OPG/RANKL/RANK Axis Is a Critical Inflammatory Signaling System in Ischemic Brain in Mice

OPG/RANKL/RANK axis is a critical inflammatory signaling system in ischemic brain in mice

Munehisa Shimamuraa, Hironori Nakagamia, Mariana K. Osakoa,1, Hitomi Kurinamia, Hiroshi Koriyamaa, Pang Zhengdab, Hideki Tomiokac, Akiko Tenmaa, Kouji Wakayamad, and Ryuichi Morishitac,2

aDivision of Vascular Medicine and Epigenetics, Osaka University United Graduate School of Child Development, Osaka 565-0871, Japan; bDepartment of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Japan; cDepartment of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; and dDepartment of Advanced Clinical Science and Therapeutics, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan

Edited by Michael Karin, University of California, San Diego School of Medicine, La Jolla, CA, and approved April 24, 2014 (received for review January 13, 2014) Osteoprotegerin (OPG) is a soluble secreted protein and a decoy inflammatory disease, and postischemic inflammation affects the receptor, which inhibits a receptor activator of nuclear factor κB outcome, i.e., the infarct volume (17). These inflammatory cyto- (NF-κB) ligand (RANKL)/the receptor activator of NF-κB (RANK) kines are produced mainly from microglia in the early phase and signaling. Recent clinical studies have shown that a high-serum- from mixed microglia/macrophage (M/M) in the delayed phase, OPG level is associated with unfavorable outcome in ischemic i.e., 12–24 h after ischemia, whereas neutrophilic granulocytes are stroke, but it is unclear whether OPG is a culprit or an innocent not responsible for the production of those inflammatory cyto- bystander. Here we demonstrate that enhanced RANKL/RANK sig- kines (17). Because OPG/RANKL/RANK is expressed in mac- OPG−/− naling in mice or recombinant RANKL-treated mice contrib- rophages (6, 9–12) and affects inflammatory responses (13), we uted to the reduction of infarct volume and brain edema via hypothesized that one of the mechanisms of poor outcome in reduced postischemic inflammation. On the contrary, infarct vol- high-level OPG might reflect the modulation of such postischemic ume was increased by reduced RANKL/RANK signaling in OPG−/− inflammations by the OPG/RANKL/RANK signaling system. mice and WT mice treated with anti-RANKL neutralizing antibody. Interestingly, OPG, RANKL, and RANK have also been re- OPG, RANKL, and RANK mRNA were increased in the acute stage ported to be expressed in normal brain in rodents, although the and were expressed in activated microglia and macrophages. MEDICAL SCIENCES Although enhanced RANKL/RANK signaling had no effects in distribution of their expression is controversial. Early reports showed that RANKL mRNA was expressed in neurons in the glutamate, CoCl2,orH2O2-stimulated neuronal culture, enhanced RANKL/RANK signaling showed neuroprotective effects with re- cerebral cortex (18) and that RANK (19) and OPG (5) mRNA duced expression in inflammatory cytokines in LPS-stimulated was expressed in normal brain. However, a recent report re- neuron-glia mixed culture, suggesting that RANKL/RANK signaling vealed that the RANK protein was specifically expressed in can attenuate inflammation through a Toll-like receptor signaling neurons and astrocytes in the preoptic area and the medial septal pathway in microglia. Our findings propose that increased OPG nucleus, whereas RANKL mRNA was expressed in the lateral could be a causal factor of reducing RANKL/RANK signaling and in- septal nucleus (20). In normal brain, RANKL/RANK signaling creasing postischemic inflammation. Thus, the OPG/RANKL/RANK axis was reported to be associated with fever and body temperature plays critical roles in controlling inflammation in ischemic brains. Significance cerebral ischemia | neuroprotection | immune cells Although a high-serum osteoprotegerin (OPG) level is associ- n elevated serum osteoprotegerin (OPG) level has been ated with an unfavorable outcome in ischemic stroke, it is un- Areported to be associated with the severity (1, 2), subtype (2), clear whether OPG is a culprit or an innocent bystander. Here we poor functional outcome (1), and long-term mortality of ischemic show that the deletion of OPG and enhanced RANKL/RANK stroke (2, 3). However, it is still unclear why a high-serum-OPG signaling contribute to the reduction of infarct volume with level could result in a poor prognosis in ischemic stroke. lower brain edema, whereas infarct volume is increased by re- OPG is a soluble secreted protein that lacks transmembrane duced RANKL/RANK signaling in OPG−/− mice and WT mice and cytoplasmic domains. It binds to a receptor activator of nu- treated with anti-RANKL neutralizing antibody. OPG, RANKL, clear factor-кB ligand (RANKL) (4, 5), whose receptor is the re- and RANK mRNA were increased in ischemic brain and were ceptor activator of NF-κB (RANK), and inhibits RANKL/RANK expressed in activated microglia and macrophages. Enhanced signaling. The OPG/RANKL/RANK system in bone (6) and RANKL/RANK signaling showed neuroprotective effects with vasculature (7) is well known to work on bone metabolism (8) and reduced expression in inflammatory cytokines in LPS-stimulated vascular calcification (7). In addition, immune cells (6, 9–12) ex- neuron-glia mixed culture. Our findings propose anti-inflammatory press these molecules, and this system is believed to be associated roles for RANKL/RANK signaling in ischemic brains. with the regulation of inflammatory and immune responses (13, + 14). RANKL is expressed in CD4 T cells (6) and macrophages (9, Author contributions: M.S. and R.M. designed research; M.S., M.K.O., and H. Kurinami 10), whereas OPG is expressed in mature B cells (6) and macro- performed research; H.N., H. Koriyama, P.Z., H.T., A.T., and K.W. analyzed data; and M.S., phages (11, 12). RANK is expressed in macrophage and dendritic H.N., and R.M. wrote the paper. cells (6). One of the functions of RANKL/RANK signaling in the Conflict of interest statement: The Department of Clinical Gene Therapy is financially supported by AnGes MG, Novartis, Shionogi, Boeringher, and Rohto. The Division of immune system is to control the thymocyte-mediated medulla Vascular Medicine and Epigenetics is financially supported by Bayer. R.M. is a founder formation and the formation of self-tolerance in T cells (14), as and stockholder of AnGes MG and a former board member. well as the number of regulatory T cells (Treg) (15). Additionally, This article is a PNAS Direct Submission. RANKL directly contributes to the regulation of proinflammatory 1Present address: Department of Cell and Molecular Biology, Ribeirão Preto Medical cytokine production in macrophages (13, 16). School, University of São Paulo, Ribeirão Preto, 04044-010 São Paulo, Brazil. Despite these characteristics of the OPG/RANKL/RANK 2To whom correspondence should be addressed. E-mail: [email protected]. system, its action on inflammation in central nervous system This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. diseases has yet to be studied. Ischemic stroke is a typical acute 1073/pnas.1400544111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1400544111 PNAS | June 3, 2014 | vol. 111 | no. 22 | 8191–8196 Downloaded by guest on October 2, 2021 control (20). Because body temperature is associated with the outcome in ischemic stroke, we also hypothesized that the modulation of body temperature by the OPG/RANKL/RANK system might be another mechanism behind a poor outcome in ischemic stroke. To clarify these hypotheses, we examined the action of the OPG/RANKL/RANK signaling system in a transient − − middle cerebral artery occlusion (MCAo) model using OPG / mice and treated RANK fragment crystalizable (Fc) chimera to inhibit the function of RANKL or recombinant RANKL. Results Effect of OPG/RANKL/RANK Signaling on Infarct Volume. Because a high-serum-OPG level is associated with stroke severity in clinical trials (1–3), we examined the effects of OPG in ischemic − − − − brain using OPG / mice (Fig. 1A). The OPG / mice showed a significant reduction in infarct volume compared with WT mice (Fig. 1A), which was associated with a significant decrease in cerebral edema (Fig. 1B). Although the differences in the Fig. 2. Temporal expression profile of RANK, RANKL, or OPG mRNA in the structure of cerebral vessels and microcirculations could affect ischemic brain. The expression of RANK (A), RANKL (B), or OPG (C) mRNA stroke outcome, no differences were observed in the pial arterial was analyzed by real-time RT-PCR. RANK mRNA was increased starting at 4 h anastomoses in middle cerebral arteries and anterior cerebral after MCAo and peaked at 12 h after MCAo. RANKL and OPG mRNA showed arteries and the microcirculations in cerebral cortex between a biphasic profile. The peak of RANKL expression was at 7 and 48 h after −/− MCAo, and OPG expression peaked at 12–24 and 72 h after MCAo in WT normal OPG mice and WT mice (Fig. S1). In addition, ce- −/− −/− mice. The expression of RANK and RANKL in OPG mice was also shown. rebral blood flow (CBF) was also similar between OPG mice (n = 3 in each time point.) and WT mice during surgery (Fig. S2A). Although body temperature could be a critical factor for stroke outcome, there was − − no significant difference in the circadian rhythm of rectal tem- in OPG / mice. To elucidate the role of this system, we ex- − − perature between OPG / and WT mice (Fig. S2B) or the amined the inhibition of RANKL/RANK signaling using an i.c.v. change in rectal temperature after MCAo (Fig. S2C). These data injection of a neutralizing anti-RANKL antibody, RANK Fc/ − − − − indicate that OPG might work as an exacerbation factor for is- chimera, in OPG / mice. As expected, the OPG / mice treated chemic insult, independent of body temperature and CBF. with RANK Fc/chimera exhibited a significant increase in infarct Because OPG is a decoy receptor for RANKL and because volume (Fig. 1C). To examine whether RANKL/RANK signal- − − RANKL/RANK signaling was augmented in OPG / mice (21), ing would be associated with the outcome even in WT mice, we hypothesized that an enhancement of RANKL/RANK sig- RANK Fc/chimera was also injected i.c.v. in a mild ischemic model. naling might be responsible for the reduction of infarct volume The infarct volume was also significantly exacerbated (Fig. 1D), − − although the extent was less than that observed in OPG / mice (Fig. S3A). These results suggest that RANKL/RANK signaling might act as a protective signal in the ischemic brain − − in both OPG / and WT mice. However, RANKL/RANK signaling might not be fully stimulated in WT mice due to the possibility that OPG partly blocks the RANKL/RANK signaling in WT mice. To further examine the action of RANKL, WT mice in a severe ischemic model were treated with i.c.v. of RANKL starting at 4 h after MCAo (Fig. 1 E and F). The mice treated with RANKL showed a tendency to have a low mortality rate (Fig. S3B), a significantly lower infarct volume (Fig. 1E), and less − − formation of cerebral edema (Fig. 1F). Similar to OPG / mice, no differences in CBF were observed during the surgery (Fig. S4A). Because a single i.c.v. injection of RANKL was reported to affect the core body temperature (20), we examined the change of core body temperature using data loggers (Fig. S4 B–D). The

−/− core body temperature was not affected by the i.c.v. injection of Fig. 1. Infarct volume and brain edema in OPG mice and mice treated RANKL in normal mice (Fig. S4B) and mice exposed to MCAo with anti-RANKL neutralizing antibody or RANKL. The infarct volume at 72 h after 45 min middle cerebral artery occlusion (MCAo) (A–C), 35 min MCAo (Fig. S4 C and D). These results indicated that the stimulation of (D), and 70 min MCAo (E and F). (A and B) OPG−/− mice showed a reduced RANKL/RANK signaling resulted in a reduction of ischemic infarct volume and brain edema compared with WT mice. **P < 0.01 vs. WT injury without changes in body temperature or CBF. − − mice. n = 8 in WT mice and n = 7inOPG / mice. (C) Intracerebroventricular injection of anti-RANKL neutralizing antibody, which inhibited RANKL/RANK Temporal Profile of RANK, RANKL, and OPG mRNA Expression. Next, − − signaling, exacerbated the infarct volume in OPG / mice. *P < 0.05 vs. BSA- we examined the temporal expression of RANK, RANKL, and injected mice. n = 6 in each group. (D) Treatment with anti-RANKL neu- OPG mRNA in the ischemic brain in WT mice (Fig. 2). RANK tralizing antibody in WT mice also showed an increased infarct volume. mRNA was increased starting at 4 h after MCAo and peaked at < < = = **P 0.01; *P 0.05 vs. BSA injected mice. n 10 in BSA-treated mice; n 11 12 h after MCAo. RANKL and OPG mRNA showed a biphasic in RANKL-treated mice. (E) WT mice treated with RANKL showed lower in- profile. The peak of RANKL expression occurred at 7 and 48 h farct volume when the mice survived to 72 h after MCAo (Fig. S3B)were – compared. **P < 0.01; *P < 0.05 vs. BSA injected mice. (F) There was a lower after MCAo, and OPG expression peaked at 12 24 and 72 h −/− formation of brain edema in RANKL-treated mice. n = 8inRANKL-treated after MCAo. In OPG mice, RANK and RANKL mRNA were mice and n = 5 in BSA-treated mice. also increased after MCAo (Fig. 2 A and B) although the

8192 | www.pnas.org/cgi/doi/10.1073/pnas.1400544111 Shimamura et al. Downloaded by guest on October 2, 2021 expression level of RANK was lower than that in WT mice (Fig. 2A). Thus, OPG, RANKL, and RANK were up-regulated in the acute phase of cerebral ischemia. Immunohistochemistry showed that RANK, RANKL, and OPG were expressed in F4/80-positive or Iba1-expressing M/M in the border between the infarct and intact areas, whereas the nonischemic hemisphere showed − − no expression of RANK, RANKL, and OPG (Fig. 3). OPG / mice also showed expression of RANK and RANKL in M/M in the ischemic hemisphere (Fig. S5). These data indicate that OPG, RANKL, and RANK work primarily in M/M in the acute stage of ischemic brain.

Expression of Inflammatory Cytokines in the Ischemic Brain. Because inflammatory cytokines produced from microglia have modifying effects on the infarct volume in the early phase of cerebral ischemia (17), we hypothesized that RANKL/RANK signaling in microglia modulated the inflammatory cytokine expression. As − − expected, OPG / mice showed a significantly lower expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) and a lower expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and monocyte chemotactic protein-1 (MCP- 1), whereas the expression of arginase 1 (Arg1) was higher (Fig. 4A). Moreover, the number of F4/80 positive cells in ischemic − − brain was significantly lower in the OPG / mice (Fig. 5). When RANK Fc/chimera was intracerebroventricularly injected in − − OPG / mice, a higher expression of mRNA was observed in α β

RANK Fc/chimera-treated mice in IL-6, TNF , IL-1 , MCP-1, MEDICAL SCIENCES and iNOS, but not in Arg1 (Fig. S6). RANKL-treated mice in WT Fig. 4. Cytokine expressions in MCAo mice. Cytokine expression was ex- mice showed a significant reduction of IL-1β and MCP-1 and amined using real-time RT-PCR at 48 h after MCAo in OPG−/− mice (A) and at a lower expression in both IL-6 and TNFα (Fig. 4B). However, 24 h after MCAo in RANKL-treated WT mice (B). *P < 0.05 vs. WT mice RANKL treatment did not affect the expression of iNOS and subjected to MCAo (A), *P < 0.05 vs. BSA-treated mice (B). n = 4 in each Arg1. These results indicate that RANKL/RANK signaling had group. BI, brain infarction. anti-inflammatory properties in postischemic injury. RANKL Inhibited Toll-like receptor 4-Mediated Neuronal Death in Neuron-Glia Mixed Cultures. In postischemic inflammation, en- dogenous danger signals, such as high-mobility group box 1 (HMGB-1) and peroxiredoxin family proteins, were reported to be responsible for exacerbating the ischemic insult (22). Because Toll-like receptor 4 (TLR-4) is one of the receptors for these molecules (23), we examined the effect of RANKL on neuronal cell death induced by a TLR-4 ligand and LPS using neuron-glia mixed culture. In the neuron-glia culture, RANK expression was observed in Iba1-positive microglia (Fig. S7). When LPS was added to the mixed culture, the number of MAP-2 neurons that survived was decreased, whereas RANKL pretreatment for 24 h prevented neuronal death (Fig. 6 A and B). Because LPS in- directly causes neuronal death by increasing the expression of inflammatory cytokines produced from activated microglia (24), we examined the expression of IL-6 and TNFα in cultured medium (Fig. 6C). Although RANKL itself did not affect the expression of those cytokines, RANKL pretreatment prevented the production of IL-6 and TNFα stimulated by LPS (Fig. 6C). To exclude the possibility of neuroprotection of RANKL through the direct action on neurons, the effect of RANKL was examined in primary neuronal cultures (Fig. S8). We examined the direct neuroprotective effects of RANKL in a hypoxic model induced Fig. 3. Expression of RANK, RANKL, or OPG in the ischemic brain at 48 h after MCAo in WT mice. Immunohistochemistry of RANK (A–E), RANKL (F–J), by CoCl2. However, no neuroprotective effects of RANKL were or OPG (K–O) expression at 48 h after MCAo. Although RANK, RANKL, and observed (Fig. S8A). We further examined the effects of RANKL OPG were not expressed in intact regions in ischemic cortex (A, F, and K)as in glutamate toxicity and H2O2-induced oxidative stress using an well as nonischemic cortex (E, J, and O), these molecules were expressed in increased dose of RANKL (Fig. S8 B and C). Similarly, no direct – – – – F4/80-positive (A D) or Iba-1 positive (F I and K N) activated M/M in the neuroprotective effects of RANKL were observed. These data border zone between the intact and infarct region. Higher magnification of the images in the rectangle area (A, B, F, G, K, and L) were shown in C, D, H, I, suggest that the neuroprotective effects of RANKL are not through M, and N. (Scale bar: 100 μminA, B, E, F, G, J, K, L, and O;20μminC, D, H, I, its direct actions on neurons but instead through the inhibition of M, and N.) inflammatory cytokines from microglia.

Shimamura et al. PNAS | June 3, 2014 | vol. 111 | no. 22 | 8193 Downloaded by guest on October 2, 2021 A B RANKL in WT mice did not affect the expression of iNOS and WT OPG-/- WT OPG-/- Arg1 mRNA. This finding suggests that the temporal stimulation of RANKL might have anti-inflammatory effects without changes in M/M polarization. Because the previous study showed that Intact Intact myeloid differentiation primary response 88 (Myd88) expression was Infarct Infarct decreased in bone-marrow–derived macrophages by recombinant RANKL treatment (13), RANKL might work downstream of TLR signaling in both microglia and macrophages. Further C 2000 studies are necessary to clarify the detailed mechanisms. 1500 In the present study, the expression level of RANK mRNA was − − ** lower in OPG / mice, probably due to the chronic stimulation 1000 − − of RANKL in OPG / mice. Previous reports showed an in-

positive cells − − 500 consistent expression level of RANK and RANKL in OPG / Total number of F4/80Total number of 0 WT OPG-/--/- WT OPGOPG-/--/- mice compared with WT mice: increased mRNA levels of RANK Ischemic Nonischemic and RANKL in the aorta (21); the same expression level of RANKL Fig. 5. Activated macrophage/microglia in the peri-infarct region in WT and mRNA expression in osteoblasts (26, 27); and a decreased ex- OPG−/− mice. Immunohistochemistry for F4/80 was shown in ischemic cortex pression level of RANK mRNA in osteoclasts in 12-wk-old mice (A) and nonischemic brain (B)inWT(Left)orOPG−/− mice (Right). The followed by an increased level at 20 and 28 wk (28). From this −/− number of F4/80-positive cells was counted at 0.7 mm from the bregma (C). viewpoint, the expression level of RANK and RANKL in OPG − − n = 8 in WT mice and n = 7inOPG / mice. **P < 0.01 vs. WT mice. Ischemic, might be dependent in cells or tissue. ischemic hemisphere; Nonischemic, nonischemic hemisphere. Compared with a previous report showing the site-specific expression of RANK and RANKL in the normal brain, i.e., the preoptic area and medial septal nucleus or the lateral septal Discussion nucleus (20), the present study showed that these molecules In the present study, we demonstrate that the stimulation of started to be diffusely expressed in the cerebral cortex in the RANKL/RANK signaling through the deletion of OPG or ex- ischemic border zone. These data indicate that the RANKL/ ogenous RANKL addition prevented the further exacerbation of RANK axis might not work in neurons and astrocytes in the infarct volume and cerebral edema by inhibiting the production cortex but that this axis might work in the activated microglia of inflammatory cytokines. This stimulation was independent of when the brain is exposed to ischemia. This speculation is sup- body temperature and CBF, whereas the blockade of RANKL/ ported by the data indicating that RANKL exhibited neuro- RANK signaling resulted in the exacerbation of infarct volume. protective effects in the mixed glia-neuron culture but not in OPG, RANKL, and RANK were up-regulated in M/M in the cultured neurons from the cerebral cortex. In addition, because ischemic border. Because microglia are responsible for the pro- OPG is expressed in activated M/M with the expression of duction of inflammatory cytokines in the early phase of ischemic RANK, the OPG/RANKL/RANK axis might work through auto- brain (17) and because RANK expression starts to be up-regu- crine and/or paracrine action in M/M. Thus, the RANKL/RANK lated at 4 h after MCAo, RANKL could act on microglia first. Thereafter, RANKL could work on M/M in the later phase because both macrophages and microglia produce inflammatory cytokines 12–24 h after MCAo (17). Because LPS-induced neuronal death in mixed neuronal glia was prevented by RANKL through the inhibition of inflammatory cytokine production, one possible mechanism might involve an inhibitory effect of RANKL/ RANK signaling on TLR-4 signaling in activated microglia, which has been reported to be responsible for the receptor of intrinsic danger signals in the ischemic brain (25). Similarly, in the later phase, inflammatory cytokines from macrophages might be inhibited by RANKL because a previous report demonstrated the anti-inflammatory action of RANKL in LPS-stimulated bone- marrow–derived macrophages and peritoneal macrophages (13). Therefore, the association of high OPG with poor functional outcome in clinics (1–3) could result from the attenuated anti- inflammatory function of RANKL/RANK signaling due to the higher level of OPG, although the effects of the OPG/RANKL/ RANK axis on bone metabolism (8) and vascular calcification (7) should be carefully considered. The molecular mechanisms that determine how RANKL affects TLR signaling in microglia have yet to be clarified, but one possible − − mechanism in the OPG / model could be associated with driving the alternative activation of M/M (M2) because iNOS mRNA was decreased and Arg1 mRNA was increased in the ischemic brain Fig. 6. Prevention of LPS-triggered neuronal death by RANKL treatment. − − in OPG / mice. Unexpectedly, the blockade of RANKL/RANK Typical immunohistochemical images for MAP2 in mixed neuron-glia cul- signaling with RANK Fc chimera did not decrease the expression tures at 5 d after LPS stimulation (A). The cells were pretreated with 100 ng/mL − − μ of Arg1 in OPG / mice, but we speculate that this might be due RANKL for 24 h and exposed to 10 g/mL LPS for 5 d. The LPS-treated culture showed a lower number of MAP2-positive cells compared with RANKL-treated to the functional compensation in the knockout mouse instead of culture (B, n = 4 in each group). The expression of TNFα andIL-6inmediumat RANKL/RANK signaling. Further studies are necessary to clarify 24 h after exposure to LPS was less in the group treated with 100 ng/mL RANKL the mechanisms that determine how OPG deletion influences (C). n = 3 in the cells without LPS; n = 10 in LPS-stimulated cells. #P < 0.05 vs. on phenotype of M/M. On the contrary, the i.c.v. injection of cells without LPS and RANKL; *P < 0.05 vs. LPS-stimulated cells without RANKL.

8194 | www.pnas.org/cgi/doi/10.1073/pnas.1400544111 Shimamura et al. Downloaded by guest on October 2, 2021 axis could be associated with other central nervous system dis- Measurement of Infarction Volume. Ischemic damage was evaluated using eases such as multiple sclerosis and Alzheimer’s disease, where sections stained with cresyl violet at 72 h after MCAo. Coronal sections (12-μm − − activated microglia work as a deteriorative factor. thickness) were made at 1.4, 0.7, 0, 0.7, and 1.4 mm from the bregma, Although the role of the RANKL/RANK axis in the patho- mounted on the stereomicroscope, and photographed. The hemispheric lesion area (HLA) in the coronal sections was calculated. The corrected HLA logical brain has not been reported, except in the present study, was calculated as HLA (%) = [LT-(RT-RI)]/LT × 100, where LT is the area of the its role in the control of body temperature and fever has been left hemisphere, RT is the area of the right hemisphere, and RI is the infarcted reported in normal rodent brain (20). In this previous study, area (23). Brain edema was calculated as brain edema (%) = [RT-LT]/LT × 100. RANKL-injected mice (1, 10, or 100 ng single i.c.v. injection) − − showed the induction of fever at 6 h after injection, with the Measurement of Body Temperature. In OPG / mice, body temperature was activation of specific brain regions involved in thermoregula- measured by rectal probe thermometer (Unique Medical) without anesthe- tion and induction via the COX2-PGE2/EP3R pathway (20). In sia. Core body temperature was continuously recorded using temperature contrast, the present study demonstrated that RANKL-injected data loggers (KN Laboratories) implanted into the peritoneal cavity. The − − WT mice and OPG / mice did not show any differences in data loggers were programmed to record body temperature every 3 min core body temperature in either normal mice or mice subjected with a resolution of 0.1 °C. Following the implant, animals were allowed 4 d to recover. to MCAo. Although the cause of discrepancy between the previous report (20) and the present study is still unclear, the better −/− Fluorescein Angiography. The structure of cerebral vessels, capillary density, outcome in OPG mice and RANKL-treated WT mice could and blood–brain barrier leakage was assessed using an angiographic tech- not be explained by the differences in body temperature. nique with FITC-conjugated albumin (31). The depicted brain was fixed in Although we focused on the OPG/RANKL/RANK axis in 4% (wt/vol) paraformaldehyde (PFA), and the image was acquired using M/M in the acute stage of ischemic brain, it is possible that the a fluorescence stereoscope (SZX 12, Olympus). The brains were frozen, cut into OPG/RANKL/RANK axis might influence both the control of 20-μm-long sections, and examined using a Nikon A1 confocal scanning laser T-cell–mediated immune responses because RANKL regulates microscope. The images were analyzed using NIS Elements software (Nikon). the formation of self-tolerance in T cells (14) and any immuno- suppressive effects by controlling the number of Treg in allergic Administration of Recombinant RANKL and RANK/Fc Chimera. Recombinant mouse RANKL [5 ng in 2 μL artificial cerebrospinal fluid (aCSF)] was purchased contact hypersensitivity responses and the development of sys- from Peprotech EC. Recombinant mouse RANK/TNFRSF11a Fc Chimera temic autoimmunity (15). Because T lymphocytes are significantly μ μ + (neutralizing anti-RANKL antibody, 4 gin2 L aCSF) was obtained from increased at day 3 (29) and because Foxp3 Treg cells work as R&D Systems. Intracerebroventricle injection was performed using a pulled cerebroprotective immunomodulators in the more delayed phase, glass micropipette (anteroposterior 1.0 mm, lateral 1.2 mm from bregma, MEDICAL SCIENCES i.e., 3–7 d after MCAo (30), the OPG/RANKL/RANK axis could depth 1.7 mm). Recombinant RANKL was injected at 4, 24, and 48 h after also be associated with the regulation of T-cell–mediated immune MCAo. Recombinant mouse RANK/TNFRSF11a Fc Chimera was intra- responses in the delayed phase. Further studies are necessary to cerebroventricularly injected 2 h before MCAo. For the controls, BSA (Nacalai clarify the actions of the OPG/RANKL/RANK axis on the T-cell– Tesque) was used at the same concentration with each reagent. FITC- conju- μ μ mediated immune response. gated albumin (10 g/2 l, Sigma-Aldrich) was intracerebroventricularly injected at 4 or 24 h after MCAo to examine whether the injected proteins could In conclusion, in the present study we demonstrate that the penetrate the cortex in ischemic brain. The concentration of FITC-conjugated OPG/RANKL/RANK system might be one of the key signals albumin was half that in the previous report examining the fluorescein angi- regulating M/M-derived inflammatory responses in ischemic ography (31). At 2 h after injection, the fluorescence was observed around the brain. Because the i.c.v. injection of RANKL a minimum of 4 h microglia in the ischemic cortex (Fig. S9). after MCAo decreased the infarct volume, RANKL/RANK signaling might be a therapeutic target to treat ischemic stroke. Real-Time RT-PCR. The ischemic hemisphere was collected at 48 h after MCAo − − Although the development of a delivery system and a modifi- in OPG / mice. With the RANKL-treatment being 70-min ischemia in WT cation of RANKL are needed to avoid RANKL-induced osteo- mice, the sample was collected at 24 h because the survival rate was low in mice at 48 h (Fig. S3B). The mRNAs were isolated using QIAGEN RNeasy Lipid porosis, further studies on the OPG/RANKL/RANK system in ’ the brain might shed light on the molecular mechanism involved Tissue Mini Kit (Qiagen), according to the manufacturer s recommendations. The cDNA reaction was performed using a High-Capacity cDNA Archive kit in postischemic inflammation and lead to the development of (Applied Biosystems) according to the manufacturer’s instructions. The oli- new therapeutic options for ischemic stroke. gonucleotide primers used exclusively in the in vitro experiments were purchased according to the identification RANK: Mm00437135; RANKL: Materials and Methods Mm01313944; OPG: Mm00435451; MCP1: Mm00441243; IL-6: Mm00446190; Surgical Procedure. All procedures were approved by the Institutional Animal Arg1: Mm00475988; iNOS: Mm00440502; IL-17a: Mm004329618; IL-10: − − Care and Use Committee of Osaka University. OPG / mice on C57Bl6/J back- Mm01288386; IL-1β: Mm99999061; and GAPDH: Mm99999915 (Applied ground and C57Bl6/J mice (wild-type) were obtained from CLEA Japan, Inc. Biosystems). The 5′ nuclease assay PCRs were performed in a MicroAmp Optical 384-well reaction plate using ABI PRISM 7900 Sequence Detection Transient Middle Cerebral Artery Occlusion. Mice were anesthetized with iso- System. The levels of the target genes were quantified by comparing the flurane (1.4%). Cerebral blood flow was measured using a laser Doppler fluorescence generated by each sample with that of the serially diluted flowmeter (Unique Acquisition software; Unique Medical). A 6.0 monofilament standard, and the target gene expressions were normalized by the level of surgical suture was advanced into the internal carotid artery to obstruct the GAPDH expression in each individual sample. origin of the middle cerebral artery. The filament was left in place for 35, 45, or 70 min and then withdrawn. Because the effect of deleting OPG was not clear Cell Culture. Primary neuron-glial cultures were prepared from postnatal days in the first experiment, we chose 45 min of ischemia, which caused moderate 1–2 in C57BL/6J mice using the method of Araki with some modifications ischemia and was suitable for observing both deteriorating and improving (32). The entire cerebral cortex was dissected and minced in Neurobasal-A effects. To examine the deteriorative effect of neutralizing anti-RANKL anti- (Invitrogen) with B-27 supplement (Invitrogen, Neurobasal-A/B-27) after − − body both in WT mice and OPG / mice, 45- and 35-min ischemic models were removing the meninx. Cells were treated with papain (25 U/mL) and DNaseI − − applied in OPG / and WT mice because the average infarct volume between (25 U/mL) for 30 min at 30 °C. The cells were dissociated mechanically in − − OPG / and WT mice was almost the same in that ischemic duration. To assess Neurobasal-A/B-27 with 0.25 mM Glutamax (Invitrogen) and 10% horse serum the therapeutic effect of RANKL, 70 min of ischemia to cause a severe infarct by using a siliconized Pasteur pipette. Cells were plated on polyethyleneimine- was also applied. Only animals that exhibited a typical reduction pattern and coated 24-well plates at 7 × 105/well. Cultures were maintained at 37 °C in a – more than 82% reduction in CBF during MCAo, in which CBF recovered by 30 humidified atmosphere containing 5% CO2. Half of the medium was replaced 80% after 5 min of reperfusion, were included in the study. In all mice, rectal with Neurobasal-A/B-27 and 5% horse serum twice per week. After 9 d of temperature was kept at 37.0 ± 0.5 °C during surgery and in the recovery plating, the cells were composed of 42 ± 5% neurons, 56 ± 5% astrocytes, and period until animals regained consciousness. The overall mortality was 27% at 2 ± 1% microglia. In the experiment on neural death, cells were treated with 72 h after ischemia. RANKL (100 ng/mL) for 24 h, and the medium was replaced with Neurobasal-A

Shimamura et al. PNAS | June 3, 2014 | vol. 111 | no. 22 | 8195 Downloaded by guest on October 2, 2021 containing N2 supplement (Invitrogen, Neurobasa-A/N2), 1% horse serum, LPS (1:500 for F4/80 and CD11b, Alexa Flour 546, Invitrogen) or anti-rabbit fluores- (10 μg/mL), and RANKL (100 ng/mL). Cells were immunostained with MAP-2 to cent antibody (1:500 for Iba-1, Alexa Flour 546, Invitrogen). The sections were determine the survival of the neurons at 5 d after the addition of LPS. The blocked again and were incubated in streptavidin/biotin blocking kit (Vector images were then digitized by microscope (FSX-100, Olympus). The acquired Lab, Vector Laboratories). Then they were incubated with anti-RANK antibody images were converted to grayscale with GIMP 2.8.6, and the stained area was (1:13, R&D systems) or RANKL (1:50, eBioscience). As a negative control, the calculated using ImageJ (National Institutes of Health). In the experiment ex- same concentration of normal control IgG (Santa Cruz) was applied. Then they amining the expression of TNFα or IL-6, the medium was collected at 24 h after were incubated in biotinylated anti-goat IgG antibody (1:200 for RANK; Vector LPS stimulation. The concentration of these cytokines was checked using Lab) or anti-rat IgG antibody (1:200 for RANKL; Vector Lab) followed by fluo- commercially available ELISA kits [TNFα: Quantikine Mouse TNF-α ELISA Kit rescent streptavidin conjugates (1:1,000, Alexa 488; Invitrogen). In the immu- (R&D systems); IL-6: Quantikine Mouse IL-6 ELISA Kit (R&D Systems)]. nohistochemistry for OPG, the sections were blocked with 20% horse serum and For the primary neuronal culture, mouse embryonic cerebral cortex then incubated with streptavidin/biotin blocking kit (Vector Lab), followed by neurons were obtained from pregnant C57BL/6J mice on the 18th day of incubation with biotin affinity-purified polyclonal antibody for OPG (1:10, R&D gestation and cultured (33). The cerebral cortex was dissected, and individual Systems). Then the sections were incubated with fluorescent streptavidin cells were isolated by treatment with papain and triturated in Leibovitz’s conjugates (1:1,000, Alexa 488; Invitrogen). L-15 medium (Invitrogen). Cells were plated on polyethyleneimine-coated Immunohistochemical staining was examined using Nikon A1 confocal 24-well plastic culture dishes with Neurobasal (Invitrogen)/B-27 (Invitrogen) scanning laser microscope, and the images were analyzed using NIS Elements with 0.25 nM Glutamax (Invitrogen) at 37 °C in a humidified atmosphere of software (Nikon). All parameters were set in a similar manner when the signal 95% air-5% CO2. The medium was changed on the fourth day. On day 9, the intensity was compared. medium was changed to Neurobasal/N2 (Invitrogen) containing BSA or μ RANKL (10 or 100 ng/mL). Thereafter, CoCl2 (100 M, 48 h), glutamate (100 Statistical Analysis. All values are expressed as the mean ± SEM. Multiple μ μ M, 10 min), or H2O2 (100 M, 24 h) was added to the medium. To check cell comparisons were evaluated by ANOVA followed by Dunnett’s Multiple viability, cells were assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-car- Comparison Test. Two groups were compared using an unpaired t test. boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay follow- Survival rates were evaluated using a log-rank test. The core body temper- ’ ing the manufacturer s protocol (CellTiter 96 AQueous One Solution Cell atures with data loggers were analyzed by a two-way repeated-measures Proliferation Assay) at the following time points after the stimulation: ANOVA with Bonferroni posttests. Differences were considered significant — — — CoCl2 48 h; H2O2 24 h; and glutamate 24 h. at P < 0.05. The statistical analysis were performed with the software Prism 5.0 (GraphPad Software). Immunohistochemical Staining. Mice were perfused with 4% PFA, and the brain μ was cut into 12- m-thick sections. For double immunostaining, these sections ACKNOWLEDGMENTS. This work was supported by the grants from The were fixed and then blocked. The sections were incubated with anti-F4/80 Ichiro Kanehara Foundation (M.S.), the SENSHIN Medical Research Founda- (1:50, AbD Serotec), anti-Iba1 (1:1,000, Wako), or anti-CD11b (1:100, AbD tion (M.S.), and Japan Society for the Promotion of Science Grants-in-Aid for Serotec). Then, the sections were incubated with anti-rat fluorescent antibody Scientific Research (KAKENHI) Grant 25462214 (to M.S.).

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