- Email: [email protected]
CNS TLR2 mediates innate neuroinflammation but not autoimmune pathogenesis and demyelination
Abstracts
damage to the lesioned spinal cord, whereas central etanercept treatment had no effect. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI. These studies suggest that by selectively blocking soITNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. doi:10.1016/j.jneuroim.2014.08.305
100 Characterization of the humoral immune response in a Lewis rat model of CIDP Susana Brun, Jérôme De Seze, Elisabeth Trifilieff INSERM U1119 — Biopathologie de la Myéline, Neuroprotection et Stratégies Thérapeutiques, Université de Strasbourg, Strasbourg, France Objective: Recently we have developed a reliable and reproducible preclinical chronic-EAN model in the Lewis rat that may prove useful for translational drug studies for CIDP. We have clearly shown that it is a T-cell mediated disease with an accumulation of IL-17+ cells and macrophages in its late chronic phase. Since pathophysiological mechanisms involved in CIDP are believed to involve not only cellular but also humoral immunity, we therefore investigated the humoral response in our chronic-EAN model induced in the Lewis rat after injection of thiopalmitoylated P0(180–199) peptide in comparison to the classical acute EAN model induced with P0(180–199). Methods: We investigated by ELISA (at 18, 31, 43 and 57 dpi) in the sera of EAN and chronic-EAN rats the levels of antibodies directed against peptide P0(180–199), the inducing antigen. The reactivity of the antibodies obtained was tested on sciatic nerve by immunohistochemistry. To check the possibility of an epitope spreading at the late phase of the chronic disease, the reactivity of the sera at 60 dpi was tested by ELISA against different peptides described as neuritogenic: P0(56–71), P0(152–171), P0(180–199) and P2(57– 81). We also investigated the presence of B-cells in the sciatic nerve by immunohistochemistry using a mouse anti-rat CD45RA MnAb. Results: High levels of antibodies against P0(180–199) were found in chronic-EAN and EAN rats, but the antibody reactivity remained high in the chronic group for the duration of the disease, whereas it started to decline in the EAN group by day 57. At the late phase of the chronic disease (60 dpi) we found in the sera significant levels of antibodies against peptide P0(152–171) but this was not statistically different from the EAN group. No reactivity was detected against the other peptides. The rat polyclonal anti P0(180–199) antibodies labeled nicely the PNS myelin in a control sciatic nerve, highly comparable to the myelin labeling with the commercial anti-P0 MnAb. This indicates that the anti-P0(180–199) is able to recognize the corresponding peptide sequence when protein P0 is inserted in the myelin membrane. The presence of B cells was detected in the sciatic nerve of EAN rats and also of chronic-EAN rats but in greater number. Conclusion: We have indeed shown that there is a humoral response in our chronic model, but further studies are needed to clarify the roles of the antibodies and B-cells in the persistence of the chronicity of the disease. doi:10.1016/j.jneuroim.2014.08.306
115
60 Treatment with FTY-720 reverses social recognition deficits without affecting clinical scores or impaired motor performance in EAE in SJL mice Natasja M.W.J. De Bruina, Katja Schmitzb, Irmgard Tegederb, Sonja Talmona, Holger Jordana, Mike Schmidta, Annett Haeusslerb, Gerd Geisslingerb, Michael J. Parnhama a
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group Translational Medicine & Pharmacology (TMP), Frankfurt am Main, Germany; bInstitute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany
Experimental autoimmune encephalomyelitis (EAE) serves as a disease model for aspects of human multiple sclerosis (MS). Our aim was to explore additional parameters that might be predictive for the study of disease progression, including score-free intervals. The effects of FTY-720 (0.5 mg/kg/day in drinking water) were investigated in rodent EAE models. Dark Agouti (DA) rats were immunized with rat spinal cord homogenate (rSCH), Lewis rats with guinea pig myelin basic protein (gpMBP)69–88 and C57Bl/6J mice with myelin oligodendrocyte glycoprotein (MOG)35–55. SJL/J mice were immunized with myelin proteolipid protein (PLP)139–151 peptide. Clinical scores were registered in all models. In SJL mice with relapsingremitting EAE, we also performed behavioral tests: rotarod, gait analysis and grip strength. Between days 26–28, mice were examined, according to Crawley's sociability and preference for social novelty test, in a rectangular, three-chamber box. Prophylactic treatment (3 days post-immunization, p.i.) with FTY-720 prevented clinical scores in 3 of the EAE rodent models: DA and Lewis rats and C57Bl/6J mice. In contrast, neither prophylactic nor late therapeutic (18 days p.i.) treatment with FTY-720 reduced clinical scores nor reversed deficits in the rotarod test in SJL mice. However, FTY-720 had some subtle effects on motor functions and sociability in SJL mice. Prophylactic treatment with FTY-720 improved the gait, specifically it normalized the distance between the tip of the tail and floor. FTY-720 also improved manifestations of reduced social (re)cognition or preference for social novelty which were evident in vehicle treated EAE SJL mice. The data suggest that changes in behavioral parameters can be detected in absence of clinical scores, may be indicative of subtle drug effects and may have translational value for human MS. We acknowledge support by the State of Hessen (LOEWE), Germany doi:10.1016/j.jneuroim.2014.08.307
577 CNS TLR2 mediates innate neuroinflammation but not autoimmune pathogenesis and demyelination Nina Fainsteina, Avital Luzb, Ofira Einsteinb, Tamir Ben-Hura a
Department of Neurology, Hadassah Hebrew University Medical Center, Jerusalem, Israel; bPhysiotherapy Department, Faculty of Health Sciences, Ariel University Center of Samaria, Ariel, Israel Background: Toll-like receptor 2 (TLR2) is expressed on immune cells in the periphery and the CNS and mediates innate immune responses. Recent studies have implicated TLR2 in systemic pathogenesis of adaptive immunity in experimental autoimmune encephalomyelitis
116
Abstracts
(EAE). In addition, TLR2 is expressed on oligodendrocyte progenitor cells and its activation inhibits their differentiation and remyelination. These suggest a possible role of CNS TLR2 in progressive autoimmune demyelination. Methods: We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymozan, a TLR2 agonist, on the clinical and pathological course of EAE. The survival and clinical scores were monitored; demyelination and axonal loss were quantified by gold-black and Bielschowsky stains, and the nature of neuro-inflammatory response was characterized by TLR2, IBA-1 and CD3 stainings and PCR for immune cytokines. Immune cells were isolated from EAE brain tissue and their proliferative response to the autoantigen (PLP peptide) or Concavallin A was examined in vitro. Results: Zymozan injection in naïve mice induced a strong neuroinflammatory response without any clinical manifestations. In EAE mice, ICV Zymozan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease and CNS inflammatory parameters were not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymozan injections. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide and ConA indicated an increase in innate but not adaptive immune response. Conclusions: (1) EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. (2) CNS TLR2 activation does not alter the clinical and pathological course of EAE. (3) These findings imply that CNS TLR2 activation affects the innate but not adaptive brain immune responses.
Y-maze alternation test, serum autoantibody levels were determined and mice were then sacrificed. Golgi–Cox staining was used to analyze hippocampal neurons, dendrites and dendritic spines (n = 7/group). Immunohistochemistry was performed with antibodies against microtubule-associated protein 2 (MAP2), synaptobrevin 2 and synaptopodin, representing molecular markers of total dendritic integrity, presynaptic plasticity and dendritic spines (n = 7/group). eAPS resulted in increased aPL titers, abnormal behavior and impaired short term memory. This was further accompanied by reduced dendritic complexity of hippocampal CA1 neurons in the Golgi–Cox analysis as reflected by decreased dendritic length, arborization and spine density, respectively. Further the spine-associated protein expression of synaptopodin was reduced. In conclusion, our results are the first to indicate a role for the dendrites and in particular the dendritic spines in the pathogenesis of aPL-mediated central nervous system dysfunction in eAPS. The autoimmune-induced neurodegeneration in the absence of ischemia could also be a possible contributing factor to the central nervous system dysfunction in APS patients.
doi:10.1016/j.jneuroim.2014.08.308
Division of Neurology, Department of Internal Medicine, Toho University Oohashi Medical Centre, Tokyo, Japan
38
Phosphodiesterase (PDE) is involved in homeostasis, inflammation, tissue regeneration through degradation of cyclic AMP. PDE inhibitor is known to suppress autoimmune inflammatory neurological disease, however, very few report has been published concerning autoimmune peripheral nerve disease. PDE has several isoenzymes and respective selective inhibitors. Some of these inhibitors are already in therapeutical use. PDE-3 inhibitor cilostazole (CLZ) has antiplatelet action and widely used for the prevention of thrombosis. The anti-inflammatory effect of CLZ has also been shown in experimental autoimmune encephalomyelitis (EAE) however, its effect on peripheral nerve disease is unclear. To elucidate the effect of CLZ on neuroinflammation, rats with experimental autoimmune neuritis (EAN) were treated with CLZ. Female Lewis rats were immunized with synthetic peptides from bovine peripheral nerve P2 protein. After immunization, CLZ (high dose group: 30 mg·kg or low dose group: 10 mg/kg) or vehicle was given by gastric tube daily. Symptomatic score was checked daily. 11 days post-immunization (dpi) all rats developed initial symptom of EAN namely weakness of tip of tail, thereafter ascending flaccid paralysis developed. Vehicle-treated rats peaked at 17 dpi followed by gradual recovery. High dose CLZ group showed significantly less severity at 13, 14, 15, 18, 20 dpi. Low dose CLZ group showed tendency of amelioration without statistical significance. Some rats in vehicle group were randomly selected for receiving high dose CLZ after disease onset. This group showed significantly less severity at 14 dpi compared to vehicle group. At 14 dpi, rats were euthanatized to remove cauda equina (CE). Histological examination of hematoxylin eosin-stained frozen section of CE revealed intense intraneural, perivascular cell infiltration in vehicle group. High dose CLZ group showed mild cell infiltration. Semiquantitative real time PCR for
The experimental antiphospholipid syndrome: an invaluable tool to study autoimmunity-induced neurodegeneration Katrin Frauenknechta, Aviva Katzavb, Ronen Weiss Lavib, Avishag Sabagb, Susanne Ottena, Joab Chapmanb, Clemens Sommera a
Department of Neuropathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; bDepartment of Neurology, Chaim Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel The antiphospholipid syndrome (APS) is an autoimmune disease characterized by high titers of auto-antibodies against phospholipids leading to thrombosis and consecutive infarcts. Many patients develop neurological symptoms in the absence of ischemia but the underlying mechanisms leading to these manifestations are not yet identified. In experimental APS (eAPS) mice with high aPL serum levels consistently develop behavioral abnormalities despite lack of ischemic brain injury. Therefore, it seems to be a valuable model to study principle mechanisms. In previous experiments we could link alterations of hippocampal neurotransmitter receptor binding densities to altered behavior. The present study was designed to identify possible underlying structural alterations of hippocampal neurons. Adult female Balb/C mice (n = 28) were subjected to either induction of eAPS (n = 14) by immunization with beta2-Glycoprotein 1 emulsified in Freund's adjuvant (CFA) or to a control group (n = 14) by immunization with CFA alone. Sixteen weeks post immunization mice were behaviorally tested in the staircase and
doi:10.1016/j.jneuroim.2014.08.309
108 The effects of cilostazol on experimental autoimmune neuritis in Lewis rats Wataru Hagiwara, Masashi Inoue, Shingo Konno, Toshiki Fujioka
damage to the lesioned spinal cord, whereas central etanercept treatment had no effect. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI. These studies suggest that by selectively blocking soITNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. doi:10.1016/j.jneuroim.2014.08.305
100 Characterization of the humoral immune response in a Lewis rat model of CIDP Susana Brun, Jérôme De Seze, Elisabeth Trifilieff INSERM U1119 — Biopathologie de la Myéline, Neuroprotection et Stratégies Thérapeutiques, Université de Strasbourg, Strasbourg, France Objective: Recently we have developed a reliable and reproducible preclinical chronic-EAN model in the Lewis rat that may prove useful for translational drug studies for CIDP. We have clearly shown that it is a T-cell mediated disease with an accumulation of IL-17+ cells and macrophages in its late chronic phase. Since pathophysiological mechanisms involved in CIDP are believed to involve not only cellular but also humoral immunity, we therefore investigated the humoral response in our chronic-EAN model induced in the Lewis rat after injection of thiopalmitoylated P0(180–199) peptide in comparison to the classical acute EAN model induced with P0(180–199). Methods: We investigated by ELISA (at 18, 31, 43 and 57 dpi) in the sera of EAN and chronic-EAN rats the levels of antibodies directed against peptide P0(180–199), the inducing antigen. The reactivity of the antibodies obtained was tested on sciatic nerve by immunohistochemistry. To check the possibility of an epitope spreading at the late phase of the chronic disease, the reactivity of the sera at 60 dpi was tested by ELISA against different peptides described as neuritogenic: P0(56–71), P0(152–171), P0(180–199) and P2(57– 81). We also investigated the presence of B-cells in the sciatic nerve by immunohistochemistry using a mouse anti-rat CD45RA MnAb. Results: High levels of antibodies against P0(180–199) were found in chronic-EAN and EAN rats, but the antibody reactivity remained high in the chronic group for the duration of the disease, whereas it started to decline in the EAN group by day 57. At the late phase of the chronic disease (60 dpi) we found in the sera significant levels of antibodies against peptide P0(152–171) but this was not statistically different from the EAN group. No reactivity was detected against the other peptides. The rat polyclonal anti P0(180–199) antibodies labeled nicely the PNS myelin in a control sciatic nerve, highly comparable to the myelin labeling with the commercial anti-P0 MnAb. This indicates that the anti-P0(180–199) is able to recognize the corresponding peptide sequence when protein P0 is inserted in the myelin membrane. The presence of B cells was detected in the sciatic nerve of EAN rats and also of chronic-EAN rats but in greater number. Conclusion: We have indeed shown that there is a humoral response in our chronic model, but further studies are needed to clarify the roles of the antibodies and B-cells in the persistence of the chronicity of the disease. doi:10.1016/j.jneuroim.2014.08.306
115
60 Treatment with FTY-720 reverses social recognition deficits without affecting clinical scores or impaired motor performance in EAE in SJL mice Natasja M.W.J. De Bruina, Katja Schmitzb, Irmgard Tegederb, Sonja Talmona, Holger Jordana, Mike Schmidta, Annett Haeusslerb, Gerd Geisslingerb, Michael J. Parnhama a
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group Translational Medicine & Pharmacology (TMP), Frankfurt am Main, Germany; bInstitute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany
Experimental autoimmune encephalomyelitis (EAE) serves as a disease model for aspects of human multiple sclerosis (MS). Our aim was to explore additional parameters that might be predictive for the study of disease progression, including score-free intervals. The effects of FTY-720 (0.5 mg/kg/day in drinking water) were investigated in rodent EAE models. Dark Agouti (DA) rats were immunized with rat spinal cord homogenate (rSCH), Lewis rats with guinea pig myelin basic protein (gpMBP)69–88 and C57Bl/6J mice with myelin oligodendrocyte glycoprotein (MOG)35–55. SJL/J mice were immunized with myelin proteolipid protein (PLP)139–151 peptide. Clinical scores were registered in all models. In SJL mice with relapsingremitting EAE, we also performed behavioral tests: rotarod, gait analysis and grip strength. Between days 26–28, mice were examined, according to Crawley's sociability and preference for social novelty test, in a rectangular, three-chamber box. Prophylactic treatment (3 days post-immunization, p.i.) with FTY-720 prevented clinical scores in 3 of the EAE rodent models: DA and Lewis rats and C57Bl/6J mice. In contrast, neither prophylactic nor late therapeutic (18 days p.i.) treatment with FTY-720 reduced clinical scores nor reversed deficits in the rotarod test in SJL mice. However, FTY-720 had some subtle effects on motor functions and sociability in SJL mice. Prophylactic treatment with FTY-720 improved the gait, specifically it normalized the distance between the tip of the tail and floor. FTY-720 also improved manifestations of reduced social (re)cognition or preference for social novelty which were evident in vehicle treated EAE SJL mice. The data suggest that changes in behavioral parameters can be detected in absence of clinical scores, may be indicative of subtle drug effects and may have translational value for human MS. We acknowledge support by the State of Hessen (LOEWE), Germany doi:10.1016/j.jneuroim.2014.08.307
577 CNS TLR2 mediates innate neuroinflammation but not autoimmune pathogenesis and demyelination Nina Fainsteina, Avital Luzb, Ofira Einsteinb, Tamir Ben-Hura a
Department of Neurology, Hadassah Hebrew University Medical Center, Jerusalem, Israel; bPhysiotherapy Department, Faculty of Health Sciences, Ariel University Center of Samaria, Ariel, Israel Background: Toll-like receptor 2 (TLR2) is expressed on immune cells in the periphery and the CNS and mediates innate immune responses. Recent studies have implicated TLR2 in systemic pathogenesis of adaptive immunity in experimental autoimmune encephalomyelitis
116
Abstracts
(EAE). In addition, TLR2 is expressed on oligodendrocyte progenitor cells and its activation inhibits their differentiation and remyelination. These suggest a possible role of CNS TLR2 in progressive autoimmune demyelination. Methods: We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymozan, a TLR2 agonist, on the clinical and pathological course of EAE. The survival and clinical scores were monitored; demyelination and axonal loss were quantified by gold-black and Bielschowsky stains, and the nature of neuro-inflammatory response was characterized by TLR2, IBA-1 and CD3 stainings and PCR for immune cytokines. Immune cells were isolated from EAE brain tissue and their proliferative response to the autoantigen (PLP peptide) or Concavallin A was examined in vitro. Results: Zymozan injection in naïve mice induced a strong neuroinflammatory response without any clinical manifestations. In EAE mice, ICV Zymozan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease and CNS inflammatory parameters were not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymozan injections. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide and ConA indicated an increase in innate but not adaptive immune response. Conclusions: (1) EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. (2) CNS TLR2 activation does not alter the clinical and pathological course of EAE. (3) These findings imply that CNS TLR2 activation affects the innate but not adaptive brain immune responses.
Y-maze alternation test, serum autoantibody levels were determined and mice were then sacrificed. Golgi–Cox staining was used to analyze hippocampal neurons, dendrites and dendritic spines (n = 7/group). Immunohistochemistry was performed with antibodies against microtubule-associated protein 2 (MAP2), synaptobrevin 2 and synaptopodin, representing molecular markers of total dendritic integrity, presynaptic plasticity and dendritic spines (n = 7/group). eAPS resulted in increased aPL titers, abnormal behavior and impaired short term memory. This was further accompanied by reduced dendritic complexity of hippocampal CA1 neurons in the Golgi–Cox analysis as reflected by decreased dendritic length, arborization and spine density, respectively. Further the spine-associated protein expression of synaptopodin was reduced. In conclusion, our results are the first to indicate a role for the dendrites and in particular the dendritic spines in the pathogenesis of aPL-mediated central nervous system dysfunction in eAPS. The autoimmune-induced neurodegeneration in the absence of ischemia could also be a possible contributing factor to the central nervous system dysfunction in APS patients.
doi:10.1016/j.jneuroim.2014.08.308
Division of Neurology, Department of Internal Medicine, Toho University Oohashi Medical Centre, Tokyo, Japan
38
Phosphodiesterase (PDE) is involved in homeostasis, inflammation, tissue regeneration through degradation of cyclic AMP. PDE inhibitor is known to suppress autoimmune inflammatory neurological disease, however, very few report has been published concerning autoimmune peripheral nerve disease. PDE has several isoenzymes and respective selective inhibitors. Some of these inhibitors are already in therapeutical use. PDE-3 inhibitor cilostazole (CLZ) has antiplatelet action and widely used for the prevention of thrombosis. The anti-inflammatory effect of CLZ has also been shown in experimental autoimmune encephalomyelitis (EAE) however, its effect on peripheral nerve disease is unclear. To elucidate the effect of CLZ on neuroinflammation, rats with experimental autoimmune neuritis (EAN) were treated with CLZ. Female Lewis rats were immunized with synthetic peptides from bovine peripheral nerve P2 protein. After immunization, CLZ (high dose group: 30 mg·kg or low dose group: 10 mg/kg) or vehicle was given by gastric tube daily. Symptomatic score was checked daily. 11 days post-immunization (dpi) all rats developed initial symptom of EAN namely weakness of tip of tail, thereafter ascending flaccid paralysis developed. Vehicle-treated rats peaked at 17 dpi followed by gradual recovery. High dose CLZ group showed significantly less severity at 13, 14, 15, 18, 20 dpi. Low dose CLZ group showed tendency of amelioration without statistical significance. Some rats in vehicle group were randomly selected for receiving high dose CLZ after disease onset. This group showed significantly less severity at 14 dpi compared to vehicle group. At 14 dpi, rats were euthanatized to remove cauda equina (CE). Histological examination of hematoxylin eosin-stained frozen section of CE revealed intense intraneural, perivascular cell infiltration in vehicle group. High dose CLZ group showed mild cell infiltration. Semiquantitative real time PCR for
The experimental antiphospholipid syndrome: an invaluable tool to study autoimmunity-induced neurodegeneration Katrin Frauenknechta, Aviva Katzavb, Ronen Weiss Lavib, Avishag Sabagb, Susanne Ottena, Joab Chapmanb, Clemens Sommera a
Department of Neuropathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; bDepartment of Neurology, Chaim Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel The antiphospholipid syndrome (APS) is an autoimmune disease characterized by high titers of auto-antibodies against phospholipids leading to thrombosis and consecutive infarcts. Many patients develop neurological symptoms in the absence of ischemia but the underlying mechanisms leading to these manifestations are not yet identified. In experimental APS (eAPS) mice with high aPL serum levels consistently develop behavioral abnormalities despite lack of ischemic brain injury. Therefore, it seems to be a valuable model to study principle mechanisms. In previous experiments we could link alterations of hippocampal neurotransmitter receptor binding densities to altered behavior. The present study was designed to identify possible underlying structural alterations of hippocampal neurons. Adult female Balb/C mice (n = 28) were subjected to either induction of eAPS (n = 14) by immunization with beta2-Glycoprotein 1 emulsified in Freund's adjuvant (CFA) or to a control group (n = 14) by immunization with CFA alone. Sixteen weeks post immunization mice were behaviorally tested in the staircase and
doi:10.1016/j.jneuroim.2014.08.309
108 The effects of cilostazol on experimental autoimmune neuritis in Lewis rats Wataru Hagiwara, Masashi Inoue, Shingo Konno, Toshiki Fujioka