Intrinsic DNA damage repair deficiency results in progressive microglia loss and replacement

The DNA excision repair protein Ercc1 is important for nucleotide excision, double strand DNA break, and interstrand DNA crosslink repair. In constitutiveErcc1-knockout mice, microglia display increased phagocytosis, proliferation and an enhanced responsiveness to lipopolysaccharide (LPS)-induced peripheral inflammation. However, the intrinsic effects ofErcc1-deficiency on microglia are unclear. In this study,Ercc1was specifically deleted from Cx3cr1-expressing cells and changes in microglia morphology and immune responses at different times after deletion were determined. Microglia numbers were reduced with approximately 50% at 2-12 months afterErcc1deletion. Larger and more ramified microglia were observed followingErcc1deletion both in vivo and in organotypic hippocampal slice cultures.Ercc1-deficient microglia were progressively lost, and during this period, microglia proliferation was transiently increased.Ercc1-deficient microglia were gradually replaced by nondeficient microglia carrying a functionalErcc1allele. In contrast to constitutiveErcc1-deficient mice, microglia-specific deletion ofErcc1did not induce microglia activation or increase their responsiveness to a systemic LPS challenge. Gene expression analysis suggested thatErcc1deletion in microglia induced a transient aging signature, which was different from a priming or disease-associated microglia gene expression profile.</p

Characterizing microglial gene expression in a model of secondary progressive multiple sclerosis

Multiple sclerosis (MS) is the most common inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults. Chronic-relapsing experimental autoimmune encephalomyelitis (crEAE) in Biozzi ABH mice is an experimental model of MS. This crEAE model is characterized by an acute phase with severe neurological disability, followed by remission of disease, relapse of neurological disease and remission that eventually results in a chronic progressive phase that mimics the secondary progressive phase (SPEAE) of MS. In both MS and SPEAE, the role of microglia is poorly defined. We used a crEAE model to characterize microglia in the different phases of crEAE phases using morphometric and RNA sequencing analyses. At the initial, acute inflammation phase, microglia acquired a pro-inflammatory phenotype. At the remission phase, expression of standard immune activation genes was decreased while expression of genes associated with lipid metabolism and tissue remodeling were increased. Chronic phase microglia partially regain inflammatory gene sets and increase expression of genes associated with proliferation. Together, the data presented here indicate that microglia obtain different features at different stages of crEAE and a particularly mixed phenotype in the chronic stage. Understanding the properties of microglia that are present at the chronic phase of EAE will help to understand the role of microglia in secondary progressive MS, to better aid the development of therapies for this phase of the disease

CCL21-induced calcium transients and proliferation in primary mouse astrocytes:CXCR3-dependent and independent responses

CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in damaged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10(-9) M and 10(-8) M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high concentrations of CCL21 (10(-7) M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 promoted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (chemokine-) receptor, which is activated at high CCL21 concentrations. (C) 2009 Elsevier Inc. All rights reserved

Proton therapy induces a local microglial neuroimmune response

BACKGROUND AND PURPOSE: Although proton therapy is increasingly being used in the treatment of paediatric and adult brain tumours, there are still uncertainties surrounding the biological effect of protons on the normal brain. Microglia, the brain-resident macrophages, have been shown to play a role in the development of radiation-induced neurotoxicity. However, their molecular and hence functional response to proton irradiation remains unknown. This study investigates the effect of protons on microglia by comparing the effect of photons and protons as well as the influence of age and different irradiated volumes.MATERIALS AND METHODS: Rats were irradiated with 14 Gy to the whole brain with photons (X-rays), plateau protons, spread-out Bragg peak (SOBP) protons or to 50 % anterior, or 50 % posterior brain sub-volumes with plateau protons. RNA sequencing, validation of microglial priming gene expression using qPCR and high-content imaging analysis of microglial morphology were performed in the cortex at 12 weeks post irradiation.RESULTS: Photons and plateau protons induced a shared transcriptomic response associated with neuroinflammation. This response was associated with a similar microglial priming gene expression signature and distribution of microglial morphologies. Expression of the priming gene signature was less pronounced in juvenile rats compared to adults and slightly increased in rats irradiated with SOBP protons. High-precision partial brain irradiation with protons induced a local microglial priming response and morphological changes.CONCLUSION: Overall, our data indicate that the brain responds in a similar manner to photons and plateau protons with a shared local upregulation of microglial priming-associated genes, potentially enhancing the immune response to subsequent inflammatory challenges.</p

Systemic administration of beta-glucan induces immune training in microglia

BACKGROUND: An innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal β-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo. METHODS: Two experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array. RESULTS: Microglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1β, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia. CONCLUSIONS: Our results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia

Additional file 4 of Systemic administration of β-glucan induces immune training in microglia

Additional file 4. Raw morphometric information for all cells analyzed in acute design, cortex and hippocampus regions. Statistical comparisons between experimental groups are also provided

Additional file 7 of Systemic administration of β-glucan induces immune training in microglia

Additional file 7. Raw morphometric information for all cells analyzed in preconditioning design, cortex and hippocampus regions. Statistical comparisons between experimental groups are also provided