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JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice
Biochemical and Biophysical Research Communications xxx (2017) 1e7
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JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice Ka rpa ti a, Takuro Matsuzawa a, Tomomitsu Iida a, Takeo Yoshikawa a, *, Aniko Haruna Kitano a, Asuka Mogi a, Ryuichi Harada a, Fumito Naganuma a, b, Tadaho Nakamura a, b, Kazuhiko Yanai a a
Department of Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan Division of Pharmacology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 April 2017 Accepted 14 May 2017 Available online xxx
Brain histamine acts as a neurotransmitter and regulates various physiological functions, such as learning and memory, sleep-wake cycles, and appetite regulation. We have recently shown that histamine H3 receptor (H3R) is expressed in primary mouse microglia and has a strong influence on critical functions in microglia, including chemotaxis, phagocytosis, and cytokine secretion in vitro. However, the importance of H3R in microglial activity in vivo remains unknown. Here, we examined the effects of JNJ10181457 (JNJ), a selective and potent H3R inverse agonist, on microglial functions ex vivo and in vivo. First, we injected ATP, which is a typical chemoattractant, into hippocampal slices to investigate the effect of JNJ on chemotaxis. ATP-induced microglial migration toward the injected site was significantly suppressed by JNJ treatment. Next, we examined whether JNJ affected microglial phagocytosis in hippocampal slices and in the prefrontal cortex. Microglial engulfment of dead neurons induced by Nmethyl-D-aspartate was inhibited in the presence of JNJ. The increase in zymosan particle uptake by activated microglia in the prefrontal cortex was prevented by JNJ administration. Finally, we determined the importance of JNJ in a lipopolysaccharide (LPS)-induced depression model. JNJ reduced the LPSinduced upregulation of microglial pro-inflammatory cytokines and improved depression-like behaviour in the tail-suspension test. These results demonstrate the inhibitory effects of JNJ on chemotaxis, phagocytosis, and cytokine production in microglia inside the brain, and highlight the importance of microglial H3R for brain homeostasis. © 2017 Elsevier Inc. All rights reserved.
Keywords: Histamine Histamine H3 receptor Microglia Depression
1. Introduction Histamine, which is a bioactive amine, regulates various physiological events, such as allergic reactions, gastric acid secretion, and itch sensation. In the central nervous system (CNS), histamine acts as a neurotransmitter. Neuronal histamine is enzymatically synthesized in histaminergic neurons [1], which are located in the tuberomammilary nucleus in the posterior hypothalamus and project their axons to the entire brain [2]. Among the four G protein-coupled histamine receptors (H1ReH4R), H1R, H2R, and H3R are widely distributed in the CNS and regulate various physiological functions. H1R and H2R are expressed as postsynaptic
* Corresponding author. E-mail address: [email protected] (T. Yoshikawa).
receptors in various areas of the CNS and control sleep-wake cycles and aggression [3,4]. In contrast, H3R, which is an inhibitory G protein-coupled receptor, is an autoreceptor in presynaptic membranes of histaminergic neurons and regulates histamine release [5]. H3R also acts as a heteroreceptor in non-histaminergic neurons and modulates the release of various neurotransmitters, including GABA and acetylcholine [6]. In addition, H3R is the most abundant histamine receptor in the CNS. Consistent with its abundance and strong impact on neurotransmitter release, H3R is involved in diverse brain functions, such as memory, cognition, appetite, and arousal [7]. These findings have attracted attention toward H3R in the CNS from a broad range of disciplines. Preclinical and clinical trials have studied the therapeutic effects of H3R inverse agonists on various neurological disorders, such as epilepsy and attentiondeficit hyperactivity disorder [8e10]. Indeed, pitolisant, an H3R
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Please cite this article in press as: T. Iida, et al., JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.05.081
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T. Iida et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
inverse agonist, was approved for the treatment of narcolepsy by the European Medicine Agency in 2016 [11]. Recent studies have revealed that H3R is also expressed in glial cells. Astrocytes, which are the most abundant cells in the CNS, express H3R, and activation of H3R enhances the expression and synthesis of neurotrophin-3 [12]. However, H1R, and not H3R, has a predominant role in astrocytic functions [13,14]. Our recent findings indicate that microglia, which are brain-resident macrophages, express H2R and H3R. Although we were not able to confirm the importance of H2R in primary mouse microglia, H3R was shown to regulate the levels of second messengers, such as intracellular Ca2þ and cyclic adenosine monophosphate, which in turn affect critical microglial functions including chemotaxis, phagocytosis, and cytokine secretion [15]. Thus, we hypothesize that H3R in microglia also plays a role in the CNS, and that H3R inverse agonists might exert therapeutic effects by regulating microglial H3R as well as neuronal H3R. Here we investigated the effects of a selective and potent H3R inverse agonist, JNJ10181457 (JNJ) [16], on microglial chemotaxis, phagocytosis, and cytokine production in ex vivo and in vivo assays, including in mouse models of human depression.
HBSS, 25% HBSS (Life Technologies), 25% horse serum (HyClone Laboratories; Logan, UT, USA), and 25 mM HEPES supplemented with 100 IU/mL penicillin G potassium and 100 mg/ml streptomycin sulphate. After stimulation with 50 mM N-methyl-D-aspartate (NMDA) (Tokyo Chemical Industry; Tokyo, Japan) in the presence or absence of 1 mM JNJ for 3 h, the slices were incubated with 0.5 mg/ml of propidium iodide (PI) (Tokyo Chemical Industry), which is a membrane-impermeable fluorescent dye for DNA, with or without 1 mM JNJ for 21 h. Fluorescent images were captured using a Nikon C2si microscope (Nikon; Tokyo, Japan). Microglial phagocytosis was evaluated using Bz-9000 (Keyence; Osaka, Japan), and the images were analysed using Hybrid Cell Count image analysis software (Keyence). 2.5. Immunohistochemistry for CD68 Fixed slices were incubated with a primary antibody for CD68 (Abcam; Cambridge, UK) in 20% bovine serum albumin (SigmaAldrich; St. Louis, MO, USA) in phosphate-buffered saline (Wako) overnight at 4 C. The sections were then treated with a secondary antibody.
2. Materials and methods 2.1. Mice CX3C chemokine receptor 1 (CX3CR1)-green fluorescent protein (GFP) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) [17]. All experiments were performed according to the Principles for Care and Use of Research Animals of Tohoku University, Sendai, Japan. All experiments involving animals are reported in accordance with Animal Research: Reporting of in vivo Experiments guidelines [18,19]. 2.2. Organotypic hippocampal slice cultures Organotypic hippocampal slices were prepared as described previously [20]. The hippocampus was cut into 120- (for chemotaxis assay) or 350 mm-thick (for ex vivo phagocytosis assay) sections using a MacIlwain tissue chopper (Mickle Laboratory Engineering; Gomshall Lane, UK). 2.3. Chemotaxis assay Hippocampal slices were pre-incubated with or without 10 mM JNJ (Tocris Bioscience; Bristol, UK) containing dissection buffer with the following composition: Modified Eagle's Medium (MEM)/ Hank's Balanced Salt Solution (HBSS) (Life Technologies; Carlsbad, CA, USA), 10 mM Tris-HCl (pH 7.2), 25 mM HEPES (Life Technologies) supplemented with 100 IU/mL penicillin G potassium (Wako Pure Chemical Industry; Osaka, Japan) and 100 mg/ml streptomycin sulphate (Wako). Injection of 3 mM ATP (Wako) was performed using a microinjector (BJ-110; BEX; Tokyo, Japan) with the following specifications: diameter of pipette tip, 8 mm; pressure, 0.04 MPa; duration, 100 m sec. Images were captured every 30 s using a fluorescent microscope (IX-71; Olympus; Tokyo, Japan). Microglial chemotaxis was analysed as described previously [21]. Briefly, the fluorescent intensity in region Rx (100 mm in radius from the injected site) at each time point Rx(t) was subtracted from the first image Rx(0). Microglial chemotaxis at any time point is calculated using the equation Rx(t) e Rx(0). 2.4. Ex vivo phagocytosis assay The hippocampal slices were pre-incubated for 1 h at 37 C with slice culture medium with the following composition: 50% MEM/
2.6. Quantitative real-time PCR RNA isolation, reverse transcription, and PCR were performed as described previously [15]. The sequences of the primers are listed in Table 1. 2.7. In vivo phagocytosis assay In vivo phagocytosis was assayed as described previously [22]. Yeast-derived zymosan particles conjugated with Alexa 568 (Thermo Fisher Scientific Inc.; Waltham, MA, USA) was stereotaxically injected into the prefrontal cortex at the following coordinates: anterior, 0.3 mm; lateral, ± 1.2 mm; and ventral, 1.6 mm from bregma. We intracranially co-administered 10 mg/uL of lipopolysaccharide (LPS) (Sigma-Aldrich) and/or 10 mM JNJ with the zymosan particles. We injected the mice with 10 mg/kg JNJ or saline intraperitoneally for the next 3 consecutive days. Four days after the zymosan injection, brain coronal sections (50 mm-thick) were prepared using a vibratome (Leica; Wetzlar, Germany) after perfusion with 4% paraformaldehyde (Wako). Phagocytosis was quantified by counting the numbers of engulfed beads in microglia using a fluorescent microscope and appropriate software (Keyence).
Table 1 Primer sequences for PCR. Gene GAPDH Sense Antisense CD68 Sense Antisense IL-1b Sense Antisense IL-6 Sense Antisense TNF-a Sense Antisense
Primer sequence
Product size (bp)
50 -AGAACATCATCCCTGCATCC-30 50 -CACATTGGGGGTAGGAACAC-30
91
50 -ACTGGTGTAGCCTAGCTGGT-30 50 -CCTTGGGCTATAAGCGGTCC-30
85
50 -TGCCACCTTTTGACAGTGATG-30 50 -TGATGTGCTGCTGCGAGATT30
138
50 -CCGGAGAGGAGACTTCACAG-30 50 -CAGAATTGCCATTGCACAAC-30
134
50 -CCCTCACACTCACAAACCAC-30 50 -ACAAGGTACAACCCATCGGC-30
133
GAPDH, glyceraldehyde-6-phosphate dehydrogenase; IL-1b, interleukin-1b; TNF-a, tumour necrosis factor a.
Please cite this article in press as: T. Iida, et al., JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.05.081
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2.8. Tail suspension test
2.9. Measurement of interleukin-1b protein (IL-1b) levels
Mice were injected with saline, LPS (1 mg/kg), JNJ (10 mg/kg), or both LPS and JNJ. Four hours after the injection, a tail suspension test was performed as previously described, with minor modifications [23]. Mice were suspended 50 cm above the floor by holding their tails using a clip. The immobility time over the 6 min test was recorded and analysed.
Brain tissues were homogenized in 50 mM Tris-HCl buffer containing 1 mM EDTA (pH 7.4) and centrifuged at 1000 g for 10 min at 4 C. The supernatants were centrifuged at 20,000 g for 40 min at 4 C to remove remaining debris. Total protein and IL-1b levels was determined using a bicinchoninic acid assay kit (Thermo) and an IL-1b ELISA kit (R & D systems; Minneapolis, MN,
Fig. 1. JNJ suppressed microglial chemotaxis. (A) Representative images of microglial migration induced by the application of 3 mM ATP in hippocampal slices from CX3CR1-GFP mice without (upper panels) or with 10 mM JNJ pre-treatment (lower panels). ATP was locally applied (T: 0 min, left side) and microglial responses were observed for 30 min (T: 30 min, right side). Microglial process motility was tracked using coloured lines. Scale bars ¼ 100 mm. (B) Quantitative analysis of microglial migration. Medium without ATP was applied (black line, n ¼ 3). ATP was applied without (blue line, n ¼ 4) or with 10 mM JNJ pre-treatment (Orange line, n ¼ 4). Cumulative ratio of fluorescent intensity at each time point was calculated as a bar graph. Data are presented as mean ± standard error of the mean. Differences were identified using one-way ANOVA and multiple comparisons tests; *P < 0.05; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. JNJ suppressed microglial phagocytosis in hippocampal slices. A, Representative images of CX3CR1- GFP microglia (green) and PI (red) in hippocampal slices treated with 50 mM NMDA in the presence or absence of JNJ (1 mM). Scale bars ¼ 100 mm. B, PI-positive area in GFP-positive microglia with or without JNJ (1 mM) treatment was measured (n ¼ 8). C, Representative images of CX3CR1-GFP microglia (green) and immunostaining for CD68 (red) in hippocampal slices treated with 50 mM NMDA in the presence or absence of JNJ (1 mM). Scale bars ¼ 100 mm. D, CD68-stained area in GFP-positive microglia with or without JNJ (1 mM) treatment was measured (n ¼ 4). E, mRNA expression levels of CD68 in hippocampal slices treated with 50 mM NMDA in the presence or absence of JNJ (1 mM) (n ¼ 4). mRNA expression levels of CD68 were measured using quantitative PCR. Data are presented as mean ± standard error of the mean. Differences were identified using Student's t-tests; *P < 0.05; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: T. Iida, et al., JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.05.081
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USA), respectively. 2.10. Microglial isolation Microglial isolation was performed as previously described, with minor modifications [24]. Brain tissues were minced and exposed to collagenase (final concentration: 1 mg/mL) (Roche; Basel, Switzerland) for 20 min at 37 C. The tissues were then incubated with DNase І solution (Roche) for 5 min at 37 C. Dissociated cells were passed through a 100-mm nylon mesh cell strainer (BD Falcon; Franklin Lakes, NJ, USA) and centrifuged at 600 g for 5 min at 4 C. After removal of debris, cells were resuspended in 30% percoll (GE Healthcare; Princeton, NJ, USA) solution and centrifuged for 10 min at 700 g. At least 90% of the pelleted cells were immunopositive for the microglial marker CD11b (data not shown). 2.11. Statistical analysis Data analysis was performed using Student's t-tests or one-way ANOVA using GraphPad Prism 5 software (GraphPad; La Jolla, CA, USA). P-values less than 0.05 were considered significant. 3. Results 3.1. JNJ suppressed microglial chemotaxis in hippocampal slices Microglial movement towards the injured site is required to start the removal of wounded tissues in the CNS [21]. Since ATP leakage from injured cells is a strong chemoattractant and induces microglial migration, we examined the effects of JNJ on chemotaxis in response to local ATP injection into acute hippocampal slices. Although capillary insertion slightly induced microglial movement, ATP administration significantly promoted microglial migration to the injection site (Fig. 1A and B). In the presence of JNJ, microglial responses to ATP were significantly reduced. This demonstrates that JNJ has an inhibitory effect on microglial chemotaxis. 3.2. JNJ suppressed phagocytosis of dead cells in hippocampal slices Engulfment of dead cells by phagocytosis is essential to the repair of damaged tissue [25]. To investigate the effects of JNJ on microglial phagocytosis in hippocampal slices, we induced neuronal death using NMDA and evaluated the microglial engulfment of dead neurons. The numbers of PI-positive neuronal nuclei in the microglia were reduced following JNJ administration, suggesting that JNJ inhibits phagocytosis activity of microglia (Fig. 2A and B). We also evaluated the expression of CD68, which is a marker of phagocytic microglia [26]. Protein and mRNA expression levels of CD68 were also suppressed in JNJ-treated hippocampal slices (Fig. 2CeE). These results suggest that JNJ inhibits the phagocytic activity of microglia. 3.3. In vivo phagocytosis was inhibited by JNJ We injected yeast-derived zymosan particles into the prefrontal cortex of the mice to investigate the effects of JNJ on in vivo phagocytosis. LPS significantly increased the numbers of zymosan particles engulfed by microglia (Fig. 3A and B). JNJ did not affect zymosan engulfment in the absence of LPS stimulation, but completely abolished the increase in phagocytosis following LPS administration. These results indicate that JNJ reduces microglial engulfment in the inflammatory conditions induced by LPS treatment.
Fig. 3. JNJ suppressed microglial phagocytosis in vivo. A, CX3CR1-GFP microglia (green) engulfed fluorescent zymosan particles (red) in control, JNJ-, LPS-, or LPS- and JNJ-treated mouse prefrontal cortex. Scale bars ¼ 200 mm. B, Quantitative analysis of the numbers of engulfed zymosan particles by microglia (n ¼ 3). Data are presented as mean ± standard error of the mean. Differences were identified using one-way ANOVA and multiple comparisons tests; *P < 0.05; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. JNJ treatment suppressed microglial activation and improved depression-like behaviour Abnormal microglial activation exacerbates various neurological disorders, including depression [27]. In contrast, normalisation of excessive microglial activity has therapeutic effects on neuropsychiatric diseases [28]. We examined whether inhibitory effects of JNJ on microglia could improve depression-like behaviour in mice. LPS-induced depression-like behaviour is mediated by cytokine secretion from abnormally activated microglia. JNJ attenuated LPS-induced increases in the expression levels of IL-1b, IL-6, and TNF-a in purified microglia (Fig. 4A). IL-1b protein, which is a key cytokine responsible for depression-like behaviours, was especially significantly decreased in the presence of JNJ (Fig. 4B). Moreover, the prolongation of immobile time induced by LPS in
Please cite this article in press as: T. Iida, et al., JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.05.081
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the tail-suspension test was attenuated following JNJ treatment (Fig. 4C). These results indicate that JNJ has an inhibitory effect on microglial activation and improves depression-like behaviour in mice.
4. Discussion Here, we report the inhibitory effects of JNJ on microglial chemotaxis and phagocytosis using ex vivo and in vivo assays. We also report that JNJ reduces inflammatory cytokine expression and improves depression-like behaviour. This is the first report on the importance of H3R in critical microglial functions in the CNS. Contrary to the results of this study, our previous report using primary mouse microglia revealed that H3R agonists, but not inverse agonists, inhibit in vitro microglial functions [15]. In the brain, neurons around microglia secrete neurotransmitters, such as norepinephrine and serotonin, which considerably alter microglial activity [29,30]. Neurons are also in direct contact with microglia via the interaction between CD200 and the CD200 receptor, which leads to the inhibition of microglia [31]. Indeed, brain-resident microglia and primary microglia isolated from the neonatal cortex have different properties, including gene expression patterns [32]. These lines of evidence demonstrate the strong impact of the local environment on microglial activity, suggesting that distinct extracellular microenvironments might contribute to the different roles of H3R in microglial functions in vitro and in vivo. Microglial functions, such as chemotaxis and phagocytosis, are essential for the maintenance of a healthy CNS. However, under
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pathological conditions, such as Alzheimer's disease and schizophrenia, abnormally activated microglia have devastating effects on neurons through excessive synaptic pruning and elimination of healthy neurons, which in turn leads to disease progression [33e35]. Thus, silencing abnormal microglia enhances neuronal viability and survival and alleviates various neuropsychiatric disorders [33]. Indeed, Webster et al. have shown that excessive activation of microglia during brain ischemia exacerbates brain damage, and that the inhibition of microglial chemotaxis to ischemic regions prevents neuronal death [36]. Moreover, blockade of abnormal phagocytosis in animal models of Alzheimer's disease dramatically reduces neuronal loss and improves neurotransmission [33,37]. Previous studies using H3R inverse agonists have shown their therapeutic potential in CNS diseases, and the present study describes the inhibitory effects of JNJ on chemotaxis and phagocytosis. These lines of evidence indicate that microglial inhibition by H3R inverse agonists might have therapeutic benefit in neuropsychiatric disorders, such as brain ischemia and Alzheimer's disease, although further studies are necessary to examine the roles of microglial H3R in animal models of human brain diseases. Recent studies indicated that the increase in inflammatory cytokines secreted from activated microglia played causative roles in depression [27,38]. Especially, IL-1b, a key pro-inflammatory cytokine, is up-regulated in the brain of patients with depression and inhibition of IL-1b signalling is sufficient to prevent depression-like behaviour [39,40]. We demonstrated that JNJ suppressed LPSinduced microglial IL-1b, IL-6 and TNFa expression, indicating that JNJ inhibited the activation of microglia. We also showed that
Fig. 4. JNJ treatment suppressed microglial activation and improved depression-like behaviour. A, mRNA expression levels of IL-1b, IL-6, and TNFa in purified microglia from mouse brain after injection of saline, LPS (1 mg/kg), JNJ (10 mg/kg), or LPS plus JNJ (n ¼ 3e4). mRNA expression levels of IL-1b, IL-6, and TNFa in purified microglia were measured using quantitative PCR. B, IL-1b protein levels in the mouse brain after injection of saline, LPS (1 mg/kg), JNJ (10 mg/kg), or LPS plus JNJ (n ¼ 3e4). The levels of IL-1b protein were measured using an enzyme-linked immunosorbent assay. C, Total immobility time in the tail suspension test after injection of saline, LPS (1 mg/kg), JNJ (10 mg/kg), or LPS plus JNJ (n ¼ 6e8). Data are presented as mean ± standard error of the mean. Differences were identified using one-way ANOVA and multiple comparisons tests; *P < 0.05; **P < 0.01.
Please cite this article in press as: T. Iida, et al., JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.05.081
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JNJ administration reduced IL-1b protein levels and decreased depression-like behaviours. Thus, the inhibitory effect of JNJ on cytokine secretion from activated microglia contributed to the reduction of depression-like behaviours, although we could not rule out the possibility that JNJ also affected other cell types and altered the behaviours. In conclusion, to our knowledge, this is the first study to reveal that JNJ10181457, a H3R inverse agonist, has an inhibitory role in in vivo microglial functions. In addition, LPS-induced depressionlike behaviour was improved with decreasing IL-1b protein expression in the mouse brain. Our finding could promote better understandings of CNS disorders involved in the brain histaminergic system and might lead to the development of new drugs that target microglial H3R.
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Acknowledgement The authors have no conflicts of interest. This work was supported by a Grant-in-Aid for JSPS Research Fellow (No. 16J05282) and Grant-in-Aid for Scientific Research (A) (No. 26253016) from the Japan Society for the Promotion of Science, and by Nishinomiya Basic Research Fund, Japan, and by Tohoku University Division For Interdisciplinary Advanced Research and Education. We also acknowledge the support of the Biomedical Research Core of Tohoku University Graduate School of Medicine and the Biomedical Research Unit of Tohoku University Hospital.
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JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice Ka rpa ti a, Takuro Matsuzawa a, Tomomitsu Iida a, Takeo Yoshikawa a, *, Aniko Haruna Kitano a, Asuka Mogi a, Ryuichi Harada a, Fumito Naganuma a, b, Tadaho Nakamura a, b, Kazuhiko Yanai a a
Department of Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan Division of Pharmacology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 April 2017 Accepted 14 May 2017 Available online xxx
Brain histamine acts as a neurotransmitter and regulates various physiological functions, such as learning and memory, sleep-wake cycles, and appetite regulation. We have recently shown that histamine H3 receptor (H3R) is expressed in primary mouse microglia and has a strong influence on critical functions in microglia, including chemotaxis, phagocytosis, and cytokine secretion in vitro. However, the importance of H3R in microglial activity in vivo remains unknown. Here, we examined the effects of JNJ10181457 (JNJ), a selective and potent H3R inverse agonist, on microglial functions ex vivo and in vivo. First, we injected ATP, which is a typical chemoattractant, into hippocampal slices to investigate the effect of JNJ on chemotaxis. ATP-induced microglial migration toward the injected site was significantly suppressed by JNJ treatment. Next, we examined whether JNJ affected microglial phagocytosis in hippocampal slices and in the prefrontal cortex. Microglial engulfment of dead neurons induced by Nmethyl-D-aspartate was inhibited in the presence of JNJ. The increase in zymosan particle uptake by activated microglia in the prefrontal cortex was prevented by JNJ administration. Finally, we determined the importance of JNJ in a lipopolysaccharide (LPS)-induced depression model. JNJ reduced the LPSinduced upregulation of microglial pro-inflammatory cytokines and improved depression-like behaviour in the tail-suspension test. These results demonstrate the inhibitory effects of JNJ on chemotaxis, phagocytosis, and cytokine production in microglia inside the brain, and highlight the importance of microglial H3R for brain homeostasis. © 2017 Elsevier Inc. All rights reserved.
Keywords: Histamine Histamine H3 receptor Microglia Depression
1. Introduction Histamine, which is a bioactive amine, regulates various physiological events, such as allergic reactions, gastric acid secretion, and itch sensation. In the central nervous system (CNS), histamine acts as a neurotransmitter. Neuronal histamine is enzymatically synthesized in histaminergic neurons [1], which are located in the tuberomammilary nucleus in the posterior hypothalamus and project their axons to the entire brain [2]. Among the four G protein-coupled histamine receptors (H1ReH4R), H1R, H2R, and H3R are widely distributed in the CNS and regulate various physiological functions. H1R and H2R are expressed as postsynaptic
* Corresponding author. E-mail address: [email protected] (T. Yoshikawa).
receptors in various areas of the CNS and control sleep-wake cycles and aggression [3,4]. In contrast, H3R, which is an inhibitory G protein-coupled receptor, is an autoreceptor in presynaptic membranes of histaminergic neurons and regulates histamine release [5]. H3R also acts as a heteroreceptor in non-histaminergic neurons and modulates the release of various neurotransmitters, including GABA and acetylcholine [6]. In addition, H3R is the most abundant histamine receptor in the CNS. Consistent with its abundance and strong impact on neurotransmitter release, H3R is involved in diverse brain functions, such as memory, cognition, appetite, and arousal [7]. These findings have attracted attention toward H3R in the CNS from a broad range of disciplines. Preclinical and clinical trials have studied the therapeutic effects of H3R inverse agonists on various neurological disorders, such as epilepsy and attentiondeficit hyperactivity disorder [8e10]. Indeed, pitolisant, an H3R
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inverse agonist, was approved for the treatment of narcolepsy by the European Medicine Agency in 2016 [11]. Recent studies have revealed that H3R is also expressed in glial cells. Astrocytes, which are the most abundant cells in the CNS, express H3R, and activation of H3R enhances the expression and synthesis of neurotrophin-3 [12]. However, H1R, and not H3R, has a predominant role in astrocytic functions [13,14]. Our recent findings indicate that microglia, which are brain-resident macrophages, express H2R and H3R. Although we were not able to confirm the importance of H2R in primary mouse microglia, H3R was shown to regulate the levels of second messengers, such as intracellular Ca2þ and cyclic adenosine monophosphate, which in turn affect critical microglial functions including chemotaxis, phagocytosis, and cytokine secretion [15]. Thus, we hypothesize that H3R in microglia also plays a role in the CNS, and that H3R inverse agonists might exert therapeutic effects by regulating microglial H3R as well as neuronal H3R. Here we investigated the effects of a selective and potent H3R inverse agonist, JNJ10181457 (JNJ) [16], on microglial chemotaxis, phagocytosis, and cytokine production in ex vivo and in vivo assays, including in mouse models of human depression.
HBSS, 25% HBSS (Life Technologies), 25% horse serum (HyClone Laboratories; Logan, UT, USA), and 25 mM HEPES supplemented with 100 IU/mL penicillin G potassium and 100 mg/ml streptomycin sulphate. After stimulation with 50 mM N-methyl-D-aspartate (NMDA) (Tokyo Chemical Industry; Tokyo, Japan) in the presence or absence of 1 mM JNJ for 3 h, the slices were incubated with 0.5 mg/ml of propidium iodide (PI) (Tokyo Chemical Industry), which is a membrane-impermeable fluorescent dye for DNA, with or without 1 mM JNJ for 21 h. Fluorescent images were captured using a Nikon C2si microscope (Nikon; Tokyo, Japan). Microglial phagocytosis was evaluated using Bz-9000 (Keyence; Osaka, Japan), and the images were analysed using Hybrid Cell Count image analysis software (Keyence). 2.5. Immunohistochemistry for CD68 Fixed slices were incubated with a primary antibody for CD68 (Abcam; Cambridge, UK) in 20% bovine serum albumin (SigmaAldrich; St. Louis, MO, USA) in phosphate-buffered saline (Wako) overnight at 4 C. The sections were then treated with a secondary antibody.
2. Materials and methods 2.1. Mice CX3C chemokine receptor 1 (CX3CR1)-green fluorescent protein (GFP) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) [17]. All experiments were performed according to the Principles for Care and Use of Research Animals of Tohoku University, Sendai, Japan. All experiments involving animals are reported in accordance with Animal Research: Reporting of in vivo Experiments guidelines [18,19]. 2.2. Organotypic hippocampal slice cultures Organotypic hippocampal slices were prepared as described previously [20]. The hippocampus was cut into 120- (for chemotaxis assay) or 350 mm-thick (for ex vivo phagocytosis assay) sections using a MacIlwain tissue chopper (Mickle Laboratory Engineering; Gomshall Lane, UK). 2.3. Chemotaxis assay Hippocampal slices were pre-incubated with or without 10 mM JNJ (Tocris Bioscience; Bristol, UK) containing dissection buffer with the following composition: Modified Eagle's Medium (MEM)/ Hank's Balanced Salt Solution (HBSS) (Life Technologies; Carlsbad, CA, USA), 10 mM Tris-HCl (pH 7.2), 25 mM HEPES (Life Technologies) supplemented with 100 IU/mL penicillin G potassium (Wako Pure Chemical Industry; Osaka, Japan) and 100 mg/ml streptomycin sulphate (Wako). Injection of 3 mM ATP (Wako) was performed using a microinjector (BJ-110; BEX; Tokyo, Japan) with the following specifications: diameter of pipette tip, 8 mm; pressure, 0.04 MPa; duration, 100 m sec. Images were captured every 30 s using a fluorescent microscope (IX-71; Olympus; Tokyo, Japan). Microglial chemotaxis was analysed as described previously [21]. Briefly, the fluorescent intensity in region Rx (100 mm in radius from the injected site) at each time point Rx(t) was subtracted from the first image Rx(0). Microglial chemotaxis at any time point is calculated using the equation Rx(t) e Rx(0). 2.4. Ex vivo phagocytosis assay The hippocampal slices were pre-incubated for 1 h at 37 C with slice culture medium with the following composition: 50% MEM/
2.6. Quantitative real-time PCR RNA isolation, reverse transcription, and PCR were performed as described previously [15]. The sequences of the primers are listed in Table 1. 2.7. In vivo phagocytosis assay In vivo phagocytosis was assayed as described previously [22]. Yeast-derived zymosan particles conjugated with Alexa 568 (Thermo Fisher Scientific Inc.; Waltham, MA, USA) was stereotaxically injected into the prefrontal cortex at the following coordinates: anterior, 0.3 mm; lateral, ± 1.2 mm; and ventral, 1.6 mm from bregma. We intracranially co-administered 10 mg/uL of lipopolysaccharide (LPS) (Sigma-Aldrich) and/or 10 mM JNJ with the zymosan particles. We injected the mice with 10 mg/kg JNJ or saline intraperitoneally for the next 3 consecutive days. Four days after the zymosan injection, brain coronal sections (50 mm-thick) were prepared using a vibratome (Leica; Wetzlar, Germany) after perfusion with 4% paraformaldehyde (Wako). Phagocytosis was quantified by counting the numbers of engulfed beads in microglia using a fluorescent microscope and appropriate software (Keyence).
Table 1 Primer sequences for PCR. Gene GAPDH Sense Antisense CD68 Sense Antisense IL-1b Sense Antisense IL-6 Sense Antisense TNF-a Sense Antisense
Primer sequence
Product size (bp)
50 -AGAACATCATCCCTGCATCC-30 50 -CACATTGGGGGTAGGAACAC-30
91
50 -ACTGGTGTAGCCTAGCTGGT-30 50 -CCTTGGGCTATAAGCGGTCC-30
85
50 -TGCCACCTTTTGACAGTGATG-30 50 -TGATGTGCTGCTGCGAGATT30
138
50 -CCGGAGAGGAGACTTCACAG-30 50 -CAGAATTGCCATTGCACAAC-30
134
50 -CCCTCACACTCACAAACCAC-30 50 -ACAAGGTACAACCCATCGGC-30
133
GAPDH, glyceraldehyde-6-phosphate dehydrogenase; IL-1b, interleukin-1b; TNF-a, tumour necrosis factor a.
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2.8. Tail suspension test
2.9. Measurement of interleukin-1b protein (IL-1b) levels
Mice were injected with saline, LPS (1 mg/kg), JNJ (10 mg/kg), or both LPS and JNJ. Four hours after the injection, a tail suspension test was performed as previously described, with minor modifications [23]. Mice were suspended 50 cm above the floor by holding their tails using a clip. The immobility time over the 6 min test was recorded and analysed.
Brain tissues were homogenized in 50 mM Tris-HCl buffer containing 1 mM EDTA (pH 7.4) and centrifuged at 1000 g for 10 min at 4 C. The supernatants were centrifuged at 20,000 g for 40 min at 4 C to remove remaining debris. Total protein and IL-1b levels was determined using a bicinchoninic acid assay kit (Thermo) and an IL-1b ELISA kit (R & D systems; Minneapolis, MN,
Fig. 1. JNJ suppressed microglial chemotaxis. (A) Representative images of microglial migration induced by the application of 3 mM ATP in hippocampal slices from CX3CR1-GFP mice without (upper panels) or with 10 mM JNJ pre-treatment (lower panels). ATP was locally applied (T: 0 min, left side) and microglial responses were observed for 30 min (T: 30 min, right side). Microglial process motility was tracked using coloured lines. Scale bars ¼ 100 mm. (B) Quantitative analysis of microglial migration. Medium without ATP was applied (black line, n ¼ 3). ATP was applied without (blue line, n ¼ 4) or with 10 mM JNJ pre-treatment (Orange line, n ¼ 4). Cumulative ratio of fluorescent intensity at each time point was calculated as a bar graph. Data are presented as mean ± standard error of the mean. Differences were identified using one-way ANOVA and multiple comparisons tests; *P < 0.05; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. JNJ suppressed microglial phagocytosis in hippocampal slices. A, Representative images of CX3CR1- GFP microglia (green) and PI (red) in hippocampal slices treated with 50 mM NMDA in the presence or absence of JNJ (1 mM). Scale bars ¼ 100 mm. B, PI-positive area in GFP-positive microglia with or without JNJ (1 mM) treatment was measured (n ¼ 8). C, Representative images of CX3CR1-GFP microglia (green) and immunostaining for CD68 (red) in hippocampal slices treated with 50 mM NMDA in the presence or absence of JNJ (1 mM). Scale bars ¼ 100 mm. D, CD68-stained area in GFP-positive microglia with or without JNJ (1 mM) treatment was measured (n ¼ 4). E, mRNA expression levels of CD68 in hippocampal slices treated with 50 mM NMDA in the presence or absence of JNJ (1 mM) (n ¼ 4). mRNA expression levels of CD68 were measured using quantitative PCR. Data are presented as mean ± standard error of the mean. Differences were identified using Student's t-tests; *P < 0.05; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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USA), respectively. 2.10. Microglial isolation Microglial isolation was performed as previously described, with minor modifications [24]. Brain tissues were minced and exposed to collagenase (final concentration: 1 mg/mL) (Roche; Basel, Switzerland) for 20 min at 37 C. The tissues were then incubated with DNase І solution (Roche) for 5 min at 37 C. Dissociated cells were passed through a 100-mm nylon mesh cell strainer (BD Falcon; Franklin Lakes, NJ, USA) and centrifuged at 600 g for 5 min at 4 C. After removal of debris, cells were resuspended in 30% percoll (GE Healthcare; Princeton, NJ, USA) solution and centrifuged for 10 min at 700 g. At least 90% of the pelleted cells were immunopositive for the microglial marker CD11b (data not shown). 2.11. Statistical analysis Data analysis was performed using Student's t-tests or one-way ANOVA using GraphPad Prism 5 software (GraphPad; La Jolla, CA, USA). P-values less than 0.05 were considered significant. 3. Results 3.1. JNJ suppressed microglial chemotaxis in hippocampal slices Microglial movement towards the injured site is required to start the removal of wounded tissues in the CNS [21]. Since ATP leakage from injured cells is a strong chemoattractant and induces microglial migration, we examined the effects of JNJ on chemotaxis in response to local ATP injection into acute hippocampal slices. Although capillary insertion slightly induced microglial movement, ATP administration significantly promoted microglial migration to the injection site (Fig. 1A and B). In the presence of JNJ, microglial responses to ATP were significantly reduced. This demonstrates that JNJ has an inhibitory effect on microglial chemotaxis. 3.2. JNJ suppressed phagocytosis of dead cells in hippocampal slices Engulfment of dead cells by phagocytosis is essential to the repair of damaged tissue [25]. To investigate the effects of JNJ on microglial phagocytosis in hippocampal slices, we induced neuronal death using NMDA and evaluated the microglial engulfment of dead neurons. The numbers of PI-positive neuronal nuclei in the microglia were reduced following JNJ administration, suggesting that JNJ inhibits phagocytosis activity of microglia (Fig. 2A and B). We also evaluated the expression of CD68, which is a marker of phagocytic microglia [26]. Protein and mRNA expression levels of CD68 were also suppressed in JNJ-treated hippocampal slices (Fig. 2CeE). These results suggest that JNJ inhibits the phagocytic activity of microglia. 3.3. In vivo phagocytosis was inhibited by JNJ We injected yeast-derived zymosan particles into the prefrontal cortex of the mice to investigate the effects of JNJ on in vivo phagocytosis. LPS significantly increased the numbers of zymosan particles engulfed by microglia (Fig. 3A and B). JNJ did not affect zymosan engulfment in the absence of LPS stimulation, but completely abolished the increase in phagocytosis following LPS administration. These results indicate that JNJ reduces microglial engulfment in the inflammatory conditions induced by LPS treatment.
Fig. 3. JNJ suppressed microglial phagocytosis in vivo. A, CX3CR1-GFP microglia (green) engulfed fluorescent zymosan particles (red) in control, JNJ-, LPS-, or LPS- and JNJ-treated mouse prefrontal cortex. Scale bars ¼ 200 mm. B, Quantitative analysis of the numbers of engulfed zymosan particles by microglia (n ¼ 3). Data are presented as mean ± standard error of the mean. Differences were identified using one-way ANOVA and multiple comparisons tests; *P < 0.05; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. JNJ treatment suppressed microglial activation and improved depression-like behaviour Abnormal microglial activation exacerbates various neurological disorders, including depression [27]. In contrast, normalisation of excessive microglial activity has therapeutic effects on neuropsychiatric diseases [28]. We examined whether inhibitory effects of JNJ on microglia could improve depression-like behaviour in mice. LPS-induced depression-like behaviour is mediated by cytokine secretion from abnormally activated microglia. JNJ attenuated LPS-induced increases in the expression levels of IL-1b, IL-6, and TNF-a in purified microglia (Fig. 4A). IL-1b protein, which is a key cytokine responsible for depression-like behaviours, was especially significantly decreased in the presence of JNJ (Fig. 4B). Moreover, the prolongation of immobile time induced by LPS in
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the tail-suspension test was attenuated following JNJ treatment (Fig. 4C). These results indicate that JNJ has an inhibitory effect on microglial activation and improves depression-like behaviour in mice.
4. Discussion Here, we report the inhibitory effects of JNJ on microglial chemotaxis and phagocytosis using ex vivo and in vivo assays. We also report that JNJ reduces inflammatory cytokine expression and improves depression-like behaviour. This is the first report on the importance of H3R in critical microglial functions in the CNS. Contrary to the results of this study, our previous report using primary mouse microglia revealed that H3R agonists, but not inverse agonists, inhibit in vitro microglial functions [15]. In the brain, neurons around microglia secrete neurotransmitters, such as norepinephrine and serotonin, which considerably alter microglial activity [29,30]. Neurons are also in direct contact with microglia via the interaction between CD200 and the CD200 receptor, which leads to the inhibition of microglia [31]. Indeed, brain-resident microglia and primary microglia isolated from the neonatal cortex have different properties, including gene expression patterns [32]. These lines of evidence demonstrate the strong impact of the local environment on microglial activity, suggesting that distinct extracellular microenvironments might contribute to the different roles of H3R in microglial functions in vitro and in vivo. Microglial functions, such as chemotaxis and phagocytosis, are essential for the maintenance of a healthy CNS. However, under
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pathological conditions, such as Alzheimer's disease and schizophrenia, abnormally activated microglia have devastating effects on neurons through excessive synaptic pruning and elimination of healthy neurons, which in turn leads to disease progression [33e35]. Thus, silencing abnormal microglia enhances neuronal viability and survival and alleviates various neuropsychiatric disorders [33]. Indeed, Webster et al. have shown that excessive activation of microglia during brain ischemia exacerbates brain damage, and that the inhibition of microglial chemotaxis to ischemic regions prevents neuronal death [36]. Moreover, blockade of abnormal phagocytosis in animal models of Alzheimer's disease dramatically reduces neuronal loss and improves neurotransmission [33,37]. Previous studies using H3R inverse agonists have shown their therapeutic potential in CNS diseases, and the present study describes the inhibitory effects of JNJ on chemotaxis and phagocytosis. These lines of evidence indicate that microglial inhibition by H3R inverse agonists might have therapeutic benefit in neuropsychiatric disorders, such as brain ischemia and Alzheimer's disease, although further studies are necessary to examine the roles of microglial H3R in animal models of human brain diseases. Recent studies indicated that the increase in inflammatory cytokines secreted from activated microglia played causative roles in depression [27,38]. Especially, IL-1b, a key pro-inflammatory cytokine, is up-regulated in the brain of patients with depression and inhibition of IL-1b signalling is sufficient to prevent depression-like behaviour [39,40]. We demonstrated that JNJ suppressed LPSinduced microglial IL-1b, IL-6 and TNFa expression, indicating that JNJ inhibited the activation of microglia. We also showed that
Fig. 4. JNJ treatment suppressed microglial activation and improved depression-like behaviour. A, mRNA expression levels of IL-1b, IL-6, and TNFa in purified microglia from mouse brain after injection of saline, LPS (1 mg/kg), JNJ (10 mg/kg), or LPS plus JNJ (n ¼ 3e4). mRNA expression levels of IL-1b, IL-6, and TNFa in purified microglia were measured using quantitative PCR. B, IL-1b protein levels in the mouse brain after injection of saline, LPS (1 mg/kg), JNJ (10 mg/kg), or LPS plus JNJ (n ¼ 3e4). The levels of IL-1b protein were measured using an enzyme-linked immunosorbent assay. C, Total immobility time in the tail suspension test after injection of saline, LPS (1 mg/kg), JNJ (10 mg/kg), or LPS plus JNJ (n ¼ 6e8). Data are presented as mean ± standard error of the mean. Differences were identified using one-way ANOVA and multiple comparisons tests; *P < 0.05; **P < 0.01.
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JNJ administration reduced IL-1b protein levels and decreased depression-like behaviours. Thus, the inhibitory effect of JNJ on cytokine secretion from activated microglia contributed to the reduction of depression-like behaviours, although we could not rule out the possibility that JNJ also affected other cell types and altered the behaviours. In conclusion, to our knowledge, this is the first study to reveal that JNJ10181457, a H3R inverse agonist, has an inhibitory role in in vivo microglial functions. In addition, LPS-induced depressionlike behaviour was improved with decreasing IL-1b protein expression in the mouse brain. Our finding could promote better understandings of CNS disorders involved in the brain histaminergic system and might lead to the development of new drugs that target microglial H3R.
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Acknowledgement The authors have no conflicts of interest. This work was supported by a Grant-in-Aid for JSPS Research Fellow (No. 16J05282) and Grant-in-Aid for Scientific Research (A) (No. 26253016) from the Japan Society for the Promotion of Science, and by Nishinomiya Basic Research Fund, Japan, and by Tohoku University Division For Interdisciplinary Advanced Research and Education. We also acknowledge the support of the Biomedical Research Core of Tohoku University Graduate School of Medicine and the Biomedical Research Unit of Tohoku University Hospital.
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Please cite this article in press as: T. Iida, et al., JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.05.081