Impact of X-irradiation on microglia.

Irradiation is widely used to treat brain tumors, and also to create bone marrow (BM) chimeras. BM chimeras are widely used to dissect functions and origin of microglia and blood-derived mononuclear cells under homeostatic or pathological conditions. This is facilitated by the fact that microglia survive irradiation and are thus regarded radio-resistant. In this study, we tested whether microglia are indeed radio-resistant and looked for potential mechanisms that might explain this phenomenon. We analyzed the radio-resistance of microglia independently of their physiological brain environment compared to other mononuclear cells from spleen and brain after X-irradiation with 7 Gy or 30 Gy. Furthermore, we investigated long-term effects of X-irradiation on microglia using organotypic hippocampal slice cultures (OHSCs). We found a significant higher survival rate of isolated microglia 4 hr after X-irradiation with 30 Gy accompanied by a decreased proliferation rate. Investigations of apoptosis-related genes revealed no regulation of a specific antiapoptotic pathway but ataxia telangiectasia mutated (ATM), a DNA-repair-related gene, was significantly upregulated in isolated microglia 4 hr after 30 Gy. Irradiation of OHSCs with 7 and 30 Gy revealed a highly and significantly decreased cell number, morphological changes and an increase in migration velocity of microglia. Furthermore, cell loss, increased soma size and process length of microglia was also found in BM chimeras irradiated with 9.5 Gy 5 weeks after irradiation. Here, we present new evidence implying that microglia are not a homogeneous population of radio-resistant cells and report on long-term alterations of microglia that survived irradiation.

Indications for cellular migration from the central nervous system to its draining lymph nodes in CD11c-GFP+ bone-marrow chimeras following EAE.

The concept as to how the brain maintains its immune privilege has initially been based on observations that it is lacking classical lymph vessels and later, the absence of dendritic cells (DC). This view has been challenged by several groups demonstrating drainage/migration of injected tracers and cells into cervical lymph nodes (CLNs) and the presence of brain antigens in CLNs in the course of various brain pathologies. Using CD11c-diphtheria toxin receptor (DTR)-green fluorescent protein (GFP) transgenic (tg) mice, we have shown the existence of CD11c+ cells, a main DC marker, within the brain parenchyma. Since injecting tracers or cells may cause barrier artefacts, we have now transplanted wild type (wt)-bone marrow (BM) to lethally irradiated CD11c-DTR-GFP tg mice to restrict the CD11c-DTR-GFP+ population to the brain and induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We observed ramified GFP+ cells in the olfactory bulb, the cribriform plate, the nasal mucosa and superficial CLNs. We measured a significant increase of host gfp genomic DNA (gDNA) levels in lymph nodes (LNs) previously described as draining stations for the central nervous system (CNS). Using flow cytometry analysis, we observed an increase of the percentage of CD11c-GFP+ cells in brain parenchyma in the course of EAE which is most likely due to an up-regulation of CD11c of resident microglial cells since levels of gfp gDNA did not increase. Our data supports the hypothesis that brain-resident antigen presenting cells (APC) are capable of migrating to CNS-draining LNs to present myelin-associated epitopes.

Neurons exhibit Lyz2 promoter activity in vivo: Implications for using LysM-Cre mice in myeloid cell research.

To characterize LysM-Cre mediated gene targeting in mice, we crossed LysM-Cre mice to two independent reporter-mouse lines (tdTomato or YFP). Surprisingly, we found that more than 90% of cells with LysM-Cre mediated recombination in the brain were neurons, rather than myeloid cells, such as microglia. Hence, by using the LysM-Cre mouse line for conditional knockout approaches, a significant neuronal recombination needs to be considered.

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.

Blood-brain barrier breakdown involves four distinct stages of vascular damage in various models of experimental focal cerebral ischemia.

Ischemic stroke not only impairs neuronal function but also affects the cerebral vasculature as indicated by loss of blood-brain barrier (BBB) integrity. Therefore, therapeutical recanalization includes an enhanced risk for hemorrhagic transformation and bleeding, traditionally attributed to a 'reperfusion injury'. To investigate the mechanisms underlying ischemia-/reperfusion-related BBB opening, we applied multiple immunofluorescence labeling and electron microscopy in a rat model of thromboembolic stroke as well as mouse models of permanent and transient focal cerebral ischemia. In these models, areas exhibiting BBB breakdown were identified by extravasation of intravenously administered fluorescein isothiocyanate (FITC)-albumin. After 24 hours, expression of markers for tight and adherens junctions in areas of FITC-albumin leakage consistently remained unaltered in the applied models. However, lectin staining with isolectin B4 indicated structural alterations in the endothelium, which were confirmed by electron microscopy. While ultrastructural alterations in endothelial cells did not differ between the applied models including the reperfusion scenario, we regularly identified vascular alterations, which we propose to reflect four distinct stages of BBB breakdown with ultimate loss of endothelial cells. Therefore, our data strongly suggest that ischemia-related BBB failure is predominantly caused by endothelial degeneration. Thus, protecting endothelial cells may represent a promising therapeutical approach in addition to the established recanalizing strategies.

Altered microglial phagocytosis in GPR34-deficient mice.

GPR34 is a Gi/o protein-coupled receptor (GPCR) of the nucleotide receptor P2Y12 -like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34-deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34-deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis.

Natural compounds with P2X7 receptor-modulating properties.

The adenosine 5'-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca(2+) response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca(2+) entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs.

Local proliferation of macrophages in adipose tissue during obesity-induced inflammation.

AIMS/HYPOTHESIS: Obesity is frequently associated with low-grade inflammation of adipose tissue (AT), and the increase in adipose tissue macrophages (ATMs) is linked to an increased risk of type 2 diabetes. Macrophages have been regarded as post-mitotic, but recent observations have challenged this view. In this study, we tested the hypothesis that macrophages proliferate within AT in diet-induced obesity in mice and humans. METHODS: We studied the expression of proliferation markers by immunofluorescence, PCR and flow cytometry in three different models of mouse obesity as well as in humans (n = 239). The cell fate of dividing macrophages was assessed by live imaging of AT explants. RESULTS: We show that ATMs undergo mitosis within AT, predominantly within crown-like structures (CLS). We found a time-dependent increase in ATM proliferation when mice were fed a high-fat diet. Upregulation of CD206 and CD301 in proliferating ATMs indicated preferential M2 polarisation. Live imaging within AT explants from mice revealed that macrophages emigrate out of the CLS to become resident in the interstitium. In humans, we confirmed the increased expression of proliferation markers of CD68(+) macrophages in CLS and demonstrated a higher mRNA expression of the proliferation marker Ki67 in AT from obese patients. CONCLUSIONS/INTERPRETATION: Local proliferation contributes to the increase in M2 macrophages in AT. Our data confirm CLS as the primary site of proliferation and a new source of ATMs and support a model of different recruitment mechanisms for classically activated (M1) and alternatively activated (M2) macrophages in obesity.

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

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