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Microglia are the myeloid cells residing in the central nervous system, quickly responding to pathological alterations. Microglia polarization refers to establishment of a specific phenotype which can produce detrimental or beneficial effects. The inflammatory (M1) and alternative, anti-inflammatory (M2) phenotype are the extreme phenotypes. It is poorly understood how microglia are reprogrammed in a responses to challenges and how signals are converted into sustained patterns of gene expression in brain pathologies. Transcriptome analysis of microglial cultures exposed to glioma (GCM) or lipopolysaccharide (LPS) shows activation of distinct signaling and metabolic pathways resulting in different patterns of gene expression. Studies of activating and repressive histone marksrevealed the early decrease of histone acetylation in microglia exposed to GCM, while changes in repressive histone modifications after GCM or LPS were delayed and correlated to transcription down-regulation. HDAC inhibitors blocked morphological changes associated with GCM or LPS treatment and reduced GCMinduced gene expression. Those results demonstrate that the inflammatory genes are epigenetically “primed” and easy to be induced in microglia, while the erasure of histone acetylation marksis prerequisite to put the repressive marks on M1 inflammatory genes and induce activating histone acetylation at the M2 genes. Studies of epigenetic patternsin sorted microglia were extended to a cerebral ischemia (MCAo) model, in which microglia activation is associated with the prolonged inflammation and subsequent neurodegeneration. Our results demonstrate that microglial polarization is mediated by alterations in gene expression and changes in their epigenetic patterns. Epigenetic modifications provide an additional step for the control of long-lasting changes in transcriptional programs in stimulated microglia.
Excessive, uncontrolled infl ammation and gliosis contribute to a majority of neurologic disorders despite of their different etiology. Activated microglial cells release pro-infl ammatory cytokines, infl ammation mediators, matrix proteinases and toxic factors. Reactive astrogliosis involves astrocyte proliferation, activation and hypertrophy accompanied by production of cytokines, growth factors and metabolic alterations. Infl ammatory signalling involves activation of transcription factors NFκB and MAP kinases as critical signal transducers. We demonstrate activation of JAK/STAT (signal transducers and activators of transcription) signaling pathway associated with infl ammatory microglia and ìreactive astrogliosisî in vitro when primary rat astrocyte cultures were stimulated with the pro-infl ammatory cytokines (IL1-β, IFN-γ and TNF-α). Global gene expression profi ling revealed a coordinated and strong upregulation of infl ammatory and immune response genes in infl ammatory microglia in vitro and after transient focal ischemia. Moreover, we found a large representation of STAT responsive genes in both conditions suggesting a strong contribution of STAT pathway to stroke-induced infl ammation. We studied whether inhibition of signal transduction mediated by MAP kinases and JAK/STAT pathway interferes with expression/release of infl ammatory cytokines, mediators, matrix proteinases and toxic factors by activated glial cells. Our fi ndings establish immunosuppressants as effective therapeutic candidate for use in the treatment of human neurologic disorders. Identifi cation of STAT dependent events underlying infl ammation and development specifi c inhibitors may facilitate development of innovative strategies blocking hostile microglial responses to control infl ammation.
Microglia are myeloid cells residing in the central nervous system (CNS) that rapidly respond to signals originating in the injured or infected brain. Microglia respond differently to challenges and acquire different functional phenotypes with extremes: an inflammatory, detrimental M1 or immunosuppressive, cytoprotective M2 phenotype. Post‑ischemic inflammation plays a key role in secondary and delayed neuronal damage and death, and involves activation of microglia, perivascular and peripheral macrophages, that accumulate in the ischemic brain and contribute to disease. The molecular signature of myeloid cells in the inflammed brain, and specific roles of different populations are unclear. Lack of robust markers that differentiate microglia from macrophages makes it less feasible. We developed effective, flow cytometry‑based methods for sorting myeloid subpopulations that accumulate in the hemispheres of sham-operated and ischemic rats undergoing transient 90 min MCA (middle cerebral artery) occlusion. We analyzed transcriptomes of specific populations by RNAseq and evaluated their functional phenotypes. This analysis revealed a prevalence of inflammatory M1 microglia 1 day after MCAo and accumulation of M2-immunosuppressive macrophages 3–7 days after MCAo. Ablation of peripheral macrophages reduced abundance of M2 immunosuppressive, Arg1 expressing macrophages. Using immunofluorescence and confocal microscopy we demonstrated that with time Arg1+ macrophages lose their M2‑phenotype and become inflammatory iNos expressing cells. Moreover, we found that macrophages of perivascular space and meninges (defined by CD163+ staining) are a distinct myeloid subpopulation in the brain, and these cells proliferate and migrate to the ischemic parenchyma after MCAo. Altogether, we demonstrate distinct functional properties and transcriptional networks in brain macrophages, that suggests functional plasticity and distinctive functions in neuroinflammation and repair.
Microglia are multifunctional immune cells of the brain executing various functions and rapidly responding to pathological insults. Brain injury, hypoxia, infection or aberrant protein accumulation may lead to chronic infl ammation with a progressive shift in microglia function towards infl ammatory phenotype and accumulation of immune cells. Under pathological conditions, the interplay of extrinsic signals directs microglia towards neuroprotective or detrimental phenotype. Molecular mechanisms of initiation, progression and termination of microglia-initiated infl ammatory responses in the brain, in particular gene networks and signaling pathways are poorly understood. Characterization of the global transcriptome of microglia exposed to infl ammatory or cytoprotective signals and analysis of signalling pathways revealed differences in expression of genes encoding cytokines/ chemokines and transcription regulators. Identifi cation of signalling pathways contributing to discrete microglia phenotypes and discovery of transcription regulators which may serve as “master switches” for induction of an infl ammatory phenotype, will allow to target specifi c functions of microglia. Therapeutic approaches targeting signal transduction in microglia will be discussed. A greater understanding of microglia functions coupled with advances in pharmacology and gene therapy will support development of functionally “engineered” microglia able to convey neuroprotection.
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