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Fingolimod may support neuroprotection via blockade of astrocyte S1P and cytokine signaling cascades in Multiple Sclerosis
144
Abstracts
226 Influence of effector molecules released by Th1 and Th17 cells on glial cells Prajeeth Chittappen Kandiyila, Julius Kornischa, Reza Khorooshib, Henrik Toft-hansenb, Kirsten Löhrc, Trevor Owensb, Jochen Huehnc, Martin Stangela a
Clinical Neuroimmunology and Neurochemistry, Department of Neurology, Hannover Medical School, Hannover, Germany; bDepartment of Neurobiology, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark; cExperimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany In neurodegenerative diseases like multiple sclerosis and experimental autoimmune encephalomyelitis, pathogenic CD4+ T helper cell subsets that characteristically secrete IFN-gamma (Th1) or IL-17 (Th17) infiltrate from the periphery into the central nervous system (CNS) and cause tissue damage. Their interaction with astrocytes and microglia is crucial in the regulation of neuroinflammation. The potential effects of distinct effector molecules released by pathogenic Th1 and Th17 cells in this process are poorly understood. In this study, we tested the ability of factors secreted by in vitro generated Th1 and Th17 cells to induce glial cell activation. Recently, we have shown that only effector molecules secreted by Th1 cells activate and polarize microglia to an M1-like phenotype, whereas the effectors released by Th17 cells had no direct influence on microglia. To further characterize the interaction between T cells and glia we investigated the effect of Th1- and Th17-derived factors on astrocytes. For this purpose, we treated astrocytes harvested from mixed glial preparations with Th1- and Th17-derived culture supernatants and assessed the expression of factors that are known to be involved in mediating neuroinflammation. Measurement of culture supernatants revealed significantly higher amounts of monocyte chemoattractant protein-1 (MCP-1/CCL2) when astrocytes were exposed to Th1 and Th17 derived factors. RT-PCR analysis of astrocytes revealed that both Th1- and Th17derived factors significantly upregulated the expression of inducible nitric oxide synthase (NOS2). Interestingly, expression of IL6 and CCL20 that are involved in differentiation and recruitment of Th17 cells was upregulated only in those astrocytes that were treated with Th17derived factors. Furthermore, transwell migration assays demonstrate that Th17-activated astrocytes efficiently attracted microglia which reflects the situation in the CNS wherein astrocytes have been shown to be crucial in recruiting microglia. Taken together, in our attempt to dissect the individual contribution of Th1 and Th17 cells in propagating neuroinflammation we show that Th1-derived factors can directly influence both microglia and astrocytes, whereas the targets of Th17derived factors in this process seem to be mainly astrocytes. doi:10.1016/j.jneuroim.2014.08.385
246 The impact of neuroinflammation on tau pathology in healthy aging and neurodegeneration Alexander W. Collcutt, Jessica L. Teeling, Ayodeji A. Asuni Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom Systemic infections in a population accelerate the progression of neurodegenerative conditions such as Alzheimer’s Disease (AD) but
reasons for this remain unclear. Pathology of AD is characterised by accumulating misfolded protein aggregates of amyloid-beta and Tau neurofibrillary tangles, as well as neuroinflammation. Although amyloid and tau pathology are synergistic, therapeutically A(beta) has received the most attention to date and has not as yet yielded a viable treatment. Neuroinflammation and Tau pathology positively correlate with cognitive deficits and there is some evidence to suggest the former accelerates the latter. To explore this mechanism we injected LPS or saline unilaterally into the hippocampus of C57BL/6 wild-type mice. Using histological methods we found markers of microglial activation such as Fc(gamma)RI and CD11b had increased after 1 day. In addition, there was an increase in tau phosphorylation (PHF-1, CP13) at the same time point. Both of these increases had returned to control levels after 28 days. In comparison, a pronounced immune activation was also observed in tau transgenic mice (Tg4510) at 1 year of age, with significant tau phosphorylation and neurodegeneration seen previously in this model. We have shown that an acute inflammatory insult can cause tau phosphorylation in the brain, which is resolved quickly, but the immune cells may subsequently remain primed. We hypothesise that many of these acute insults or a (systemic) low-grade chronic inflammatory state may increase an individual’s risk to a neurodegenerative disease or worsen a current one, through modulation of tau phosphorylation. Components of this mechanism may act as targets for novel therapeutics against neurodegenerative diseases like AD. doi:10.1016/j.jneuroim.2014.08.386
367 Fingolimod may support neuroprotection via blockade of astrocyte S1P and cytokine signaling cascades in Multiple Sclerosis Emanuela Colomboa, Marco Di Darioa, Eleonora Capitoloa, Linda Chaabanea, Jia Newcombeb, Gianvito Martinoa, Cinthia Farinaa a
Institute of Experimental Neurology (INSpe), San Raffaele Scientific Institute, Milan, Italy; bNeuroResource, UCL Institute of Neurology, London, United Kingdom Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid that regulates different cellular processes such as growth, survival and differentiation. S1P signaling supports growth factor signal transduction and triggers activation of nuclear transcription factor NFκB and of nitric oxide (NO) synthase. S1P acts via binding to specific G protein-coupled receptors (S1P1-5), which may be expressed by resident cells in the central nervous system (CNS), including astrocytes. Upon CNS injury astrocytes actively participate in glial scar formation and tissue repair, and dysregulation of their activity can have detrimental effects on CNS function. Recent reports suggest that activation of pathways leading to NFκB activation and NO production in astrocytes contributes to multiple sclerosis (MS) pathology. Published studies have shown that the proinflammatory cytokines IL1 and IL17 are produced in the CNS during neuroinflammation and that cytokines may induce inside-out S1P signaling. Nevertheless, the possibility and relevance of the crosstalk between the signaling cascades of S1P and of IL1 and IL17 in MS remain largely unaddressed. Here we show that, while absent in normal appearing and control white matter, IL1R, IL17RA and S1P receptors S1P1 and S1P3 are all upregulated on astrocytes in MS lesions and in the spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. In vitro assays demonstrate that IL1, IL17 and S1P induce NFκB translocation and NO production in astrocytes, and that the astrocyte conditioned media trigger neuronal death.
Abstracts
Importantly, in vitro targeting of S1P signaling by Fingolimod blocks NFκB translocation and NO production in astrocytes evoked by either S1P or inflammatory cytokines and this effect results in the inhibition of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of Fingolimod to EAE mice hampers astrocyte activation and nitrosative stress in vivo. In conclusion these data indicate that neurotoxic mediators released by astrocytes following activation of S1P and cytokine signalling cascades may contribute to neuronal damage in MS via NFκB pathway activation and NO production, and that the neuroprotective effect of Fingolimod in vivo may be the indirect result of its action on astrocyte activation. This study was supported by Fondazione Italiana Sclerosi Multipla (FISM) (2012/R/7), Amici Centro Sclerosi Multipla (Acesm) and Italian Ministry of Health.
doi:10.1016/j.jneuroim.2014.08.387
102 Shedding microvesicles released by a human microglial cell line (CHME-5): A good platform for studying the biology of the system Federico Colomboa, Annamaria Finardib, Gabriella Racchettib, Roberto Furlanb a
San Raffaele Scientific Institute, Università Vita-Salute San Raffaele, Milano, Italy; bSan Raffaele Scientific Institute, Ospedale San Raffaele, Milano, Italy Cell-to-cell communication is a field of growing interest in the current cell biology since the recent discovery of multiple and unexpected mechanisms by which cells can propagate information among them through active biomolecules. One of these mechanisms is based on the release of membrane particles of different size, generally called microvesicles (MVs), filled by either proteins or nucleic acids whose composition seems to precisely reflect that of the cell of origin. Until now two main microvesicle populations have been isolated and characterized: exosomes and shedding vesicles (SVs). The former are nanoparticles (diameter: 30–100 nm) of endocytic origin constitutively released by cells upon the fusion of multivesicular bodies (MVBs) with the plasma membrane whereas the latter are larger vesicles (diameter: 100–1500 nm) formed by the outward bending of the plasma membrane (called budding or ectocytosis) triggered by few specific signals as ATP. The mechanisms by which the deformation of membranes occurs are known only in part and the biology of this process is still very far from be fully understood. For a long time MVs were considered as mere cell artifacts until recent evidences demonstrating the importance of their ability in carrying and protecting molecular cargoes in the extracellular space to be crucial importance either in physiological or in pathological conditions ranging from tumor cell migration and metastasis dissemination, to coagulation and inflammation. Recently our group has reported a massive increase of shedding vesicles of myeloid origin in the cerebrospinal fluid of multiple sclerosis patients respect to the healthy controls and that their number correlates with the severity of the disease. In order to elucidate some aspects of SVs biology in the contest of human physiology and pathology we chose as a model a microglia cell line of human embryonal origin, namely CHME-5. Here I report the detailed characterization of the ability of this cell line to release detectable amounts of SVs upon ATP or cytokine
145
stimulation (conditions mimic the inflamed tissues) by using multiple approaches ranging from western blotting to immunofluorescence, flow cytometry and electron microscopy. Thus this cell line will offer the crucial advantage of producing enough microvesicles to perform OMICS analyses in order to reveal their content and possibly also their functions.
doi:10.1016/j.jneuroim.2014.08.388
420 Neutrophils induce Alzheimer's-like disease via LFA-1-integrin and neutrophil extracellular traps Elena Zenaroa, Enrica Pietronigroa, Vittorina Della Biancaa, Gennj Piacentinoa, Alessio Montresora, Ermanna Turanob, Bruno Bonettib, Gabriela Constantina a
Department of Pathology and Diagnostics, University of Verona, Verona, Italy; bDepartment of Neurological Sciences, University of Verona, Verona, Italy
Inflammation has been correlated to Alzheimer's disease (AD) and a better understanding of inflammation mechanisms may potentially help to develop new approaches to treat this disorder. The aim of our study was to investigate the role of vascular inflammation and immune cell trafficking in animal models of AD. We detected enhanced expression of vascular adhesion molecules and increased accumulation of neutrophils in 5XFAD and 3xTg-AD transgenic mice with cognitive impairment during early stage of AD-like disease compared to age-matched wild-type controls. Using twophoton laser-scanning microscopy we observed that neutrophils crawl in blood vessels and transmigrate in high numbers in areas with amyloid plaques in both AD-like models. Interestingly, neutrophils released intravascular and intraparenchymal neutrophil extracellular traps (NETs), suggesting that NETosis may represent a potential neutrophildependent disease mechanism in AD. Amyloid beta peptide 1-42 induced high affinity state of LFA-1 integrin and LFA-1-dependent adhesion of neutrophils. Moreover, two-photon microscopy experiments showed that LFA-1 integrin blockade prevented neutrophil extravasation, and inhibited intraparenchymal motility. Notably, neutrophil depletion or blockade of neutrophil trafficking by an anti-LFA-1 integrin antibody inhibited amyloid deposition and tau phosphorylation, and rescued cognitive deficits in behavioral tests in 5XFAD and 3xTg mice, suggesting that neutrophils play a key role in AD-like disease. Importantly, restoration of cognitive function in mice with temporary inhibition of neutrophil function during early disease was maintained also at later time points in aged animals. To understand the relevance of our data in humans, we analyzed human cortical brain samples from subjects with AD. Our results showed that neutrophils adhered and spread inside brain venules or migrated into the parenchyma in high numbers and release NETs in human AD brains compared to control subjects. Taken together our results demonstrate that neutrophils induce cognitive impairment in AD-like disease and suggest that inhibition of neutrophil trafficking may represent a new therapeutic strategy in AD. This work was supported by the European Research Council (ERC) (261079) grant Neurotrafficking (to G.C.).
doi:10.1016/j.jneuroim.2014.08.389
Abstracts
226 Influence of effector molecules released by Th1 and Th17 cells on glial cells Prajeeth Chittappen Kandiyila, Julius Kornischa, Reza Khorooshib, Henrik Toft-hansenb, Kirsten Löhrc, Trevor Owensb, Jochen Huehnc, Martin Stangela a
Clinical Neuroimmunology and Neurochemistry, Department of Neurology, Hannover Medical School, Hannover, Germany; bDepartment of Neurobiology, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark; cExperimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany In neurodegenerative diseases like multiple sclerosis and experimental autoimmune encephalomyelitis, pathogenic CD4+ T helper cell subsets that characteristically secrete IFN-gamma (Th1) or IL-17 (Th17) infiltrate from the periphery into the central nervous system (CNS) and cause tissue damage. Their interaction with astrocytes and microglia is crucial in the regulation of neuroinflammation. The potential effects of distinct effector molecules released by pathogenic Th1 and Th17 cells in this process are poorly understood. In this study, we tested the ability of factors secreted by in vitro generated Th1 and Th17 cells to induce glial cell activation. Recently, we have shown that only effector molecules secreted by Th1 cells activate and polarize microglia to an M1-like phenotype, whereas the effectors released by Th17 cells had no direct influence on microglia. To further characterize the interaction between T cells and glia we investigated the effect of Th1- and Th17-derived factors on astrocytes. For this purpose, we treated astrocytes harvested from mixed glial preparations with Th1- and Th17-derived culture supernatants and assessed the expression of factors that are known to be involved in mediating neuroinflammation. Measurement of culture supernatants revealed significantly higher amounts of monocyte chemoattractant protein-1 (MCP-1/CCL2) when astrocytes were exposed to Th1 and Th17 derived factors. RT-PCR analysis of astrocytes revealed that both Th1- and Th17derived factors significantly upregulated the expression of inducible nitric oxide synthase (NOS2). Interestingly, expression of IL6 and CCL20 that are involved in differentiation and recruitment of Th17 cells was upregulated only in those astrocytes that were treated with Th17derived factors. Furthermore, transwell migration assays demonstrate that Th17-activated astrocytes efficiently attracted microglia which reflects the situation in the CNS wherein astrocytes have been shown to be crucial in recruiting microglia. Taken together, in our attempt to dissect the individual contribution of Th1 and Th17 cells in propagating neuroinflammation we show that Th1-derived factors can directly influence both microglia and astrocytes, whereas the targets of Th17derived factors in this process seem to be mainly astrocytes. doi:10.1016/j.jneuroim.2014.08.385
246 The impact of neuroinflammation on tau pathology in healthy aging and neurodegeneration Alexander W. Collcutt, Jessica L. Teeling, Ayodeji A. Asuni Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom Systemic infections in a population accelerate the progression of neurodegenerative conditions such as Alzheimer’s Disease (AD) but
reasons for this remain unclear. Pathology of AD is characterised by accumulating misfolded protein aggregates of amyloid-beta and Tau neurofibrillary tangles, as well as neuroinflammation. Although amyloid and tau pathology are synergistic, therapeutically A(beta) has received the most attention to date and has not as yet yielded a viable treatment. Neuroinflammation and Tau pathology positively correlate with cognitive deficits and there is some evidence to suggest the former accelerates the latter. To explore this mechanism we injected LPS or saline unilaterally into the hippocampus of C57BL/6 wild-type mice. Using histological methods we found markers of microglial activation such as Fc(gamma)RI and CD11b had increased after 1 day. In addition, there was an increase in tau phosphorylation (PHF-1, CP13) at the same time point. Both of these increases had returned to control levels after 28 days. In comparison, a pronounced immune activation was also observed in tau transgenic mice (Tg4510) at 1 year of age, with significant tau phosphorylation and neurodegeneration seen previously in this model. We have shown that an acute inflammatory insult can cause tau phosphorylation in the brain, which is resolved quickly, but the immune cells may subsequently remain primed. We hypothesise that many of these acute insults or a (systemic) low-grade chronic inflammatory state may increase an individual’s risk to a neurodegenerative disease or worsen a current one, through modulation of tau phosphorylation. Components of this mechanism may act as targets for novel therapeutics against neurodegenerative diseases like AD. doi:10.1016/j.jneuroim.2014.08.386
367 Fingolimod may support neuroprotection via blockade of astrocyte S1P and cytokine signaling cascades in Multiple Sclerosis Emanuela Colomboa, Marco Di Darioa, Eleonora Capitoloa, Linda Chaabanea, Jia Newcombeb, Gianvito Martinoa, Cinthia Farinaa a
Institute of Experimental Neurology (INSpe), San Raffaele Scientific Institute, Milan, Italy; bNeuroResource, UCL Institute of Neurology, London, United Kingdom Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid that regulates different cellular processes such as growth, survival and differentiation. S1P signaling supports growth factor signal transduction and triggers activation of nuclear transcription factor NFκB and of nitric oxide (NO) synthase. S1P acts via binding to specific G protein-coupled receptors (S1P1-5), which may be expressed by resident cells in the central nervous system (CNS), including astrocytes. Upon CNS injury astrocytes actively participate in glial scar formation and tissue repair, and dysregulation of their activity can have detrimental effects on CNS function. Recent reports suggest that activation of pathways leading to NFκB activation and NO production in astrocytes contributes to multiple sclerosis (MS) pathology. Published studies have shown that the proinflammatory cytokines IL1 and IL17 are produced in the CNS during neuroinflammation and that cytokines may induce inside-out S1P signaling. Nevertheless, the possibility and relevance of the crosstalk between the signaling cascades of S1P and of IL1 and IL17 in MS remain largely unaddressed. Here we show that, while absent in normal appearing and control white matter, IL1R, IL17RA and S1P receptors S1P1 and S1P3 are all upregulated on astrocytes in MS lesions and in the spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. In vitro assays demonstrate that IL1, IL17 and S1P induce NFκB translocation and NO production in astrocytes, and that the astrocyte conditioned media trigger neuronal death.
Abstracts
Importantly, in vitro targeting of S1P signaling by Fingolimod blocks NFκB translocation and NO production in astrocytes evoked by either S1P or inflammatory cytokines and this effect results in the inhibition of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of Fingolimod to EAE mice hampers astrocyte activation and nitrosative stress in vivo. In conclusion these data indicate that neurotoxic mediators released by astrocytes following activation of S1P and cytokine signalling cascades may contribute to neuronal damage in MS via NFκB pathway activation and NO production, and that the neuroprotective effect of Fingolimod in vivo may be the indirect result of its action on astrocyte activation. This study was supported by Fondazione Italiana Sclerosi Multipla (FISM) (2012/R/7), Amici Centro Sclerosi Multipla (Acesm) and Italian Ministry of Health.
doi:10.1016/j.jneuroim.2014.08.387
102 Shedding microvesicles released by a human microglial cell line (CHME-5): A good platform for studying the biology of the system Federico Colomboa, Annamaria Finardib, Gabriella Racchettib, Roberto Furlanb a
San Raffaele Scientific Institute, Università Vita-Salute San Raffaele, Milano, Italy; bSan Raffaele Scientific Institute, Ospedale San Raffaele, Milano, Italy Cell-to-cell communication is a field of growing interest in the current cell biology since the recent discovery of multiple and unexpected mechanisms by which cells can propagate information among them through active biomolecules. One of these mechanisms is based on the release of membrane particles of different size, generally called microvesicles (MVs), filled by either proteins or nucleic acids whose composition seems to precisely reflect that of the cell of origin. Until now two main microvesicle populations have been isolated and characterized: exosomes and shedding vesicles (SVs). The former are nanoparticles (diameter: 30–100 nm) of endocytic origin constitutively released by cells upon the fusion of multivesicular bodies (MVBs) with the plasma membrane whereas the latter are larger vesicles (diameter: 100–1500 nm) formed by the outward bending of the plasma membrane (called budding or ectocytosis) triggered by few specific signals as ATP. The mechanisms by which the deformation of membranes occurs are known only in part and the biology of this process is still very far from be fully understood. For a long time MVs were considered as mere cell artifacts until recent evidences demonstrating the importance of their ability in carrying and protecting molecular cargoes in the extracellular space to be crucial importance either in physiological or in pathological conditions ranging from tumor cell migration and metastasis dissemination, to coagulation and inflammation. Recently our group has reported a massive increase of shedding vesicles of myeloid origin in the cerebrospinal fluid of multiple sclerosis patients respect to the healthy controls and that their number correlates with the severity of the disease. In order to elucidate some aspects of SVs biology in the contest of human physiology and pathology we chose as a model a microglia cell line of human embryonal origin, namely CHME-5. Here I report the detailed characterization of the ability of this cell line to release detectable amounts of SVs upon ATP or cytokine
145
stimulation (conditions mimic the inflamed tissues) by using multiple approaches ranging from western blotting to immunofluorescence, flow cytometry and electron microscopy. Thus this cell line will offer the crucial advantage of producing enough microvesicles to perform OMICS analyses in order to reveal their content and possibly also their functions.
doi:10.1016/j.jneuroim.2014.08.388
420 Neutrophils induce Alzheimer's-like disease via LFA-1-integrin and neutrophil extracellular traps Elena Zenaroa, Enrica Pietronigroa, Vittorina Della Biancaa, Gennj Piacentinoa, Alessio Montresora, Ermanna Turanob, Bruno Bonettib, Gabriela Constantina a
Department of Pathology and Diagnostics, University of Verona, Verona, Italy; bDepartment of Neurological Sciences, University of Verona, Verona, Italy
Inflammation has been correlated to Alzheimer's disease (AD) and a better understanding of inflammation mechanisms may potentially help to develop new approaches to treat this disorder. The aim of our study was to investigate the role of vascular inflammation and immune cell trafficking in animal models of AD. We detected enhanced expression of vascular adhesion molecules and increased accumulation of neutrophils in 5XFAD and 3xTg-AD transgenic mice with cognitive impairment during early stage of AD-like disease compared to age-matched wild-type controls. Using twophoton laser-scanning microscopy we observed that neutrophils crawl in blood vessels and transmigrate in high numbers in areas with amyloid plaques in both AD-like models. Interestingly, neutrophils released intravascular and intraparenchymal neutrophil extracellular traps (NETs), suggesting that NETosis may represent a potential neutrophildependent disease mechanism in AD. Amyloid beta peptide 1-42 induced high affinity state of LFA-1 integrin and LFA-1-dependent adhesion of neutrophils. Moreover, two-photon microscopy experiments showed that LFA-1 integrin blockade prevented neutrophil extravasation, and inhibited intraparenchymal motility. Notably, neutrophil depletion or blockade of neutrophil trafficking by an anti-LFA-1 integrin antibody inhibited amyloid deposition and tau phosphorylation, and rescued cognitive deficits in behavioral tests in 5XFAD and 3xTg mice, suggesting that neutrophils play a key role in AD-like disease. Importantly, restoration of cognitive function in mice with temporary inhibition of neutrophil function during early disease was maintained also at later time points in aged animals. To understand the relevance of our data in humans, we analyzed human cortical brain samples from subjects with AD. Our results showed that neutrophils adhered and spread inside brain venules or migrated into the parenchyma in high numbers and release NETs in human AD brains compared to control subjects. Taken together our results demonstrate that neutrophils induce cognitive impairment in AD-like disease and suggest that inhibition of neutrophil trafficking may represent a new therapeutic strategy in AD. This work was supported by the European Research Council (ERC) (261079) grant Neurotrafficking (to G.C.).
doi:10.1016/j.jneuroim.2014.08.389