MicroRNA-146a: A Key Regulator of Astrocyte-Mediated Inflammatory Response Anand Iyer1., Emanuele Zurolo1., Avanita Prabowo 1 , Kees Fluiter2, Wim G. M. Spliet3, Peter C. van Rijen4, Jan A. Gorter5,6, Eleonora Aronica1,6* 1 Department of (Neuro) Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Genome Analysis, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 3 Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands, 4 Department of Neurosurgery/Rudolf Magnus Institute for Neuroscience, University Medical Center Utrecht, Utrecht, The Netherlands, 5 Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, Amsterdam, The Netherlands, 6 SEIN-Epilepsy Institute in the Netherlands Foundation, Heemstede, The Netherlands Abstract Increasing evidence supports the involvement of microRNAs (miRNA) in the regulation of inflammation in human neurological disorders. In the present study we investigated the role of miR-146a, a key regulator of the innate immune response, in the modulation of astrocyte-mediated inflammation. Using Taqman PCR and in situ hybridization, we studied the expression of miR-146a in epilepsy-associated glioneuronal lesions which are characterized by prominent activation of the innate immune response. In addition, cultured human astrocytes were used to study the regulation of miR-146a expression in response to proinflammatory cytokines. qPCR and western blot were used to evaluate the effects of overexpression or knockdown of miR-146a on IL-1b signaling. Downstream signaling in the IL-1b pathway, as well as the expression of IL-6 and COX-2 were evaluated by western blot and ELISA. Release several cytokines was evaluated using a human magnetic multiplex cytokine assay on a LuminexH 100TM/200TM platform. Increased expression of miR-146a was observed in glioneuronal lesions by Taqman PCR. MiR-146a expression in human glial cell cultures was strongly induced by IL-1b and blocked by IL-1b receptor antagonist. Modulation of miR-146a expression by transfection of astrocytes with anti- miR146a or mimic, regulated the mRNA expression levels of downstream targets of miR-146a (IRAK-1, IRAK-2 and TRAF-6) and the expression of IRAK-1 protein. In addition, the expression of IL-6 and COX-2 upon IL-1b stimulation was suppressed by increased levels of miR-146a and increased by the reduction of miR-146a. Modulation of miR-146a expression affected also the release of several cytokines such as IL-6 and TNF-a. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation and suggests that this miR may represent a novel target for therapeutic strategies. Citation: Iyer A, Zurolo E, Prabowo A, Fluiter K, Spliet WGM, et al. (2012) MicroRNA-146a: A Key Regulator of Astrocyte-Mediated Inflammatory Response. PLoS ONE 7(9): e44789. doi:10.1371/journal.pone.0044789 Editor: Pablo Villoslada, Institute Biomedical Research August Pi Sunyer (IDIBAPS) - Hospital Clinic of Barcelona, Spain Received May 29, 2012; Accepted August 13, 2012; Published September 13, 2012 Copyright: 2012 Iyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Epilepsy Fund - ‘‘Power of the Small’’, the Hersenstichting Nederland (NEF 09-05; NEF 13-01); EU FP7 project NeuroGlia (Grant Agreement Nu 202167) and EU FP7 project DEVELAGE (Grant Agreement Nu 278486) EA; KIKA (Stichting Kinderen Kankervrij; AP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: e.aronica@amc.uva.nl . These authors contributed equally to this work. Introduction During the last decade, both experimental and clinical studies have demonstrated the relevance of inflammation in the patho- physiology of epilepsy (for reviews see [1–3]. Neuropathological examination of surgical epilepsy specimens provides evidence of a complex and sustained inflammatory phenomenon with pro- duction of pro-inflammatory molecules in patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS). Activation of cells of the microglia/macrophage lineage and astrocytes, and concomitant induction of various inflammatory pathways, also have been observed in focal malformations of cortical develop- ment (MCD; such as focal cortical dysplasia FCD), which represent major causes of pediatric epilepsy [4]. Both the innate and the adaptive immune responses are activated in these lesions ([5]; reviewed in [3]). In particular, both in vitro and in vivo data, point to the role of astrocytes as a major source and/or targets of pro-epileptogenic inflammatory signaling, such as the interleuki- n(IL)-1, High Mobility Group Box 1 (HMGB1) and Toll-like receptor (TLR) signaling pathways [1,3,6–9]. An increasing body of literature supports the critical role of miRNAs in post-transcriptional gene regulation in several bi- ological processes of the central nervous system (CNS), as well as in the pathogenesis of different disorders (including developmental, neurodegenerative, vascular and neuroinflammatory disorders), and in oncogenesis [10–12]. Various miRNAs are also considered to represent a new class of mediators of inflammation [13–16]. In particular, miRNA-146a has been associated with the regulation of Toll-like and interleukin-1 receptors (TIRs) signaling [15,17–19]. Interestingly, this miRNA has been shown to be upregulated in experimental models of epilepsy, as well as in human TLE [20– 22]. miRNA-146a is expressed in human brain in astrocytes which PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44789