CSF contents of IL-17 predicts disability progression and neurodegeneration in relapsing remitting Multiple Sclerosis L. Brambilla1, C. Zecca2, V. Torri Clerici1, L. Pareja1, G.
Camera1, J. Perugini1, R. Frangiamore1, F. Andreetta1, P. Confalonieri1, G. Brenna1, F. Torelli3, C. Gobbi2, R. Mantegazza1, S. Rossi1
1IRCCS Fondazione Istituto Neurologico Carlo Besta, MIlan, 2Neurology Department, Regional Hospital Lugano (EOC), Lugano, Switzerland, Lugano, 3Merck Spa, via Casilina 125, Rome, Italy
Background: Multiple sclerosis (MS) is characterized by inflam- mation driven by Interleukin 17 (IL-17). However the recently developed monoclonal antibody therapies against IL-17 have not still reached solid efficacy results.
Objective: To investigate the role of cerebrospinal fluid (CSF) levels of IL-17 in the inflammatory and degenerative processes of MS.
Methods: CSF determination of IL-17 in 200 treatment-naïve relapsing-remittent MS (RRMS) patients with at least 2 years of follow-up at the Neurological Institute Carlo Besta in Milan (Italy) and at the Hospital of Lugano (Switzerland). All RRMS patients started immunomodulatory treatment (interferon beta-1a 43%, interferon beta-1b 8%, glatiramer acetate 39%, fingolimod 10%) at the time of diagnosis, after CSF withdrawal. Treatment was considered as covariate in regression analysis. Clinical, Magnetic Resonance Imaging (MRI) and optical coherence tomography follow-up was performed, after patients stratification according to their CSF IL-17 levels. Average retinal nerve fiber layer thickness (RNFL) for 360° around optic disk was recorded and analyzed using the Fast RNFL analysis.
Results: IL-17 levels were not different between patients with active/not active MRI scans performed at the time of lumbar puncture (p>0.05). No significant difference between IL-17+ and IL-17- were revealed by survival analysis considering time to first clinical relapse (p>0.05) and the time to detect an active MRI scan at the follow-up (p>0.05). Conversely, mean progression index was significantly higher among subjects with detectable CSF IL-17 (p< 0.05). In line with this, the frequency of subjects with sustained EDSS worsening was higher in IL-17+ group (p< 0.05). A multiple logistic regression model showed that the probability to reach EDSS 3.0 or 4.0 was significantly affected by the CSF presence of IL-17, together with the duration of the disease. A significant main effect of IL-17 CSF contents was revealed ana- lyzing both RNFL thickness (p< 0.05) and macular volume (p< 0.05), indicating a damage of neuronal structures among IL-17+ group.
Conclusion: CSF IL-17 detection is not associated to early inflammatory activity, but to neurodegenerative processes in relapsing-remittent MS.
Disclosure
This work was supported by Merck Serono.
Dr. Rossi acted as an Advisory Board member of Biogen Idec, Bayer Schering, Merck Serono, Teva, Novartis and Genzyme, and received funding for traveling and honoraria for speaking or writ- ing from Biogen Idec, Merck Serono, Teva, Novartis, Bayer Schering, Genzyme, Almirall. She received support for research project by Teva, Merck Serono and Bayer Schering and is involved as principal investigator in clinical trials for Teva and Roche. Dr Mantegazza acted as an Advisory Board member of Biomarin. He received funding for traveling and honoraria for speaking from
Sanofi-Aventis, Grifols, Teva, Bayer, Biomarin, Alexion, Argenx. He is involved as principal investigator in clinical trials for Alexion, Merck Serono, Hoffman-La Roche, Teva, Biogen, Biomarin, Almirall, Novartis, Genzyme, Catalyst.
Dr. Torri Clerici acted as an Advisory Board member of Biogen Idec and Novartis, and received funding for traveling and hono- raria for speaking or writing from Teva, Novartis, Genzyme, Almirall. She received support for research project by Almirall. Dr. Confalonieri acted as an Advisory Board member of Biogen Idec and received funding for traveling from Biogen Idec, Novartis and Teva.
Dr Torelli is a full time employee at Merk Italy.Dr Zecca received financial support from Teva, Merck Serono, Biogen Idec, Genzyme and Novartis.
Dr Gobbi received financial support from Teva, Merck Serono, Biogen Idec, Genzyme and Novartis.
The other authors have nothing to disclose. P929
IL-10 in multiple sclerosis: unexpected excitotoxic effects and clinical implications
J. Perugini1, V. Torri Clerici1, C. Zecca2, L. Pareja1, G. Camera1, L. Brambilla1, S. Bonanno1, F. Andreetta1, C. Antozzi1, F. Torelli3, C. Gobbi2, R. Mantegazza1, S. Rossi1
1Neuroimmunology and Neuromuscular diseases, IRCCS Fondazione Isitituto Neurologico Carlo Besta, Milan, Italy, 2Regional Hospital Lugano, Lugano, Switzerland, 3Merck Spa, via Casilina 125, Rome, Italy
Background: Relapsing-remitting multiple sclerosis (RRMS) has extremely variable clinical course, and limited availability of dis- ease severity predictors at an early stage of the disease challenges the clinical practice. We have previously shown that pro-inflam- matory cytokines, as Interleukin-1β (IL-1β), have the potential to drive inflammatory neurodegeneration, and their levels are useful for the identification of rapidly progressing patients. The effects of central anti-inflammatory cytokines are still elusive.
Objective: To investigate the role of IL-10 in the inflammatory and degenerative processes of MS.
Methods: Cerebrospinal fluid (CSF) determination of IL-10 in 200 RRMS patients with at least 2 years of follow-up at the Neurological Institute Carlo Besta in Milan, Italy, and at the Hospital of Lugano, Switzerland. Clinical, MRI and optical coher- ence tomography (OCT) follow-up in patients stratified according to their CSF IL-10 levels
Whole-cell patch clamp recordings and cell swelling measure- ments in mouse brain slices to assess neuronal effects of cytokines. Results: The mean annualized relapse rate CSF withdrawal, the number of subjects with 2 or more clinical relapses in the first two years of the disease, the number of subjects with an active MRI scan in the first two years of the disease, were slightly but not significantly lower in group with detectable CSF levels of IL-10 (IL10+ group; p>0.05 for each comparisons). Mean progression index was significantly lower among subjects with undetectable IL-10 (p< 0.05) and the frequency of subjects with sustained EDSS worsening was higher in IL-10+ group (p< 0.05). RNFL and macular volume were lower in IL-10+ group (p< 0,05), sug- gesting a major damage of neuronal structures. Given these effects of central IL-10 on neurodegenerative processes and disability
progression in RR MS, we investigated the direct effects of Il-10 on neurons. The duration of glutamate-mediated excitatory post- synaptic currents increased in the presence of IL-10. In parallel, time-dependent neuronal swelling was significantly more pro- nounced in slices incubated with IL-10.
Conclusion: IL-10 showed an unexpected excitotoxic effect in vitro in parallel to association with markers of disability progres- sion and neurodegeneration in RRMS patients, despite its anti- inflammatory action.
Disclosure
Dr. Rossi acted as an Advisory Board member of Biogen Idec, Bayer Schering, Merck Serono, Teva, Novartis and Genzyme, and received funding for traveling and honoraria for speaking or writ- ing from Biogen Idec, Merck Serono, Teva, Novartis, Bayern Schering, Genzyme, Almirall. She received support for research project by Teva, Merck Serono and Bayer Schering and is involved as principal investigator in clinical trials for Teva and Roche. Dr Mantegazza acted as an Advisory Board member of Biomarin. He received funding for traveling and honoraria for speaking from Sanofi-Aventis, Grifols, Teva, Bayer, Biomarin, Alexion, Argenx. He is involved as principal investigator in clinical trials for Alexion, Merck Serono, Hoffman-La Roche, Teva, Biogen, Biomarin, Almirall, Novartis, Genzyme, Catalyst.
Dr. Torri Clerici acted as an Advisory Board member of Biogen Idec and Novartis, and received funding for travelling and hono- raria for speaking or writing from Teva, Novartis, Genzyme, Almirall. She received support for research project by Almirall. Dr. Antozzi received funding for travelling from Teva, Merck and Biogen
Dr Torelli is a full time employee at Merk Italy.
Dr. Zecca received financial support from Teva, Merck Serono, Biogen Idec, Genzyme and Novartis
Dr. Gobbi received financial support from Teva, merck Serono, Biogeon Idec, Genzyme and Novartis.
The other authors have nothing to disclose P930
Signature of neurodegeneration due to primary cytodegeneration and adaptive immune responses R. Haag, T. Hochstrasser, C. Roggenkamp, F. Kramer, C. Schmitz, M. Kipp
Department of Anatomy II, Ludwig Maximilian University, Munich, Germany
Multiple sclerosis (MS) represents more than an inflammatory dis- ease: it harbors several characteristic aspects of a classical neuro- degenerative disorder, i.e. damage to axons, synapses and nerve cell bodies. While several sophisticated treatment options exist to dampen inflammatory-driven tissue damage, modalities to prevent early and late neurodegeneration are still in their infancy. A better understanding of the neurodegenerative signature in MS patients and respective animal models is therefore urgently needed.
In this study we use three distinct gold-standard methods to investigate the magnitude and morphological appearance of neuro- degeneration in the cuprizone model. In this model, neurodegen- eration occurs in the absence of peripheral immune cells. Furthermore, such foci can form the basis for secondary immune cell recruitment (see Scheld et al and Kipp; J Neurosci. 2016 Jan
27;36(4):1410-5). Loss of neuronal cell bodies was addressed by design-based stereology. Dendritic trees, spines and synapses were reconstructed in a 3-dimensional (3D) environment. Serial block- face scanning electron microscopy was used to detect different morphological features of axonal damage. Finally, genome-wide array analyses were performed to correlate morphological altera- tions with the transcriptome.
First analyses show that neuronal elements are subjected to degenerative processes in both, the grey and white matter. Demyelination and adaptive immune responses are not just evident in the white matter (corpus callosum) but as well occur in the cor- tex in a layer-specific fashion. Several pro-inflammatory genes are up-regulated in the cortex including chemokines, protease inhibi- tors, complement component receptors, and synaptic proteins. 3D-reconstruction of callosal fibers on the ultrastructural level reveals different types of axonal injury among (i) focal swellings of axon with intact myelin sheaths, (ii) splitting of the innermost myelin lamella from the axon with focal swelling, or (iii) complete axonal transection. Furthermore, morphological and transcrip- tomal changes suggestive of synaptic plasticity were detected in the cortex.
This study, which is ongoing, shows the complex nature of neurodegeneration in a progressive, non-immune cell driven, MS animal model. A better understanding of how such morphological changes occur, and which factors regulate them, will pave the way for the development of novel, holistic neuroprotective strategies. Disclosure
- Robert Haag: nothing to disclose - Tanja Hochstrasser: nothing to disclose - Caroline Roggenkamp: nothing to disclose - Franziska Kramer: nothing to disclose - Christoph Schmitz: nothing to disclose
- Markus Kipp: This study was supported by SFZ ProMyelo (MK). The technical support by Renovo Neural is acknowledged. P931
At clinical onset CXCL13 identifies MS patients with higher intrathecal IgG production and cortical atrophy
M. Puthenparampil1, L. Federle1, S. Miante1, E. Toffanin1, S. Ruggero1, M. Ermani2, F. Rinaldi1, P. Gallo1
1Multiple Sclerosis Centre of Padua, Department of Neuroscience, University of Padua, 2Department of Neuroscience, University of Padua, Padua, Italy
Background: Since B-cells play a relevant role in multiple scle- rosis (MS) pathology, CXCL13 may be a marker for naïve B cells recruitment in the CNS.
Objective: To evaluate the correlation between locally produced IgG, CXCL13 and MRI cortical parameter in patients with clini- cally isolated syndromes (CIS) suggestive of MS or early relapse- onset MS (eRRMS) at clinical presentation.
Methods: Paired serum and CSF were obtained from 44 patients with CIS/eRRMS and 18 healthy controls (HC). Routine exami- nation of CSF and serum included Intrathecal IgG synthesis eval- uation by means of quantitative formulae (IgG Index, IgG Hyp. Function for IgG intrathecal synthesis fraction (IgGIF) and Local Production (IgGLoc)) and demonstration of IgG oligoclonal bands (IgGOB). CXCL13 were detected by means of a highly
sensitive ELISA. CXCL13 ratio (CSF-CXCL13/serum-CXCL13, QCXCL13) and Indexes (QCXCL13/QAlb, CXCL13Index) were evalu- ated. Global Cortical Thickness (gCTh) were also calculated on 3D-T1 sequences by means of Freesurfer.
Results: Compared to HC, MS patients presented higher intrathe- cal IgG indexes (higher IgG-CSF, IgG Index and IgGIF values (all p-values < 0.01); higher frequency of IgGOB, p< 0.0001)). We confirmed the lower BAFF-Index value in MS patients (12.3±5.5) compared to HC (17.5±5.2, p< 0.005), while CXCL13-CSF con- centration and Index were higher in MS patients (22.3±31.6pg/mL and 82.8±121.3 respectively) than in HC (0.8±1.5 pg/mL and 4.2±6.3 respectively, p< 0.001 for both comparisons). CXCL13 values explained CSF features (IgG intrathecal synthesis) better than the presence of IgGOB. Therefore, we divide MS patients in 2 groups: 23 CXCL13+ patients (CSF-CXCL13 concentration > 7.8 pg/uL) and 21 CXCL13-. CXCL13+ presented higher IgG- CSF, IgG Index and IgGIF values (all p-values < 0.05) and lower CSF-BAFF concentrations (65.5±26.0 pg/uL) when compared to CXCL13- (49.3±18.7 pg/uL, p< 0.05). While IgG intrathecal syn- thesis strongly correlated with CSF-BAFF in CXCL13- (r: -0.7, p< 0.001 for IgGIndex; r: -0.5, p< 0.05 for IgGLoc and IgGIF), in CXCL13+ they correlated with QCXCL13 (r: +0.6, p< 0.005 for IgGIndex; r: +0.5, p< 0.05 for IgGLoc and r: + 0.6, < 0.005 for IgGIF). Finally, CXCL13+ presented lower gCTh (2.41±0.1 mm) compared to CXCL13- (2.49±0.1 mm, p< 0.05).
Conclusions: CSF-CXCL13 can be considered a promising bio- marker to distinguish early MS patients with higher B-cell recruit- ment, higher IgG parameters in the CSF and higher cortical atrophy.
Disclosure
Puthenparampil Marco received travel grant from Novartis, Genzyme, Biogen Idec, Teva and Sanofi Aventis; he has been consultant for Genzyme.
Federle Lisa has received funding for travel from Novartis, Merck Serono, Biogen Idec, Sanofi-Aventis, Bayer Schering Pharma, Almirall, Genzyme, Teva and honoraria from Genzyme, Merk Serono, Teva and Almirall.
Miante Silvia received travel grant form Biogen Idec, Novartis, Sanofi Aventis and Teva. Toffanin Elisabetta, Ruggiero Susanna and Ermani Mario have nothing to disclose. Rinaldi Francesca serves as an advisory board member of Biogen-Idec and has received funding for travel and speaker honoraria from Merck Serono, Biogen Idec, Sanofi-Aventis, Teva and Bayer Schering Pharma.
Gallo Paolo has been a consultant for Bayer Schering, Biogen Idec, Genzyme, Merck Serono and Novartis; has received funding for travel and speaker honoraria from Merck-Serono, Biogen Idec, Sanofi-Aventis, Novartis Pharma and Bayer-Schering Pharma, Teva; has received research support from Bayer, Biogen Idec/Elan, Merk Serono, Genzyme and Teva; and has received research grant from the University of Padova, Veneto Region of Italy, the Italian Association for Multiple Sclerosis, the Italian Ministry of Public Health.
P932
Bone morphogenetic protein signaling as a potential therapeutic target for Multiple Sclerosis
H. Eixarch1, L. Calvo-Barreiro1, M. Castillo1, A. Gutiérrez- Franco1, V. Gil2, J.A. De Río2, X. Montalban1, C. Espejo1
1Multiple Sclerosis Centre of Catalonia/Neuroimmunology Department, Vall d’Hebron University Hospital, 2Department of Cell Biology, Universitat de Barcelona, Barcelona, Spain
Introduction: Bone morphogenic proteins (BMPs) are critical regulators of brain development, cell proliferation and cell fate. BMPs are involved in neurogenesis and in oligodendrocyte matur- ing. Moreover, they are differentially expressed in the immune system of multiple sclerosis (MS) patients. Thus, BMP signaling might be a potential therapeutic target for MS.
Objective: i) To elucidate the effect of BMP4 and noggin in neu- rogenesis and the immune response. ii) To characterize the expres- sion of BMP2, 4, 5, 6, 7, BMPRII and their antagonist, noggin, in the experimental autoimmune encephalomyelitis (EAE) model, in both the CNS and the immune system.
Methods: For in vitro studies, neurospheres were obtained from P15 pups. The effect of BMP4 and noggin on cell lineage commit- ment was assessed by immunochemistry. Splenocytes were cul- tured, challenged with BMP4 and/or noggin, and their proliferation capacity was evaluated. In in vivo studies, EAE was induced in C57BL/6J mice with the MOG35-55 peptide. Spinal cords and spleens were removed before EAE induction (basal condition), and in the induction (7 post-immunization; p.i.), inflammatory (15 p.i.), early chronic (30 p.i.) and late chronic (50 p.i.) phases of the disease. After RNA extraction, levels of expression of BMP2, 4, 5, 6, 7, BMPRII and noggin were assessed by RTqPCR.
Results: In vitro, neurosphere cultures challenged with BMP4 presented more differentiated astrocytes, and a higher amount and complexity of neurons. In the immune system, BMP4 and espe- cially noggin promoted the proliferation of splenocytes, although the combination of both proteins exerted a more pronounced effect. In vivo, we found a general reduction in the expression of BMPs and noggin in the different phases of the experimental dis- ease in the CNS. On the contrary, the expression of BMPs in the immune system was elevated upon EAE induction, reaching its maximum increase at the induction and inflammatory phases of the disease. Altogether point out a role of these proteins in the induction of the inflammatory response in EAE.
Conclusions: Our findings confirmed the role of BMP4 in astro- glial commitment and found a positive effect on the differentia- tion and maturation of neural cells. Moreover, our results suggest that BMP signaling is involved in the regulation of the peripheral and probably in the local immune response. Thus, it seems to be relevant in the pathogenesis of EAE and could be a potential ther- apeutic target for MS.
Disclosure
- Herena Eixarch declares no competing financial interests. - Laura C Barreiro declares no competing financial interests. - Mireia Castillo declares no competing financial interests. - Ana Gutierrez-Franco declares no competing financial
interests.
- Vanessa Gil declares no competing financial interests. - Jose Antonio Del Rio declares no competing financial interests. - Xavier Montalban has received speaking honoraria and travel
expenses for scientific meetings, has been a steering committee member of clinical trials or participated in advisory boards of clinical trials in the past with Bayer Schering Pharma, Biogen Idec, EMD Merck Serono, Genentech, Genzyme, Novartis, Sanofi-Aventis, Teva Pharmaceuticals and Almirall.
- Carmen Espejo is partially supported by the “Miguel Servet” programme (CP07/00146; CP13/00028) of the Fondo de Investigación Sanitaria, the Instituto de Salud Carlos III, the Ministry of Economy and Competitiveness of Spain.
P933
The genetic multiple sclerosis (MS) risk variant rs7665090-G enhances NF-κB signaling responses in astrocytes
G. Ponath, D. Pitt
Neurology, Yale University, New Haven, CT, United States
The NF-κB pathway has been strongly implicated by genome wide association studies as a central defect in MS pathology with more than 18% of genetic variants affecting genes involved in the NF-κB pathway. NF-κB regulates responses of many different cell types including astrocytes, where NF-κB signaling is critical for propagation of CNS inflammation. Therefore, genetic MS risk variants that affect the NF-κB pathway may lead to unchecked activation of astrocytes in response to inflammatory stimuli lead- ing to excess production of cytokines, enhanced attraction of peripheral lymphocytes and impaired homeostatic and neuro-sup- portive functions.
We tested this hypothesis by modeling the impact of a com- mon NF-κB-relevant genetic risk variant on astrocyte function using induced pluripotent stem cells (iPSCs). We obtained skin biopsies from MS patients homozygous for the rs7665090-G risk variant or the protective variant and generated iPSCs that we dif- ferentiated into astrocytes. Stimulation of astrocytes with the risk variant resulted in increased NF-κB signaling and secretion of TNF-α and IL-6 and reduced uptake of glutamate and lactate/glu- cose utilization ratios.
Thus, we demonstrate that the rs7665090-G MS risk variant enhances inflammatory responses of astrocytes and impairs gluta- mate homeostasis and energy supply to neurons. In MS, this might lead to a lower threshold for lesion formation and enhanced neu- rotoxicity. MS patients with this and other NF-κB-relevant risk variants might thus benefit from therapeutic blockade of NF-κB or NF-κB-activating cytokines.
Disclosure
Gerald Ponath: nothing to disclose David Pitt: nothing to disclose P934
Investigating the role of astrocytes’ anti-excitotoxicity potential for neuronal damage formation in experimental autoimmune encephalomyelitis - an in vivo two-photon imaging approach
K.S. Rosiewicz1, T. Crowley1, M. Alisch1, A. Margineanu2, V. Siffrin1
1ECRC - Neuroimmunology Lab, Charité - Universitätsmedizin Berlin, 2Advanced Light Microscopy, Max Delbrueck Center for Molecular Medicine, Berlin, Germany
Background and goals: Neuronal damage in autoimmune neuro- inflammation is the correlate for long-term disability in patients suffering from Multiple Sclerosis (MS). Recent reports suggest