ABSTRACT
Brain metastasis in breast cancer remains difficult to treat and its incidence is increasing. Therefore, the development of new therapies is of utmost clinical relevance. Recently, toll-like receptor (TLR) 4 was correlated with IL6 expression and poor prognosis in 1 215 breast cancer primaries. In contrast, we demonstrated that TLR4 stimulation reduces microglia-assisted breast cancer cell invasion. However, the expression, prognostic value, or therapeutic potential of TLR signaling in breast cancer brain metastasis have not been investigated. We thus tested the prognostic value of various TLRs in two brain-metastasis gene sets. Furthermore, we investigated different TLR agonists, as well as MyD88 and TRIF-deficient microenvironments in organotypic brain-slice ex vivo co-cultures and in vivo colonization experiments. These experiments underline the ambiguous roles of TLR4, its adapter MyD88, and the target nitric oxide (NO) during brain colonization. Moreover, analysis of the gene expression datasets of breast cancer brain metastasis patients revealed associations of TLR1 and IL6 with poor overall survival. Finally, our finding that a single LPS application at the onset of colonization shapes the later microglia/macrophage reaction at the macro-metastasis brain-parenchyma interface (MMPI) and reduces metastatic infiltration into the brain parenchyma may prove useful in immunotherapeutic considerations.
Subject(s)
Brain Neoplasms , Breast Neoplasms , Humans , Female , Toll-Like Receptor 4/metabolism , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Interleukin-6/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/genetics , Brain/pathology , Brain Neoplasms/drug therapy , Adaptor Proteins, Vesicular Transport/metabolism , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Immunity, Innate/drug effects , Immunocompromised Host/immunology , Meningitis, Escherichia coli/immunology , Poly I-C/pharmacology , Animals , Immunity, Innate/immunology , Male , Mice , Mice, Inbred C57BL , Neutropenia/immunology , Poly I-C/immunology , Toll-Like Receptor 3/agonists , Toll-Like Receptor 3/drug effectsABSTRACT
More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.
Subject(s)
Brain Neoplasms/pathology , Brain Neoplasms/secondary , Breast Neoplasms/pathology , Glutathione/metabolism , Lymphoid Enhancer-Binding Factor 1/metabolism , Reactive Oxygen Species/metabolism , Brain/pathology , Cell Line, Tumor , Cell Movement/physiology , Epithelial-Mesenchymal Transition/physiology , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Parenchymal Tissue/pathologyABSTRACT
Mutations and activation of the PI3K signaling pathway in breast cancer cells have been linked to brain metastases. However, here we describe that in some breast cancer brain metastases samples the protein expression of PI3K signaling components is restricted to the metastatic microenvironment. In contrast to the therapeutic effects of PI3K inhibition on the breast cancer cells, the reaction of the brain microenvironment is less understood. Therefore we aimed to quantify the PI3K pathway activity in breast cancer brain metastasis and investigate the effects of PI3K inhibition on the central nervous system (CNS) microenvironment. First, to systematically quantify the PI3K pathway activity in breast cancer brain metastases, we performed a prospective biomarker study using a reverse phase protein array (RPPA). The majority, namely 30 out of 48 (62.5%) brain metastatic tissues examined, revealed high PI3K signaling activity that was associated with a median overall survival (OS) of 9.41 months, while that of patients, whose brain metastases showed only moderate or low PI3K activity, amounted to only 1.93 and 6.71 months, respectively. Second, we identified PI3K as a master regulator of metastasis-promoting macrophages/microglia during CNS colonization; and treatment with buparlisib (BKM120), a pan-PI3K Class I inhibitor with a good blood-brain-barrier penetrance, reduced their metastasis-promoting features. In conclusion, PI3K signaling is active in the majority of breast cancer brain metastases. Since PI3K inhibition does not only affect the metastatic cells but also re-educates the metastasis-promoting macrophages/microglia, PI3K inhibition may hold considerable promise in the treatment of brain metastasis and the respective microenvironment.
Subject(s)
Brain Neoplasms/pathology , Brain Neoplasms/secondary , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic/physiology , Macrophages/enzymology , Microglia/enzymology , Adult , Aged , Aminopyridines/therapeutic use , Animals , Calcium-Binding Proteins/metabolism , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Humans , Macrophages/drug effects , Mice , Mice, Inbred BALB C , Microfilament Proteins/metabolism , Microglia/drug effects , Middle Aged , Morpholines/therapeutic use , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
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.
Subject(s)
Activins/pharmacology , Cytokines/metabolism , Lipopolysaccharides/pharmacology , Microglia/drug effects , Phagocytosis/drug effects , Toll-Like Receptors/agonists , Animals , Animals, Newborn , Brain/cytology , Cells, Cultured , Dose-Response Relationship, Drug , Escherichia coli/physiology , Humans , Infant, Newborn , Mice , Mice, Inbred C57BL , Nitric Oxide/metabolism , Plant Lectins/metabolism , Toll-Like Receptors/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , Xenograft Model Antitumor Assays , Animals , Apoptosis/drug effects , Azacitidine/administration & dosage , Azacitidine/pharmacology , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Female , Glioblastoma/pathology , Humans , Mice, Inbred NOD , Mice, Knockout , Mice, Nude , Mice, SCID , Tumor Burden/drug effectsABSTRACT
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.
Subject(s)
Hemorrhagic Fevers, Viral/immunology , Interferon-gamma/immunology , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/physiology , Nitric Oxide Synthase Type II/immunology , Animals , Disease Models, Animal , Female , Hemorrhagic Fevers, Viral/genetics , Hemorrhagic Fevers, Viral/virology , Humans , Interferon-gamma/genetics , Lymphocytic Choriomeningitis/genetics , Lymphocytic Choriomeningitis/virology , Lymphocytic choriomeningitis virus/genetics , Male , Mice , Mice, Inbred C57BL , Nitric Oxide/immunology , Nitric Oxide Synthase Type II/geneticsABSTRACT
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.
Subject(s)
Lipopolysaccharides/pharmacology , Microglia/drug effects , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Animals, Newborn , Brain/cytology , Cells, Cultured , Corpus Striatum/drug effects , Cytokines/metabolism , Dose-Response Relationship, Drug , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Macrophages/drug effects , Macrophages/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myelin Sheath/drug effects , Myelin Sheath/pathology , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Phagocytosis/drug effects , Phagocytosis/physiology , Toll-Like Receptor 4/genetics , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
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.
Subject(s)
Meningitis, Pneumococcal/pathology , Methionine Sulfoxide Reductases/metabolism , Streptococcus pneumoniae/pathogenicity , Thioredoxins/metabolism , Virulence Factors/metabolism , Animals , Blood/microbiology , Brain/microbiology , Disease Models, Animal , Female , Gene Deletion , Meningitis, Pneumococcal/immunology , Methionine Sulfoxide Reductases/genetics , Mice, Inbred C57BL , Oxidation-Reduction , Oxidative Stress , Spleen , Streptococcus pneumoniae/genetics , Survival Analysis , Thioredoxins/genetics , Virulence , Virulence Factors/geneticsABSTRACT
AIM: To analyse bone remodeling in regard to the age of scaphoid non-unions (SNU) with immunohistochemistry. METHODS: Thirty-six patients with symptomatic SNU underwent surgery with resection of the pseudarthrosis. The resected material was evaluated histologically after staining with hematoxylin-eosin (HE), tartrate resistant acid phosphatase (TRAP), CD 68, osteocalcin (OC) and osteopontin (OP). Histological examination was performed in a blinded fashion. RESULTS: The number of multinuclear osteoclasts in the TRAP-staining correlated with the age of the SNU and was significantly higher in younger SNU (P = 0.034; r = 0.75). A higher number of OP-immunoreactive osteoblasts significantly correlated with a higher number of OC-immunoreactive osteoblasts (P = 0.001; r = 0.55). Furthermore, a greater number of OP-immunoreactive osteoblasts correlated significantly with a higher number of OP-immunoreactive multinuclear osteoclasts (P = 0.008; r = 0.43). SNU older than 6 mo showed a significant decrease of the number of fibroblasts (P = 0.04). Smoking and the age of the patients had no influence on bone remodeling in SNU. CONCLUSION: Multinuclear osteoclasts showed a significant decrease in relation to the age of SNU. However, most of the immunhistochemical findings of bone remodeling do not correlate with the age of the SNU. This indicates a permanent imbalance of bone formation and resorption as indicated by a concurrent increase in both osteoblast and osteoclast numbers. A clear histological differentiation into phases of bone remodeling in SNU is not possible.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Brain Injuries/immunology , Brain Ischemia/immunology , Escherichia coli Infections/metabolism , Lipopolysaccharide Receptors/metabolism , Microglia/immunology , Stroke/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Brain/immunology , Brain/pathology , Brain Injuries/complications , Brain Injuries/pathology , Brain Ischemia/pathology , Cells, Cultured , Disease Models, Animal , Escherichia coli , Escherichia coli Infections/complications , Escherichia coli Infections/pathology , Feedback, Physiological/physiology , Infarction, Middle Cerebral Artery , Interferon-beta/metabolism , Lipopolysaccharide Receptors/genetics , Lipopolysaccharides/toxicity , Macrophages/immunology , Male , Mice, Inbred C57BL , Mice, Knockout , Neuroimmunomodulation , Stroke/pathology , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
BACKGROUND: The examination of as large a number of lymph nodes as possible in rectal carcinoma resectates is important for exact staging. However, after neoadjuvant radiochemotherapy (RCT), it can be difficult to obtain a sufficient number of lymph nodes. We therefore investigated whether staining with methylene blue via the inferior mesenteric artery can lead to an increase in the yield of lymph nodes in rectal carcinoma tissue after neoadjuvant RCT. METHODS: In a prospective, unicentric study rectal carcinoma resectates from three consecutive groups of patients were examined (Group I, no staining; Group II, staining with methylene blue; Group III, again no staining). The numbers of lymph nodes examined were compared (a) between the groups and (b) between patients who had not, or who had, received neoadjuvant RCT. RESULTS: In all, 75 rectal carcinoma preparations were assessed. The yield of lymph nodes investigated before the use of staining (Group I) increased when staining was introduced (Group II), both for the patients without neoadjuvant RCT (20.9 vs. 31.3, p = 0.018) and for those who did receive this (15.0 vs. 35.1; p = 0.003). After withdrawal of the staining procedure (Group III), the lymph-node yield remained high for the patients without neoadjuvant RCT (31.3 vs. 30.4; p = 0.882), but it reverted to a lower value for those who did receive neoadjuvant RCT (35.1 vs. 24.2; p = 0.029). Before the introduction of staining (Group I), significantly fewer lymph nodes were examined for patients who received neoadjuvant RCT (15.0 vs. 20.9; p = 0.039). However, with staining (Group II), no difference was found associated with the use or non-use of neoadjuvant RCT (31.3 vs. 35.1; p = 0.520). CONCLUSION: The use of methylene blue staining of rectal carcinoma preparations leads to a significant increase in the number of lymph nodes examined after neoadjuvant RCT. This can be expected to improve the accuracy of lymph-node staging of neoadjuvant-treated rectal carcinoma.
Subject(s)
Coloring Agents/administration & dosage , Lymph Node Excision , Lymph Nodes/pathology , Methylene Blue/administration & dosage , Rectal Neoplasms/pathology , Rectal Neoplasms/therapy , Adult , Aged , Chemoradiotherapy, Adjuvant , Female , Humans , Injections, Intra-Arterial , Male , Mesenteric Artery, Inferior , Middle Aged , Neoadjuvant Therapy , Prospective Studies , Staining and LabelingABSTRACT
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.
Subject(s)
GABAergic Neurons/immunology , GABAergic Neurons/pathology , Interferon-gamma/immunology , Microglia/immunology , Neurodegenerative Diseases/immunology , Toll-Like Receptor 4/immunology , Animals , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Astrocytes/immunology , CD11b Antigen/metabolism , Cell Death/immunology , Cells, Cultured , Hippocampus/immunology , Hippocampus/pathology , Inflammation/immunology , Inflammation/pathology , Interferon-gamma/agonists , Interleukin-1beta/immunology , Interleukin-6/immunology , Interneurons/immunology , Interneurons/pathology , Lipopolysaccharides/immunology , Neuronal Plasticity/immunology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Rats , Rats, Wistar , Receptors, Interferon/immunology , Toll-Like Receptor 4/agonists , Tumor Necrosis Factor-alpha/immunologyABSTRACT
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.
Subject(s)
Brain/cytology , Brain/physiology , Neurons/physiology , Proteome/genetics , Animals , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/pharmacology , Brain Neoplasms/pathology , Breast Neoplasms/pathology , Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptor, Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Animals , Antibodies, Monoclonal/immunology , Brain/cytology , Brain/immunology , Brain/pathology , Brain Neoplasms/secondary , Breast Neoplasms/immunology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Humans , Interleukin-1/pharmacology , MCF-7 Cells , Macrophage Colony-Stimulating Factor/biosynthesis , Macrophage Colony-Stimulating Factor/immunology , Macrophages/immunology , Mice , Mice, Inbred BALB C , Microglia/cytology , Microglia/immunology , Microglia/pathology , Monocytes/immunology , Neoplasm Invasiveness/immunology , Neoplasm Invasiveness/pathology , Receptor, Macrophage Colony-Stimulating Factor/biosynthesisABSTRACT
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.
Subject(s)
Cell Movement/drug effects , Erythropoietin/pharmacology , Microglia/metabolism , Neuroprotective Agents/pharmacology , Brain Injuries/drug therapy , Brain Injuries/metabolism , Brain Injuries/pathology , Cells, Cultured , Humans , Recombinant Proteins/pharmacologyABSTRACT
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.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Neuroprotective Agents/pharmacology , Tyrphostins/pharmacology , Agammaglobulinaemia Tyrosine Kinase , Animals , Cells, Cultured , Cytokines/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Female , Hydrolysis , Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/drug effects , Microglia/physiology , Myeloid Differentiation Factor 88/metabolism , Neuroprotective Agents/chemistry , Nitriles/chemistry , Nitriles/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Spleen/cytology , Spleen/drug effects , Spleen/physiopathology , Th17 Cells/drug effects , Th17 Cells/pathology , Th17 Cells/physiology , Tyrphostins/chemistryABSTRACT
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.
Subject(s)
Cholecalciferol/deficiency , Escherichia coli Infections/complications , Escherichia coli/pathogenicity , Meningoencephalitis/etiology , Meningoencephalitis/mortality , Vitamin D Deficiency , Analysis of Variance , Animals , Bacterial Load/methods , Body Weight , Central Nervous System/metabolism , Central Nervous System/microbiology , Central Nervous System/pathology , Cholecalciferol/administration & dosage , Cholecalciferol/blood , Cytokines/metabolism , Dietary Supplements , Disease Models, Animal , Gene Expression Regulation/drug effects , Meningoencephalitis/pathology , Mice , Mice, Inbred C57BL , Spleen/metabolism , Spleen/microbiology , Spleen/pathology , Time FactorsABSTRACT
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.