Pre-treatment with the viral Toll-like receptor 3 agonist poly(I:C) modulates innate immunity and protects neutropenic mice infected intracerebrally with Escherichia coli.

BACKGROUND: Individuals with impaired immunity are more susceptible to infections than immunocompetent subjects. No vaccines are currently available to induce protection against E. coli meningoencephalitis. This study evaluated the potential of poly(I:C) pre-treatment to induce trained immunity. Poly(I:C) was administered as a non-specific stimulus of innate immune responses to protect immunocompetent and neutropenic wild-type mice from a subsequent challenge by the intracranial injection of E. coli K1. METHODS: Three days prior to infection, mice received an intraperitoneal injection of poly(I:C) or vehicle. Kaplan-Meier survival curves were analyzed. In short-term experiments, bacterial titers and the inflammatory response were characterized in the blood, cerebellum, and spleen homogenates. NK cell subpopulations in the brain and spleen were analyzed by flow cytometry. Numbers of microglia and activation scores were evaluated by histopathology. RESULTS: Pre-treatment with 200 µg poly(I:C) increased survival time, reduced mortality, and enhanced bacterial clearance in the blood, cerebellum, and spleen at early infection in neutropenic mice. Poly(I:C)-mediated protection correlated with an augmented number of NK cells (CD45+NK1.1+CD3-) and Iba-1+ microglial cells and a higher production of IFN-γ in the brain. In the spleen, levels of CCL5/RANTES and IFN-γ were increased and sustained in surviving poly(I:C)-treated animals for 14 days after infection. In immunocompetent animals, survival time was not significantly prolonged in poly(I:C)-treated animals although poly(I:C) priming reduced brain bacterial concentrations compared with vehicle-injected animals at early infection. CONCLUSIONS: Pre-treatment with the viral TLR3 agonist poly(I:C) modulated innate immune responses and strengthened the resistance of neutropenic mice against E. coli K1 meningoencephalitis.

Activin A increases phagocytosis of Escherichia coli K1 by primary murine microglial cells activated by toll-like receptor agonists.

BACKGROUND: Bacterial meningitis is associated with high mortality and long-term neurological sequelae. Increasing the phagocytic activity of microglia could improve the resistance of the CNS against infections. We studied the influence of activin A, a member of the TGF-ß family with known immunoregulatory and neuroprotective effects, on the functions of microglial cells in vitro. METHODS: Primary murine microglial cells were treated with activin A (0.13 ng/ml-13 µg/ml) alone or in combination with agonists of TLR2, 4, and 9. Phagocytosis of Escherichia coli K1 as well as release of TNF-α, IL-6, CXCL1, and NO was assessed. RESULTS: Activin A dose-dependently enhanced the phagocytosis of Escherichia coli K1 by microglial cells activated by agonists of TLR2, 4, and 9 without further increasing NO and proinflammatory cytokine release. Cell viability of microglial cells was not affected by activin A. CONCLUSIONS: Priming of microglial cells with activin A could increase the elimination of bacteria in bacterial CNS infections. This preventive strategy could improve the resistance of the brain to infections, particularly in elderly and immunocompromised patients.

Treatment with 5-azacitidine delay growth of glioblastoma xenografts: a potential new treatment approach for glioblastomas.

PURPOSE: Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults. The epigenetically active ribonucleoside analog 5-azacitidine is a new therapy option that changes tumor cell chromatin, which is frequently modified by methylation and deacetylation in malignant gliomas. METHODS: In vitro, we analyzed cell viability, cell apoptosis, and migration of human GBM cells. In vivo, we established subcutaneous and intracerebral GBM mouse models originating from U87MG, U373MG, and primary GBM cells as well as one patient-derived xenograft. Xenografts were treated with 5-azacitidine as well as valproic acid, bevacizumab, temozolomide, and phosphate buffered saline. The tumor sizes and Ki67 proliferation indices were determined. Glioma angiogenesis was examined immunohistochemically by expression analysis of endothelial cells (CD31) and pericytes (PDGFRß). RESULTS: In vitro, 5-azacitidine treatment significantly reduced human glioblastoma cell viability, increased cellular apoptosis, and reduced cellular migration. In vivo, 5-azacitidine significantly reduced growth in two intracerebral GBM models. Notably, this was also shown for a xenograft established from a patient surgery sample; whereas, epigenetically acting valproic acid did not show any growth reduction. Highly vascularized tumors responded to treatment, whereas low-vascularized xenografts showed no response. Furthermore, intracerebral glioblastomas treated with 5-azacitidine showed a clearly visible reduction of tumor angiogenesis and lower numbers of endothelial cells and tumor vessel pericytes. CONCLUSIONS: Our data show significant growth inhibition as well as antiangiogenic effects in intracerebral as well as patient-derived GBM xenografts. This encourages to investigate in detail the multifactorial effects of 5-azacitidine on glioblastomas.

Interferon-γ-Driven iNOS: A Molecular Pathway to Terminal Shock in Arenavirus Hemorrhagic Fever.

Arenaviruses such as Lassa virus (LASV) cause hemorrhagic fever. Terminal shock is associated with a systemic cytokine storm, but the mechanisms are ill defined. Here we used HLA-A2-expressing mice infected with a monkey-pathogenic strain of lymphocytic choriomeningitis virus (LCMV-WE), a close relative of LASV, to investigate the pathophysiology of arenavirus hemorrhagic fever (AHF). AHF manifested as pleural effusions, edematous skin swelling, and serum albumin loss, culminating in hypovolemic shock. A characteristic cytokine storm included numerous pro-inflammatory cytokines and nitric oxide (NO) metabolites. Edema formation and terminal shock were abrogated in mice lacking inducible nitric oxide synthase (iNOS), although the cytokine storm persisted. iNOS was upregulated in the liver in a T cell- and interferon-γ (IFN-γ)-dependent fashion. Accordingly, blockade of IFN-γ or depletion of T cells repressed hepatic iNOS and prevented disease despite unchecked high-level viremia. We identify the IFN-γ-iNOS axis as an essential and potentially druggable molecular pathway to AHF-induced shock.

A presumed antagonistic LPS identifies distinct functional organization of TLR4 in mouse microglia.

Microglia as principle innate immune cells of the central nervous system (CNS) are the first line of defense against invading pathogens. They are capable of sensing infections through diverse receptors, such as Toll-like receptor 4 (TLR4). This receptor is best known for its ability to recognize bacterial lipopolysaccharide (LPS), a causative agent of gram-negative sepsis and septic shock. A putative, naturally occurring antagonist of TLR4 derives from the photosynthetic bacterium Rhodobacter sphaeroides. However, the antagonistic potential of R. sphaeroides LPS (Rs-LPS) is no universal feature, since several studies suggested agonistic rather than antagonistic actions of this molecule depending on the investigated mammalian species. Here we show the agonistic versus antagonistic potential of Rs-LPS in primary mouse microglia. We demonstrate that Rs-LPS efficiently induces the release of cytokines and chemokines, which depends on TLR4, MyD88, and TRIF, but not CD14. Furthermore, Rs-LPS is able to regulate the phagocytic capacity of microglia as agonist, while it antagonizes Re-LPS-induced MHC I expression. Finally, to our knowledge, we are the first to provide in vivo evidence for an agonistic potential of Rs-LPS, as it efficiently triggers the recruitment of peripheral immune cells to the endotoxin-challenged CNS. Together, our results argue for a versatile and complex organization of the microglial TLR4 system, which specifically translates exogenous signals into cellular functions. Importantly, as demonstrated here for microglia, the antagonistic potential of Rs-LPS needs to be considered with caution, as reactions to Rs-LPS not only differ by cell type, but even by function within one cell type.

Thioredoxins and Methionine Sulfoxide Reductases in the Pathophysiology of Pneumococcal Meningitis.

BACKGROUND: Pneumococcal proteins involved in the resistance against oxidative stress are present in all strains and therefore are potential antigens that could be suitable for new therapies and/or vaccines. Their role in the pathogenesis of pneumococcal meningitis has not been addressed. METHODS: We investigated the individual contributions of extracellular thioredoxin lipoproteins (Etrx1 and Etrx2) and the intracellular and extracellular methionine sulfoxide reductases (SpMsrAB1 and SpMsrAB2, respectively) in the progression and outcome of pneumococcal meningitis, using Kaplan-Meier survival curves, bacteriological and histological studies, and measurements of proinflammatory mediators. RESULTS: The absence of Etrx1, Etrx2, or SpMsrAB1 moderately attenuated virulence as compared to the wild-type strain but did not significantly affect bacterial growth in the brain and bloodstream. Loss of function of SpMsrAB2 alone, both Etrx proteins, or both SpMsrAB proteins resulted in a less severe course of infection, with low numbers of animals dying of infection, a lower risk of associated meningeal inflammation, and reduced bacterial densities in the cerebellum, blood, and spleen. CONCLUSIONS: Our data support the importance of the extracellular redox repair system in virulence and its potential as a target for the design of new antimicrobials and vaccine formulations against Streptococcus pneumoniae.

Laquinimod prevents cuprizone-induced demyelination independent of Toll-like receptor signaling.

OBJECTIVE: To test whether Toll-like receptor (TLR) signaling plays a key role for reduced nuclear factor B (NF-κB) activation after laquinimod treatment in the model of cuprizone-induced demyelination, oligodendrocyte apoptosis, inflammation, and axonal damage. METHODS: Ten-week-old C57BL/6J, TLR4(-/-), and MyD88(-/-) mice received 0.25% cuprizone for 6 weeks and were treated daily with 25 mg/kg laquinimod or vehicle. After 6 weeks of demyelination, extent of demyelination, oligodendrocyte density, microglia infiltration, and axonal damage were analyzed in the corpus callosum. Additionally, we analyzed primary mouse astrocytes from C57BL/6J, TLR4(-/-), MyD88(-/-), and TRIF(-/-) mice for alteration in NF-κB signaling. RESULTS: Vehicle-treated controls from C57BL/6J, TLR4(-/-), and MyD88(-/-) mice displayed extensive callosal demyelination as well as microglial activation. In contrast, mice treated with 25 mg/kg laquinimod showed mainly intact callosal myelin. The demyelination score was significantly higher in all untreated mice compared to mice treated with laquinimod. There were significantly fewer APP-positive axonal spheroids, Mac3-positive macrophages/microglia, and less oligodendrocyte apoptosis in the corpus callosum of laquinimod-treated mice in comparison to untreated controls. Stimulated primary mouse astrocytes from laquinimod-treated groups show reduced NF-κB activation compared to vehicle-treated controls. CONCLUSIONS: Our results confirm that laquinimod prevents demyelination in the cuprizone mouse model for multiple sclerosis via downregulation of NF-κB activation. This laquinimod effect, however, does not involve upstream Toll-like receptor signaling.

CD14 is a key organizer of microglial responses to CNS infection and injury.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury-associated factors. We show that its co-receptor CD14 serves three non-redundant functions in microglia. First, it confers an up to 100-fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon ß-mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage-associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo- or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non-TLR systems to thereby fine-tune microglial damage-sensing capacity upon infectious and non-infectious CNS challenges.

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ.

Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia-neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K(+) concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1ß, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30-100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.

Cell type- and brain region-resolved mouse brain proteome.

Brain transcriptome and connectome maps are being generated, but an equivalent effort on the proteome is currently lacking. We performed high-resolution mass spectrometry-based proteomics for in-depth analysis of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type-specific proteins defined as tenfold more abundant than average expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.

Anti-CSF-1 treatment is effective to prevent carcinoma invasion induced by monocyte-derived cells but scarcely by microglia.

The mononuclear phagocytic system is categorized in three major groups: monocyte-derived cells (MCs), dendritic cells and resident macrophages. During breast cancer progression the colony stimulating factor 1 (CSF-1) can reprogram MCs into tumor-promoting macrophages in the primary tumor. However, the effect of CSF-1 during colonization of the brain parenchyma is largely unknown. Thus, we analyzed the outcome of anti-CSF-1 treatment on the resident macrophage population of the brain, the microglia, in comparison to MCs, alone and in different in vitro co-culture models. Our results underline the addiction of MCs to CSF-1 while surprisingly, microglia were not affected. Furthermore, in contrast to the brain, the bone marrow did not express the alternative ligand, IL-34. Yet treatment with IL-34 and co-culture with carcinoma cells partially rescued the anti-CSF-1 effects on MCs. Further, MC-induced invasion was significantly reduced by anti-CSF-1 treatment while microglia-induced invasion was reduced to a lower extend. Moreover, analysis of lung and breast cancer brain metastasis revealed significant differences of CSF-1 and CSF-1R expression. Taken together, our findings demonstrate not only differences of anti-CSF-1 treatment on MCs and microglia but also in the CSF-1 receptor and ligand expression in brain and bone marrow as well as in brain metastasis.

Erythropoietin dampens injury-induced microglial motility.

Traumatic brain injury causes progressive brain atrophy and cognitive decline. Surprisingly, an early treatment with erythropoietin (EPO) prevents these consequences of secondary neurodegeneration, but the mechanisms have remained obscure. Here we show by advanced imaging and innovative analytical tools that recombinant human EPO, a clinically established and neuroprotective growth factor, dampens microglial activity, as visualized also in vivo by a strongly attenuated injury-induced cellular motility.

Tyrphostin AG126 exerts neuroprotection in CNS inflammation by a dual mechanism.

The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.

Vitamin D deficiency decreases survival of bacterial meningoencephalitis in mice.

BACKGROUND: Meningoencephalitis caused by Escherichia coli is associated with high rates of mortality and risk of neurological sequelae in newborns and infants and in older or immunocompromised adults. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. METHODS: In vivo, we studied the effects of vitamin D3 on survival and the host's immune response in experimental bacterial meningoencephalitis in mice after intracerebral E. coli infection. To produce different systemic vitamin D3 concentrations, mice received a low, standard, or high dietary vitamin D3 supplementation. Bacterial titers in blood, spleen, and brain homogenates were determined. Leukocyte infiltration was assessed by histological scores, and tissue cytokine or chemokine concentrations were measured. RESULTS: Mice fed a diet with low vitamin D3 concentration died earlier than control animals after intracerebral infection. Vitamin D deficiency did not inhibit leukocyte recruitment into the subarachnoid space and did not lead to an increased density of bacteria in blood, spleen, or brain homogenates. The release of proinflammatory interleukin (IL)-6 was decreased and the release of anti-inflammatory IL-10 was increased in mice fed a diet with high vitamin D3 supplementation. CONCLUSION: Our observations suggest a detrimental role of vitamin D deficiency in bacterial central nervous system infections. Vitamin D may exert immune regulatory functions.

CD11c-positive cells from brain, spleen, lung, and liver exhibit site-specific immune phenotypes and plastically adapt to new environments.

The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double-positive cells from lung, liver, and spleen in healthy mice using seven-color flow cytometry. We identified unique, site-specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC-II. Furthermore, we observed the two known CD45-positive populations (CD45(high) and CD45(int) ) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45(high) population. CD11c-positive microglia lacked MHC-II expression and CD45(high) /CD11c-positive cells from the brain have a lower percentage of MHC-II-positive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC-II-positive splenocytes is paralleled by down-regulation of MHC-II, whereas brain-derived mononuclear cells neither ramified nor up-regulated MHC-II in OSSCs. Thus, brain-derived mononuclear cells maintain their MHC-II-negative phenotype within the environment of an immune organ. Intraparenchymal CD11c-positive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC-II expression.

Higher mortality and impaired elimination of bacteria in aged mice after intracerebral infection with E. coli are associated with an age-related decline of microglia and macrophage functions.

Incidence and mortality of bacterial meningitis are strongly increased in aged compared to younger adults demanding new strategies to improve prevention and therapy of bacterial central nervous system (CNS) infections the elderly. Here, we established a geriatric mouse model for an intracerebral E. coli infection which reflects the clinical situation in aged patients: After intracerebral challenge with E. coli K1, aged mice showed a higher mortality, a faster development of clinical symptoms, and a more pronounced weight loss. Elimination of bacteria and systemic inflammatory response were impaired in aged mice, however, the number of infiltrating leukocytes and microglial cells in the CNS of aged and young mice did not differ substantially. In vitro, primary microglial cells and peritoneal macrophages from aged mice phagocytosed less E. coli and released less NO and cyto-/chemokines compared to cells from young mice both without activation and after stimulation by agonists of TLR 2, 4, and 9. Our results suggest that the age-related decline of microglia and macrophage functions plays an essential role for the higher susceptibility of aged mice to intracerebral infections. Strategies to improve the phagocytic potential of aged microglial cells and macrophages appear promising for prevention and treatment of CNS infections in elderly patients.

Influence of intrathecal delivery of bone marrow-derived mesenchymal stem cells on spinal inflammation and pain hypersensitivity in a rat model of peripheral nerve injury.

BACKGROUND: Multipotent mesenchymal stem (stromal) cells (MSCs) have been credited with immunomodulative properties, supporting beneficial outcomes when transplanted into a variety of disease models involving inflammation. Potential mechanisms include the secretion of paracrine factors and the establishment of a neurotrophic microenvironment. To test the hypothesis that MSCs release soluble mediators that can attenuate local inflammation, we here analysed the influence of MSCs on the activation of microglia cells, as well as on inflammatory parameters and pain behaviour in a surgical rat model of neuropathic pain. METHODS: We focussed on an experimental model of partial sciatic nerve ligation (PSNL), characterised by a rapid and persistent inflammation in the dorsal lumbar spinal cord where sensory inputs from the sciatic nerve are processed. Via indwelling intrathecal catheters, MSCs were repetitively grafted into the intrathecal lumbar space. Animals were evaluated for mechanical and thermal hypersensitivity over a period of 21 days after PSNL. Afterwards, spinal cords were processed for immunohistochemical analysis of the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) and quantification of inflammatory markers in ipsilateral dorsal horns. We hypothesised that injections on postsurgical days 2 to 4 would interfere with microglial activation, leading to a reduced production of pro-inflammatory cytokines and amelioration of pain behaviour. RESULTS: PSNL-induced mechanical allodynia or heat hyperalgesia were not influenced by MSC transplantation, and spinal cord inflammatory processes remained largely unaffected. Indeed, the early microglial response to PSNL characterised by increased Iba1 expression in the lumbar dorsal horn was not significantly altered and cytokine levels in the spinal cord at 21 days after surgery were similar to those found in vehicle-injected animals. Grafted MSCs were detected close to the pia mater, but were absent within the spinal cord parenchyma. CONCLUSIONS: We conclude that intrathecal administration is not an appropriate route to deliver cells for treatment of acute spinal cord inflammation as it leads to entrapment of grafted cells within the pia mater. We propose that the early inflammatory response triggered by PSNL in the lumbar spinal cord failed to effectively recruit MSCs or was insufficient to disturb the tissue integrity so as to allow MSCs to penetrate the spinal cord parenchyma.

Microglial diversity by responses and responders.

Microglia are the principal resident innate immune cells of the CNS. Their contributions to the normal development of the CNS, the maintenance and plasticity of neuronal networks and the safeguarding of proper functionality are becoming more and more evident. Microglia also survey the tissue homeostasis to respond rapidly to exogenous and endogenous threats, primarily with a protective outcome. However, excessive acute activation, chronic activity or an improper adaptation of their functional performance can foster neuropathologies. A key to the versatile response behavior of these cells is their ability to commit to reactive phenotypes, which reveal enormous complexity. Yet the respective profiles of induced genes and installed functions may build up on heterogeneous contributions of cellular subsets. Here, we discuss findings and concepts that consider the variety of microglial activities and response options as being based-at least in part-on a diversity of the engaged cells. Whether it is the production of proinflammatory cytokines, clearance of tissue debris, antigen presentation or the ability to sense neurotransmitters, microglial cells present with an unanticipated heterogeneity of their constitutive and inducible features. While the organizational principles of this heterogeneity are still largely unknown, functional implications are already perceptible.

Vitamin d deficiency reduces the immune response, phagocytosis rate, and intracellular killing rate of microglial cells.

Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality and neurological sequelae. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides (AMPs) and suppression of T-cell proliferation, but its influence on microglial cells is unknown. The purpose of the present study was to determine the effects of vitamin D deficiency on the phagocytosis rate, intracellular killing, and immune response of murine microglial cultures after stimulation with the Toll-like receptor (TLR) agonists tripalmitoyl-S-glyceryl-cysteine (TLR1/2), poly(I·C) (TLR3), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9). Upon stimulation with high concentrations of TLR agonists, the release of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was decreased in vitamin D-deficient compared to that in vitamin D-sufficient microglial cultures. Phagocytosis of E. coli K1 after stimulation of microglial cells with high concentrations of TLR3, -4, and -9 agonists and intracellular killing of E. coli K1 after stimulation with high concentrations of all TLR agonists were lower in vitamin D-deficient microglial cells than in the respective control cells. Our observations suggest that vitamin D deficiency may impair the resistance of the brain against bacterial infections.

Overexpression of protease-activated receptor type 1 (PAR-1) in glioblastoma multiforme WHO IV cells and blood vessels revealed by NCAM-assisted glioblastoma border labeling.

Glioblastomas are neuroepithelial tumors with lost cellular differentiation and tenfold increased growth rates compared to low-grade gliomas. Despite of very aggressive treatment options based on surgery, irradiation, and chemotherapy, the prognosis of affected patients has remained poor and showed only slight improvements during the last 30 years. Research on glioblastoma border zone was hindered by the tumor's intense invasion into the brain parenchyma and the lack of suitable tumor cell markers. Nevertheless, the compact tumor mass and tumor invasion zone are composed of distinct cell types that need to be distinguished from each other to be addressed selectively. As the isoform 140 of the neural cell adhesion molecule (NCAM-140) was recently demonstrated to be lost in human gliomas with rising WHO grade, human multiform glioblastomas were characterized as a NCAM-140 negative entity displaying three main distinct invasion patterns. Evaluation of putative therapy targets within the tumor tissue and tumor invasion zone has been made possible through NCAM-140 negativity. In the present study, brain tissue controls and human glioblastoma samples with compact tumor mass and invasion areas were analyzed for their vascularization at the tumor border and the expression of thrombin receptor protease-activated receptor type 1 (PAR-1) within tumor tissue and vascular vessel walls. Use of NCAM-140 enabled the identification of the tumor invasion zone and its experimental investigation. Tissue vascularization was found to be significantly increased in the compact tumor mass of glioblastomas compared to their invasion zone and tumor-free controls with a significantly high and specific overexpression of PAR-1 within tumor cells and within tumor blood vessels depending upon the tumor area. This suggests thereby a functional role of the thrombin receptor PAR-1 in glioma cell malignancy and glioblastoma neoangiogenesis.