyelinating disease of the CNS. 1 The disease course in MS is highly heterogeneous. Monitoring subclinical disease activity is a major challenge for clinicians caring for patients with MS. 1 Identification of biomarkers that indicate disease activity in individual MS patients is largely an unmet need. Immune cell migration across the blood-brain barrier plays an important role in the pathogenesis of MS and depends on integrins, including lymphocyte function-associated antigen 1 (LFA-1). 2 The spatially limited extension and temporal resolution of most MS lesions over time 3 suggest that anti-inflammatory mechanisms counteract the proinflammatory processes during lesion evolution.
Growth differentiation factor 15 (GDF-15) is a transforming growth factor-beta-related cytokine. 4 Under homeostatic conditions, GDF-15 expression is weak in most tissues and increases following injury in various tissues 4 including the CNS. 5 Data from animal models indicate that GDF-15 counteracts LFA-1-dependent extravasation of leukocytes into inflamed tissues, hereby limiting tissue destruction. 4 In addition, inflammation-induced GDF-15 was recently shown to protect tissues against inflammatory damage by promoting a metabolic adaptation. 6 In the current study, we hypothesized that increased serum GDF-15 reflects subclinical tissue injury in MS. We therefore measured GDF-15 concentrations in sera and also in the CSF of various cohorts of patients with MS, including patients with clinically stable MS with or without radiologic disease activity.

Methods
Standard protocol approvals, registrations, and patient consents The study was approved by the Ethical Committee Northwest and Central Switzerland, University of Basel, Basel, Switzerland, and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants.

Study subjects
Serum and CSF concentrations of GDF-15 were measured cross-sectionally in a cohort of patients with relapsing MS (rMS) and controls during routine diagnostic workup according to international consensus guidelines at the University Hospital Basel after informed consent (table 1). Serum concentrations of GDF-15 were measured longitudinally in a cohort of patients with rMS (table 2). All subjects provided written informed consent, and the study was approved by the local ethics committee.
GDF-15 measurements GDF-15 concentrations were measured in serum with a commercially available ELISA according to the protocol of the manufacturer (DuoSet, Cat.Nr. DY957, R&D Systems).

Statistical analysis
Data were tested for normality with the D'Agostino-Pearson normality test. The Mann-Whitney test was performed in case of non-normality and/or differing variance among study groups; the unpaired t test was performed in case of normally distributed data. Non-normally distributed variables are presented as median with interquartile range (IQR). Correlation of data was calculated by Pearson R. Multivariate analyses were performed by analysis of covariance. GraphPad-Prism v7.0b was used for statistical analyses.

Data availability
The sample collection procedure of subjects of cohort 1 has been described earlier. 7 Cohort 2 contained longitudinally sampled patients with rMS as part of a prospective multicenter study initiated in 2003. 8 Our study included patients with rMS who were recruited at the Neurologic Clinic and Policlinic, University Hospital Basel (Switzerland), as part of a prospective multicenter study.

Results
Analytical performance of the GDF-15 assay and stability of the analyte The mean coefficients of variation (CVs) of concentration of duplicates (intraassay variability) ranged among the different cohorts from 1.4% to 2.6%. There was no significant change in measured GDF-15 concentration in sera analyzed following 5 freeze-thaw-cycles (median % CV 4.48 [1.20-5.78]). The CV of the interassay controls ranged from 9.8% to 13.8% and thus remained well below the widely accepted CV 20% for interassay controls.

Longitudinal assessment of serum GDF-15 in patients with MS
To assess the role of GDF-15 in MS in more detail, we measured the concentration of serum GDF-15 in patients treated with interferon-beta that had been sampled longitudinally and were divided into a group of patients with clinically and radiologically stable disease course (n = 20) and into a group of patients with MS with intermittent radiologic disease activity (n = 22). MRI activity was defined as intermittently occurring gadolinium-enhancing lesions (GELs) or appearance of one or more new T2 hyperintense lesion(s) in MRI in comparison to previous scans. In these patients, GDF-15 concentrations were measured annually in all available serum samples (mean observation time 4.6 years). In the control group without radiologic disease activity (mean observation time 5.9 years),

Discussion
Several cytokines and chemokines have been proposed as biomarkers for inflammatory disease activity in MS. In the current study, we found increased concentrations of the cytokine GDF-15 in acute inflammatory CNS disorders. However, we could not confirm our initial hypothesis that increased serum concentrations of GDF-15 are associated with inflammatory disease activity in MS. On the contrary, in longitudinally sampled, well-characterized patient groups, we observed higher GDF-15 concentrations in those patients with MS with no clinical or imaging signs of acute or recent inflammatory disease activity. Increased GDF-15 concentrations may reflect an endogenous anti-inflammatory mechanism in patients with stable disease, counteracting evolving inflammatory lesions in their early stage while being still below the threshold for detection in MRI. Our data suggest that GDF-15 is not involved in the pathogenesis of MS. Alternatively, nonincreased concentrations in patients with active MS may relate to increased GDF-15 consumption during inflammation or less effective counterregulation due to impaired production of GDF-15. The mechanisms resulting in changes of serum GDF-15 in neuroinflammation remain to be determined.
Three-fold higher concentrations of GDF-15 in serum compared with CSF are compatible with an endothelial or subendothelial source of GDF-15 production. 10 GDF-15 is known to inhibit LFA-1-dependent adhesion of immune cells to endothelial cells 9 and would thus locally counteract immune cell infiltration into the CNS.
Pending further elucidation of the mechanism underlying our observation, our data suggest that elevated GDF-15 may serve as an indicator for disease stability in general or for a favorable response to disease-modifying treatments in a subgroup of patients with MS. With the advent of more and more effective treatments with different mechanisms of action, biomarkers allowing to better characterize inflammatory diseases activity are urgently needed. Our data suggest that GDF-15 deserves being further explored as a biomarker for stable disease or even response to treatment.

Study funding
This study was supported by the Swiss Multiple Sclerosis Society. AA was supported by the Goldschmidt-Jacobson Foundation. MM is supported by the Swiss National Science Foundation (PZ00P3_179662).

Disclosure
L. Kappos's institution (University Hospital Basel) has received in the last 3 years and used exclusively for research support: steering committee, advisory board, and consultancy fees (Actelion, Addex, Bayer HealthCare, Biogen Idec, Biotica, Genzyme, Lilly, Merck, Mitsubishi, Novartis, Ono Pharma, Figure 2 Longitudinal measurements of serum GDF-15 concentrations in patients with MS Concentrations of GDF-15 in patients treated with interferon-beta with a continuously stable disease course (stable) and patients with intermittent MRI activity during a median follow-up of 5.9 years (A) (mean + SD). GDF-15 serum concentrations of patients with T2 activity at time points with or without new T2 lesions (B) and patients with intermittent GEL with actual GEL and at time points without GEL (C) (median and IQR). Longitudinal changes of GDF-15 concentrations in stable patients (D) and patients with intermittent MRI activity (E) (red symbols indicate time points with MRI activity). *p < 0.05. GDF-15 = growth differentiation factor 15; GEL = gadolinium-enhancing lesion; SD = standard deviation.