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Schwann cell locomotion during peripheral nerve inflammation
72
Abstracts
Methods: Acute brain lesions were induced by transient focal brain ischemia in WT and caspase-1−/− mice. A different group of mice was treated with the specific caspase-1 inhibitor z-YVAD-FMK or control. We analyzed differential cell percentages of lymphocyte and monocyte subpopulations at 6 h and 3 days after experimental stroke by flow cytometry of blood and spleen. Serum cytokine levels were analyzed by ELISA and cellular activation after stroke detected by intracellular cytokine assays. Pyroptosis as a cause of immune cell death was measured by intracellular caspase-1 labeling and membrane disruption by 7-AAD. Results: An increase in caspase-1 activation was detected after 3 days of stroke in splenic monocytes and lymphocytes. However, caspase-1 activation in circulating leukocytes was evident only in lymphocytes but not in monocytes. Furthermore, differential blood cell counts were unaffected by caspase-1 expression after stroke. Additionally, we detected an enhanced expression of intracellular inflammatory cytokines in monocytes during the acute phase after stroke. This differential activation of caspase-1 in leukocyte subpopulations was associated with enhanced inflammatory cytokine concentrations. Genetic as well as pharmacological disruption of caspase-1 signaling abrogated leukocyte pyroptosis and the post-stroke inflammatory reaction. Conclusion: We revealed caspase-1 as a key molecule in the regulation of post-stroke immune cell death and activation. It induces early immune activation as well as delayed pyroptotic immune cell death, thereby, suggesting a novel and comprehensive molecular target to treat the complex immune dysregulation after acute brain injuries. doi:10.1016/j.jneuroim.2014.08.188
414 Schwann cell locomotion during peripheral nerve inflammation Mark Stettner, Sandra Labus, Jan-philipp Weinberger, Thomas Dehmel, Angelika Derksen, Anne K. Mausberg, Bernd C. Kieseier Department of Neurology, Heinrich-Heine-University, Dusseldorf, Germany Background: Chronic immune-mediated neuropathies are characterized by de- and remyelination of the peripheral nervous system (PNS). Schwann cells (SCs) as glial cells of the PNS play a main role in myelin restructuring, a process requiring the ability of SCs to migrate to damaged peripheral nerves. The specific impact of cytokines on SC mobility, however, is not fully understood so far. Methods: SCs were prepared and purified from sciatic nerves of neonatal rats or mice. Migration was recorded using transwells as well as chemotaxis-migration-slides to analyze directed horizontal and vertical migration, and stamp plates as well as scratch assays in order to record undirected migration. Interleukin 4 (IL-4), interferon gamma (IFNgamma) as key cytokines of an inflammatory TH1/TH2 response, as well as the toll-like receptor 4 ligand lipopolysaccharides (LPS), were analyzed as attractants and modulators of migration. Matrix metalloproteinases (MMP) were analyzed by zymography and blocked using specific inhibitors. Long-term imaging was performed in a conditioned microscope chamber up to two days. Results: We observed an increase of undirected SC mobility after treatment with LPS, whereas inhibition of MMP-9 diminished this elevation. Stimulation with IL-4 and IFN-gamma attracted SCs concerning directed migration and caused center of mass relocation. Furthermore, both cytokines altered total migration distance in undirected migration detection. CXCR2 expression as well as intracellular calcium concentrations were confirmed as underlying molecular mechanisms. Conclusions: Unspecific inflammatory stimuli increased SC mobility whereas a specific pattern of cytokines during inflammation and
regeneration is crucial to conduct and regulate SC locomotion, which was detected to be MMP-9 and CXCR2-dependent and regulated via calcium-homeostasis. This study provides new insights into restorative mechanisms of the PNS, which may identify useful novel therapeutic pathways. doi:10.1016/j.jneuroim.2014.08.189
354 Th1 polarization of T cells injected into the cerebrospinal fluid induces brain immunosurveillance Itai Strominger, Yair Fisher, Shva Biton, Anna Nemirovsky, Rona Baron, Alon Monsonego The Shraga Segal Dept. of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel Although CD4 T cells reside within the cerebrospinal fluid (CSF), it is yet unclear whether and how they enter the brain parenchyma and migrate to target specific antigens. We examined the ability of Th1, Th2 and Th17 CD4 T cells injected intracerebroventricularly to migrate from the lateral ventricles into the brain parenchyma in mice. We show that primarily Th1 cells cross the ependymal layer of the ventricle and migrate within the brain parenchyma by stimulating an IFN-gamma-dependent dialogue with neural cells, which maintains the effector function of the T cells. When injected into a mouse model of Alzheimer's disease, A-beta-specific Th1 cells target A-beta plaques, increase A-beta uptake and promote neurogenesis with no evidence of pathogenic autoimmunity or neuronal loss. Overall, we provide a mechanistic insight to the migration of CSF CD4 T cells into the brain parenchyma and highlight implications on brain immunity and repair. Keywords: Th1 CD4 T cells, cerebrospinal fluid, ependymal layer, Alzheimer's disease. doi:10.1016/j.jneuroim.2014.08.190
231 The inflammasome pyrin contributes to pertussis toxin-induced IL-1β synthesis, neutrophil intravascular crawling and autoimmune encephalomyelitis Luc Vallieresa, Aline Dumasa, Nathalie Amiablea, Juan Pablo De Rivero Vaccarib, Jae Jin Chaec, Robert W. Keaned, Steve Lacroixa Laval University, CHU de Quebec, Quebec, Canada; bThe Miami Project to Cure Paralysis, University of Miami, Miami, United States; cMedical Genetics Branch, National Human Genome Research Institute, Bethesda, United States; dDepartment of Physiology and Biophysics, University of Miami, Miami, United States a
Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in
Abstracts
Methods: Acute brain lesions were induced by transient focal brain ischemia in WT and caspase-1−/− mice. A different group of mice was treated with the specific caspase-1 inhibitor z-YVAD-FMK or control. We analyzed differential cell percentages of lymphocyte and monocyte subpopulations at 6 h and 3 days after experimental stroke by flow cytometry of blood and spleen. Serum cytokine levels were analyzed by ELISA and cellular activation after stroke detected by intracellular cytokine assays. Pyroptosis as a cause of immune cell death was measured by intracellular caspase-1 labeling and membrane disruption by 7-AAD. Results: An increase in caspase-1 activation was detected after 3 days of stroke in splenic monocytes and lymphocytes. However, caspase-1 activation in circulating leukocytes was evident only in lymphocytes but not in monocytes. Furthermore, differential blood cell counts were unaffected by caspase-1 expression after stroke. Additionally, we detected an enhanced expression of intracellular inflammatory cytokines in monocytes during the acute phase after stroke. This differential activation of caspase-1 in leukocyte subpopulations was associated with enhanced inflammatory cytokine concentrations. Genetic as well as pharmacological disruption of caspase-1 signaling abrogated leukocyte pyroptosis and the post-stroke inflammatory reaction. Conclusion: We revealed caspase-1 as a key molecule in the regulation of post-stroke immune cell death and activation. It induces early immune activation as well as delayed pyroptotic immune cell death, thereby, suggesting a novel and comprehensive molecular target to treat the complex immune dysregulation after acute brain injuries. doi:10.1016/j.jneuroim.2014.08.188
414 Schwann cell locomotion during peripheral nerve inflammation Mark Stettner, Sandra Labus, Jan-philipp Weinberger, Thomas Dehmel, Angelika Derksen, Anne K. Mausberg, Bernd C. Kieseier Department of Neurology, Heinrich-Heine-University, Dusseldorf, Germany Background: Chronic immune-mediated neuropathies are characterized by de- and remyelination of the peripheral nervous system (PNS). Schwann cells (SCs) as glial cells of the PNS play a main role in myelin restructuring, a process requiring the ability of SCs to migrate to damaged peripheral nerves. The specific impact of cytokines on SC mobility, however, is not fully understood so far. Methods: SCs were prepared and purified from sciatic nerves of neonatal rats or mice. Migration was recorded using transwells as well as chemotaxis-migration-slides to analyze directed horizontal and vertical migration, and stamp plates as well as scratch assays in order to record undirected migration. Interleukin 4 (IL-4), interferon gamma (IFNgamma) as key cytokines of an inflammatory TH1/TH2 response, as well as the toll-like receptor 4 ligand lipopolysaccharides (LPS), were analyzed as attractants and modulators of migration. Matrix metalloproteinases (MMP) were analyzed by zymography and blocked using specific inhibitors. Long-term imaging was performed in a conditioned microscope chamber up to two days. Results: We observed an increase of undirected SC mobility after treatment with LPS, whereas inhibition of MMP-9 diminished this elevation. Stimulation with IL-4 and IFN-gamma attracted SCs concerning directed migration and caused center of mass relocation. Furthermore, both cytokines altered total migration distance in undirected migration detection. CXCR2 expression as well as intracellular calcium concentrations were confirmed as underlying molecular mechanisms. Conclusions: Unspecific inflammatory stimuli increased SC mobility whereas a specific pattern of cytokines during inflammation and
regeneration is crucial to conduct and regulate SC locomotion, which was detected to be MMP-9 and CXCR2-dependent and regulated via calcium-homeostasis. This study provides new insights into restorative mechanisms of the PNS, which may identify useful novel therapeutic pathways. doi:10.1016/j.jneuroim.2014.08.189
354 Th1 polarization of T cells injected into the cerebrospinal fluid induces brain immunosurveillance Itai Strominger, Yair Fisher, Shva Biton, Anna Nemirovsky, Rona Baron, Alon Monsonego The Shraga Segal Dept. of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel Although CD4 T cells reside within the cerebrospinal fluid (CSF), it is yet unclear whether and how they enter the brain parenchyma and migrate to target specific antigens. We examined the ability of Th1, Th2 and Th17 CD4 T cells injected intracerebroventricularly to migrate from the lateral ventricles into the brain parenchyma in mice. We show that primarily Th1 cells cross the ependymal layer of the ventricle and migrate within the brain parenchyma by stimulating an IFN-gamma-dependent dialogue with neural cells, which maintains the effector function of the T cells. When injected into a mouse model of Alzheimer's disease, A-beta-specific Th1 cells target A-beta plaques, increase A-beta uptake and promote neurogenesis with no evidence of pathogenic autoimmunity or neuronal loss. Overall, we provide a mechanistic insight to the migration of CSF CD4 T cells into the brain parenchyma and highlight implications on brain immunity and repair. Keywords: Th1 CD4 T cells, cerebrospinal fluid, ependymal layer, Alzheimer's disease. doi:10.1016/j.jneuroim.2014.08.190
231 The inflammasome pyrin contributes to pertussis toxin-induced IL-1β synthesis, neutrophil intravascular crawling and autoimmune encephalomyelitis Luc Vallieresa, Aline Dumasa, Nathalie Amiablea, Juan Pablo De Rivero Vaccarib, Jae Jin Chaec, Robert W. Keaned, Steve Lacroixa Laval University, CHU de Quebec, Quebec, Canada; bThe Miami Project to Cure Paralysis, University of Miami, Miami, United States; cMedical Genetics Branch, National Human Genome Research Institute, Bethesda, United States; dDepartment of Physiology and Biophysics, University of Miami, Miami, United States a
Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in