MicroRNAs are a family of regulatory molecules involved in many physiological processes, including differentiation and activation of cells of the immune system. We found that brainspecific miR-124 is expressed in microglia but not in peripheral monocytes or macrophages. When overexpressed in macrophages, miR-124 directly inhibited the transcription factor CCAAT/ enhancer-binding protein-α (C/EBP-α) and its downstream target PU.1, resulting in transformation of these cells from an activated phenotype into a quiescent CD45 low , major histocompatibility complex (MHC) class II low phenotype resembling resting microglia. During experimental autoimmune encephalomyelitis (EAE), miR-124 was downregulated in activated microglia. Peripheral administration of miR-124 in EAE caused systemic deactivation of macrophages, reduced activation of myelin-specific T cells and marked suppression of disease. Conversely, knockdown of miR-124 in microglia and macrophages resulted in activation of these cells in vitro and in vivo. These findings identify miR-124 both as a key regulator of microglia quiescence in the central nervous system and as a previously unknown modulator of monocyte and macrophage activation.MicroRNAs (miRNAs) belong to a family of small non-protein-coding RNAs that regulate expression of multiple target genes and are involved in many fundamental biological processes, such as embryonic development, cell proliferation, differentiation and apoptosis [1][2][3][4][5] . miRNAs promote degradation of mRNA or prevent translation of the target genes, and they can be viewed as endogenous mediators of RNA interference (RNAi) 1 . miRNAs have been identified as crucial regulators of differentiation of various cell types, © 2011 Nature America, Inc. All rights reserved. Correspondence should be addressed to H.L.W. (hweiner@rics.bwh.harvard.edu) and A.M.K. (akrichevsky@rics.bwh.harvard.edu).. 3 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS E.D.P. performed all flow-cytometry assays, EAE experiments, experiments with chimeric, knockout and transgenic mice, in vivo injections of oligonucleotides, cell isolations, cell cultures, coculture assays and immunohistochemistry; collected and analyzed the data; and wrote the manuscript. T.V. performed in vitro transfections, miRNA and mRNA expression assays and data analysis, western blots, luciferase target validation assay, immunohistochemistry and in silico target prediction analysis and helped to write the manuscript. N.B. performed imaging cytometry. E.D.P. and A.M.K. conceived the project. E.D.P., T.V. and A.M.K. developed the hypothesis and designed the experiments. A.M.K. and H.L.W. discussed the hypothesis, helped with data interpretation, coordinated and directed the project and wrote the manuscript.Note: Supplementary information is available on the Nature Medicine website. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. 2,6 . Furthermore, deregulated expression of specific miRNAs is associated with many patholo...

Microglial cells are central nervous system (CNS) resident cells that are thought to become activated and contribute to the inflammation that occurs in the human autoimmune disease multiple sclerosis (MS). This has never been proven, however, because microglial cells cannot be phenotypically distinguished from peripheral macrophages that accumulate in MS inflammatory lesions. To study the kinetics and nature of microglial cell activation in the CNS, we used the animal model of MS, experimental autoimmune encephalomyelitis (EAE), and induced EAE in bone marrow (BM) chimera mice generated using major histocompatibility complex (MHC)-mismatched donor BM, allowing the separation of microglial cells and peripheral monocytes/macrophages. We found that microglial cell activation was evident before onset of disease symptoms and infiltration of peripheral myeloid cells into the CNS. Activated microglial cells underwent proliferation and upregulated the expression of CD45, MHC class II, CD40, CD86, and the dendritic cell marker CD11c. At the peak of EAE disease, activated microglial cells comprised 37% of the total macrophage and dendritic cell populations and colocalized with infiltrating leukocytes in inflammatory lesions. Our findings thus definitively demonstrate that during EAE, microglial cells become activated early in EAE disease and then differentiate into both macrophages and dendritic-like cells, suggesting they play an active role in the pathogenesis of EAE and MS.

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB 2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB 2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB 2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB 2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB 2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB 2 receptor, we cultured microglial cells with combinations of c-interferon (IFN-c) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB 2 receptor. We found no difference in the amount of the CB 2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB 2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB(2) receptor.