Author Of 3 Presentations
P0976 - Lymphotoxin alpha overexpression in the meninges causes lymphoid-like tissue development and subsequent neurodegeneration (ID 1450)
Abstract
Background
Lymphotoxin alpha (LTa) plays a role in both lymphoid organ development and in cell cytotoxicity mechanisms in the immune system. Levels of LTa expression are increased in the post-mortem meninges and CSF from MS patients with high levels of meningeal inflammation and subpial cortical pathology. Tertiary lymphoid-like structures (TLS) are discrete, structured organisations of infiltrating immune cells that form in nonlymphoid tissue and share structural and functional characteristics with secondary lymphoid structures such as lymph nodes.
Objectives
Here we aimed to study whether chronically increased levels of LTa in the cortical meninges in a rat model could give rise to tertiary lymphoid-like structures in the meninges and subsequent underlying cortical pathology.
Methods
To do this we stereotactically injected HIV-1 based VSV-g pseudotyped lentiviral (LV) transfer vectors into the sagittal sulcus (SS) of MOG peptide immunised DA rats to deliver continuous transgene expression (LTα + IFNγ) in the meninges. A neuropathological analysis was conducted at chronic time points up to 3 months.
Results
In LTα/IFNγ LV injected animals accumulation of meningeal inflammation in the midline subarachnoid space and lateral surface of the cortex was found at 28 and 90 days post injection. Meningeal aggregates were formed of CD4 and CD8+ T-cells and CD79a+ B-cells. CD4+ and CD8+ cells were evenly distributed throughout the space whilst CD79a+ B-cells formed into densely, packed focal clusters, lacking T-cells but containing IgG plasma cells. Within CD79a+ B-cell clusters a proportion co-expressed the proliferation marker Ki67.Throughout the infiltrates we identified the formation of channels similar to lymphatic vessels or that stained for a marker of high endothelial venules (HEVs), mucosal addressin cell adhesion molecule ((MAdCAM)-1). Within T-cell rich zones staining for laminin and podoplanin revealed a dense network of stromal cells that are most likely fibroblastic reticular cells (FRC). A population of follicular dendritic cells (FDC) could be stained with the marker ED5. Immunostaining showed high levels of lymphoid chemokines CCL19, CCL21, CXCL13 and CXCL12 and their receptors CCR7 and CXCR4, which organise lymphocyte compartmentalisation. The lymphotoxin beta receptor, through which LTa could induce chemokine and MAdCAM-1 production, was highly expressed throughout the infiltrates. In the underlying cortical parenchyma there were expansive regions that demonstrated marked neuronal loss (40% loss neun+ cells) extending from the pial surface into deeper cortical layer V.
Conclusions
The segregation of meningeal infiltrates into discrete T-cell and B-cell regions, the presence of FRC and FDC cell networks, HEVs and lymphoid chemokine production show LTα overexpression is sufficient to induce formation of meningeal TLS. The loss of subpial neurons shows TLS formation can lead to underlying cortical pathology.
P0984 - Neuron-specific activation of necroptosis signaling in multiple sclerosis cortical grey matter (ID 1341)
Abstract
Background
The cause of cortical neurodegeneration and subsequent grey matter (GM) atrophy is not completely understood, although it is suggested to be stimulated by a cascade of events triggered by chronic inflammation. Transcriptomics analysis of cortical GM suggests a dysregulation of TNF signaling towards activation of necroptosis in the presence of increased meningeal inflammation.
Objectives
To explore the role of sustained production of TNF in the subarachnoid space/meninges as a trigger for the activation of TNFR1-dependent necroptosis and subsequent neurodegeneration in progressive MS.
Methods
Quantitation of levels of proteins involved in necroptotic signaling was carried out in post-mortem brains of MS cases (n=28) and controls (n=11) by western blotting, together with localisation by immunohistochemistry. A rat model of cortical pathology in which TNF and IFNg are chronically over-expressed in the cortical meninges was used to determine the consequences of elevated CSF cytokines on cortical neurons. Primary rat cortical neurons were used of in vitro validation studies.
Results
A substantial significant increase in protein levels for TNFR1 and the subsequent key steps of necroptotic signaling, namely the RIPK1, RIPK3 and MLKL kinase cascade, was present in the MS GM, accompanied by a decrease in the caspase 8 cleaved p18 subunit. Upregulation of these proteins occurred predominantly in pyramidal neurons in cortical layers II-III and V-VI, together with an increase in the percentage of positive neurons, with negligible expression in the control brain. When MS cases were classified according to the severity of meningeal inflammation, pMLKL and MLKL levels were both significantly increased in cases with more abundant meningeal inflammation (6.5 and 8.0-fold respectively). Oligomeric forms of MLKL, which indicate the final stages of necroptosis, were found exclusively in MS GM. Chronic overexpression of TNF and IFNg in the rat cortical meninges gave rise to neuronal loss in cortical layers II-III and a >20-fold increase in neurons expressing necroptotic markers. Finally, exposure of rat primary cultured cortical neurons to TNF induced necroptosis, but only when apoptosis was inhibited.
Conclusions
Taken together, our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated by chronic meningeal inflammation, which contributes to the accumulation of clinical deficit in MS.
P0999 - The role of neuronal CXCL13 chemokine expression in Multiple Sclerosis Pathology (ID 907)
Abstract
Background
Chemokine CXCL13 is suggested as a potential prognostic marker for patient outcomes. Cerebrospinal fluid (CSF) levels of CXCL13 are increased at all stages of MS and correlate with CSF B-cell counts, disability, cortical thinning and markers of neurodegeneration. A recent GWAS study identified a SNP in the promoter region of its receptor CXCR5 (rs630923) was a MS susceptibility loci. However, little is known concerning its expression pattern or mechanisms of action within the CNS.
Objectives
We aimed to study the expression of CXCL13 and its receptor CXCR5 in the grey matter (GM) of post-mortem SPMS brains and how this might contribute to cortical pathology and clinical outcomes. To investigate the occurrence of the rs630923 SNP in 240 cases in the MS tissue bank ask whether having either the common (C:C) or minor (A:C) 'protective' genotype correlated to CXCL13 or CXCR5 levels or with any clinical outcomes
Methods
Levels of CXCL13 and CXCR5 were assessed in cortical GM from 50 MS post-mortem brains using ELISA and western blotting and immunofluorescence with cell specific markers. We examined the role of CXCL13 and CXCR5 expression in primary cultures of rat neurons and astrocytes. Finally, we used a rat model of cortical pathology in which LTα and IFNγ are chronically over-expressed in the cortical subarachnoid space of DA rats.
Results
Protein levels for both CXCL13 and CXCR5 were increased in MS GM and correlated with the extent of GM demyelination and neuronal loss. Patients with the shortest disease duration had the highest GM CXCL13 levels. CXCL13 was expressed primarily by neurons in layers II/III and IV. CXCR5 was expressed in neurons and astrocytes, mainly in the subpial layers and in deep layer VI near the white matter. Patients with C:C allele had significantly shorted time to progressive than A:C genotypes. Both CXCL13 protein in tissue and CSF and CXCR5 tissue levels are higher in C:C genotype than A:C. Chronic overexpression of LTα and IFNγ in the rat meninges increased the neuronal expression of CXCL13, suggesting CSF cytokines can influence parenchymal CXCL13 expression. Stimulation of primary neurons with LTα, TNF or IFNγ lead to an increase in CXCL13 gene and protein expression. Recombinant CXCL13 alone was not toxic to neurons, but it increased the level of cell death when added to neuronal cultures together with L-glutamate. This was accompanied by increased levels of caspase-8/9 mRNA and was inhibited by a caspase inhibitor Z-VAD-FMK. Protein arrays of primary astrocyte cultures stimulated with CXCL13 showed increased expression of CCL5, CCL3, CCL20, CXCL10 and CXCL5.
Conclusions
We suggest that neuronal expression of CXCL13 is stimulated by cytokines present in the GM and CSF and may signal through CXCR5 receptors on astrocytes to increase production of chemokines linked to MS progression. Neuronally produced or CSF derived CXCL13 may act on CXCR5 on neurons making them more vulnerable to cell death when they are exposed to stressors.
Presenter Of 2 Presentations
P0976 - Lymphotoxin alpha overexpression in the meninges causes lymphoid-like tissue development and subsequent neurodegeneration (ID 1450)
Abstract
Background
Lymphotoxin alpha (LTa) plays a role in both lymphoid organ development and in cell cytotoxicity mechanisms in the immune system. Levels of LTa expression are increased in the post-mortem meninges and CSF from MS patients with high levels of meningeal inflammation and subpial cortical pathology. Tertiary lymphoid-like structures (TLS) are discrete, structured organisations of infiltrating immune cells that form in nonlymphoid tissue and share structural and functional characteristics with secondary lymphoid structures such as lymph nodes.
Objectives
Here we aimed to study whether chronically increased levels of LTa in the cortical meninges in a rat model could give rise to tertiary lymphoid-like structures in the meninges and subsequent underlying cortical pathology.
Methods
To do this we stereotactically injected HIV-1 based VSV-g pseudotyped lentiviral (LV) transfer vectors into the sagittal sulcus (SS) of MOG peptide immunised DA rats to deliver continuous transgene expression (LTα + IFNγ) in the meninges. A neuropathological analysis was conducted at chronic time points up to 3 months.
Results
In LTα/IFNγ LV injected animals accumulation of meningeal inflammation in the midline subarachnoid space and lateral surface of the cortex was found at 28 and 90 days post injection. Meningeal aggregates were formed of CD4 and CD8+ T-cells and CD79a+ B-cells. CD4+ and CD8+ cells were evenly distributed throughout the space whilst CD79a+ B-cells formed into densely, packed focal clusters, lacking T-cells but containing IgG plasma cells. Within CD79a+ B-cell clusters a proportion co-expressed the proliferation marker Ki67.Throughout the infiltrates we identified the formation of channels similar to lymphatic vessels or that stained for a marker of high endothelial venules (HEVs), mucosal addressin cell adhesion molecule ((MAdCAM)-1). Within T-cell rich zones staining for laminin and podoplanin revealed a dense network of stromal cells that are most likely fibroblastic reticular cells (FRC). A population of follicular dendritic cells (FDC) could be stained with the marker ED5. Immunostaining showed high levels of lymphoid chemokines CCL19, CCL21, CXCL13 and CXCL12 and their receptors CCR7 and CXCR4, which organise lymphocyte compartmentalisation. The lymphotoxin beta receptor, through which LTa could induce chemokine and MAdCAM-1 production, was highly expressed throughout the infiltrates. In the underlying cortical parenchyma there were expansive regions that demonstrated marked neuronal loss (40% loss neun+ cells) extending from the pial surface into deeper cortical layer V.
Conclusions
The segregation of meningeal infiltrates into discrete T-cell and B-cell regions, the presence of FRC and FDC cell networks, HEVs and lymphoid chemokine production show LTα overexpression is sufficient to induce formation of meningeal TLS. The loss of subpial neurons shows TLS formation can lead to underlying cortical pathology.
P0999 - The role of neuronal CXCL13 chemokine expression in Multiple Sclerosis Pathology (ID 907)
Abstract
Background
Chemokine CXCL13 is suggested as a potential prognostic marker for patient outcomes. Cerebrospinal fluid (CSF) levels of CXCL13 are increased at all stages of MS and correlate with CSF B-cell counts, disability, cortical thinning and markers of neurodegeneration. A recent GWAS study identified a SNP in the promoter region of its receptor CXCR5 (rs630923) was a MS susceptibility loci. However, little is known concerning its expression pattern or mechanisms of action within the CNS.
Objectives
We aimed to study the expression of CXCL13 and its receptor CXCR5 in the grey matter (GM) of post-mortem SPMS brains and how this might contribute to cortical pathology and clinical outcomes. To investigate the occurrence of the rs630923 SNP in 240 cases in the MS tissue bank ask whether having either the common (C:C) or minor (A:C) 'protective' genotype correlated to CXCL13 or CXCR5 levels or with any clinical outcomes
Methods
Levels of CXCL13 and CXCR5 were assessed in cortical GM from 50 MS post-mortem brains using ELISA and western blotting and immunofluorescence with cell specific markers. We examined the role of CXCL13 and CXCR5 expression in primary cultures of rat neurons and astrocytes. Finally, we used a rat model of cortical pathology in which LTα and IFNγ are chronically over-expressed in the cortical subarachnoid space of DA rats.
Results
Protein levels for both CXCL13 and CXCR5 were increased in MS GM and correlated with the extent of GM demyelination and neuronal loss. Patients with the shortest disease duration had the highest GM CXCL13 levels. CXCL13 was expressed primarily by neurons in layers II/III and IV. CXCR5 was expressed in neurons and astrocytes, mainly in the subpial layers and in deep layer VI near the white matter. Patients with C:C allele had significantly shorted time to progressive than A:C genotypes. Both CXCL13 protein in tissue and CSF and CXCR5 tissue levels are higher in C:C genotype than A:C. Chronic overexpression of LTα and IFNγ in the rat meninges increased the neuronal expression of CXCL13, suggesting CSF cytokines can influence parenchymal CXCL13 expression. Stimulation of primary neurons with LTα, TNF or IFNγ lead to an increase in CXCL13 gene and protein expression. Recombinant CXCL13 alone was not toxic to neurons, but it increased the level of cell death when added to neuronal cultures together with L-glutamate. This was accompanied by increased levels of caspase-8/9 mRNA and was inhibited by a caspase inhibitor Z-VAD-FMK. Protein arrays of primary astrocyte cultures stimulated with CXCL13 showed increased expression of CCL5, CCL3, CCL20, CXCL10 and CXCL5.
Conclusions
We suggest that neuronal expression of CXCL13 is stimulated by cytokines present in the GM and CSF and may signal through CXCR5 receptors on astrocytes to increase production of chemokines linked to MS progression. Neuronally produced or CSF derived CXCL13 may act on CXCR5 on neurons making them more vulnerable to cell death when they are exposed to stressors.