[Show abstract][Hide abstract] ABSTRACT: Microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network. Here, we used a digital tool for the quantitative morphometric characterization of fine cortical microglial structures in mice, and the changes they undergo with aging and in Alzheimer's-like disease. We show that, compared with microglia in young mice, microglia in old mice are less ramified and possess fewer branches and fine processes along with a slightly increased proinflammatory cytokine expression. A similar microglial pathology appeared 6–12 months earlier in mouse models of Alzheimer's disease (AD), along with a significant increase in brain parenchyma lacking coverage by microglial processes. We further demonstrate that microglia near amyloid plaques acquire unique activated phenotypes with impaired process complexity. We thus show that along with a chronic proinflammatory reaction in the brain, aging causes a significant reduction in the capacity of microglia to scan their environment. This type of pathology is markedly accelerated in mouse models of AD, resulting in a severe microglial process deficiency, and possibly contributing to enhanced cognitive decline.
[Show abstract][Hide abstract] ABSTRACT: Although CD4 T cells reside within the cerebrospinal fluid, it is yet unclear whether and how they enter the brain parenchyma and migrate to target specific Ags. 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-γ-dependent dialogue with neural cells, which maintains the effector function of the T cells. When injected into a mouse model of Alzheimer's disease, amyloid-β (Aβ)-specific Th1 cells target Aβ plaques, increase Aβ uptake, and promote neurogenesis with no evidence of pathogenic autoimmunity or neuronal loss. Overall, we provide a mechanistic insight to the migration of cerebrospinal fluid CD4 T cells into the brain parenchyma and highlight implications on brain immunity and repair.
The Journal of Immunology 12/2013; 192(1). DOI:10.4049/jimmunol.1301707 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.
PLoS ONE 10/2013; 8(10):e76092. DOI:10.1371/journal.pone.0076092 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stress activates the hypothalamic-pituitary-adrenocortical (HPA) axis to promote the release of corticosterone (CORT), which consequently suppresses pathogenic stimulation of the immune system. Paradoxically, however, stress often promotes autoimmunity through yet unknown mechanisms. Here we investigated how chronic variable stress (CVS), and the associated alterations in urine CORT levels, affect the susceptibility to experimental autoimmune encephalomyelitis (EAE) in female and male C57BL6 mice. Under baseline (non-stressed) conditions, females exhibited substantially higher CORT levels and an attenuated EAE with less mortality than males. However, CVS induced a significantly worsened EAE in females, which was prevented if CORT signaling was blocked. In addition, females under CVS conditions showed a shift towards proinflammatory Th1/Th17 versus Th2 responses and a decreased proportion of CD4(+) CD25(+) Treg cells. This demonstrates that whereas C57BL6 female mice generally exhibit higher CORT levels and an attenuated form of EAE than males, they become less responsive to the immunosuppressive effects of CORT under chronic stress and thereby prone to a higher risk of destructive autoimmunity.
European Journal of Immunology 03/2013; 43(3). DOI:10.1002/eji.201242613 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proper function of the neurovasculature is required for optimal brain function and preventing neuroinflammation and neurodegeneration. Within this review, we discuss alterations of the function of the blood–brain barrier in neurologic disorders such as multiple sclerosis, epilepsy, and Alzheimer’s disease and address potential underlying mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Microglia are myeloid-derived cells that colonize the central nervous system (CNS) at early stages of development and constitute up to 20% of the glial populations throughout life. While extensive progress has been recently made in identifying the cellular origin of microglia, the mechanism whereby the cells acquire the unique ramified and quiescent phenotype within the CNS milieu remains unknown. Here, we show that upon co-culturing of either CD117(+) /Lin(-) hematopoietic progenitors or CD11c(+) bone marrow derived cells with organotypic hippocampal slices or primary glia, the cells acquire a ramified morphology concomitant with reduced levels of CD86, MHCII, and CD11c and up-regulation of the microglial cell-surface proteins CX(3) CR1 and Iba-1. We further demonstrate that the transforming growth factor beta (TGF-β) signaling pathway via SMAD2/3 phosphorylation is essential for both primary microglia and myeloid-derived cells in order to acquire their quiescent phenotype. Our study suggests that the abundant expression of TGF-β within the CNS during development and various inflammatory processes plays a key role in promoting the quiescent phenotype of microglia and may thus serve as a target for therapeutic strategies aimed at modulating the function of microglia in neurodegenerative diseases such as Alzheimer's and prion.
[Show abstract][Hide abstract] ABSTRACT: Aβ vaccination has been shown to induce remarkable clearance of brain amyloid plaques in mouse models of Alzheimer's disease (AD). However, the extent to which antibody-mediated Aβ clearance is affected by predominant formation of Aβ42 over Aβ40 is unclear. Here we demonstrate for the first time that in a mouse model carrying the human APP mutations KM670/671NL and the human PS1 mutation P166L, Aβ vaccination does not result in plaque clearance. This was in spite of the strong T- and B-cell immune responses evoked under the DR1501 genetic background and the activation of microglia at sites of Aβ plaques. Our findings suggest the existence of antibody-resistant forms of Aβ deposits in the brain consisting of primarily Aβ42, and shed light on the mechanisms of antibody-dependent amyloid clearance as well as novel therapeutic strategies for AD.
Journal of neuroimmunology 04/2012; 247(1-2):95-9. DOI:10.1016/j.jneuroim.2012.03.017 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amyloid-β (Aβ) accumulation in the brain is one of the hallmarks of Alzheimer's disease (AD). T-cell entry into vascular and parenchymal brain areas loaded with Aβ has been observed with both beneficial as well as detrimental effects. Using a new AD mouse model, we studied the molecular mechanisms allowing CD4 T cells to specifically target Aβ-loaded brain areas. We observed that following Aβ immunization, CD11c+ dendritic cells (DCs) and CD4 T cells occurred primarily in the perivascular and leptomeningial spaces of cerebral vessels deposited with Aβ. CD11c+ cells expressed high levels of the DC maturation markers DEC-205, MHC class II and CD86. Notably, the majority of cerebral blood vessels were found adjacent to Aβ plaques, expressing high levels of the ICAM-1 and VCAM-1 adhesion molecules. We propose that the drainage of Aβ to the leptomeningeal and perivascular spaces and its deposition there provide the antigenic source for DCs to stimulate Aβ-specific T cells on their way to target amyloid plaques within the brain tissue.
[Show abstract][Hide abstract] ABSTRACT: Active vaccination with amyloid beta peptide (Aβ) to induce beneficial antibodies was found to be effective in mouse models of Alzheimer's disease (AD), but human vaccination trials led to adverse effects, apparently caused by exuberant T-cell reactivity. Here, we sought to develop a safer active vaccine for AD with reduced T-cell activation. We treated a mouse model of AD carrying the HLA-DR DRB1*1501 allele, with the Aβ B-cell epitope (Aβ 1-15) conjugated to the self-HSP60 peptide p458. Immunization with the conjugate led to the induction of Aβ-specific antibodies associated with a significant reduction of cerebral amyloid burden and of the accompanying inflammatory response in the brain; only a mild T-cell response specific to the HSP peptide but not to the Aβ peptide was found. This type of vaccination, evoking a gradual increase in antibody titers accompanied by a mild T-cell response is likely due to the unique adjuvant and T-cell stimulating properties of the self-HSP peptide used in the conjugate and might provide a safer approach to effective AD vaccination.
[Show abstract][Hide abstract] ABSTRACT: Engineering of cell-based constructs for treating a variety of immune-related diseases by local transplantation of the cells in a pre-designed matrix is an emerging therapeutic approach, which can potentially reduce the side effects associated with systemic cell injection. Stromal cells have been shown to exert immunosuppressive properties and thus can be exploited for autoimmune regulation and cell transplantation. Here, we demonstrate the fabrication of a stromal cell-based construct, which serves as a lymphoid-like organ with immune regulatory characteristics. In the proposed system, stromal cells are co-seeded with dendritic cells (DC) in a macro-porous alginate scaffold containing the encephalitogenic myelin-derived peptide, proteolipid protein (PLP). We demonstrate that the presence of stromal cells attenuates DC maturation upon lipopolysaccharide stimulus. In vitro, we show that while the migration of pathogenic PLP-specific T cells to construct cultivated with or without stromal cells does not differ, their activation and proliferation are significantly suppressed in the presence of stromal cells. Upon in vivo transplantation, under the kidney capsule of mice, the pathogenic activation and proliferation of T cells which were drawn into the construct were suppressed in the co-seeded constructs. This system thus serves as a lymphoid-like organ with regulatory characteristics, which can be applied for local tolerance induction, for application in cell transplantations as well as autoimmune diseases.
[Show abstract][Hide abstract] ABSTRACT: The Gene Ontology (GO) is used to describe genes and gene products from many organisms. When used for functional annotation of microarray data, GO is often slimmed by editing so that only higher level terms remain. This practice is designed to improve the summarizing of experimental results by grouping high level terms and the statistical power of GO term enrichment analysis. Here, we propose a new approach to editing the gene ontology, clipping, which is the editing of GO according to biological relevance. Creation of a GO subset by clipping is achieved by removing terms (from all hierarchal levels) if they are not functionally relevant to a given domain of interest. Terms that are located in levels higher to relevant terms are kept, thus, biologically irrelevant terms are only removed if they are not parental to terms that are relevant.
Using this approach, we have created the Neural-Immune Gene Ontology (NIGO) subset of GO directed for neurological and immunological systems. We tested the performance of NIGO in extracting knowledge from microarray experiments by conducting functional analysis and comparing the results to those obtained using the full GO and a generic GO slim. NIGO not only improved the statistical scores given to relevant terms, but was also able to retrieve functionally relevant terms that did not pass statistical cutoffs when using the full GO or the slim subset.
Our results validate the pipeline used to generate NIGO, suggesting it is indeed enriched with terms that are specific to the neural/immune domains. The results suggest that NIGO can enhance the analysis of microarray experiments involving neural and/or immune related systems. They also directly demonstrate the potential such a domain-specific GO has in generating meaningful hypotheses.
[Show abstract][Hide abstract] ABSTRACT: Patients with Alzheimer's disease (AD) exhibit substantial accumulation of amyloid-beta (Abeta) plaques in the brain. Here, we examine whether Abeta vaccination can facilitate the migration of T lymphocytes to specifically target Abeta plaques and consequently enhance their removal. Using a new mouse model of AD, we show that immunization with Abeta, but not with the encephalitogenic proteolipid protein (PLP), results in the accumulation of T cells at Abeta plaques in the brain. Although both Abeta-reactive and PLP-reactive T cells have a similar phenotype of Th1 cells secreting primarily IFN-gamma, the encephalitogenic T cells penetrated the spinal cord and caused experimental autoimmune encephalomyelitis (EAE), whereas Abeta T cells accumulated primarily at Abeta plaques in the brain but not the spinal cord and induced almost complete clearance of Abeta. Furthermore, while a single vaccination with Abeta resulted in upregulation of the phagocytic markers triggering receptors expressed on myeloid cells-2 (TREM2) and signal regulatory protein-beta1 (SIRPbeta1) in the brain, it caused downregulation of the proinflammatory cytokines TNF-alpha and IL-6. We thus suggest that Abeta deposits in the hippocampus area prioritize the targeting of Abeta-reactive but not PLP-reactive T cells upon vaccination. The stimulation of Abeta-reactive T cells at sites of Abeta plaques resulted in IFN-gamma-induced chemotaxis of leukocytes and therapeutic clearance of Abeta.
PLoS ONE 05/2010; 5(5):e10830. DOI:10.1371/journal.pone.0010830 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One of the more substantial hurdles to be overcome in realizing the exciting potential of siRNA molecules as therapeutic agents for a wide range of diseases is the intact delivery of the active molecule into its target cell. Here, we present a platform for in vitro and in vivo delivery and intracellular release of siRNA in peritoneal macrophages (Mvarphis). The delivery platform is based on the encapsulation of siRNA in biodegradable poly(d,l-lactide) (PLA) microspheres, which are targeted to Mvarphis by the simple principle of size exclusion. Proof of concept was achieved using siRNAs targeting TNFalpha and CD86 in macrophages. We show that the release of the siRNA in peritoneal-derived macrophages in vitro occurs intracellularly, and is abrogated by cytochalasin B, a phagocytosis inhibitor. Silencing in these cells is potent and lasts for at least one week. In vivo, we prove that siRNA encapsulated in biodegradable PLA microspheres can be delivered to peritoneal-residing Mvarphis and can induce potent silencing of TNFalpha secretion for at least one week. The PLA microspheres hold great potential for in vivo use, due to their biocompatibility and degradability, and can potentially be used for in vivo immunomodulation of Mvarphis for treatment of autoimmune and chronic inflammatory conditions.
[Show abstract][Hide abstract] ABSTRACT: The signal regulatory protein-beta1 (SIRPbeta1) is a DAP12-associated transmembrane receptor expressed in a subset of hematopoietic cells. Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Here, we found that SIRPbeta1 was up-regulated and acted as a phagocytic receptor on microglia in amyloid precursor protein J20 (APP/J20) transgenic mice and in Alzheimer's disease (AD) patients. Interferon (IFN)-gamma and IFN-beta stimulated gene transcription of SIRPbeta1 in cultured microglia. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase-2. Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta (Abeta). Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells. Thus, SIRPbeta1 is a novel IFN-induced microglial receptor that supports clearance of neural debris and Abeta aggregates by stimulating phagocytosis.
American Journal Of Pathology 11/2009; 175(6):2528-39. · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Active amyloid beta-peptide (Abeta) immunization of patients with Alzheimer's disease (AD) caused meningoencephalitis in approximately 6% of immunized patients in a clinical trial. In addition, long-term studies of AD patients show varying degrees of Abeta Ab responses, which correlate with the extent of Abeta clearance from the brain. In this study, we examined the contribution of various HLA-DR alleles to these immune-response variations by assessing Abeta T cell reactivity, epitope specificity, and immunogenicity. Analysis of blood samples from 133 individuals disclosed that the abundant DR haplotypes DR15 (found in 36% of subjects), DR3 (in 18%), DR4 (12.5%), DR1 (11%), and DR13 (8%) were associated with Abeta-specific T cell responses elicited via distinct T cell epitopes within residues 15-42 of Abeta. Because the HLA-DRB1*1501 occurred most frequently, we examined the effect of Abeta challenge in humanized mice bearing this allele. The observed T cell response was remarkably strong, dominated by secretion of IFN-gamma and IL-17, and specific to the same T cell epitope as that observed in the HLA-DR15-bearing humans. Furthermore, following long-term therapeutic immunization of an AD mouse model bearing the DRB1*1501 allele, Abeta was effectively cleared from the brain parenchyma and brain microglial activation was reduced. The present study thus characterizes HLA-DR alleles directly associated with specific Abeta T cell epitopes and demonstrates the highly immunogenic properties of the abundant allele DRB1*1501 in a mouse model of AD. This new knowledge enables us to explore the basis for understanding the variations in naturally occurring Abeta-reactive T cells and Abeta immunogenicity among humans.
The Journal of Immunology 09/2009; 183(5):3522-30. DOI:10.4049/jimmunol.0900620 · 4.92 Impact Factor