Pierrot Tremblay

Identification of genes preferentially expressed by microglia and upregulated during cuprizone‐induced inflammation

Microglia, monocytes, and peripheral macrophages share a common origin and many characteristics, but what distinguishes them from each other at the level of gene expression remains largely unknown. In this study, we compared the transcriptional profiles of freshly purified microglia, monocytes, and spleen macrophages using Affymetrix Mouse Genome arrays to identify genes predominantly expressed by microglia. Among tens of thousands of genes assayed, 127 potential candidates were found, including nine newly discovered genes encoding plasma membrane and extracellular proteins. In the brain, the latter were selectively expressed by microglia, as revealed by in situ hybridization. Three of them were confirmed to be exclusively (MSR2) or predominantly (GPR12, GPR34) expressed in the brain compared to the other tissues examined. Furthermore, all of these genes were upregulated in activated microglia after treatment with the demyelinating toxin cuprizone, suggesting that they play roles in neuroinflammation. In conclusion, this study reports the identification of new selective markers for microglia, which should prove useful not only to identify and isolate these cells, but also to better understand their distinctive properties.

G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.

G protein‐coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin‐1 (IL‐1), because mice lacking either one or both of these molecules have fewer GPR84‐expressing cells in their cerebral cortex than wild‐type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone‐insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.

Tumor Necrosis Factor Reduces Brain Tumor Growth by Enhancing Macrophage Recruitment and Microcyst Formation

Recent findings implicate macrophages and some of their secreted products, especially tumor necrosis factor (TNF), as tumor promoters. Inhibitors of these inflammatory components are currently regarded as potential therapeutic tools to block tumor progression. Here, we show that infiltrating macrophages represented a significant population of nonneoplastic cells within malignant gliomas, in which they were the exclusive producers of TNF. Contrary to the reported pro-oncogenic effects of TNF in other types of solid tumors, glioma-bearing mice deficient in TNF developed larger tumors and had reduced survival compared with their wild-type controls. Histologic examinations revealed that glioma volume was negatively correlated with the number of macrophages and small cavities called microcysts. Overall, our results support the concept that macrophages alter brain tumor development through a TNF-dependent process that culminates in the formation of microcysts. This raises the question of whether anti-inflammatory drugs, such as those commonly administrated to patients with brain cancer, could interfere with antitumor mechanisms.

Reduced Glioma Growth Following Dexamethasone or Anti-Angiopoietin 2 Treatment

All patients with glioblastoma, the most aggressive and common form of brain cancer, develop cerebral edema. This complication is routinely treated with dexamethasone, a steroidal anti‐inflammatory drug whose effects on brain tumors are not fully understood. Here we show that dexamethasone can reduce glioma growth in mice, even though it depletes infiltrating T cells with potential antitumor activity. More precisely, T cells with helper or cytotoxic function were sensitive to dexamethasone, but not those that were negative for the CD4 and CD8 molecules, including gammadelta and natural killer (NK) T cells. The antineoplastic effect of dexamethasone was indirect, as it did not meaningfully affect the growth and gene expression profile of glioma cells in vitro. In contrast, hundreds of dexamethasone‐modulated genes, notably angiopoietin 2 (Angpt2), were identified in cultured cerebral endothelial cells by microarray analysis. The ability of dexamethasone to attenuate Angpt2 expression was confirmed in vitro and in vivo. Selective neutralization of Angpt2 using a peptide‐Fc fusion protein reduced glioma growth and vascular enlargement to a greater extent than dexamethasone, without affecting T cell infiltration. In conclusion, this study suggests a mechanism by which dexamethasone can slow glioma growth, providing a new therapeutic target for malignant brain tumors.

PRODUCTION OF TISSUE-ENGINEERED THREE-DIMENSIONAL HUMAN BRONCHIAL MODELS

SummaryWe have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serium-free medium supplemented with retinoic acid (5×10−8M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models with be useful for studying human bronchial properites in vitro.

Crawling Phagocytes Recruited in the Brain Vasculature after Pertussis Toxin Exposure through IL6, ICAM1 and ITGαM

The cerebral vasculature is constantly patrolled by rod‐shaped leukocytes crawling on the luminal endothelial surface. These cells are recruited in greater numbers after exposure to bacterial lipopolysaccharide (LPS) by a mechanism involving tumor necrosis factor (TNF), interleukin‐1β (IL1β) and angiopoietin‐2 (Angpt2). Here, we report that the population of crawling leukocytes, consisting mainly of granulocytes, is also increased in the brains of mice suffering from experimental autoimmune encephalomyelitis (EAE) or injected with pertussis toxin (PTX), which is commonly used to induce EAE. However, this recruitment occurs through an alternative mechanism, independent of Angpt2. In a series of experiments using DNA microarrays, knockout mice and neutralizing antibodies, we found that PTX acts indirectly on the endothelium in part through IL6, which is essential for the post‐transcriptional upregulation of intercellular adhesion molecule 1 (ICAM1) in response to PTX but not to LPS. We also found that phagocytes adhere to brain capillaries through the interaction of integrin αM (ITGαM) with ICAM1 and an unidentified ligand. In conclusion, this study supports the concept that PTX promotes EAE, at least in part, by inducing vascular changes necessary for the recruitment of patrolling leukocytes.

Matrix metalloproteinase 2 attenuates brain tumour growth, while promoting macrophage recruitment and vascular repair

Matrix metalloproteinase 2 (MMP2) is an extracellular protein‐degrading enzyme widely believed to be involved in the invasion of brain tumour cells. However, this assumption is mainly based on in vitro studies. By characterizing the transcriptome and in vivo properties of 20 astrocytoma cell lines, we found that the levels of MMP2 were higher in GFAP− astrocytoma cells and correlated with their ability to induce vascular changes, a common complication of malignant tumours. To study the relationship between MMP2 expression and vascular alteration, we intracerebrally implanted immunodeficient mice with human astrocytoma cells stably transduced with lentiviral vectors expressing either MMP2 or a short hairpin RNA against MMP2. We found that the tumours depleted of MMP2 were larger, contained more proliferating cells and fewer macrophages, and had a vasculature that was more destabilized and regressed with fewer capillary sprouts. In contrast, the tumours overexpressing MMP2 were smaller and showed no histological difference compared to the controls. We therefore suggest that MMP2 is not the cause of vascular atypia in malignant brain tumours, but is involved in a tissue repair response that tends to limit the growth of these tumours. This study argues against MMP2 inhibition as a therapeutic approach for brain cancer and provides a comprehensive characterization of popular astrocytoma cell lines that should help to identify alternative targets. Copyright