Chronic Neuroinflammatory Disease Research Articles

Enzyme Selective Flim in Microglia: Studying the Role of NADPH Oxidase in Chronic Neuroinflammation

Understanding the pathological mechanisms in the brain is the decisive step in developing novel specific therapeutic strategies and in curing chronic neuroinflammatory or neurodegenerative diseases like multiple sclerosis (MS). For this purpose, techniques which confer high molecular specificity and spatial resolution, which are non-invasive and allow for dynamics visualization in vivo are necessary. We already showed that NAD(P)H-based fluorescence lifetime imaging (FLIM) is the appropriate technique to detect the activation of NADPH oxidase in murine polymorphonuclear cells and in cells of Nicotiana tabacum during phagocytosis. The fluorescence lifetime of NAD(P)H if bound to NADPH oxidase increases to approx. 3600 ps as compared to the case of NAD(P)H-dependent metabolic enzymes, i.e. approx. 2000 ps. A central yet still not fully understood role in neuronal injury is assigned to microglial NADPH oxidase: this might be a neurotoxic effect due to direct production of reactive oxygen species or, indirectly, due to the induction of NO synthase or a protective role if NADPH oxidase is involved in the phagocytosis of noxious supra-molecular structures like beta-amyloids. By means of NAD(P)H-based FLIM we investigate the activation of NADPH oxidase in murine and human peripheral macropahges as compared to microglia, i.e. macrophages of the central nervous system. The same response to specific chemical activation and to phagocytosis of Stafiloccocus aureus functionalized beads is demonstrated for macrophages and microglia of new-born mice. However, this response is different for microglia of adult, healthy mice as also reflected by their reduced phagocytotic capacity. In pathological context, we investigate the activation of NADPH oxidase in microglia of adult, healthy mice as compared to microglia of mice affected by a murine model of MS. Thus, we were able to identify a new mechanism in chronic neuroinflammation.

CD11c-expressing cells reside in the juxtavascular parenchyma and extend processes into the glia limitans of the mouse nervous system

Recent studies demonstrated that primary immune responses can be induced within the brain depending on vessel-associated cells expressing markers of dendritic cells (DC). Using mice transcribing the green fluorescent protein (GFP) under the promoter of the DC marker CD11c, we determined the distribution, phenotype, and source of CD11c+ cells in non-diseased brains. Predilection areas of multiple sclerosis (MS) lesions (periventricular area, adjacent fibre tracts, and optical nerve) were preferentially populated by CD11c+ cells. Most CD11c+ cells were located within the juxtavascular parenchyma rather than the perivascular spaces. Virtually all CD11c+ cells co-expressed ionized calcium-binding adaptor molecule 1 (IBA-1), CD11b, while detectable levels of major histocompatibility complex II (MHC-II) in non-diseased mice was restricted to CD11c+ cells of the choroid plexus. Cellular processes project into the glia limitans which may allow transport and/or presentation of intraparenchymal antigens to extravasated T cells in perivascular spaces. In chimeric mice bearing CD11c-GFP bone marrow, fluorescent cells appeared in the CNS between 8 and 12 weeks after transplantation. In organotypic slice cultures from CD11c-GFP mice, the number of fluorescent cells strongly increased within 72 h. Strikingly, using anti-CD209, an established marker for human DC, a similar population was detected in human brains. Thus, we show for the first time that CD11c+ cells can not only be recruited from the blood into the parenchyma, but also develop from an intraneural precursor in situ. Dysbalance in their recruitment/development may be an initial step in the pathogenesis of chronic (autoimmune) neuroinflammatory diseases such as MS.

Assessing Activation States in Microglia

Since the original identification of microglia as a principal player in the brain's innate immune response, microglial activation has been widely studied. Recent studies suggest that microglial responses are heterogeneous, requiring a more precise definition of the functional outcomes of their participation in disease. Similarly to other tissue macrophages, microglia respond to inflammatory or injurious stimuli in the CNS in a pre-programmed manner that is designed to both kill and to set the stage for repair and resolution of the disease. In vitro studies on acute immune responses have provided key information on the initiation, signaling pathways and products of activated macrophages. However, in chronic neurodegenerative diseases such as Alzheimer's disease where in vivo analyses are critical to understanding the long-term disease processes, our knowledge of the integrated tissue immune response and the outcome of this immune activity to neurons and other glia over the extended course of disease is more limited. This is due in part to the complexity of microglial activation states and to the location of microglia in a dense neuronal network. Classical activation, alternative activation and acquired deactivation are each found in the brain during chronic neuroinflammatory diseases and may demonstrate regional differences in expression levels. This review will identify "markers" that can be used to explore inflammatory states in the brain and will discuss the likely functional outcomes when these cytoactive factors are expressed. A broad-based functional view is provided that is designed to more fully explore the balance between inflammo-toxic and inflammo-resolution factors that govern chronic disease progression.

Circular compartmentalized microfluidic platform: Study of axon–glia interactions

We describe a compartmentalized circular microfluidic platform that enables directed cell placement within defined microenvironments for the study of axon-glia interactions. The multi-compartment platform consists of independent units of radial microchannel arrays that fluidically isolate somal from axonal compartments. Fluidic access ports punched near the microchannels allow for direct pipetting of cells into the device. Adjacent somal or axonal compartments can be readily merged so that independent groups of neurons or axons can be maintained in either separate or uniform microenvironments. We demonstrate three distinct modes of directed cell placement in this device, to suit varying experimental needs for the study of axon-glia interactions: (1) centrifugation of the circular platform can result in a two-fold increase in axonal throughput in microchannels and provides a new technique to establish axon-glia interactions; (2) microstencils can be utilized to directly place glial cells within areas of interest; and (3) intimate axon-glia co-culture can be attained via standard pipetting techniques. We take advantage of this microfluidic platform to demonstrate a two-fold preferential accumulation of microglia specifically near injured CNS axons, an event implicated in the maintenance and progression of a number of chronic neuroinflammatory and neurodegenerative diseases.

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.Principal FindingsHere, we demonstrate that CD4+CD25+CCR4+ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4+CD25+CCR4+ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4+CD25+CCR4+Foxp3− T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.ConclusionsWe have defined a unique T cell subset—IFN-γ+CCR4+CD4+CD25+ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

Continuing on the road to health: a short history of the Charité–universitätsmedizin Berlin from a plague house in the past to a medical school with a future

We are currently witnesses to and authors of a paradigm shift in neuropathology. While classical acute and chronic neuroinflammatory diseases such as meningitis or multiple sclerosis (MS) present aspects of neurodegeneration, the disease course of progressive degenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), or stroke-mediated neuronal deficit are demonstrably affected by inflammation. These insights have immediate consequences both for research methods and for the development of novel, more efficient therapies for these diseases. In this review, we analyze the inflammatory and degenerative pathological mechanisms in the brain with particular emphasis on the classical chronic inflammatory disease MS. We demonstrate that the latest pathological considerations not only require the application of advanced research technologies to investigate new pathomechanistic pathways, but also affect the investigation, development, and monitoring of novel potential therapeutic tools.

New developments in understanding and treating neuroinflammation

We are currently witnesses to and authors of a paradigm shift in neuropathology. While classical acute and chronic neuroinflammatory diseases such as meningitis or multiple sclerosis (MS) present aspects of neurodegeneration, the disease course of progressive degenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), or stroke-mediated neuronal deficit are demonstrably affected by inflammation. These insights have immediate consequences both for research methods and for the development of novel, more efficient therapies for these diseases. In this review, we analyze the inflammatory and degenerative pathological mechanisms in the brain with particular emphasis on the classical chronic inflammatory disease MS. We demonstrate that the latest pathological considerations not only require the application of advanced research technologies to investigate new pathomechanistic pathways, but also affect the investigation, development, and monitoring of novel potential therapeutic tools.

The Glial Response to CNS HIV Infection Includes p53 Activation and Increased Expression of p53 Target Genes

HIV-associated dementia (HAD) is a chronic neuroinflammatory disease that remains an important clinical problem without available rational treatment. As HIV does not infect neurons, the pathogenesis of HAD is thought to be secondary to the impact of infected leukocytes, including parenchymal microglia, which can secrete inflammatory mediators and viral products that alter the function of surrounding uninfected cells. We previously reported that the transcription factor p53 accumulates in neurons, microglia, and astrocytes of HAD patients. We have also shown that microglia from p53-deficient mice fail to induce neurotoxicity in response to the HIV coat protein gp120 in a coculture system, supporting the hypothesis that p53 plays a pathogenic role in the chronic neuroinflammatory component of HIV-associated neurodegeneration. We analyzed the extent and cell type specificity of p53 accumulation in subcortical white matter of ten AIDS patients that had previously been shown to demonstrate white matter p53 accumulation. To determine if p53 activation functioned to alter gene expression in HAD, cortical tissue sections were also immunolabeled for the p53 target genes Bax and p21(WAF1). These studies reveal that microglia, astrocytes, and oligodendrocytes all demonstrate p53 activation in response to HIV infection. We observed immunoreactivity for both Bax and p21(WAF1) in neurons and glia from patients demonstrating elevated p53 immunoreactivity. Our findings demonstrate that widespread increased p53 expression is present in HAD. Activation of p53 mediated pathways in the glia of HAD patients may contribute to the neuroinflammatory processes that promote neurodegeneration by inhibiting glial proliferation and/or promoting glial cell dysfunction.

Noninvasive ventilation or paradigm paralysis?

<h3>ABSTRACT</h3> Multiple sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease. MS prevalence varies geographically, both between and within countries and is notably high in Scotland. Disease trajectory varies significantly between individuals and the causes for this are largely unclear. Biomarkers predictive of disease course are urgently needed to allow improved stratification for current disease modifying therapies and future targeted treatments aimed at neuroprotection and remyelination, and as endpoints for clinical trials. Magnetic resonance imaging (MRI) can detect disease activity and underlying neurological damage non-invasively <i>in vivo</i> at the micro and macro structural level. FutureMS is a prospective Scottish longitudinal multi-centre cohort study which focuses on deeply phenotyping and genotyping patients with recently diagnosed relapsing-remitting MS (RRMS) to identify predictors of disease activity and severity. Neuroimaging is a central component of the study and provides two main primary endpoints for disease activity and neurodegeneration. The aim of the current paper is to provide an overview of MRI data acquisition, management and processing in FutureMS. MRI is acquired at baseline (N=431) and 1-year follow-up, in Dundee, Glasgow and Edinburgh (3T Siemens) and in Aberdeen (3T Philips), and managed and processed in Edinburgh. The core structural MRI protocol comprises T1-weighted, T2-weighted, 2D/3D FLAIR and proton density images. The original study primary imaging outcome measures are new/enlarging white matter lesions (WML) assessed by neuroradiological visual read and reduction in brain volume over one year. Secondary imaging outcomes comprise WML volume as an additional quantitative structural MRI outcome measure, rim lesions on susceptibility-weighted imaging (SWI), and microstructural MRI measures, including diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) metrics, relaxometry, magnetisation transfer (MT) ratio, MT saturation and derived g-ratio measures. FutureMS aims to reduce uncertainty around disease course and allow for targeted treatment in RRMS by exploring the role of conventional and advanced MRI measures as biomarkers of disease severity and progression in a large population of RRMS patients in Scotland.

Nitric oxide-induced mitochondrial dysfunction: implications for neurodegeneration.

Excessive generation of nitric oxide (NO) has been implicated in the pathogenesis of several neurodegenerative disorders. Damage to the mitochondrial electron transport chain has also been implicated in these disorders. NO and its toxic metabolite peroxynitrite (ONOO(-)) can inhibit the mitochondrial respiratory chain, leading to energy failure and ultimately cell death. There appears to be a differential susceptibility of brain cell types to NO/ONOO(-), which may be influenced by factors including cellular antioxidant status and the ability to maintain energy requirements in the face of marked respiratory chain damage. Although formation of NO/ONOO(-) following cytokine exposure does not affect astrocyte survival, these molecules may diffuse out and cause mitochondrial damage to neighboring NO/ONOO(-)-sensitive cells such as neurons. Evidence suggests that NO/ONOO(-) causes release of neuronal glutamate, leading to glutamate-induced activation of neuronal NO synthase and generation of further damaging species. While neurons appear able to recover from short-term exposure to NO/ONOO(-), extending the period of exposure results in persistent damage to the respiratory chain and cell death ensues. These findings have important implications for acute infection vs. chronic neuroinflammatory disease states. The evidence for NO/ONOO(-)-mediated mitochondrial damage in neurodegenerative disorders is reviewed and potential therapeutic strategies are discussed.

Elevated serum titers of proinflammatory cytokines and CNS autoantibodies in patients with chronic spinal cord injury.

This study characterized the proinflammatory cytokines, interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFalpha), the antiinflammatory cytokines, IL-4 and IL-10, autoantibodies specific for GM1 ganglioside (anti-GM1), IgG and IgM, and myelin-associated glycoprotein (anti-MAG), in the sera of infection-free, chronic (>12 months), traumatically injured SCI patients (n = 24). Healthy able-bodied subjects (n = 26) served as controls. The proinflammatory cytokines and anti-GM1 antibodies were of particular interest as they have been implicated in an autoimmune "channelopathy" component to central and peripheral conduction deficits in various chronic neuroinflammatory diseases. Antibody and cytokine titers were established using enzyme-linked immunosorbent assays (ELISA). The mean anti-GM(1) (IgM) titer value for the SCI group was significantly higher (p < 0.05) than controls. The SCI group also demonstrated significantly higher titers (p < 0.05) of IL-2 and TNF alpha than controls. No differences were found between the SCI group and control group mean levels of IL-4 or IL-10. Overall, the serum of 57% of SCI patients contained increased levels of autoantibodies or proinflammatory cytokines relative to control values. These results provide preliminary support for the hypothesis that chronic immunological activation in the periphery occurs in a subpopulation of chronic SCI patients. It remains to be established whether elevated serum titers of proinflammatory cytokines and autoantibodies against GM1 are beneficial to the patients or whether they are surrogate markers of a channelopathy that compounds the neurological impairment associated with traumatic axonopathy or myelinopathy.