ISNI 2012 Abstracts

2012 Journal of Neuroimmunology  
Objective: To dissect the relative pathogenic relevance of myelin-specific antibodies (ab) from myelin-specific B cells in the MS model experimental autoimmune encephalomyelitis (EAE). Background: B cells and B cell-derived ab may both play a pathogenic role in CNS autoimmune disease. Transgenic mice in which a majority of B cells recognizes myelin oligodendrocyte glycoprotein (MOG) and plasma cells secrete high titres of MOG-specific ab (MOG BCR knockin mice) experience a fulminant course of
more » ... tively-induced EAE. Double-transgenic mice which further contain myelin-reactive T cells (MOG BCR knockin× 2D2) spontaneously develop EAE. Utilizing both models in combination with anti (α)-CD20, which depletes B cells but spares CD20 negative plasma cells, we dissected the relative pathogenic contribution of myelin-reactive B cells from endogenously produced myelin-reactive ab. Methods: MOG BCR knockin mice received 0.2 mg α-CD20 i.p./week starting 3 weeks prior to immunization with mouse MOG 1-117 or after EAE was established. MOG BCR knockin × 2D2 mice were injected with 0.2 mg α-CD20/week starting at the age of 4 weeks. T cell phenotype was determined by FACS for IFN-γ and IL-17. Serum from MOG BCR knockin mice immunized with MOG 1-117 was transferred into recipient wild-type (WT) mice with established EAE or naïve 2D2 mice. Serum from WT mice immunized with MOG p35-55 served as control. Results: MOG BCR knockin mice developed fulminant EAE compared to WT mice, which was not affected by depletion of B cells. In doubletransgenic MOG BCR knockin×2D2 mice, α-CD20 treatment did not interfere with development of encephalitogenic T cells or incidence/ severity of spontaneous EAE. While all peripheral compartments were efficiently depleted of B cells, α-CD20 did not affect constitutive secretion of α-MOG ab in either model. Further corroborating the deduced pathogenic role of myelin-reactive ab, serum from BCR MOG-knockin mice containing high titres of pathogenic α-MOG ab exacerbated established EAE in WT recipients and triggered spontaneous EAE in naïve 2D2 recipients. Conclusion: Taken together, these data indicate that endogenously produced self-reactive ab contribute to CNS autoimmune disease independent of myelin-reactive B cells. Ongoing mechanistic experiments suggest that besides promoting CNS demyelination α-MOG ab may enhance myelin-recognition of antigen-presenting cells resulting in accentuated activation of myelin-reactive T cells. 57 Clonally expanded and autoantibody producing B cells are present both in peripheral blood and cerebrospinal fluid of multiple sclerosis patients Objective: B cells and oligoclonal antibodies are present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) and clinically isolated syndrome (CIS) patients but their target antigens remain unknown. The focus of this study was to characterize B cells from the peripheral blood (PB) and CSF of MS and CIS patients following B cell immortalization. Methods: Peripheral blood mononuclear cells (PBMC), peripheral immunoglobulin G + (IgG + ) B cells or CSF cells were infected with Epstein-Barr virus (EBV) to obtain continually dividing B cell lines. Molecular analysis of the Ig heavy chain variable region (V H ) genes was used to analyze clonality and diversity of the immortalized B cells. Binding of the produced antibodies to human oligodendroglioma (HOG), astrocytoma (U251), control epithelial alveolar carcinoma (A549) cell lines and PBMC was analyzed by flow cytometry and immunocytochemistry. Journal of Neuroimmunology xxx (2012) xxx-xxx No of Pages 195 Results: We generated 301 immortalized B cell lines from 18 MS patients, 45 from 4 CIS patients, 7 from 4 patients with a non-inflammatory neurological disease (NIND) and 40 from 2 healthy subjects (HC). Most B cell lines were obtained from the PB although 7 B cell lines were isolated from the CSF of 1 MS patient and 2 from 1 NIND patient. A monoclonality rate of more than 82% was obtained for these B cell lines, eliminating the need for subcloning. Antigen-stimulated clonal expansion and affinity maturation were evidenced both in PB and CSF of MS and CIS patients by the occurrence of B cell lines expressing identical CDR3 sequences and increased V H mutation frequencies when compared to B cells from HC. Moreover, a proportion of peripheral B cells from MS and CIS patients displayed autoreactivity, as demonstrated by analysis of antibody binding to HOG cells, U251 cells, PBMC and A549 cells. Intracellular specificity to HOG cells was demonstrated for antibodies from 4 B cell lines of 3 MS patients. General intracellular binding to 2 or more tested cell lines was demonstrated for 75 other B cell lines from both MS and CIS patients. Interestingly, the major myelin lipid phosphatidyl choline was identified as specific target for 1 CSF B cell line of a MS patient. Conclusions: In conclusion, clonal expansion and affinity maturation were demonstrated for the first time in peripheral B cells of MS and CIS patients. As several B cell lines showed reactivity to human brain cell lines, the antigen reactivity of the generated monoclonal antibodies is now further examined to identify the specific target antigens. Objective: Viral infections of the CNS are often characterized by accumulation of antibody secreting cells (ASC) within the nervous tissue. These ASC play a critical role in clearance or controlling viral recrudescence by providing a local source of antibody (Ab). The signals fostering ASC recruitment into the inflamed CNS are poorly understood. Accumulation of ASC within the CNS during gliatropic coronavirus induced encephalomyelitis is CXCR3 dependent and associated with the sustained expression of CXCL9 and CXCL10 mRNA. The present study determined if either CXCR3 ligand was equally capable of recruiting ASC into the CNS. Methods: To investigate the potential role(s) of CXCR3 ligand(s) on ASC recruitment during virus-induced encephalomyelitis ASC accumulation in the CNS of wild-type, CXCL9 deficient (CXCL9 −/− ), CXCL10 −/− and CXCR3 −/− mice were compared. Mice were infected intracranially with the gliatropic JHM variant of mouse hepatitis virus (JHMV). Real-time quantitative PCR was used to measure parameters of CNS inflammation, while anatomical distribution of CD138 + ASC, CXCL9 and CXCL10 in the CNS were examined by immunohistochemical analysis. Results: Parenchymal CD138 + ASC in the CNS were specifically reduced in CXCL10 −/− , but not in CXCL9 −/− mice. Impaired ASC recruitment in CXCL10 −/− mice coincided with reduced CNS levels of virus specific IgG relative to wt mice, but elevated levels relative to CXCR3 −/− mice. Neutralizing serum Ab levels were not affected by CXCL9 or CXCL10 deficiency. Moreover transcript expression of BCMA and TACI, B cell receptors that function to support ASC survival and differentiation, remained near naïve levels throughout infection in CXCL10 −/− mice. Consistent with similar T cell accumulation and effector function, control of infectious virus was not affected in either CXCL9 −/− or CXCL10 −/− mice. Immunohistochemical analysis revealed CXCL10 expression in parenchymal cells consistent with astrocyte morphology. On the other hand, CXCL9 expression was primarily confined to the perivascular spaces surrounding blood vessels. Increased magnitude and distinct focal expression of CXCL10 compared to CXCL9 may thus underlie preferential in vivo migration of ASC by CXCL10. Conclusions: These results highlight the distinct localization of CXCR3 ligands in the CNS during JHMV infection and demonstrate CXCL10 is indispensable for driving ASC migration into the inflamed CNS parenchyma. 423 Astrocytic autoantibody of neuromyelitis optica (NMO-IgG) binds to aquaporin-4 extracellular loops, monomers, tetramers and high order arrays Blood brain barrier 15 Endothelial miR-155 promotes blood-brain barrier dysfunction in neuroinflammation Objective: Blood-brain barrier (BBB) dysfunction is thought to contribute to disease progression of many neurological conditions with an inflammatory component such as multiple sclerosis (MS). However, the molecular mechanisms leading to breakdown of the brain endothelial barrier 3 Abstracts are largely unknown. Here, we have investigated the role of brain endothelial microRNAs (miRNAs), known post-transcriptional regulators of gene expression, in BBB dysfunction during neuroinflammation. Methods: Initially, we used the immortalized human cerebral microvascular endothelial cell line, hCMEC/D3, to determine altered miRNAs after stimuli with cytokines at different time points using microarray analysis. We confirmed brain endothelial deregulated miRNAs at the neurovascular unit in MS brain tissue, using laser capture microdissection (LCM), and in spinal cords of the experimental autoimmune encephalomyelitis (EAE), by in situ hybridization (ISH). RT-qPCR was carried out to validate altered miRNAs in vitro and in vivo. To identify the molecular mechanisms mediating miR-155-induced brain endothelial breakdown we used transcriptome analysis and novel targets were validated using 3′ UTR analysis. Results: Pro-inflammatory cytokines induced altered levels of many miRNAs in cultured human cerebral microvascular endothelial cells. Amongst the deregulated miRNAs, miR-155 showed the highest and earliest increases in brain endothelium during inflammation both in vitro and in vivo. Changes in the levels of selected brain-endothelial deregulated miRNAs, including miR-155, were confirmed in spinal cord of mice at different stages of EAE and in human MS brain tissue. MiR-155-deficient mice were partially resistant to develop EAE, an effect associated with a reduction in BBB leakage to paracellular tracers at the inflammatory plaque. In addition, miR-155 induced increases in brain endothelial paracellular permeability by targeting not only set of genes that maintain brain endothelial cell-cell junctions but also those that regulate focal adhesion contacts. Conclusions: We propose miR-155 as a novel negative regulator of BBB function in inflammation-mediated neurodegenerative diseases. Thus investigating the roles of miR-155 at the BBB have a number of implications including uncovering new regulators that mediate BBB integrity and identifying potential therapeutic targets to ameliorate the progression of CNS inflammatory disorders. Objective: The blood-brain barrier (BBB) is the regulated interface between the peripheral circulation and the central nervous system (CNS). One of the key protective features of the BBB is that it strictly regulates the efflux of various toxic compounds through specialized ATP-binding cassette (ABC) transporters, thereby maintaining brain homeostasis. ABC transporters can drive cellular exclusion of a wide variety of exogenous compound and drugs, but recent data indicate that endogenous substrates may include inflammatory mediators like cytokines, chemokines and bioactive lipids, suggesting a potential role in (neuro)inflammatory diseases like multiple sclerosis (MS), topic of study in the current project. Methods: To study the role of ABC transporters in MS pathology in more detail, we investigated their expression pattern in well-characterized human post-mortem MS tissue and their expression/function in relevant CNS cells. Moreover, proof of principal was obtained in vivo by inducing experimental autoimmune encephalomyelitis (EAE).in mice that lack one of the ABC transporters P-glycoprotein. Results: Here we demonstrate that in active and chronic inactive MS lesions ABC transporter expression is severely altered not only at the level of the blood-brain barrier (BBB) but also at reactive astrocytes, in which they play a role in regulating immune cell migration across in vitro BBB models. Moreover, we have provided conclusive in vivo evidence indicating that animals that lack P-glycoprotein experience significantly reduced clinical symptoms of experimental autoimmune encephalomyelitis (EAE). Conclusions: Together, these data highlight a novel immunomodulatory capacity of ABC transporters during neuroinflammation and indicate their therapeutic potential to prevent MS lesion formation. 162 microRNAs control brain endothelial cell barrier function and immune quiescence, implications for MS Objective: The sphingosine 1-phosphate (S1P) receptor modulator FTY720P (Gilenya®) potently reduces relapse rate and lesion activity in the neuro-inflammatory disorder multiple sclerosis (MS). Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested 4 Abstracts Magnetic resonance imaging studies suggest that early changes in the normal-appearing white matter of multiple sclerosis (MS) patients precede appearance of gadolinium-enhancing lesions. Although these findings suggest the breakdown of the blood-brain barrier (BBB) as a central characteristic in MS pathogenesis, limited data is available regarding the changes to the CNS vasculature during lesion formation. Objective: To study the differential changes of BBB components, particularly during the early stages of lesion formation in MS and in spontaneous experimental autoimmune encephalomyelitis (EAE). Methods: CNS material from MS patients and from the novel T cell receptor (TCR)-1640 transgenic animals which develop spontaneous relapsing-remitting (RR) EAE were classified according to cellular infiltration, demyelination, astrogliosis and microglial activation. BBB disruption was determined at different levels. We first evaluated the extravasation of endogenous tracers to then examine the expression of the junctional protein molecules. These findings were correlated with changes in the basement membrane and the activation of endothelial cells was determined according to the expression of cell adhesion molecules (CAMs). Results: Preactive MS lesions displayed considerable microglia and astrocyte activation, minimal leukocyte infiltration, a relative absence of demyelination and discrete basement membrane abnormalities. However, we detected considerable redistribution of junctional proteins and increased expression of all CAMs, suggesting that barrier breach occurs at early stages of lesion formation, before significant immune cell infiltration and demyelination. The pattern of BBB disruption in RR mice was established at various stages before clinical signs and the breakdown of the barrier in these mice was nearly identical to the changes detected in preactive lesions from MS brains. Conclusion: Our findings indicate early disturbances in the expression of junctional proteins, CAMs and basement membrane proteins at the level of the BBB in non-demyelinating MS and EAE specimens. These early vascular changes coincide with perivascular immune cell infiltration and bring pathological support for important BBB disruption prior to demyelination, during the first stages of lesion formation. In addition, the involvement of peripheral immune responses in this process will be discussed.
doi:10.1016/j.jneuroim.2012.10.001 fatcat:bih44opaxndnxi3e7ursnupioe