Cell-selective Knockout and 3D Confocal Image Analysis Reveals Separate Roles for Astrocyte- and Endothelial-derived CCL2 in Neuroinflammation

Background Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood–brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). Methods Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. Results Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. Conclusions These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease

Electroceramics for High-Energy Density Capacitors: Current Status and Future Perspectives

Materials exhibiting high energy/power density are currently needed to meet the growing demand of portable electronics, electric vehicles and large-scale energy storage devices. The highest energy densities are achieved for fuel cells, batteries, and supercapacitors, but conventional dielectric capacitors are receiving increased attention for pulsed power applications due to their high power density and their fast charge–discharge speed. The key to high energy density in dielectric capacitors is a large maximum but small remanent (zero in the case of linear dielectrics) polarization and a high electric breakdown strength. Polymer dielectric capacitors offer high power/energy density for applications at room temperature, but above 100 °C they are unreliable and suffer from dielectric breakdown. For high-temperature applications, therefore, dielectric ceramics are the only feasible alternative. Lead-based ceramics such as La-doped lead zirconate titanate exhibit good energy storage properties, but their toxicity raises concern over their use in consumer applications, where capacitors are exclusively lead free. Lead-free compositions with superior power density are thus required. In this paper, we introduce the fundamental principles of energy storage in dielectrics. We discuss key factors to improve energy storage properties such as the control of local structure, phase assemblage, dielectric layer thickness, microstructure, conductivity, and electrical homogeneity through the choice of base systems, dopants, and alloying additions, followed by a comprehensive review of the state-of-the-art. Finally, we comment on the future requirements for new materials in high power/energy density capacitor applications

Resolution of Central Nervous System Astrocytic and Endothelial Sources of CCL2 Gene Expression During Evolving Neuroinflammation

Background The chemokine CCL2 is a critical mediator of neuroinflammation in diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 drives mononuclear cell infiltration into the central nervous system (CNS), alters expression and distribution of microvascular endothelial tight junction proteins, and disrupts the blood–brain and blood-spinal cord barriers. Immunohistochemistry has consistently revealed astrocytes to be a source of this chemokine during neuroinflammation, while providing less uniform evidence that CNS endothelial cells may also express CCL2. Moreover, the relative contributions of these cell types to the CNS pool of CCL2 during MS/EAE are unclear and the aim of this study was to investigate this further. Methods CCL2 gene expression was determined by qRT-PCR in different populations of CNS cells at different times following EAE induced by immunization with MOG35–55 peptide and adjuvants, or after injection with adjuvants alone. CNS cells types were isolated by two different protocols: bulk isolation to yield crude microvascular and parenchymal fractions (containing astrocytes, other glia, and neurons), or laser capture microdissection (LCM) to acquire more precisely microvascular endothelial cells, astrocytes or other parenchymal cells. Results Both CNS microvessel and parenchymal populations prepared by crude bulk isolation showed up-regulation of CCL2 mRNA following MOG immunization or injection of adjuvants alone. More exact dissection by LCM revealed microvascular endothelial cells and astrocytes to be the specific sources of CCL2 gene induction following MOG immunization, while only astrocytes showed elevated CCL2 mRNA in response to just adjuvants. Astrocytes displayed the greatest degree of stimulation of CCL2 gene expression following EAE induction. Conclusions High-precision LCM affirmed both microvascular endothelial cells and astrocytes as the major CNS sources of CCL2 gene expression during EAE. Given the high accessibility of the CNS microvascular endothelium, endothelial-derived CCL2 could prove a viable target for therapeutic intervention in neuroinflammatory disease

The CCL2 Synthesis Inhibitor Bindarit Targets Cells of the Neurovascular Unit, and Suppresses Experimental Autoimmune Encephalomyelitis

Background Production of the chemokine CCL2 by cells of the neurovascular unit (NVU) drives critical aspects of neuroinflammation. Suppression of CCL2 therefore holds promise in treating neuroinflammatory disease. Accordingly, we sought to determine if the compound bindarit, which inhibits CCL2 synthesis, could repress the three NVU sources of CCL2 most commonly reported in neuroinflammation – astrocytes, microglia and brain microvascular endothelial cells (BMEC) – as well as modify the clinical course of neuroinflammatory disease. Methods The effect of bindarit on CCL2 expression by cultured murine astrocytes, microglia and BMEC was examined by quantitative reverse transcription polymerase chain reaction (qRTPCR). Bindarit action on mouse brain and spinal cord in vivo was similarly investigated by qRT-PCR following LPS injection in mice. And to further gauge the potential remedial effects of bindarit on neuroinflammatory disease, its impact on the clinical course of experimental autoimmune encephalomyelitis (EAE) in mice was also explored. Results Bindarit repressed CCL2 expression by all three cultured cells, and antagonized upregulated expression of CCL2 in both brain and spinal cord in vivo following LPS administration. Bindarit also significantly modified the course and severity of clinical EAE, diminished the incidence and onset of disease, and evidenced signs of disease reversal. Conclusion Bindarit was effective in suppressing CCL2 expression by cultured NVU cells as well as brain and spinal cord tissue in vivo. It further modulated the course of clinical EAE in both preventative and therapeutic ways. Collectively, these result

Metabolic engineering of proanthocyanidin production by repressing the isoflavone pathways and redirecting anthocyanidin precursor flux in legume

MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying itspromotion of PA biosynthesis is not fully understood. Here, we showed that MtPAR promotes PAproduction by a direct repression of biosynthesis of isoflavones, the major flavonoids in legume,and by redirecting immediate precursors, such as anthocyanidins, flux into PA pathway. Ectopicexpression of MtPAR repressed isoflavonoid production by directly binding and suppressingisoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR up-regulatedPA-specific genes and decreased the anthocyanin levels without altering the expression ofanthocyanin biosynthetic genes. MtPAR may shift the anthocyanidin precursor flux fromanthocyanin pathway to PA biosynthesis. MtPAR complemented PA-deficient phenotype ofArabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showedthe direct interactions between MtPAR, MtTT8 and MtWD40-1 proteins from Medicagotruncatula and Glycine max, to form a ternary complex to trans-activate PA-specific ANR gene.Finally, MtPAR expression in alfalfa (Medicago sativa) hairy roots and whole plants only promotedthe production of small amount of PAs, which was significantly enhanced by co-expression ofMtPAR and MtLAP1. Transcriptomic and metabolite profiling showed an additive effect betweenMtPAR and MtLAP1 on the production of PAs, supporting that efficient PA production requiresmore anthocyanidin precursors. This study provides new insights into the role and mechanism ofMtPAR in partitioning precursors from isoflavone and anthocyanin pathways into PA pathwaysfor a specific promotion of PA production. Based on this, a strategy by combining MtPAR andMtLAP1 co-expression to effectively improve metabolic engineering performance of PAproduction in legume forage was developed

Active Induction of Experimental Autoimmune Encephalomyelitis by MOG35-55 Peptide Immunization is Associated with Differential Responses in Separate Compartments of the Choroid Plexus

Background There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood–brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process. Methods To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts. Results The CP became swollen and displayed significant molecular changes in response to MOGCFA/ PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, proinflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTXtreatment, alone, resulted in extensive, though less robust, alterations in both CP compartments. Conclusions MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease

Active induction of experimental autoimmune encephalomyelitis by MOG_35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus

<p>Abstract</p> <p>Background</p> <p>There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood–brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process.</p> <p>Methods</p> <p>To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and <it>in situ</it> expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG_35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an <it>Immune Panel</it> TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts.</p> <p>Results</p> <p>The CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments.</p> <p>Conclusions</p> <p>MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.</p