Yves Carpentier Solorio (1) – Ana Carmena Moratalla (1) – Marie-Laure Clénet (1) – Negar Farzam-kia (1) – Cyril Laurent (1) – Florent Lemaitre (1) – Annie Levert (1) – Nathalie Arbour (1)
University of Montreal-CHUM, Neuroscience, Montreal, Canada (1)

Our laboratory is focusing on Multiple Sclerosis (MS), a chronic inflammatory and demyelinating disease of the central nervous system (CNS). The etiology of this neurological disease remains elusive and no curative treatment is available. Nevertheless, it is well established that the immune system participates not only in the destruction of myelin and neural and neuronal cells but also in repair mechanisms. However, the contribution of specific immune mediators to injury and/or repair remains to be defined. The ultimate goal of our research program is to identify and characterize the immune mechanisms modulating disease development and/or progression in MS patients. More specifically, we aim to identify different molecules that could be targeted for new therapies and biomarkers that could be used for diagnosis. We are currently focused on the study of different cytokines such as IL-15 and IL-27 that have been found to be relevant in MS and in experimental allergic encephalomyelitis (EAE), a mouse model of MS. Moreover, we are investigating possible activating factors of immune effector cells, such as the activating receptor NKG2D. Our research strategy is to first identify molecules/mechanisms that are specifically altered in human samples (blood, cerebrospinal fluid, postmortem brain) obtained from MS patients. Then, we investigate the mechanistic impact of such factors using primary cultures of human immune and CNS cells. These cells are as close as we can get to the in vivo human situation. Finally, using EAE models, we confirm and dissect the role played by these identified mechanisms in the pathogenesis of MS and test in vivo strategies to correct these altered factors and thus validate them as bona fide therapeutic targets. We use various platforms and techniques available in our research center, including flow cytometry, immunohisto/cytochemistry (confocal microscopy, spinning disk) as well as molecular biology. Our ultimate goal is to identify novel tools and targets to fight and stop MS progression.

Jessica R. Allanach (1) – Blair K. Hardman (1) – Isobel C. Mouat (1) – Naomi M. Fettig (1) – Yu Gu (1) – Virginie S. E. Jean-Baptiste (1) – Iryna Shanina (1) – Lisa C. Osborne (1) – Galina Vorobeychik (2, 3) – Marc S. Horwitz (1)
Department of Microbiology & Immunology, University of British Columbia, Vancouver BC, Canada (1) – Fraser Health Multiple Sclerosis Clinic, Burnaby BC, Canada (2) – Division of Neurology – Department of Medicine, University of British Columbia, Vancouver BC, Canada (3)

Epstein-Barr virus (EBV), a human herpesvirus that establishes latent infection in B cells, is proposed to contribute to the pathogenesis of multiple sclerosis (MS) through unknown mechanisms. Evidence for a role of EBV infection in MS comes from both epidemiological and experimental studies, however, due to its narrow host tropism, there are currently few suitable animal models of MS that incorporate EBV infection. We have previously shown that latent infection of mice with the murine homologue of EBV, termed gammaherpesvirus-68, results in an altered immune response and severe clinical disease when mice are induced with experimental autoimmune encephalomyelitis (EAE), an MS-like disease in animals. Recent advancements in humanized mouse models has enabled direct infection of mice for the study of EBV-associated malignancies, including rheumatoid arthritis and systemic erythematosus lupus. We therefore chose to evaluate the role of EBV infection in humanized mice with EAE. In our studies, HuPBMC mice were first generated by engrafting immunocompromised mice with peripheral blood mononuclear cells (PBMC) derived from individuals with relapsing MS (RRMS) or from matched healthy EBV seropositive or seronegative donors. Once sufficiently reconstituted, HuPBMC mice were then induced with EAE. We observed that HuPBMC EAE mice developed ascending paralysis, weight loss and signs of discomfort consistent with classical EAE models. Further, HuPBMC EAE showed significant T cell infiltration of both the brain and spinal cord, notably of IFN gamma-expressing CD4 and CD8 T cells, resulting in spinal cord and cerebellar demyelination. HuPBMC EAE mice derived from EBV seropositive donors developed earlier disease onset with more severe clinical symptoms compared to EBV seronegative donor-derived mice. We also observed differences in EAE between HuPBMC mice derived from RRMS patients and healthy controls. Given that MS patients are majority EBV seropositive, we are quantifying the viral load and EBV-specific immune response of donors and respective HuPBMC EAE mice to evaluate this association with disease severity. With continued improvement and characterization of this novel humanized EAE model, additional environmental and genetic risk factors can be evaluated in a system with human immune-mediated pathology. Moreover, immunomodulatory therapies could be tested in this personalized preclinical model to assess the efficacy and mechanism of treatments.

Chelsea Bray (1) – Kristina Witcher (1) – Fangli Zhao (1) – Alan Gordillo (1) – Daniel McKim (2) – Xiaoyu Liu (3) – Julia Dziabis (4) – Ning Quan (3) – Candice Askwith (1) – Olga Kokiko-Cochran (1) – Daniel Eiferman (1) – Jonathan Godbout (1)
The Ohio State University, Neuroscience, Columbus, United States (1) – University of Illinois – Urbana Champaign, Neuroscience, Urbana, United States (2) – Florida Atlantic University, Neuroscience, Boca Raton, United States (3) – Duke University, Neuroscience, Durham, United States (4)

Traumatic brain injury (TBI) elicits immediate neuroinflammatory events that cause functional disturbances. Despite resolution of acute complications, chronic impairments may develop after injury. We previously identified microglia as mediators of inflammatory/immune signaling that persisted sub-acutely following diffuse TBI. It was unclear, however, if persistent microglial activation acted as reparative or neurodegenerative. Therefore, electrophysiological, histological, and transcriptome analyses were used to determine microglial contribution to neuropathology (with/without PLX5622-mediated microglial depletion) at acute (1dpi), subacute (7dpi), and chronic (30dpi) time-points. Microglia were eliminated prior to midline fluid perfusion injury and axon conduction and cortical neuropathology/inflammation was assessed. There was a persistent reduction in N2/N1 amplitude of compound action potentials in the corpus callosum 30 dpi. This TBI-associated impairment at 30 dpi was reversed by microglial elimination. To quantify cortical gene expression, Nanostring’s Neuropathology gene expression assay (760 genes) was used. Novel data revealed robust increases in genes associated with inflammation and neuropathology acutely (1 dpi) and a majority of the neuronal damage associated genes (Hmox1, Hsbp1, Fas) were microglia-independent. At 7 and 30 dpi, microglial elimination reversed TBI-related inflammatory gene expression (Itgax, Cd14, Clec7a) and neuropathology (Serpinf1). Sub-acute and chronic suppression of neuronal genes after injury were restored by microglial elimination (Ngf, Trpv1, Drd1, Drd2, Avp). To understand how cell types were influenced by TBI and continual microglia activation we used single cell sequencing. At 7dpi cortical microglia revealed a unique neurodegenerative signature not present in uninjured controls. These injury-associated microglia underwent transcriptional changes consistent with debris clearance. Furthermore, dendritic spine analysis indicates dendritic remodeling is microglia dependent following injury. Thus, microglia promote persistent neuropathological, transcriptional, and functional impairment after diffuse TBI.

Naomi Fettig (1) – Lisa Osborne (1) – Marc Horwitz (1)
University of British Columbia, Microbiology and Immunology, Vancouver, Canada (1)

Multiple sclerosis (MS) is a chronic neurodegenerative disease, in which lymphocyte infiltration into the central nervous system (CNS) induces inflammation, destruction of the myelin sheath, and eventual axonal degradation. MS is caused by both genetic and environmental factors, although the exact environmental factors and their relative contributions to disease progression remain unknown. Epidemiological data show a negative correlation between MS incidence and the prevalence of helminth infections; consequently it has been suggested that helminth infection may offer protection from neuroinflammatory disease, although the mechanism of this interaction remains unknown. One helminth, Trichinella spiralis (Ts) establishes chronic infection by forming nurse cells in the striated muscle and inducing Th2 and regulatory T cell responses, which have been suggested to be protective in MS and in the animal model experimental autoimmune encephalomyelitis (EAE). C57Bl/6 mice were infected with 500 Ts larvae by oral gavage and allowed 4 weeks to establish chronic infection. The mice were subsequently immunized with MOG35-55/CFA to induce EAE. While this model of EAE normally results in a chronic ascending paralysis, prior infection with Ts resulted in delayed disease onset, as well as a remission of symptoms that is not usually observed in C57Bl/6 EAE. Flow cytometric analysis of brain and spinal cord tissues shows decreased lymphocyte infiltration, which will be confirmed by histological analysis by H&E staining. In Ts-infected EAE, the decrease in lymphocytes present in the CNS is accompanied by a Th2-skew in the infiltrating CD4+ T cells. This Th2-skewed response may be inducing T cell-mediated neuroprotection. The type 2 immune environment may induce other immune cells to adopt regulatory or neuroprotective phenotypes, such as alternatively activated macrophages. Macrophage phenotypes within the CNS and periphery will be evaluated by qPCR to determine the cell types involved in Ts-induced attenuation of neuroinflammation and disease course.

Timothy Friedman (1) – Isabelle Tottenham (1) – Bradley Kerr (2)
University of Alberta, Neuroscience and Mental Health Institute, Edmonton, Canada (1) – University of Alberta, Department Anesthesiology and Pain Medicine, Edmonton, Canada (2)

Multiple sclerosis (MS) is an inflammatory disease with a known sex bias towards females. The bias extends into symptomatology with women reporting higher rates of chronic neuropathic pain. Using the EAE model, we have recently described significant alterations in the profiles of the dorsal root ganglia, which include the nociceptive cells responsible for generating pain signals as well as transiently located immune cells. These alterations included large increases in miR-21a-5p (miR-21) in both female and male mice at onset of symptoms, an effect previously linked to MS immune pathology. Therefore, miR-21 may be implicated in immune cell activation. However, the rate or extent at which this occurs is unclear. Hypothesis: Female immune cells are intrinsically more sensitive to inflammatory signals. Methods: Female and male C57BL/6 mouse bone-marrow derived macrophages (BMDMs) were treated with escalating doses of tumor necrosis factor alpha (TNFa) for 24 hours in order to generate an inflammatory response. The state of macrophage activation was assessed by quantitative (qPCR; miR-21, Tlr7, Tlr8, Nlrp3, and Stat1) and semi-quantitative (ICC; iNOS, Arg1, CD45) methods. To isolate the effect of miR-21, identical cultures were transfected with miR-21 mimics (mirVana, ThermoFisher) before subsequent transfection with TNFa. Results: Preliminary data suggests that miR-21 is upregulated by TNFa exposure without compromising cell viability and the level of miR-21 upregulation is consistent with previous in vivo studies. We have confirmed that BMDMs are viable after miR-21 transfection and remain responsive to TNFa stimulation. Conclusion: Sex differences in the activation of the innate immune system is important in understanding how chronic neuropathic pain may arise in MS. Here we show that miR-21 expression is stimulated by TNFa exposure in vitro and that this shift in expression is associated with a more inflammatory phenotype. Peripheral immune cells are regularly exposed to TNFa in the course of EAE, therefore the increased miR-21 seen in previous work may be a result of this immune activation.

Bradley J. Kerr is also affiliated with the Department of Psychiatry (NRU) at the University of Alberta and the Department of Pharmacology at the University of Alberta.

Revathy Guruswamy (1) – Reza Rahimian (1) – Hajer Boutej (1) – Jasna Kriz (1)
CERVO Brain Research Centre and Department of Psychiatry and Neuroscience,, Laval University, Quebec, Canada (1)

Stroke constitutes a major cause of death and disability of the adults worldwide. Until now, there is no efficient therapy for stroke patients. Ischemic stroke induces rapid activation of microglia, and as the resident immune cells of the central nervous system, microglial activation is believed to play a central role in neuroinflammation and pathological progression of ischemic tissue. Microglial activation is known to be detrimental in the early phase of ischemia, whereas in the subacute and chronic phase they are involved in the brain repair mechanisms. Therefore, strategies aiming to modulate the immune response by delaying microglial activation in the early phase of Ischemia would be an important strategy to induce brain recovery following stroke. To date, there is no clear evidence of molecular signature of activated microglia after stroke and further, the extent to which sexual dimorphism is involved in their activation profile. We have recently discovered that, there is a novel ribosome-based check point mechanism involved in the control of innate immune response and microglial activation. Serine/Arginine-Rich Splicing Factor 3, a small RNA binding protein of serine and arginine-rich (SR) family, has been shown to suppress the translation of highly induced innate immune genes in activated microglia leading to the formation of divergent mRNA and protein immune networks after Lipopolysaccharide-induce immune response. Hence, we aimed to study the molecular signature of activated microglia after ischemic stroke. We have used transgenic mice CD11brGFP in which FLAG/EGFP is fused to the N terminus of the large subunit of ribosomal protein L10a and expressed under the transcriptional control of CD11b promoter. Using translating ribosome affinity purification (TRAP) method from brain homogenates, we pull-down the ribosome-attached (both) mRNAs and peptides, thereby obtaining the active dynamic translational state of microglial ribosomes. Our data shows that, after ischemic stroke SRSF3 is differentially regulated, and the top up-regulated mRNAs and peptides is completely different in microglia derived from male and female indicating that sexual dimorphism is crucial following ischemic stroke in microglia responses. Our findings suggest that by deregulating the SRSF3 activity in microglia in early phase of post-ischemic inflammation would be a promising therapeutic approach to control the ischemic damage due to inflammation.

Giulia Fadda (1, 2*) – Patrick Waters (3*) – Mark Woodhall (3) – Sarosh Irani (3) – Robert A. Brown (1) – Julia O’Mahony (4) – Denise A. Castro (5) – Giulia Longoni (6) – E. Ann Yeh (6) – Ruth Ann Marrie (7) – Douglas L. Arnold (1) – Brenda Banwell (2, 8) – Amit Bar-Or (1, 2, 8) on behalf of the Canadian/CHOP Pediatric Demyelinating Disease Network
Montreal Neurological Institute, McGill University, Montreal, QC, Canada (1) – Center for Neuroinflammation and Neurotherapeutics, and Multiple Sclerosis Division, Department of Neurology, Perelman School of Medicine, University of Pennsylvania Philadelphia, PA, USA (2) – Neuroimmunology Unit, Montreal Neurological Institute and Hospital, McGill University, Montreal, QC, Canada (3) – Autoimmune Neurology Group, Nuffield Department of Clinical Neurosciences, University of Oxford, UK (3) – Institute of Health Policy, Management and Evaluation, the University of Toronto/The Hospital for Sick Children, Toronto, ON, Canada (4) – Department of Diagnostic Imaging, Neurosciences and Mental Health, SickKids Research Institute (5) – Department of Pediatrics, University of Toronto, Toronto, ON, Canada (6) – Departments of Internal Medicine and Community Health Sciences, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada (7) – Division of Child Neurology, Department of Neurology, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA (8)

A minority of subjects meeting the multiple sclerosis (MS) diagnostic criteria are seropositive for antibodies against myelin oligodendrocyte glycoprotein (MOGabs). Whether these patients exhibit clinical features distinct from typical MS is unclear. We characterized the clinical features and outcome of 66 children (median [IQR] age at onset 13.97 [IQR 10.88-15.06] years) with a diagnosis of MS according to 2017 international diagnostic criteria, who were assessed for the presence of MOGabs. Clinical, serological and imaging features were prospectively assessed for a median of 7 years from presentation, and compared between MOG+ and MOG- children using descriptive statistics. At clinical onset, 11/66 (17%) children were MOG+. Seropositive patients were younger (p<0.0001) than seronegative patients, and all presented at age <11 years. The presenting phenotype was optic neuritis (ON) and/or transverse myelitis (TM) for 80% of seropositive versus 41% of the seronegative patients (p =0.019). Brain MRIs at onset were atypical for MS in 9 MOG+ (3 without brain lesions, 3 with diffuse bilateral pattern and 3 with minimal lesions) and 4 MOG- patients (p<0.0001). Oligoclonal bands (OCBs) were detected in 2/8 (25%) MOG+ and 20/36 (83%) MOG- patients evaluated (p =0.0027). None of the MOG+ patients showed contrast enhancement on baseline MRI, thus none met 2010 McDonald criteria at onset; 2 MOG+ patients met the 2017 criteria due to the presence of OCBs. Of 11 MOG+ patients, 10 developed new brain lesions, 1 developed new spinal cord lesions, and 7 experienced clinical relapses (in almost all cases ON or TM). At last follow-up, total T2 lesion volume was smaller in MOG+ than MOG- patients (p<0.0001). Our findings suggest that, while meeting MS diagnostic criteria, children seropositive for MOGabs exhibit clinical and MRI features distinguishing them from MOG-negative typical relapsing MS patients.

Fernando Gonzalez Ibanez (1) – Julien Blouin (1) – Amin Benadjal (2) – Marie-Kim St-Pierre (1) – Micaël Carrier (1) – Julie Savage (1) – Mathieu Morissette (3) – Marie-Ève Tremblay (1)
Université Laval CRCHU de Québec, Université Laval, Quebec, Canada (1) – Faculté de Sciences et Ingénerie, Universtié Sorbonne, Paris, France (2) – IUCPQ, IUCPQ, Departement de médicine, Université Laval, Quebec, Canada (3)

According to World Health Organization, in 2015, there were 1.1 billion smokers worldwide. Smoking is responsible for 7 million deaths per year and represents an important risk factor for several diseases. Animal studies have shown that smoking causes increased levels of inflammatory markers and oxidative stress in several organs including the brain. Microglia are the resident immune cells of the brain. They are required for the proper functioning of the brain and are equipped with a myriad of receptors that allow them to monitor their environment, recognize damage, eliminate cells and react to insult. A lesser known function of microglia is their role in plasticity. They can promote the growth or directly eliminate synapses by phagocytosis, making them active modifiers of the neuronal network. Microglial functions are tightly regulated by their environment, suggesting possible effect of the proinflammatory condition caused by cigarette smoke. Using a model of sub-chronic cigarette smoke exposure, this project aims to study the effect cigarette smoke has on hippocampal microglia of the dentate gyrus (DG) and the CA1 radiatum. The hippocampus contains one of the two known neurogenic niches, the polymorphic layer of the dentate gyrus. Newborn neurons are sensitive to homeostatic disruptions, which have a negative impact on their survival. Microglia are also responsible of the elimination of dying newborn neurons by phagocytosis, rendering the DG a suitable region to study changes in microglial activity. The radiatum contains a high number of synapses and becoming a relevant region to study microglia-synapse interactions. With the use of immunohistochemistry, we have studied microglial density and morphology in the DG and CA1 radiatum in ventral hippocampus. With the use of a putative marker for dark microglia we have detected an increased number of positive cells in the smoking mice. Dark microglia are a subtype of microglia that show ultrastructural signs of oxidative stress and an increased phagocytic activity. Additionally, with the use array tomography and scanning electron microscopy, we are characterizing the ultrastructural changes in microglial morphology, phagocytic activity and interactions with synaptic elements in the polymorphic layer of the DG.

Ben Charpentier (1) – John Greenwell (2) – Paula Chilton (2) – Richa Signal (2) – Manicka Vadhanam (2) – Smita Ghare (2) – Scott Myers (3) – Swati Joshi-Barve (2) – Craig McClain (2, 4) – Leila Gobejishvili (2) – Scott Whittemore (1, 3) – Shirish Barve (1,2)
Departments of Anatomical Sciences and Neurobiology (1) – Departments of Medicine (2) – Departments of Neurological Surgery; University of Louisville (3) – VA Medical Center, Louisville, KY 40202 (4)

Negative effects of alcohol consumption on the nervous system include neuroinflammation that may contribute to cognitive decline and neurodegeneration. Perturbations in the gut microbiome play a role in Alzheimer’s and Parkinson’s diseases, but the role of the microbiome in ethanol-induced neuroinflammation is yet to be elucidated. In this study, we assessed the direct role of alcohol-induced gut microbial dysbiosis in the development of neuroinflammation. Fecal samples from patients with AH were well characterized by metagenomics analysis which revealed severe reduction of diversity and loss of beneficial bacteria. Development of neuroinflammation with and without chronic ethanol feeding in response to AH fecal matter transplantion (AH- FMT) was examined using conventional mice. Systemic inflammation and increased neutrophils in the circulation, liver and brain were induced by AH fecal transplantation. Particularly, immune cells isolated from the brains of AH-transplanted mice that were fed ethanol showed a decrease in the critical anti-inflammatory cytokine IL-10. Moreover, AH microbiome alone led to an increase in CD4:CD8 T-cell ratio in the brain parenchyma that was predominated by the CD4 T-cell expansion. In contrast, in AH-FMT mice fed ethanol, the ratio increased due to CD8 T cell depletion. Furthermore, immunohistochemical analysis showed that AH microbiome by itself induced neuroinflammation, as documented by an increase in reactive astrocytes and morphological changes in microglia as well as an up regulation of the inflammatory marker COX-2. These neuro inflammatory changes were further enhanced by ethanol feeding, demonstrating a more severe phenotype. Significantly, all the pathological changes induced by the AH fecal transplant were absent in animals receiving fecal transplantation from control non-alcoholic individuals. These data strongly indicate that the alcohol-induced changes in the gut microbiome play a causal role in the pathological changes in the gut-liver-brain axis and development of neuro-inflammation. Importantly, these data also indicate that the gut may be a potential therapeutic target in the treatment of alcohol-induced neuro-inflammation.
Supported by NIH/NIAAA U01 AA022618 (SB), Commonwealth of Kentucky Challenge for Excellence, Norton Healthcare (SRW)

Damon DiSabato (1) – Danny Nemeth (2) – Xiaoyu Liu (2) – Kristina Witcher (1) – Braedan Oliver (1) – Jonathan Godbout (1) – Ning Quan (2)
Institute for Behavioral Medicine Research, The Ohio State University, Columbus, United States (1) – Brain Institute, Florida Atlantic University, Jupiter, United States (2)

Stress is a well-known cause of mood disorders such as anxiety, the most prevalent psychiatric disorder affecting upwards of a third of the population. Microglia produce increased levels of pro-inflammatory cytokines such as interleukin-1 beta (IL-1) upon exposure to chronic stress. Our model is paired fighting (PF), a form of social stress in which mice are exposed one-on-one to an aggressor mouse for one hour per day for six days. To dissect the role of IL-1 in such a stress model we developed a genetic tool, the IL-1 receptor restore (IL-1R1r/r) mouse. This allows for globally knocking out IL-1R1 expression and restricting restoration via cell type-specific Cre. IL-1R1 floxed mice (IL-1R1f/f) conversely remove IL-1R1 on specific cell types. Our objectives were (1) to determine effects of PF on peripheral and central IL-1R1 responses, and (2) to identify if neuron-specific IL-1R1 was capable of eliciting anxiety-like behavior. We show that PF caused IL-1R1-dependent social withdrawal, working memory deficits, microglial reactivity, and increased brain cytokines. Cre-mediated neuronal-specific IL-1R1 knockout prevented behavioral effects, indicating its necessity for PF-induced behavior. Conversely, IL-1R1 restored solely on glutamatergic neurons reproduced behaviors from PF in wild-type mice. These data indicate IL-1R1 expression is critical for the behavioral deficits seen after social stress, and that glutamatergic neuronal IL-1R1 is both necessary and sufficient for these effects.