LETTERS Central control of fever and female body temperature by RANKL/RANK Reiko Hanada 1 , Andreas Leibbrandt 1 , Toshikatsu Hanada 1 , Shiho Kitaoka 2 , Tomoyuki Furuyashiki 2 , Hiroaki Fujihara 3 , Jean Trichereau 1 , Magdalena Paolino 1 , Fatimunnisa Qadri 4 , Ralph Plehm 4 , Steffen Klaere 5 , Vukoslav Komnenovic 1 , Hiromitsu Mimata 6 , Hironobu Yoshimatsu 6 , Naoyuki Takahashi 7 , Arndt von Haeseler 5 , Michael Bader 4 , Sara Sebnem Kilic 8 , Yoichi Ueta 3 , Christian Pifl 9 , Shuh Narumiya 2 & Josef M. Penninger 1 Receptor-activator of NF-kB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lact- ating mammary gland 1–4 . RANKL and RANK are also expressed in the central nervous system 5,6 . However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rank floxed deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rank floxed deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1b and TNFa. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE 2 /EP3R path- way. Moreover, female Nestin-Cre and GFAP-Cre rank floxed mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differ- entiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation. To test for a possible brain-specific function of the RANKL/RANK system, we performed stereotactic intracerebroventricular (i.c.v.) injections of recombinant RANKL into the lateral ventricle of rats (Supplementary Fig. 1a). Injection of RANKL did not alter serum alkaline phosphatase and serum Ca 21 levels (Supplementary Fig. 1b), suggesting that central RANKL administration has no overt effects on osteoclasts. Within minutes after i.c.v. injection, RANKL administra- tion resulted in markedly reduced activity of all animals tested. Further analyses revealed that RANKL i.c.v. injected rats developed very high fever (Fig. 1a, Supplementary Fig. 1c). Heat inactivation of RANKL abolished the fever response (Fig. 1b, Supplementary Fig. 1d), exclu- ding possible endotoxin contaminations. Similar to rats, i.c.v. injections of RANKL into mouse brains triggered hyperthermia (Fig. 1c). As expected from a febrile response 7 , i.c.v. injections of RANKL also resulted in markedly reduced activity (Fig. 1d, Supplementary Fig. 2a). In vivo inhibition of RANKL with the natural decoy receptor osteoprotegerin alleviated the fever response in mice (Fig. 1e) and rats (Supplementary Fig. 2b). Moreover, i.c.v. injections of RANKL resulted in induction of adrenocorticotropic hormone (ACTH) in the serum and Uncoupling protein 1 (UCP1) in the brown adipose tissue (Supplementary Fig. 3), required to generate heat by non-shivering thermogenesis 8 . By contrast, intraperitoneal (i.p.) delivery even of high doses of RANKL did not result in any changes in body temperature nor in activity (Supplementary Fig. 2c–e). Thus, central nervous system administration of RANKL can trigger fever. We next set out to map RANK expression in the brain. RANK protein was specifically expressed in the preoptic area (POA) and the Medial Septal nucleus (MSn) (Supplementary Figs 4 and 5). Immunostaining with neuronal (NeuN), astrocyte (GFAP), and microglia (Iba1) markers indicated that RANK is expressed on neurons and astrocytes in the POA/MSn region (Supplementary Figs 4b and 6a–c). Staining for RANKL protein revealed strong expression in the choroid plexus of the ventricles (Supplementary Fig. 7). In addition, in situ hybridization for RANKL revealed mRNA expression in the Lateral Septal nucleus (LSn) (Supplementary Figs 4b and 8). Thus, RANKL and RANK are expressed in the POA/MSn/LSn region, key brain regions involved in thermoregulation 7,9 . c-Fos expression has been previously used to map brain regions involved in fever 10,11 . In rat, i.c.v. injection of RANKL resulted in strong c-Fos activation in the POA, MSn, the ventromedial hypo- thalamus (VMH), the dorsomedial hypothalamus (DMH), and the periventricular nucleus (PVN) (Supplementary Fig. 9). The PVN, VMH, and the DMH regions relay central thermoregulation to stimulation of the sympathetic nervous system 9 . We also observed that i.c.v. RANKL triggers c-Fos activation in the suprachiasmatic nucleus (SCN) (Supplementary Fig. 9), the central regulator of cir- cadian activity. Similar to rats, i.c.v. injections of RANKL into wild- type mice resulted in nuclear accumulation of c-Fos protein in the POA, MSn, PVN, VMH, DMH, and the SCN (not shown). Using c-Fos–GFP (green fluorescent protein) reporter mice 12 , we detected increased transcriptional activation of the c-Fos promoter, in the POA, MSn, PVN, VMH, DMH, and the SCN following i.c.v. injec- tion of RANKL (Supplementary Fig. 10). Thus, RANKL/RANK trig- gers a functional response in brain areas involved in the fever response. To genetically confirm the role of the RANKL/RANK system in the central fever response, we generated a rank floxed allele that would allow us to engineer tissue-specific RANK knockout mice. (For details see Supplementary Methods and Supplementary Fig. 11). Using this line, we generated Nestin-Cre rank floxed mice to delete 1 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria. 2 Kyoto University Graduate School of Medicine, Department of Pharmacology, Kyoto 606-8501, Japan. 3 Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan. 4 Max Delbrueck Centre for Molecular Medicine, 13125 Berlin, Germany. 5 Center of Integrated Bioinformatics, Max F. Perutz Laboratories, 1030 Vienna, Austria. 6 Oita University Faculty of Medicine, Oita 879-5593, Japan. 7 Institute for Oral Science, Matsumoto Dental University, Nagano 399-0781, Japan. 8 Uludag University Medical Faculty, 16059 Bursa, Turkey. 9 Medical University of Vienna, Center for Brain Research, 1090 Vienna, Austria. Vol 462 | 26 November 2009 | doi:10.1038/nature08596 505 Macmillan Publishers Limited. All rights reserved ©2009