Contents lists available at ScienceDirect Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim TLR4 in circumventricular neural stem cells is a negative regulator for thermogenic pathways in the mouse brain Shiori Muneoka a , Saki Murayama a , Yousuke Nakano a,c , Seiji Miyata a,b, ⁎ a Department of Applied Biology, Japan b The Center for Advanced Insect Research Promotion (CAIRP), Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan c Department of Anatomy and Brain Science, Kansai Medical University, Hirakata, Japan ARTICLE INFO Keywords: Inflammation Circumventricular organs Fever NF-κB Fos ABSTRACT Toll-like receptor 4 (TLR4) recognizes bacteria-derived lipopolysaccharide (LPS). In the present study, we found that intraperitoneal LPS activated nuclear factor-κ B (NF-κB) in TLR4-expressing neural stem cells (NSCs) in the circumventricular brain regions of mice. Intracerebroventricular preadministration of low-dose TLR4 inhibitors significantly augmented hyperthermia together with the inhibition of NF-κB activation in circumventricular NSCs of LPS-inflamed animals. Moreover, intracerebroventricular administration of high-dose TLR4 inhibitors induced hyperthermia and Fos activation in circumventricular NSCs and hypothalamic neurons. These results suggest that TLR4 on circumventricular NSCs functions as a central regulator for thermogenesis under inflamed and normal conditions. 1. Introduction Recognition of infection is the first and most important process re- quired to initiate proper physiological responses to fight infection. The recognition of pathogens is mediated by several classes of receptors collectively referred to as pattern-recognition receptors, of which Toll- like receptors (TLRs) are the most widely studied (McCusker and Kelley, 2013; Gay et al., 2014). The reaction to lipopolysaccharide (LPS) de- rived from Gram-negative bacteria, which triggers severe inflammation by activating TLR4, is the best-characterized experimental in- flammatory system of bacterial infection. Administration of LPS leads to acute biological activity, including changes in body temperature, au- tonomic responses, anorexia, adipsia, modifications of sleep patterns, and decreases in locomotor activity (Roth et al., 2004; Rivest, 2003). Systemic low-dose LPS activates TLR4 to trigger inflammatory re- sponses and hyperthermia, which allows host animals to respond to invading pathogens, whereas excessive LPS leads to the development of sepsis and hypothermia (Ramachandran, 2014). LPS is able to bind with CD14 only in the presence of LPS-binding protein, and the complex of LPS-proteins is then able to interact with TLR4. The activation of TLR4 leads to a series of signaling cascades that result in the activation of two distinct signaling pathways: nuclear factor-κB (NF-κB) and activator protein-1 (Takeda and Akira, 2004; Rivest, 2003). The trimer consisting of a NF-κB dimer and monomeric inhibitor of κB (IκBα) is present in the cytosol in an inactivated state, whereas the NF-κB dimer is translocated into the nucleus after dis- sociation of IκBα (Perkins, 2007), and thereafter binds to the NF-κB motif of DNA to transcribe proinflammatory genes such as tumor ne- crosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 (Brasier, 2010). Activator protein-1 is also known to control the expression of numerous cytokines (Adcock, 1997). The cytokines IL-1β and IL-6 activate cy- clooxygenase-2 (COX-2) via the production of prostaglandin E2 (PGE2) to induce inflammatory and thermoregulatory responses (Conti et al., 2004). The selective loss of the neuronal PGE2 receptor in the preoptic area (POA) prevents PGE2- and LPS-induced hyperthermia (Lazarus et al., 2007). Selective gene deletion of PGE2-synthesizing enzymes in brain endothelial cells attenuates hyperthermia (Wilhelms et al., 2014). Hyperthermic response is dependent on PGE2 synthesis by endothelial https://doi.org/10.1016/j.jneuroim.2018.04.017 Received 25 October 2017; Received in revised form 23 April 2018; Accepted 30 April 2018 ⁎ Corresponding author at: Department of Applied Biology, Kyoto Institute of Technology, Kyoto 606-8585, Japan. E-mail address: smiyata@kit.ac.jp (S. Miyata). Abbreviations: AH, anterior hypothalamic area; AP, area postrema; Arc, arcuate nucleus; BBB, blood-brain barrier; CC, central canal; CVOs, circumventricular organs; COX2, cy- clooxygenase-2; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; FITC, fluorescein isothiocyanate isomer-I; GFP, green fluorescent protein; i.c.v., intracerebroventricular; Iba1, ionized calcium binding adapter molecule 1; LPS, lipopolysaccharide; LPS-RS, LPS from the photosynthetic bacterium Rhodobacter sphaeroides;IκBα, inhibitor of κB; ME, median eminence; MnPO, median preoptic nucleus; MPA, medial preoptic area; MW, molecular weight; NF-κB, nuclear factor-κB; NSCs, neural stem cells; NGS, normal goat serum; OVLT, organum vasculosum of the lamina terminalis; PBS, phosphate-buffered saline; PBST, PBS containing 0.3% Triton X-100; PFA, paraformaldehyde; PGE2, prostaglandin E2; PV-1, plasmalemmal vesicle-1; SFO, subfornical organ; PVN, paraventricular nucleus; TNF, tumor necrosis factor; IL, interleukin; SOX2, sex determining region Y-box 2; STAT3, signal transducer and activator of transcription factor 3; Sol, nucleus of the solitary tract; TLR, Toll-like receptor; VIPER, viral inhibitor peptide of TLR4; ZI, zona incerta Journal of Neuroimmunology 331 (2019) 58–73 0165-5728/ © 2018 Elsevier B.V. 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