Yisel Cantres-Rosario (1) – Elaine Rodriguez (1) – Valerie Wojna (1)
University of Puerto Rico, Medical Sciences Campus, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico (1)

Human immunodeficiency virus (HIV) is able to enter from the periphery to the central nervous system, triggering neurocognitive impairment which can be asymptomatic, mild cognitive and motor impairment or in a few cases severe dementia. With the availability and efficacy of antiretroviral therapy, HIV patients have a better quality of life. However, HIV-associated neurocognitive disorders (HAND) still prevail in near 50% of the treated patients. In this study, we used ELISA to measure neurofilament light chain (NF-L) and neopterin, as markers of neuronal injury and immune activation, respectively. We used plasma and cerebrospinal (CSF) samples obtained from HIV-seropositive (HIV+) patients (n=35) at different stages of HAND, compared to HIV-seronegative controls (n=10). HIV+ patients were subjected to standard laboratory tests (including viral load and CD4+ cell count) and neuropsychological tests (eight domains). Statistical tests included Mann-Whitney, Kruskal-Wallis and Spearman’s correlation. We hypothesize that HIV+ patients have increased levels of NF-L and Neopterin in plasma and CSF, compared to controls. We did not detect differences in levels of NF-L in the CSF, between controls and HIV+ patients. However, there is an inverse correlation between the levels of NF-L in CSF and the neuropsychological tests z-score (NPZ) (r = -0.345, p = 0.039). Moreover, HIV+ patients have higher levels of NF-L in plasma (p = 0.0024) compared to controls. The levels of NF-L in plasma are also inversely correlated with NPZ (r = -0.428, p = 0.0092). Neopterin is higher in the CSF of HIV+ patients compared to controls (p = 0.011), and is directly correlated with the viral load in plasma (r = 0.656, p = 0.057) and CSF (r = 0.873, p < 0.0001). Interestingly, neopterin levels were higher in the plasma of controls than HIV+ patients (p<0.0001), and where directly correlated to the viral load in plasma (r = 0.589, p = 0.0164), as well. In summary, neopterin increases and correlates with viral load in the CSF; while NF-L increases and inversely correlates with neurocognitive performance in plasma. Neopterin is a better marker of HIV-associated neuropathology in the CSF, while NF-L is a better marker in plasma. Our results point to an interesting combination of two markers of HIV-associated neuropathology and CNS immune activation.

Chieh-Hsin Lee (1) – Maryam Nakhaei-Nejad (2) – David Barilla (2) – Carlos Camara-Lemarroy (3) – Luanne Metz (3) – Claudia Silva (3) – V. Wee Yong (3) – Fabrizio Giuliani (2)
University of Alberta, Neuroscience and Mental Health Institute, Edmonton, Canada (1) – University of Alberta, Department of Medicine, Edmonton, Canada (2) – University of Calgary, Department of Clinical Neurosciences, Calgary, Canada (3)

Clinically isolated syndrome (CIS) is the prodromal phase of multiple sclerosis (MS) disease course, with patients having experienced one neurological episode. Since up to 70% of CIS patients are subsequently diagnosed with MS, it is important to determine who will convert to RRMS later. Multi-colour flow cytometry panels were used to identify up to 50 peripheral blood lymphocyte subpopulations. Samples from two sites were used, with one done on fresh whole blood and the other on cryopreserved peripheral blood mononuclear cells (PBMCs) after recovery. We compared patients with CIS who do (CIS-C) or do not (CIS-N) covert to RRMS (relapsing remitting MS) later. Comparisons were also done between the two sites. This study aims to immunophenotype PBMC subsets in CIS who do or don’t covert; identify biomarkers that predict progression to RRMS; and examine the effect of cryopreservation and recovery on cell populations. In the fresh samples, the T cell subcompartment, CIS-C patients had a higher expression level of CD127 (IL-7 receptor) in the Granzyme B expressing CD8+ T cells (CD8+CCR7-CD27-) than non-converters. A significantly lower dendritic cell population in converters than non-converters was shown as well. Converters also had a significantly higher level of CD56DimCD16+ natural killer cells. Comparisons between fresh blood and cryopreserved samples showed a variety of changes, including a higher population of classical memory and naïve & transitional B cells in the cryopreserved samples. Within the cryopreserved patient samples, no differences were detected between converters and non-converters. Partial least square discriminant analysis (PLS-DA) was used to compare the overall immunophenotypes between CIS-C and CIS-N, with no significant separation in either fresh blood or cryopreserved samples. In conclusion, some immune subpopulations are significantly different between converters and non-converters within fresh blood samples, with no differences detected in cryopreserved samples. Overall comparisons of the immunophenotypes show no separation between the groups in either fresh blood or cryopreserved samples. Our findings also showed that cryopreservation of cells can result in a wide range of differential changes in immune subpopulations, potentially obscuring differences between groups.

Camille Grasmuck (1) – Soufiane Ghannam (1) – Marc Charabati (1) – Evelyn Peelen (1) – Lyne Bourbonnière (1) – Sandra Larouche (1) – Olivier Fortin (1) – Jorge Ivan Alvarez (2) – Hania Kebir (1) – Alexandre Prat (1)
CRCHUM, Department of Neurosciences, Faculty of Medicine, Université de Montreal, Montréal, Canada (1) – Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States (2)

Disruption of the blood-brain barrier (BBB) and migration of leukocytes from the periphery to the central nervous system (CNS) are early events in lesion formation during multiple sclerosis (MS). Among CNS-infiltrated leukocytes, TH17 lymphocytes are important contributors to inflammation and tissue damage. They readily cross the BBB to infiltrate the CNS and express pro-inflammatory cytokines. Using proteomic and RNA sequencing techniques, we have identified Dual Ig domain containing Cell Adhesion Molecule (DICAM) as a new adhesion molecule expressed by human TH17 lymphocytes and BBB endothelial cells (ECs). The expression and function of DICAM in MS pathogenesis remain unexplored. The current study aims to evaluate DICAM’s role in encephalitogenic TH17 lymphocytes’ migration to the CNS. To explore the DICAM expression profile, we performed flow cytometry and qPCR on human BBB-ECs and immune cells subsets isolated from healthy control peripheral blood. Confocal microscopy, flow cytometry and qPCR have been performed to explore DICAM expression in MS lesions and on peripheral immune cells in situ and ex vivo. The role of DICAM in human TH17 lymphocytes in transmigration was assessed in vitro by blocking DICAM in a migration assay across a monolayer of human BBB-ECs. Its role in adhesion was explored in vitro by a flow adhesion assay of mouse TH17 lymphocytes on brain ECs in presence of DICAM blocking antibody or isotype control. Different animal models of MS (EAE) were also used to explore DICAM function in vivo.
We showed that DICAM expression is strongly associated with expression of ROR-gamma, IL-17 and IFN-gamma. These data demonstrate that DICAM is expressed on the surface of potentially encephalitogenic TH17 lymphocytes and that expression of DICAM is regulated by IL-23, IL-1b and IL-6, cytokines which are involved in CNS autoimmune diseases. The proportion of DICAM+ CD4+ lymphocytes is significantly increased in the blood and CNS of MS patients compared with healthy controls. Furthermore, DICAM expression is upregulated on the BBB within inflammatory lesions in the brains of MS patients. Moreover, blockade of DICAM restricts the adhesion and the migration of TH17 lymphocytes across BBB-ECs and decreases EAE severity. This study aims to characterize the interaction mediated by DICAM between TH17 lymphocytes and the BBB. This adhesion molecule might be involved in MS pathogenesis and therefore, could become a new therapeutic target for MS treatment.

Julia Hansen (1§) – Eduardo Beltrán (1§) – Lisa Ann Gerdes (1§) – Reinhard Hohlfeld (1*) – Klaus Dornmair (1*)
Institute of Clinical Neuroimmunology, Hospital of the LMU Munich, Munich, Germany (1)

Inflammatory features of multiple sclerosis (MS) include T- and B-cell lymphocyte accumulations in central nervous system (CNS) and cerebrospinal fluid (CSF), with activated CD8+ T-cells as the dominating population in the CNS. The function of the lymphocytes that invade brain tissue and the CSF is still enigmatic. We here investigated a cohort of 4 monozygotic twin pairs discordant for MS where one twin had established MS and the co-twin was clinically healthy, but carried a maximal familial risk for developing MS. Altogether we analyzed 4 MS patients, 4 healthy controls and 6 subjects with “subclinical neuro-inflammation” (SCNI). These subjects were clinically “healthy” co-twins who showed small MRI lesions, indicating a presumably preclinical form of MS. Of note, four of the SCNI patients had oligoclonal bands (OCBs), which is a hallmark of established MS. We used single-cell profiling to compare gene expression patterns of lymphocytes from CSF of MS-discordant monozygotic twin pairs. This enabled us to investigate disease-relevant changes in gene-expression patterns in genetically identical subjects and revealed insights into very early MS pathogenesis. We identified clonally expanded CD8+ T cells, plasmablasts, and – to a lesser extent – CD4+ T cells. Clonal expansions are thought to arise from sustained stimulation by antigen and are therefore presumably of pathogenic relevance. Moreover, expanded T cells showed characteristics of an activated tissue resident memory T (TRM) cell phenotype. TRM cells express the early activation marker CD69, pro-inflammatory cytokines including IFNG and IL-2 and upregulate tissue retention molecules that prevent them from leaving the CSF. In particular, molecules involved in T cell egress as S1PR1, CCR7, or SELL were strongly downregulated in expanded cells from MS patients but downregulation was also observed in SCNI. Further, expanded plasmablast clones were detected only in patients with OCBs. Our data provide evidence for very early concomitant activation of three components of the adaptive immune system in MS, with a notable contribution of activated, clonally expanded TRM-like CD8+ T cells.

Lorena Juriol (1) – Natalín Valeff (1) – María Silvia Ventimiglia (1) – Jorge Correale (2) – Federico Jensen (1)
Laboratory for Immunology of Reproduction. CEFYBO-UBA-CONICET, Faculty of Medicine. University of Buenos Aires, CABA, Argentina (1) – Department of Neuroimmunology and Demyelinating Diseases, Raúl Carrea Institute for Neurological Research, CABA, Argentina (2)

Multiple Sclerosis (MS) is an autoimmune inflammatory disease of the Central Nervous System, being 2-3 times more common in women than men. In addition, it is well known that pregnancy significantly ameliorates MS disease activity. Thus, female sex-hormones are suspected to account for this phenomenon. Human chorionic gonadotropin (hCG) is a glycoprotein hormone exclusively produced during pregnancy and it has been proved to have strong anti-inflammatory properties.
We evaluated the potential immunomodulatory effects of hCG on cytokine production by immune cells in-vitro in the context of MS.
Blood samples from 20 women diagnosed with MS (24-62 years old) were obtained. PBMC were isolated and cultivated for 24 h in the absence (control) or presence of urinary (u-hCG, 100IU/ml) or recombinant (r-hCG, 5ug/ml) hCG. During the last 5 h of culture, PBMC were stimulated with PMA and Ionomycin. Supernatants (SN) were stored at -80°C and TNF-alpha (TNF) and IL-10 were assessed by Cytometric Beads Array. Flow Cytometry was used to characterize production of TNF and IL-10 by lymphocytes: CD19+ (total B cells), CD3+CD4+ (Th cells), CD3+CD4+CD25+Foxp3+ (Treg). B cell subsets were CD19high (CD19hi) and CD19low (CD19low). Further characterization of B cells demonstrated that CD19hi expressed lower TNF/IL-10 ratio than CD19low B cells.
r-hCG in-vitro treatment significantly lowered the production of TNF in the SN of PBMC. Intracellular staining showed a reduction in the production of TNF by Th cells and total B cells after stimulation with r- and u-hCG vs control. Correlation between age of the patient and TNF production, demonstrated an increased production of TNF in older patients (Th cells, Spearman r=0.45, p<0.04; CD19low B cells, Spearman r=0.58, p<0.03). Even though there were no significant differences in IL-10 levels in SN, intracellular staining showed an increased production of IL-10 by Treg and CD19hi B cells upon hCG treatment. After stimulation with r-hCG, IL-10 production in SN was negatively correlated with age (Spearman r=-0.40, p<0.05). Moreover, this correlation was also observed in the production of IL-10 by total (Spearman r=-0.61, p<0.02) and CD19low B cells (Spearman r=-0.67, p<0.01). Overall, hCG was able to shift cytokine profile towards an anti-inflammatory milieu and this effect proved to be correlated with age. These results open new avenues to further explore the mechanisms behind pregnancy-associated improvement in MS patients.

Amandine Isenbrandt (1, 2) – Katherine Coulombe (2) – Hend Jarras (2) – Denis Soulet (1, 2)
Faculty of Pharmacy, Laval University (1) – CHU de Québec – Laval University research center (2)

Parkinson’s disease (PD) is known for its characteristic motor symptoms (MS), yet it is also important to pay close attention to the non-motor symptoms (NMS) present in the disease. These symptoms usually appear years before the onset of motor symptoms and are of more concern to the patients than MS. Gastrointestinal disorders such as constipation gastric emptying problems are some of the NMS. A new progressive beta-sisterol beta-d-glucoside (BSSG) model has recently been developed, showing a combination of several features of the disease such as behavioral disorders, a decrease in dopaminergic neurons of the substantia nigra, as well as the presence of alpha-synuclein in several regions of the brain, making it therefore the most complete PD model currently known. In order to complete the characterization of the model, analyses in the myenteric plexus of the colon were performed and showed an upward trend of CD163 and MHCII inflammatory markers in the first two months of exposure to the toxin. An analysis of VIP neurons exposes their increased number at 10 months. Although the compound does not seem to show a significant impact on the inflammatory markers tested, it seems to influence the neuronal component. Thus, it will be imperative in the future to investigate different types of neurons to understand the action of BSSG on the neuronal populations of the intestine.

Jason Fernandes (1) – Brienne McKenzie (1) – Matthew Doan (2) – William Branton (1) – Christopher Power (3)
University of Alberta, Medical Microbiology and Immunology, Edmonton, Canada (1) – University of Alberta, Neuroscience and Mental Health Institute, Edmonton, Canada (2) – University of Alberta, Department of Medicine, Edmonton, Canada (3)

Multiple sclerosis (MS) is a debilitating autoimmune disease of the CNS characterized by inflammatory demyelination and associated neurodegeneration. One of the key factors that drives the progress of MS is the loss of myelin-producing oligodendrocytes. Along with persistent activation of microglia and astrocytes, the profound reduction in myelin leads to cognitive, visual and motor deficiencies in the 2.5m people afflicted with MS globally. We previously identified pyroptosis as a key driver in neuroinflammation and neurodegeneration in MS and its animal model, experimental autoimmune encephalomyelitis. Pyroptosis (“fiery death”) is a type of highly inflammatory programmed cell death, implicated in a wide spectrum of neurological diseases. During pyroptosis, plasma membrane rupture and cell lysis are driven by inflammasome-mediated activation of the pore-forming executioner protein, gasdermin D (GSDMD). Herein, we report for the first time that GSDMD-dependent pyroptosis engages the apoptotic executioner caspases-3 and -7, using microglia as a model system. Caspase-3/7 activation is considered a prototypic hallmark of “immunologically silent” apoptotic cell death. In contrast to this paradigm, we demonstrate that caspase-3/7 activation occurred rapidly following exposure to pyroptotic stimuli and was prevented by inflammasome inhibition. Suppression of caspase-3/7 prevented GSDMD-mediated pyroptosis. We show that plasma membrane rupture, nuclear disintegration, and key morphological features associated with pyroptosis (e.g. pyroptotic bodies on the cell membrane) were rescued with caspase-3/7 inhibition. Specific substrates of caspase-3/7 were cleaved during pyroptosis in both the cytoplasm (ROCK1) and the nucleus (PARP, DFF45), confirming the proteolytic activity of caspase-3/7. Cleavage of these substrates was markedly reduced upon suppression of caspase-3/7. To verify the in vivo relevance of our findings, we also demonstrate co-expression of activated caspase-3 with GSDMD in cerebral white matter. Collectively these results highlight a putative role for caspase 3/7 in facilitating pyroptosis in human microglia.

Matthew Doan (1) – William Branton (2) – Tom Hobman (3) – Benjamin Gelman (4) – Christopher Power (2)
University of Alberta, Neuroscience and Mental Health Institute, Edmonton, Canada (1) – University of Alberta, Department of Medicine, Edmonton, Canada (2) – University of Alberta, Department of Cell Biology, Edmonton, Canada (3) – University of Texas Medical Branch, Department of Pathology, Galveston, United States (4)

Human pegivirus-1 (HPgV-1) is a positive sense, single-stranded RNA virus of the Flaviviridae family. Several viruses in this family, including Zika and West Nile viruses, have been associated with encephalitis and neuroinflammation. Recently, our group reported HPgV-1 infection in the brains of two patients with fatal leukoencephalitis, wherein HPgV-1 antigen (NS5A) was detected chiefly in glial cells (astrocytes and oligodendrocytes) in cerebral white matter (Balcom, Doan et al., 2018). Interestingly, previous studies have shown that HPgV-1 can be beneficial for patients co-infected with Human Immunodeficiency Virus (HIV). Compared to patients with HIV alone, co-infected patients show higher CD4+ counts, lower plasma HIV viral load, and increased survival. As the central nervous system (CNS) is an important reservoir for HIV and HIV causes a range of neurological symptoms in HIV-infected patients known as HIV-associated neurocognitive disorder (HAND), we hypothesized that HIV and HPgV-1 may likewise interact within the CNS. In this study, we aimed to define the neuroinflammatory profile of HPgV-1 infection both alone and in combination with HIV co-infection, and to delineate the effects of co-infection on viral load in the CNS compartment. To study the in vivo neuroinflammatory effects of HPgV-1 infection, we identified 10 patients with HPgV-1 brain infection (1000-10,000 HPgV-1 RNA copies/g of tissue) in a cohort including HIV-1+ patients, along with age-matched uninfected controls. The expression of proinflammatory cytokine genes (IL1B, TNFA, IL6) was significantly increased in the prefrontal cortex of co-infected patients (n=5) compared to uninfected (n=6) or mono-infected (HIV, n=5; HPgV-1, n=5) patients. In addition, several genes associated with pro-inflammatory regulated cell death (eg. pyroptosis) were elevated in patients co-infected with both viruses, compared to mono-infected patients. Furthermore, HIV viral copy number in the brain, detected by droplet digital PCR (ddPCR), were increased in co-infected patients compared to patients with HIV alone. The present studies indicate that HPgV-1 is a neurotropic virus with the capacity to increase HIV burden in the brain and act synergistically with HIV to promote neuroinflammation in vivo.

Deepak Kumar Kaushik (1) – Anindita Bhattacharya (1) – V. Wee Yong (1)
Hotchkiss Brain Institute, University of Calgary, Calgary, Canada (1)

Multiple sclerosis (MS) is a demyelinating condition of the central nervous system (CNS) in which the inflammatory leukocytes migrate across the post-capillary venules and meningeal barriers into the parenchyma. Migration of leukocytes from periphery to the CNS likely requires significant amounts of energy, especially as they accumulate in perivascular spaces (structures referred to as perivascular cuffs) before migrating across the blood brain barrier (BBB). Using the inflammatory mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we sought to study the metabolic requirements and its relevance to the leukocytes that cross the BBB in MS. During peak clinical disease activity in EAE, we found that leukocytes within the perivascular cuffs expressed lactate dehydrogenase A (LDHA), an inducible glycolytic enzyme that converts pyruvate to lactate. We also found that these leukocytes were strongly immuno-reactive for MCT-4, an important transporter for lactate, which was in turn regulated by extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) on macrophage membranes. These results were mirrored by strong expression of MCT-4, LDHA and EMMPRIN in inflammatory perivascular cuffs in brains from patients with MS. Functional relevance of glycolysis in the infiltrating leukocytes was demonstrated by significant reduction of macrophage transmigration in culture following exposure to the selective LDHA inhibitor, FX11, and the MCT-4 inhibitor, the cinnamon derivative alpha-cyano 4-hydroxy-cinnamic acid (CHCA). Similarly, siRNA-mediated knockdown of LDHA and MCT-4 decreased pro-inflammatory properties of macrophages in culture. Importantly, perturbation of the glycolytic machinery with CHCA in EAE mice reduced clinical severity and histopathology as compared to the PBS-treated sham group. Our results show that glycolysis plays a crucial role in conferring the infiltrating leukocytes a pro-inflammatory phenotype and identify CHCA as a potential modulator of neuroinflammation in MS with therapeutic and dietary implications.

MONIQUE MARYLIN ALVES DE ALMEIDA (1, 4) – FRANCESCA PIEROPAN (4) – Larissa de Matos Oliveira (5) – Jorge Mauricio David (2) – Juceni Pereira David (3) – Manoelito Coelho dos Santos Junior (5) – Victor Diogenes A. da Silva (1) – Cleide dos Santos Souza (6) – Silvia Lima Costa (1*) – Arthur Morgan Butt (4*)
Department of Biochemistry and Biophysics, Institute of Health Sciences, Federal University of Bahia, Brazil (1) – Departament of Inorganic Chemistry, Institute of Chemistry, Federal University da Bahia, Brazil (2) – Departament of Medication, Faculty of Pharmacy, Federal University of Bahia, Brazil (3) – School of Pharmacy and Biomedical Science, University of Portsmouth, United Kingdom (4) – Department of Health, State University of Feira de Santana, Brazil (5) – Department of Chemistry, University of Sheffield, United Kingdom (6)

Estrogen receptors (ER) are important pharmacological targets to induce remyelination. Here, we have examined the effect of the phytoestrogen agathisflavone (FAB) on demyelination and neuroinflammation, which are hallmarks of Multiple Sclerosis (MS) and is important in most neuropathologies. Cerebellar slices from Sox10-EGFP reporter mice aged postnatal day (P)10-12 were maintained for 7 days in vitro (DIV). Demyelination was induced by lysolecithin (LPC, 0.5mg/mL), followed by treatment with agathisflavone (5 or 10µM) for 2DIV and analysed by immunohistochemistry (IHC). Demyelination (MBP-IHC) and axonal degeneration (NF-IHC) were prominent following LPC treatment, compared to controls, whilst FAB significantly accelerated remyelination and protected against axonal damage, as well as protecting Purkinje neurons against apoptosis mediated death (Calbindin+/Caspase 3 cleaved+). Treatment with FAB significantly increased the number of NG2+ oligodendrocytes precursor cells (OPCs) and stimulated their proliferation (SOX10+/Ki67+), as well as increasing the number of mature oligodendrocytes (CC1+), and decreasing their apoptosis mediated death (CC1+/Caspase 3 cleaved+). Furthermore, LCT induced astrogliosis (GFAP-IHC) and microglial proliferation (IBA1+/Ki67+), as well as switching microglia phenotype from predominantly M1 to M2 (ratio of CD16/32+/CD206+), and this was reverted by FAB. RT-qPCR showed that FAB reduces Tnfa, Il1β, and Nos2 and increases Arg1, Tgfb and Acvr1b expression. Notably, the promyelinating and antigliotic effects of FAB were inhibited by blockade of alpha estrogen receptors (aER), and molecular docking analyses demonstrated intermolecular interactions between FAB and aER, which has known promyelinating and prosurvival effects. This study provides evidence that FAB acts via aER to promote oligodendrocyte regeneration and remyelination and switches microglia to a phenotype that is beneficial for repair. The results indicate FAB may constitute a promising non-toxic disease-modifying therapy in MS and other neuropathologies.
Keywords: Flavonoids, Remyelination, Microglia, Estrogen Receptors.