TLR Activation Induces TNF- Production from Adult Neural Stem/Progenitor Cells 1 Ruxandra Covacu, 2 * Lisa Arvidsson, † Åsa Andersson,* Mohsen Khademi,* Helena Erlandsson-Harris, ‡ Robert A. Harris,* Mikael A. Svensson, † Tomas Olsson,* and Lou Brundin* Adult neural stem cells (NSCs) are believed to facilitate CNS repair and tissue regeneration. However, it is not yet clear how these cells are influenced when the cellular environment is modified during neurotrauma or neuroinflammatory conditions. In this study, we determine how different proinflammatory cytokines modulate the expression of TLR2 and TLR4 in NSCs and how these cells respond to TLR2 and TLR4 agonists. Primary cultures of neural stem/progenitor cells isolated from the subventricular zone of brains from adult Dark Agouti rats were exposed to 1) supernatants from activated macrophages; 2) proinflammatory cytokines IFN-, TNF-, or both; and 3) agonists for TLR2 and TLR4. Both TLR2 and TLR4 were expressed during basal conditions and their mRNA levels were further increased following cytokine exposure. TLR4 was up-regulated by IFN- and this effect was reversed by TNF-. TLR2 expression was increased by supernatants from activated macrophages and by TNF-, which syner- gized with IFN-. TLR agonists induced the expression of TNF- mRNA. Importantly, TNF- could be translated into protein and released into the supernatants where it was quantified by cytokine ELISA. In conclusion, we demonstrate that NSCs con- stitutively express TLR2 and TLR4 and that their expression is increased as a consequence of exposure to proinflammatory mediators. Additionally, activation of these receptors can induce production of proinflammatory cytokines. These findings suggest that NSCs may be primed to participate in cytokine production during neuroinflammatory or traumatic conditions. The Journal of Immunology, 2009, 182: 6889 – 6895. T he ability of adult neural stem cells (NSCs) 3 to respond to trauma to the CNS by migration and differentiation at the site of injury has been demonstrated in numerous studies in various disease models (1– 4). However, the role of NSCs in neuroinflammatory lesions is not entirely clear. It has become ev- ident during recent years that these cells are not only a source of newly generated cells but may also supply trophic support to cells in the damaged CNS (5, 6). Moreover, in experimental autoim- mune encephalomyelitis (EAE), an animal model of multiple scle- rosis (MS), NSCs have immunomodulatory effects (7, 8) and are induced to express immune markers characteristic of APCs (9). The focus of this project is to 1) investigate the expression of the TLR2 and TLR4 on NSC/progenitor cells, both under normal and inflammatory conditions and 2) monitor the effects of TLR2 and TLR4 activation in NSC/progenitor cells. TLRs are pattern recognition receptors characteristic of immune cells and are crucial for inducing an immune response to patho- gens. Interestingly, Toll was first described in Drosophila to be involved in neural development (10, 11), and in mammals TLRs are involved in differentiation of different stem cell populations, such as hematopoietic stem cells (12–14) and mesenchymal stem cells (15). There are 13 TLRs described in mice and 10 in humans, and the best-described TLRs are TLR2 and TLR4. TLR2 associ- ates with either TLR1 (16) or TLR6 (17) and binds lipoproteins and lipopeptides. TLR4 binds to LPS (18, 19). In addition, all receptors have endogenous ligands (20 –23) that are released from damaged tissues, and there is available evidence suggesting that TLR2 and TLR4 may be involved in the disease development dur- ing EAE and MS (24 –27). Neuroinflammatory conditions, such as MS and EAE, are characterized by infiltration of immune cells into the CNS and production of proinflammatory agents such as NO, TNF-, and IFN-, which in turn contribute to destruction of CNS tissue. The most predominant location of the inflammatory lesions in MS is close to the regenerative area of the subventricular zone (SVZ) where NSCs reside. NSCs can therefore be directly influ- enced by the inflammatory process. In this study, we demonstrate evidence for TLR2 and TLR4 expression on NSCs both in vitro and in vivo. The expression of these receptors was transcriptionally up-regulated as a conse- quence to inflammatory exposure. The expression of TLR2 and TLR4 was differentially affected by the two major proinflamma- tory cytokines evident in EAE and MS, TNF- and IFN-. IFN- favored induction of TLR4. TNF- induced the expression of TLR2 and abrogated the inducing effect of IFN- on TLR4 ex- pression. Moreover, the expression of TLR2 was up-regulated fol- lowing exposure to supernatants from activated macrophages. Most importantly, NSCs exposed to TLR2 and TLR4 agonists produced and released TNF- protein. We thus demonstrate that *Department of Clinical Neurosciences, Division of Neuroimmunology, † Department of Neurosurgery, and ‡ Department of Medicine, Division of Rheumatology, Karolin- ska Institutet, Stockholm, Sweden Received for publication September 2, 2008. Accepted for publication March 24, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Torsten and Ragnar So ¨derberg Foundation, Montel Williams Foundation, The Swedish Society for Neurologically Disabled, and Karo- linska Institutet. 2 Address correspondence and reprint requests to Dr. Ruxandra Covacu, Karolinska Hospital, Center for Molecular Medicine, L8-04, Neuroimmunology Unit, Karolinska Institutet, S-171 76 Stockholm, Sweden. E-mail address: Ruxandra.Covacu@ki.se 3 Abbreviations used in this paper: NSC, neural stem cell; EAE, experimental auto- immune encephalomyelitis; MS, multiple sclerosis; LTA, lipoteichoic acid; SVZ, subventricular zone; GFAP, glial fibrillary acidic protein. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 The Journal of Immunology www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802907