Wenyuan Yin (1) – Anzela Niraula (1) – Caroline Sawicki (2) – Daniel McKim (1) – Jonathan Godbout (1) – John Sheridan (2)
The Ohio State University, Department of Neuroscience, Columbus, United States (1) – The Ohio State University, Division of Biosciences, Columbus, United States (2)
Repeated social defeat (RSD) is a murine model of psychosocial stress that causes neuronal and microglial activation, peripheral monocyte recruitment to the brain, and anxiety-like behavior. Previous studies have demonstrated that activation of neurons and endothelial cells in regions associated with threat appraisal is necessary for monocyte trafficking and the development of anxiety-like behavior following RSD. Monocytes that are recruited from the periphery express interleukin-1 (IL-1) beta and selectively interact with the reactive endothelium. Activated endothelia express higher levels of interleukin-1 receptor 1 (IL-1R1), adhesion molecules, and cyclooxygenase-2 (COX-2). In the current study, we sequenced actively translated mRNA from endothelial cells using a RiboTag approach to further characterize the reactive endothelium that develops after RSD. This approach corroborated previous findings including increased translation of IL-1R1, E-selectin, P-selectin, and prostaglandin-related proteins such as COX2 and Slco2a1. The translational changes are consistent with activation of the Nuclear Factor kappa B signaling pathway, which is downstream of IL-1 signaling. Previous reports indicate that prostaglandins, specifically PGE2, may be involved in behavioral changes after social defeat stress. Ongoing studies will determine whether PGE2 is increased specifically in regions of the brain associated with threat appraisal and anxiety after RSD. Next, we will determine if systemic pretreatment with SC-236, a selective COX-2 inhibitor, prevents the development of anxiety-like behavior after RSD. Finally, we will assess how inhibition of COX-2 impacts neuronal activation, microglia activation, and peripheral monocyte recruitment after stress. Targeting COX-2 in brain endothelia may be a promising therapeutic approach for future treatments of anxiety disorders.
Kevin Thorburn (1) – Jason Plemel (2) – Bradley Kerr (3)
University of Alberta, Pharmacology, Edmonton, Canada (1) – University of Alberta, Neurology, Edmonton, Canada (2) – University of Alberta, Anesthesiology and Pain Medicine, Edmonton, Canada (3)
Multiple Sclerosis (MS) is a central nervous system (CNS) disease characterized by inflammation, demyelination and axonal injury. In both MS and the animal model experimental autoimmune encephalomyelitis (EAE), astrocytes become activated and undergo molecular and morphological changes that can be beneficial or detrimental to CNS repair. In addition to becoming activated, astrocytes can be damaged during the course of MS and EAE. While significant progress has been made in understanding how activated astrocytes influence MS and EAE, relatively little is known about the role that injured astrocytes play in each disease. We have previously demonstrated that in mice with EAE there is a breakdown of the glia limitans that normally separates the peripheral and central nervous systems. Subsequent studies have revealed that in tissue sections with glia limitans disruption there is significantly greater T-cell infiltration, microglia/macrophage activation, demyelination and axonal injury. The increased immune cell infiltration and neural pathology can be detected in both male and female mice. However, female mice show significantly greater T-cell infiltration and axonal injury relative to males. In the tissue sections with disrupted glia limitans we also find that Iba-1 positive cells (i.e. macrophages and microglia) fill in areas devoid of astrocytes suggesting that macrophages and/or microglia create scar-like barriers similar to what has recently been described in spinal cord injury. We are currently carrying out experiments with the CX3CR1CreER mouse line to determine the relative contribution of each cell type to this scar-like barrier. We have also recently found that glial fibrillary acidic protein (GFAP) fragmentation, a marker of astrocyte injury, can be detected in the spinal cords of mice with EAE. Preliminary data suggests that there is significantly more GFAP fragmentation in animals at the peak of disease compared to animals at disease onset. Recently obtained in vitro data shows that myelin-reactivated T-cells are capable of killing astrocytes, as indicated by a reduction of GFAP staining. Current efforts are underway to characterize astrocyte function (e.g. phagocytosis) when cultured in the presence of autoreactive T-cells. Taken together, our data demonstrates that astrocyte injury in EAE is associated with more severe pathology and may be caused by infiltrating immune cells.
Rose-Marie Rebillard (1) – Lyne Bourbonnière (1) – Kathie Beland (2) – Elie Haddad (2) – Alexandre Prat (2)
CRCHUM, Département de neurosciences/Université de Montréal, Montreal, Canada (1) – Centre de recherche du CHUSJ, Département de microbiologie, infectiologie et immunologie/Université de Montréal, Montréal, Canada (2)
Introduction: Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for the study of multiple sclerosis (MS). Although instrumental in MS research, this model fails to replicate some characteristics of this highly complex demyelinating disease. Our hypothesis is that we can develop an innovative humanized mouse model of MS that can replicate the human pathology more accurately, which would be instrumental in achieving a better understanding of the disease and in facilitating the identification of novel therapeutic targets.
Methods: Peripheral blood mononuclear cells (PBMC) from MS patients and healthy donors were injected intraperitoneally into NOD/LtSz-scid IL-2Rγc(null) (NSG) mice in order to achieve immune reconstitution. 25 or 32 days following injection, immune cells were isolated from both CNS and spleen of each mouse and analyzed by flow cytometry. Data analysis was performed using a conventional gating strategy as well as using the unbiased t-SNE algorithm.
Results: Immune reconstitution was observed in all mice that received PBMC from either MS patients or healthy donors. No differences were found in the absolute number (between 1.02 x104 and 1.18 x 106 immune cells, mostly human leucocytes) or immunophenotype of CNS infiltrating cells between both groups. We did however find a significantly higher proportion of GM-CSF and IFNg expressing cells among CNS-infiltrating lymphocytes in comparison to those isolated from the spleen.
Conclusion: The intraperitoneal injection of MS patient PBMC into NSG mice alone is not sufficient to reproduce pathological features of MS in this mouse model. Nevertheless, we established that in this model, the immunophenotype of infiltrating immune cells is organ-specific, with CNS-infiltrating cells having a more inflammatory profile than cells isolated from the spleen, thereby confirming the major potential of using NSG mice in humanized models of neurological disease.
Angela Wang (1) – Karen Yeung (1) – Olga Rojas (1) – Valeria Ramaglia (1) – Jennifer Gommerman (1)
University of Toronto, Department of Immunology, Toronto, Canada (1)
B cell depletion therapy is an effective treatment for relapse-remitting Multiple Sclerosis. Anti-CD20 treatment, however, does not impact the plasma cell (PC) compartment, and oligoclonal antibody bands in the cerebrospinal fluid remain unchanged in patients who have had a positive clinical response to anti-CD20. In contrast, combined depletion of both B cells and antibody-secreting PC leads to exacerbated MS symptoms, suggesting a possible role for PCs in disease regulation. Our lab and others have demonstrated critical roles for cytokine production by PCs in regulating Experimental Autoimmune Encephalomyelitis (EAE), the animal model of MS. Specifically, we find that intestine-derived PCs can migrate into the inflamed CNS and attenuate EAE in a manner that is dependent on the anti-inflammatory cytokine IL-10. Here, I hypothesize that regulatory IgA+ PCs offer protection locally within the central nervous system (CNS) during the T cell effector phase of EAE by modulating the inflammatory cell types of the CNS. To answer this, I have used an adoptive transfer EAE approach to separate T cell priming versus effector events. I assessed the impact of encephalogenic CD4+ T cells transferred into WT recipient mice versus BAFF-transgenic (BAFF-Tg) recipient mice which have an overabundance of IgA+ PCs. Compared to WT recipient mice, BAFF-Tg recipient mice that received encephalogenic donor CD4+ T cells were protected from developing severe EAE. Protection was correlated with a lower frequency of cytokine-producing CD4+ T cells, less inflammation and demyelination, and the presence of IgA-secreting cells in the CNS. I also observed a decrease in the number of IgA+ and CD138+ cells in the small intestine of recipient mice compared to naïve mice, suggesting that inflammation may prompt IgA+ PC migration out of the gut independent of effects from immunization adjuvants. Ongoing work is directed towards adoptive transfer experiments into PC-deficient Cd19crePrdm1fl/fl mice where we expect increased EAE severity, and to identify the target cell types for IgA+ PC-derived IL-10 regulation within the CNS during inflammation.
Mitra Knezic (1) – Caitlin Lundell-Creagh (1) – Kaitlyn Tresidder (2) – Julia Segal (1) – Courtney Bannerman (1) – Ian Gilron (1, 2, 3) – Nader Ghasemlou (1, 2, 3)
Departments of Biomedical & Molecular Sciences and (1) – Departments of Anesthesiology & Perioperative Medicine (2) – Centre for Neuroscience Studies, Queen’s University, Kingston, Ontario, Canada (3)
Circadian (24-hour) rhythms affect many processes in the body including the sleep/wake cycle, hormone release, immune function, metabolic activity and core body temperature. Crosstalk between the nervous and immune systems underlie several functional and behavioural outcomes. Somatosensation – the transmission of sensory signals to the spinal cord and brain for processing – includes sensitivity to mechanical, thermal and nociceptive stimuli, propagated by distinct populations of sensory neurons. These sensory neuron subsets can be identified by their expression of specific ion channels and receptors including receptor tyrosine kinases, G-protein coupled receptors, and ion channels. Non-specific cation channels of the transient receptor potential (TRP) family confer specific modalities to sensory neurons. TRPV1, for instance, is activated by heat and capsaicin, the active ingredient in chili peppers. We sought to characterize the circadian rhythm of somatosensation in naïve mice. Our work shows that thermal somatosensation exhibits a circadian rhythm and that heat sensitivity is significantly reduced in male C57BL/6J mice during the dark phase relative to the light phase, whereas female mice do not show this effect. Due to the limited phenotypic variability associated with inbred strains, we also sought to determine if this effect is reproducible in an outbred stain, using CD1 mice, where individual mice show greater genetic and phenotypic diversity. A similar circadian variation in thermal sensitivity was also maintained in these mice, with a more prominent effect in males than females. Quantitative PCR of TRP channels in the spinal cord and dorsal root ganglia reveal few circadian changes in expression in C57BL/6J mice, suggesting that other mediators/channels may play a role in this response. Our results show a modality-specific circadian rhythm of somatosensation in inbred and outbred mouse strains, without an altered TRP channel response. Identifying potential mediators of this response may help our understanding of somatosensory function.
Nicholas Kieran (1) – Shih-Chieh Fuh (1) – Vijay Rao (1) – Manon Blain (1) – Samuel Ludwin (2) – Jack Antel (1) – Luke Healy (1)
Montreal Neurological Institute, McGill University, Montreal, Canada (1) – Pathology, Queen’s University, Kingston, Canada (2)
Astrocytes are an understudied cell type that are involved in multiple sclerosis (MS) pathology. Although astrocytes’ homeostatic functions are well-defined, their specific role in MS requires further investigation. MicroRNAs (miRNAs) are small non-coding RNA fragments that regulate cellular function by affecting translation. miRNAs have been found to be dysregulated in many diseases, and thus the differential expression of miRNAs in MS astrocytes may alter the normal phenotype of the cells and play a role in MS pathology. Studying the miRNA signature of MS astrocytes will give insight into the astrocytic response in disease. This work will also help to define the specific cellular stresses that astrocytes are exposed to in the MS brain. We hypothesize that MS associated cellular stresses drive a unique astrocytic miRNA signature that influences the functional response of astrocytes to the disease process. We aim to analyze the miRNA profile of primary human astrocytes in situ and in vitro in a stress paradigm that recapitulates the MS tissue environment. Astrocytes were laser captured from acute active MS lesions and normal appearing white matter. These cells were analyzed for their miRNA profile using qPCR. Primary human fetal astrocytes were treated in combinations of inflammatory (IL1b), metabolic (no glucose), and hypoxic (1% O2) stress conditions. qPCR analysis of canonical stress markers was used to confirm the specific response of astrocytes to the MS-like stress conditions. Next, we used qPCR to measure expression of a panel of miRNAs in astrocytes undergoing stress. Analysis of astrocytes captured from MS lesions reveals an MS-miRNA signature in these cells. We establish in vitro stress conditions that mimic the MS environment as measured by upregulation of MS-associated markers. We confirm that astrocytes respond to the individual stressors through upregulation of response genes CXCL10 (inflammatory), HMOX1 (metabolic), and SLC16A3 (hypoxic). Finally, we identify stress conditions that result in differential expression of miRNA-210, -155, and -34a compared to control cells. Overall, we have established an in vitro stress paradigm that reflects the specific cellular stresses of MS lesional tissue leading to a differential expression of specific miRNAs in these cells. This work enhances our understanding of the molecular profile and functional role of astrocytes in the MS brain.
Nataliya Tokarska (1) – Lydia Ayanwuyi (1) – Nikki M McLean (1) – Valerie MK Verge (1)
Cameco MS Neuroscience Research Center, Anatomy, Physiology and Pharmacology/College of Medicine/University of Saskatchewan, Saskatoon, Canada (1)
MS is an inflammatory disease of the central nervous system characterized by immune-mediated segmental demyelination and variable degrees of axonal and neuronal degeneration. Efficient repair of demyelinated lesions is one of the major challenges of MS. Our lab focusses on therapies that enhance intrinsic repair mechanisms of the peripheral and central nervous system following injury. We have shown that brief electrical nerve stimulation (ES), has a dramatic impact on remyelination of lysophosphatidyl choline (LPC)-induced focally demyelinated rat peripheral nerves, while also inducing an axon-protective phenotype and shifting macrophages from a predominantly pro-inflammatory (M1) toward a pro-repair (M2) phenotype. We have adapted this model to the CNS, creating a unilateral focal LPC demyelination of the dorsal column at the lumbar enlargement where the sciatic nerve afferents enter, so that subsequent sciatic nerve ES results in increased neural activity in the demyelinated axons. Preliminary data reveals a robust focal demyelination at 7 days post-LPC injection. Delivery of 1hr ES at 7d post-LPC, polarizes macrophages/microglia toward a pro-repair M2 phenotype, evident as early as 2d post-ES (12d post-LPC), and still apparent in ES animals at the latest time examined, 14d post-LPC. ES also resulted in smaller regions of inflammation compared to non-stimulated controls; the recruitment of significantly more OPCs to the demyelinated region; more effective remyelination, with the paranodal protein Caspr along demyelinated axons 14d post-LPC, shifting to a more restricted distribution, consistent with reformation of the nodes of Ranvier; and enhanced levels of phosphorylated NFMs, supporting promotion of an axon protective state. Collectively this supports that strategies that increase neural activity can be beneficial for repair following focal demyelinating insults. We are grateful for support from the MS Society of Canada, and the Colleges of Medicine and Graduate and Postdoctoral Studies at the University of Saskatchewan.
Simon Thebault (1) – Mohammad Abdoli (1) – Seyed-Mohammad Fereshtehnejad (1) – Daniel Tessier (2) – Vincent Tabard-Cossa (2) – Mark Freedman (1)
University of Ottawa, The Ottawa Hospital, Ottawa, Canada (1) – University of Ottawa, Department of Physics, Ottawa, Canada (2)
With the availability of more powerful treatments for multiple sclerosis (MS), prognostic biomarkers are badly needed. Levels of neurofilament light chains (NfL) found in serum result from the destruction of central nervous system (CNS) axons in MS and correlate with the aggressiveness of the disease. Our objective is to evaluate the prognostic values of serum NfL levels obtained close to the time of MS onset with long-term clinical outcomes.
In this prospective cohort study, we identified patients with serum collected within 5 years of first MS symptom onset (baseline) with more than 15 years of clinical follow-up. Clinical course, treatments and Expanded Disability Status Scale (EDSS) were recorded longitudinally. Levels of serum NfL were quantified in the patients as well as non-inflammatory age- and sex-matched controls using digital immunoassay technology (SiMoA HD-1 Analyzer from Quanterix).
Sixty-seven patients fit the inclusion criteria with a median follow-up period of 17.4 years (range: 15.1-26.1). Median serum NfL levels were 39.8% higher in MS patients compared to the 37 controls (p=0.004). Patients reaching EDSS≥4 had 62.0% higher baseline levels compared to the patients who did not (p=0.0001). The best NfL cutoff value for predicting progression was 7.62 pg/mL; patients with NfL >7.62 pg/mL had 8.9-times higher risk of developing progressive MS during follow-up (p = 0.034, 95% CI:1.2-68.1). Patients with the highest NfL levels (3rd-tertile) progressed most rapidly with an EDSS annual rate of 0.16 (p=0.004), remaining significant even after adjustment for sex, age, and disease-modifying treatment (p=0.022).
This study demonstrates that higher levels of serum NfL detected early in the disease are associated with poorer long-term clinical outcomes. These patients may benefit from a more aggressive initial approach to initial treatment.
Kristina Patterson (1) – Xiaolin He (2) – Elisabeth Burnor (1) – Leah Zuroff (1) – Amit Bar-Or (1) – Eric Lancaster (1)
Hospital of the University of Pennsylvania, Neurology, Philadelphia, United States (1) – Northwestern University, Molecular Pharmacology and Biological Chemistry, Chicago, United States (2)
Background: Autoantibodies to Neurofascin 155 (NF155) define a subset of patients with severe acquired demyelinating neuropathy and are usually of the IgG4 subclass. IgG4, unlike other IgG subtypes, undergoes hemi-antibody exchange making each antibody bi-specific (functionally monovalent), such that crosslinking and internalization of the target antigen is unlikely. At the paranode, NF155 interacts with contactin/Caspr complex, an association necessary for anchoring myelin to the axon. NF155 is known to dimerize in vitro, though the functional implications of this interaction are not well understood. We hypothesize that IgG4 autoantibodies disrupt important heterophilic and homophilic interactions of NF155 which, in turn, may disrupt the function of the protein.
Methods: Using cell-based assay screening, we confirmed the presence NF155 autoantibodies in a subset of patients with demyelinating neuropathy. A solid phase binding assay was used to test the effect of NF155 autoantibodies on the binding of NF155 to contactin, Caspr, and neurofascin in vitro. The effects of NF155 autoantibodies on the binding of NF155 multimers to contactin was evaluated using a clustering assay in which a NF155 Fc fusion protein was pre-clustered with an Alexa 488-conjugated antibody against human IgG then incubated with HEK293 cells transiently transfected with contactin.
Results: We identified 4 cases of acquired demyelinating neuropathy harboring serum autoantibodies to NF155. IgG4 autoantibodies to NF155 were found in all cases and typically comprised the predominant subtype. In binding assays, NF155 bound contactin and neurofascin with nanomolar affinity while NF155 and Caspr did not appear to directly interact. Formation of NF155 multimers was necessary for contactin binding. Though antibodies to neurofascin had very modest direct effects on the interaction of NF155 with contactin, these neurofascin autoantibodies modulated homophilic interactions of NF155 which, in turn, indirectly inhibited binding of NF155 to contactin.
Conclusions: Our results suggest that inhibition of important protein-protein interactions is a possible mechanism by which NF155 IgG4 autoantibodies may disrupt the paranode. This work demonstrates that NF155 self-association increases affinity of contactin for NF155 and that interference with the formation of NF155 multimers indirectly inhibits the association of NF155 with contactin. Future work will focus on further characterizing homophilic NF155 interactions and their role in myelination.
Noopur Singh (1, 2) – Aline Dumas (1) – Jean-François Richard (1) – Luc Vallières (1, 2)
Neuroscience Unit, University Hospital Centre of Quebec – Laval University, Quebec, Canada (1) – Department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec, Canada (2)
Interleukin-6 (IL-6) is essential in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis and optic neuromyelitis. In addition to its known role in the differentiation of self-reactive TH17 lymphocytes, IL-6 acts on the endothelium of blood vessels in the central nervous system (CNS) to stimulate the recruitment and activation of myeloid cells such as; neutrophils, macrophages, and dendritic cells. We tested this hypothesis by blocking the signaling of IL-6 specifically in the endothelium. Mice expressing Cre recombinase under the control of Tie2 endothelial promoter were crossed with mice in which the receptor IL-6 alpha gene had LoxP recombination sites. These mice were used to study the development of EAE, leukocyte recruitment and expression of inflammatory genes responsible for myeloid cells recruitment and activation, sequentially driving them for antigen presentation. Our findings demonstrate that endothelial cells in the CNS express the IL-6 receptor. The specific elimination of IL-6 receptor alpha in the endothelium largely delays the onset of EAE with very low incidence, and the leukocyte recruitment is blocked. It is noteworthy that the differences observed by deletion of IL-6 receptor are due to reduction in the number of ICAM1 expressing extravascular myeloid antigen presenting cells. Furthermore, we identify CXCL1, a neutrophil specific chemokine and PTGS2 (COX-2), a prostaglandin synthesizing enzyme as the key endothelial genes induced by IL-6 to recruit and activate the myeloid cells during pre-onset stages of EAE. Hence, we conclude that IL-6 plays a critical secondary role during pre-onset stages of EAE by binding to IL-6 receptor of CNS endothelium, which in turn activates the endothelial cells for up-regulation of the chemokines and prostaglandin synthesizing enzymes responsible for leukocyte recruitment and activation, a potential mechanism that could be targeted to treat autoimmune demyelinating diseases.