Regional difference in inflammatory response to LPS-injection in the brain: Role of microglia cell density (original) (raw)
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Journal of Neuroimmunology, 2006
Overall, the inflammatory potential of lipopolysaccharide (LPS) in vitro and in vivo was investigated using different omics technologies. We investigated the hippocampal response to intracerebroventricular (i.c.v) LPS in vivo, at both the transcriptional and protein level. Here, a time course analysis of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) showed a sharp peak at 4 h and a return to baseline at 16 h. The expression of inflammatory mediators was not temporally correlated with expression of the microglia marker F4/80, which did not peak until 2 days after LPS injection. Of 480 inflammation-related genes present on a microarray, 29 transcripts were robustly up-regulated and 90% of them were also detected in LPS stimulated primary microglia (PM) cultures. Further in vitro to in vivo comparison showed that the counter regulation response observed in vivo was less evident in vitro, as transcript levels in PM decreased relatively little over 16 h. This apparent deficiency of homeostatic control of the innate immune response in cultures may also explain why a group of genes comprising tnf receptor associated factor-1, endothelin-1 and schlafen-1 were regulated strongly in vitro, but not in vivo. When the overall LPS-induced transcriptional response of PM was examined on a large Affymetrix chip, chemokines and cytokines constituted the most strongly regulated and largest groups. Interesting new microglia markers included interferon-induced protein with tetratricopeptide repeat (ifit), immune responsive gene-1 (irg-1) and thymidylate kinase family LPSinducible member (tyki). The regulation of the former two was confirmed on the protein level in a proteomics study. Furthermore, conspicuous regulation of several gene clusters was identified, for instance that of genes pertaining to the extra-cellular matrix and enzymatic regulation thereof. Although most inflammatory genes induced in vitro were transferable to our in vivo model, the observed discrepancy for some genes potentially represents regulatory factors present in the central nervous system (CNS) but not in vitro.
2018
Microglia respond to CNS injuries and diseases with complex reactions, often called "activation." A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo.
Phenotyping primary human microglia: Tight regulation of LPS responsiveness
Glia, 2012
Much is still unknown about mechanisms underlying the phenotypical and functional versatility of human microglia. Therefore, we developed a rapid procedure to isolate pure microglia from postmortem human brain tissue and studied their immediate ex vivo phenotype and responses to key inflammatory mediators. Microglia were isolated, along with macrophages from the choroid plexus by tissue dissociation, density gradient separation, and selection with magnetic microbeads. By flow cytometry, microglia were identified by a CD11b 1 CD45 dim phenotype and a smaller size compared with CD11b 1 CD45 high macrophages. Interestingly, white matter microglia from donors with peripheral inflammation displayed elevated CD45 levels and increased size and granularity, but were still distinct from macrophages. The phenotype of isolated microglia was further specified by absent surface expression of CD14, CD200 receptor, and mannose receptor (MR, CD206), all of which were markedly expressed by macrophages. Microglia stimulated immediately after isolation with LPS and IFNg failed to upregulate TNFa or CCR7. Notably, responsiveness to LPS and IFNg was clearly instigated in microglia after overnight preculture, which coincided with a strong upregulation of CD14. Culture of microglia with IL-4 resulted in the induction of HLA-DR and CCL18 but not MR, whereas culture with dexamethasone did induce MR, in addition to CD163 and CCL18. In conclusion, this study demonstrates phenotypic changes of microglia associated with peripheral inflammation, and reveals tight regulation of responses to LPS and IFNg as well as distinct microglial responses to IL-4 and glucocorticoids. These findings are of high relevance to studies on human microglia functioning in health and disease. V
Scientific Reports, 2018
Numerous studies have reported the importance of microglial activation in various pathological conditions, whereas little attention has been given to the point for dynamics of microglial population under infection-induced inflammation. In the present study, the single systemic stimulation of 100 μg/kg lipopolysaccharide (LPS) induced robust microglial proliferation only in the circumventricular organs (CVOs) and their neighboring brain regions. More than half of microglia similarly showed proliferative activity in the CVOs and their neighboring brain regions after 1 mg/kg LPS stimulation, while this stimulation expanded microglia-proliferating brain regions including the hypothalamus, medulla oblongata, and limbic system. Microglia proliferation resulted in a transient increase of microglial density, since their density almost returned to basal levels within 3 weeks. Divided microglia survived at the same rate as non-divided ones. Proliferating microglia frequently expressed a resident microglia marker Tmem119, indicating that increase of microglia density is due to the proliferation of resident microglia. Thus, the present study demonstrates that transient increase in microglia density depends on the brain region and dose of LPS during infection-induced inflammation and could provide a new insight on microglia functions in inflammation and pathogenesis of brain diseases. Microglia are innate immune cells in the brain that are diffusely distributed throughout the parenchyma and function in brain immune defenses. The microglial population in the adult rodent brain accounts for 5 to 12% of the total number of cells 1. In the human brain, microglia account for 0.5 to 16.6% of the total population of brain cells and show similar regional variability to that reported in rodents 2. Microglia have the ability to respond to many types of brain homeostatic disturbances under pathological brain conditions and are rapidly transformed from a ramified to amoeboid morphology, namely "activated microglia" 3-5. Ramified microglia, composed of long branching processes and a small cellular body, function as surveying cells by actively sensing the surrounding microenvironment via dynamic fine cellular processes 4. Activated amoeboid microglia are hypertrophic, typically have a less dendritic shape, and participate in many functions including phagocytosis and cytokine release 6. Microglial proliferation in the adult rodent brains is slow with increases at a rate of only a few percent per week under physiologically healthy conditions 1,7,8. In the mouse and human brain, the microglial density remains remarkably stable, but microglia turnover several times during a lifetime 9. They further have shown that microglia turnover is maintained by coupled proliferation and apoptosis of resident microglia rather than the infiltration of bone marrow-derived immune cells 9. However, microglia increase their population by both proliferation of the resident microglia and recruitment of bone marrow-derived immune cells under pathological brain conditions: traumatic and ischemic brain injuries, Alzheimer's disease, prion diseases, and multiple sclerosis 3-5,10. The mice
Journal of Neuroinflammation, 2011
Background Chronic neurodegeneration comprises an inflammatory response but its contribution to the progression of disease remains unclear. We have previously shown that microglial cells are primed by chronic neurodegeneration, induced by the ME7 strain of prion disease, to synthesize limited pro-inflammatory cytokines but to produce exaggerated responses to subsequent systemic inflammatory insults. The consequences of this primed response include exaggerated hypothermic and sickness behavioural responses, acute neuronal death and accelerated progression of disease. Here we investigated whether inhibition of systemic cytokine synthesis using the anti-inflammatory steroid dexamethasone-21-phosphate was sufficient to block any or all of these responses. Methods ME7 animals, at 18-19 weeks post-inoculation, were challenged with LPS (500 μg/kg) in the presence or absence of dexamethasone-21-phosphate (2 mg/kg) and effects on core-body temperature and systemic and CNS cytokine production...
Journal of Neuroimmunology, 2015
Keywords: Microglia Hippocampus Inflammation Brain Fimbria LPS Microglia are resident immunocompetent cells having important roles in innate immunity in the brains. In the present study, we found that a single lipopolysaccharide (LPS) administration significantly increased microglial proliferation in the fornix and dentate gyrus (DG) but not the cerebral cortex and corpus callosum of adult mice. LPS-induced microglial proliferation was especially robust at the white matter of the fornix. The density of microglia increased in the fornix and DG for roughly one week and returned to basal levels at least 20 days after a single LPS administration. Consecutive LPS administration did not induce such dramatic increase of microglial proliferation in the fornix. The inhibition of vascular endothelial growth factor signaling by AZD2171 largely suppressed LPS-induced increase of microglial proliferation in the fornix. In conclusion, the present study indicates that the hippocampal neuronal system has a higher proliferative microglial capability against LPS-induced inflammatory administration compared with other brain regions.
Autotaxin Downregulates LPS-Induced Microglia Activation and Pro-Inflammatory Cytokines Production
Journal of Cellular Biochemistry, 2014
Inflammation is essential in defense against infection or injury. It is tightly regulated, as over-response can be detrimental, especially in immuneprivileged organs such as the central nervous system (CNS). Microglia constitutes the major source of inflammatory factors, but are also involved in the regulation of the inflammation and in the reparation. Autotaxin (ATX), a phospholipase D, converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and is upregulated in several CNS injuries. LPA, a pleiotropic immunomodulatory factor, can induce multiple cellular processes including morphological changes, proliferation, death, and survival. We investigated ATX effects on microglia inflammatory response to lipopolysaccharide (LPS), mimicking gram-negative infection. Murine BV-2 microglia and stable transfected, overexpressing ATX-BV-2 (A þ) microglia were treated with LPS. Tumor necrosis factor a (TNFa), interleukin (IL)-6, and IL-10 mRNA and proteins levels were examined by qRT-PCR and ELISA, respectively. Secreted LPA was quantified by a radioenzymatic assay and microglial activation markers (CD11b, CD14, B7.1, and B7.2) were determined by flow cytometry. ATX expression and LPA production were significantly enhanced in LPS treated BV-2 cells. LPS induction of mRNA and protein level for TNFa and IL-6 were inhibited in Aþ cells, while IL-10 was increased. CD11b, CD14, and B7.1, and B7.2 expressions were reduced in Aþ cells. Our results strongly suggest deactivation of microglia and an IL-10 inhibitory of ATX with LPS induced microglia activation.
Microglial activation has been considered as a result of neuronal damage, however, recently it becomes to recognize as a possible cause of the damage in various neurodegenerative diseases. To elucidate the mechanism of the microglial activation, we examined the time course of lipopolysaccharide (LPS)-induced change in morphology and the release of cytokines and nitric oxide (NO) in cultured microglia from neonatal rat brains. With addition of 1 mg/ml LPS, the cell morphology was drastically changed within 3 h from amoeboid shape to bipolar rod shape. The peak time of such morphological change was at 6 h and then returned to small round shape gradually. This transient change in morphology was completely inhibited by 0.1 mM dibutyryl-cAMP. On the other hand, the release of cytokines and NO showed different time courses after stimulation by LPS; at first tumor necrosis factor (TNF)-a was released within 1 h lag time, secondly interleukin (IL)-1b within 3 h, thirdly IL-6, and at last NO was released with about 6 h lag time. The addition of dibutyryl cAMP markedly inhibited the release of TNF-a and IL-1b, but not IL-6 and NO at all. These results suggest that there are at least two different intracellular signaling pathways of LPS-induced microglial activation; one for early release of TNF-a and IL-1b sensitive to dibutyryl-cAMP and the other for late release of IL-6 and NO insensitive to dibutyryl-cAMP. The transient morphological change seems to be associated with the early release based on the sensitivity to dibutyryl-cAMP.
Regulation of LPS-Induced Neuroinflammation by Targeting Microglia in vivo Using Chemogenetics
2018
Peripheral inflammation can profoundly alter motivational processes and generate behavioral symptoms of sickness. Microglia, the innate immune cells of the brain, orchestrate neuroinflammation and have long been thought to be important for sickness behavior. However, their precise roles remain obscure due to their complex bidirectional interactions with neurons in vivo, which regulate how microglia respond to inflammatory signals and interact with neurons. Neuron-microglia interactions can be at least partially mediated by various types of G-protein coupled receptors (GPCRs) expressed by microglia. Here, we generated a microglia specific hM3Gq-DREADD (designer receptors exclusively activated by designer drugs) mouse line in which hM3Gq is selectively expressed in microglia, allowing for selective stimulation of calcium signalling in vivo. In primary cultures, clozapine-N-oxide (CNO) application induced the predicted rise in intracellular Ca 2+ concentration only in hM3Gq-expressing microglia. In vivo, we validated that tamoxifen-induced hM3Gq expression was highly efficient and specific to microglia. Systemic administration of CNO (1 mg/kg, i.p.) for 3 consecutive days did not cause any changes in behavior indicative of sickness behavior, demonstrating that microglial hM3Gq signaling alone did not cause overt inflammatory response in vivo. We then challenged these mice with a low-dose lipopolysaccharide (LPS) injection (0.1 mg/kg i.p.) to trigger bona fide sickness behavior. We found that pre-treatment with CNO for 3 days alleviated classic LPS-induced sickness behavior including, depression of social interaction and locomotor activity. This treatment also suppressed LPS-induced upregulation of mRNA of proinflammatory cytokines in the hippocampus. Our work demonstrates that manipulation of GPCR signalling using this new 'microglia-DREADD' mouse line can help to reveal how microglia modulates inflammatory responses and abnormal behavior in vivo.