Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.

A majority, if not all, acute and progressive neurodegenerative diseases are accompanied by local microglia-mediated inflammation, astrogliosis, infiltration of immune cells, and activation of the adaptive immunity. These processes progress by the expression of cytokines, adhesion molecules, proteases, and other inflammation mediators. In response to brain injury or infection, intracellular signaling pathways are activated in microglia, which turn on inflammatory and antigen-presenting cell functions. Different extrinsic signals shape microglial activation toward neuroprotective or neurotoxic phenotype under pathological conditions. This review discusses recent advances regarding molecular mechanisms of inflammatory signal transduction in neurological disorders and in in vitro models of inflammation/gliosis. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases responsible for most cellular responses to cytokines and external stress signals and crucial for regulation of the production of inflammation mediators. Increased activity of MAPKs in activated microglia and astrocytes, and their regulatory role in the synthesis of inflammatory cytokines mediators, make them potential targets for novel therapeutics. MAPK inhibitors emerge as attractive anti-inflammatory drugs, because they are capable of reducing both the synthesis of inflammation mediators at multiple levels and are effective in blocking inflammatory cytokine signaling. Small molecule inhibitors targeting of p38 MAPK and JNK pathways have been developed and offer a great potential as potent modulators of brain inflammation and gliosis in neurological disorders, where cytokine overproduction contributes to disease progression. Many of the pharmacological MAPK inhibitors can be administered orally and initial results show therapeutic benefits in preclinical animal models. Anat Rec, 292:1902Rec, 292: -1913Rec, 292: , 2009

Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of pro-inflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-γ signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression.Key MessageCombined analysis of genomic Stat occupancy and transcriptome revealed novel Stat target genes in LPS-induced microglia.Jmjd3 transcription factor is a novel transcriptional target of Stat1 and Stat3.Stat1 and Stat3 cooperate with Jmjd3 to induce the expression of pro-inflammatory genes.Constitutively active Stat1 and Stat3 fully mimic the LPS-induced upregulation of inflammatory genes and secretion of cytokines.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-013-1090-5) contains supplementary material, which is available to authorized users.

Microglia are myeloid cells residing in the central nervous system that participate in inflammatory responses and could promote injury and repair. Gliomas attract microglia and polarize them into tumor-supporting cells that participate in matrix remodeling, invasion, angiogenesis, and suppression of adaptive immunity. Although signaling pathways and critical regulators underlying classical inflammation are well established, signal transduction and transcriptional circuits underlying the alternative activation of microglia are poorly known. Using primary rat microglial cultures exposed to glioma conditioned medium or lipopolysaccharide (LPS), we demonstrate that microglia adapt different fates and polarize into pro-inflammatory or alternatively activated cells. Glioma-derived factors increased cell motility, phagocytosis, and sustained proliferation of microglial cells that was mediated by enhanced focal adhesion kinase and PI-3K/Akt signaling. The signals from glioma cells induced ERK and p38 MAPK but not JNK signaling and failed to activate pro-inflammatory Stat1 and NFκB signaling in microglial cells. Transcriptome analysis of microglial cultures at 6 h after exposure to glioma-conditioned medium or LPS revealed different patterns of gene expression. Glioma-induced activation was associated with induction of genes coding for ID (inhibitor of DNA binding) 1/3 and c-Myc, markers of the alternative phenotype Arg1, MT1-MMP, CXCL14, and numerous cytokines/chemokines implicated in immune cell trafficking. Many classical inflammation-related genes and signaling pathways failed to be induced. Our study indicates for the first time molecular pathways that direct microglia toward the pro-invasive, immunosuppressive phenotype.

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