Contributions
Abstract: P418
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Pathology
Introduction: The progressive phase of multiple sclerosis (MS) is characterised by accumulating grey matter (GM) pathology. The presence of immune cell infiltrates in the meninges is associated with lymphoid tissue development, greater cortical demyelination, shorter disease duration and significant neuronal loss. Analysis of isolated meninges of MS cases has shown increased gene expression for the pro-inflammatory cytokines: tumour necrosis factor (TNF) and interferon-γ (IFNγ).
Aims: We aimed to test the hypothesis that chronic production of these cytokines in the meningeal compartment and diffusion into underlying GM can drive MS GM pathology.
Methods: To do this we stereotactically injected HIV-1 based VSV-g pseudotyped lentiviral transfer vectors into the sagittal sulcus (SS) of DA rats to deliver continuous transgene expression (TNF + IFNγ) in the meninges. Rats were either immunised with MOG peptide or IFA as a control. A neuropathological analysis was conducted at chronic time points up to 2 months.
Results: Injection of these vectors induced the formation of lymphoid follicle-like structures in the meninges by 28 dpi, which remained at 2 months, containing CD4+ and CD8+ T-cells, CD79a+ B-cells and Iba1+ macrophages, and MadCAM+ channels. Subpial demyelination underlying these aggregates was accompanied by widespread microglial activation and was partly dependant on MOG immunisation. Quantification of NeuN/HuCD co-staining showed a 23-48% decrease in neuronal numbers in cortical layers II-IV at 2 months post injection. Neuronal loss occurred in both MOG and IFA immunised animals and in the absence of demyelination. TNF/TNFR1 interactions can initiate cell death by activating pathways involved in necroptosis. Immunostaining showed TNFR1 expression by neurons. RT-PCR on cortical RNA at 28 dpi and 2 months showed an increase in expression of TNFR1 and downstream necroptotic genes, RIP3, MLKL, cIAP2 and Nox2 compared to eGFP vector control animals. RIP3+ and MLKL+ immunopositive cells with the morphology of neurons were present in TNF + IFNγ vector injected animals. Membrane staining for phosphorylated MLKL in neurons was suggestive of the final stages of necroptosis.
Conclusions: Our results suggest that TNF in the presence of IFNγ is a potent inducer of meningeal inflammation and can activate TNF signalling pathways in cortical cells leading to neuronal death and subpial demyelination and thus may contribute to clinical progression in MS.
Disclosure: Rachel James:nothing to disclose
Nicholas Mazarakis : nothing to disclose.
Richard Reynolds: nothing to disclose
Abstract: P418
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Pathology
Introduction: The progressive phase of multiple sclerosis (MS) is characterised by accumulating grey matter (GM) pathology. The presence of immune cell infiltrates in the meninges is associated with lymphoid tissue development, greater cortical demyelination, shorter disease duration and significant neuronal loss. Analysis of isolated meninges of MS cases has shown increased gene expression for the pro-inflammatory cytokines: tumour necrosis factor (TNF) and interferon-γ (IFNγ).
Aims: We aimed to test the hypothesis that chronic production of these cytokines in the meningeal compartment and diffusion into underlying GM can drive MS GM pathology.
Methods: To do this we stereotactically injected HIV-1 based VSV-g pseudotyped lentiviral transfer vectors into the sagittal sulcus (SS) of DA rats to deliver continuous transgene expression (TNF + IFNγ) in the meninges. Rats were either immunised with MOG peptide or IFA as a control. A neuropathological analysis was conducted at chronic time points up to 2 months.
Results: Injection of these vectors induced the formation of lymphoid follicle-like structures in the meninges by 28 dpi, which remained at 2 months, containing CD4+ and CD8+ T-cells, CD79a+ B-cells and Iba1+ macrophages, and MadCAM+ channels. Subpial demyelination underlying these aggregates was accompanied by widespread microglial activation and was partly dependant on MOG immunisation. Quantification of NeuN/HuCD co-staining showed a 23-48% decrease in neuronal numbers in cortical layers II-IV at 2 months post injection. Neuronal loss occurred in both MOG and IFA immunised animals and in the absence of demyelination. TNF/TNFR1 interactions can initiate cell death by activating pathways involved in necroptosis. Immunostaining showed TNFR1 expression by neurons. RT-PCR on cortical RNA at 28 dpi and 2 months showed an increase in expression of TNFR1 and downstream necroptotic genes, RIP3, MLKL, cIAP2 and Nox2 compared to eGFP vector control animals. RIP3+ and MLKL+ immunopositive cells with the morphology of neurons were present in TNF + IFNγ vector injected animals. Membrane staining for phosphorylated MLKL in neurons was suggestive of the final stages of necroptosis.
Conclusions: Our results suggest that TNF in the presence of IFNγ is a potent inducer of meningeal inflammation and can activate TNF signalling pathways in cortical cells leading to neuronal death and subpial demyelination and thus may contribute to clinical progression in MS.
Disclosure: Rachel James:nothing to disclose
Nicholas Mazarakis : nothing to disclose.
Richard Reynolds: nothing to disclose