Reduced locomotor activity and exploratory behavior in CC chemokine receptor 4 deficient mice

Accepted Manuscript Title: Reduced locomotor activity and exploratory behavior in CC chemokine receptor 4 deficient mice Author: Oliver Ambr´ee Irene Klassen Irmgard F¨orster Volker Arolt Stefanie Scheu Judith Alferink PII: DOI: Reference:

S0166-4328(16)30476-4 http://dx.doi.org/doi:10.1016/j.bbr.2016.07.041 BBR 10348

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

1-4-2016 20-7-2016 22-7-2016

Please cite this article as: Ambr´ee Oliver, Klassen Irene, F¨orster Irmgard, Arolt Volker, Scheu Stefanie, Alferink Judith.Reduced locomotor activity and exploratory behavior in CC chemokine receptor 4 deficient mice.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2016.07.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reduced locomotor activity and exploratory behavior in CC chemokine receptor 4 deficient mice

Oliver Ambrée1,2,*, Irene Klassen1, Irmgard Förster3, Volker Arolt1, Stefanie Scheu4,#, and Judith Alferink1,5, #

1 Department of Psychiatry, University of Münster, Münster, Germany 2 Department of Behavioural Biology, University of Osnabrück, Osnabrück, Germany 3 Immunology and Environment, Life & Medical Sciences Institute (LIMES), University of Bonn, Germany 4 Institute of Medical Microbiology and Hospital Hygiene, University of Düsseldorf, Düsseldorf, Germany 5 Cluster of Excellence EXC 1003, Cells in Motion, University of Münster, Münster, Germany #

equal last authorship

* Correspondence: Oliver Ambrée, Department of Psychiatry, University of Münster, AlbertSchweitzer-Campus 1, Building A9, D-48149 Münster, Germany. E-mail address: [email protected]. Present address: Department of Behavioural Biology, University of Osnabrück, Barbarastr. 11, D-49078 Osnabrück, Germany. E-mail address: [email protected]

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Highlights 

CCR4-/- mice showed reduced locomotion, object exploration, and social exploration

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Altered behavior in the elevated plus-maze might be affected by reduced locomotion

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CCR4-/- mice displayed unaffected object recognition memory compared to WT mice

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CCR4-/- mice presented differences in nest building compared to WT mice

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Except increased exploration CCL17-/- mice showed unaltered behavior compared to WT

Abstract Chemokines and their receptors are key regulators of immune cell trafficking and activation. Recent findings suggest that they may also play pathophysiological roles in psychiatric diseases like depression and anxiety disorders. The CC chemokine receptor 4 (CCR4) and its two ligands, CCL17 and CCL22, are functionally involved in neuroinflammation as well as anti-infectious and autoimmune responses. However, their influence on behavior remains unknown. Here we characterized the functional role of the CCR4-CCL17 chemokine-receptor axis in the modulation of anxiety-related behavior, locomotor activity, and object exploration and recognition. Additionally, we investigated social exploration of CCR4 and CCL17 knockout mice and wild type (WT) controls. CCR4 knockout (CCR4-/-) mice exhibited fewer anxiety-related behaviors in the elevated plus-maze, diminished locomotor activity, exploratory behavior, and social exploration, while their recognition memory was not affected. In contrast, CCL17 deficient mice did not show an altered behavior compared to WT mice regarding locomotor activity, anxiety-related behavior, social exploration, and object recognition memory. In the dark-light and object recognition tests, CCL17-/- mice even covered longer distances than WT mice. These data demonstrate a mechanistic or developmental role of CCR4 in the regulation of locomotor and exploratory behaviors, whereas the ligand CCL17 appears not to be involved in the behaviors measured here. Thus, either CCL17 and the alternative ligand CCL22 may be redundant, or CCL22 is the main activator of CCR4 in these processes. Taken together, these findings

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contribute to the growing evidence regarding the involvement of chemokines and their receptors in the regulation of behavior.

Keywords Behavior; behavioral phenotyping; knockout mouse; chemokine, chemokine receptor

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Introduction Chemokines and their G protein-coupled receptors are key modulators of leukocyte trafficking and activation of immune responses and neuroinflammation [1]. In addition, they control homeostatic chemotaxis of migratory cells within tissues. While many chemokines are widely expressed in the central nervous system (CNS) during neuroinflammation, they have also been found in the developing and adult CNS under non-inflammatory conditions. Various cells types in the CNS release chemokines under steady state conditions that mediate direct and indirect cellular effects on adjacent neurons, microglia, and glial cells [2,3]. Chemokines and their receptors play a key role in CNS development and homeostasis by controlling proliferation, differentiation, and migration of neural precursor cells, mature neurons, and glia [4]. Furthermore, they are involved in inflammatory responses associated with psychiatric disorders including depression and anxiety disorders [5,6]. There is increasing interest in understanding their pathophysiological roles by utilizing translational studies and mouse models. Based on studies in mice, several chemokines and their receptors have been reported to modulate various behaviors, including stress responses. For example, it has been shown that the CC chemokine receptor (CCR) 5 regulates olfactory and social recognition in mice [7]. The CC chemokine ligand (CCL) 2 is highly expressed by microglia during neuroinflammation and promotes migration of lymphocytes into the CNS [8]. CCL2 expression in the brain is further induced by repeated social defeat stress in mice that contributes to prolonged anxiety-related behavior [9]. Consequently, mice deficient in CCR2, which is associated with monocyte trafficking, or deficient in the microglial fractalkine receptor (CX3CR1), failed to recruit macrophages to the brain or develop anxiety-related behavior following repeated social defeat [10]. Deficiency in CXCR5 was associated with an increase in locomotor activity in an open field test and decreased stress reactivity [11]. Behavior and neuroendocrine responses to stress in mice following maternal separation was altered in the absence of CCR7, the common receptor for CCL19 and CCL21 [12]. Jaehne and Baune further demonstrated that signaling through CCR6 and CCR7 regulates locomotor activity and anxiety-related behavior in mice [13].

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CCR4 is the high-affinity receptor for CCL17 and CCL22 and expressed by various immune cells, including functionally diverse T cell subsets and myeloid cells [14-18]. It modulates T helper (Th) 2associated immune processes, but also plays a prominent role in innate immune responses. For example, CCR4 deficient (CCR4-/-) mice showed increased resistance to LPS-induced lethality or bacterial peritonitis [14,19]. During homeostasis and inflammation dendritic cells are the primary source of CCL17 and, in together with macrophages, also produce CCL22 [20-22]. CCL17 has been implicated in allograft rejection and the pathogenesis of various immune-mediated diseases such as allergic inflammation, atopic skin diseases, and atherosclerosis [4,18,21-24]. In the CNS, expression of CCR4 has been reported on activated microglia and neurons in the hippocampus and dorsal root ganglia [25,26]. We and others previously demonstrated a unique role for CCR4 to modulate neuroinflammation and disease severity in a mouse model of multiple sclerosis, underscoring the importance of chemokine ligand and receptor pairs in mediating CNS inflammation [25,27,28]. Based on its neuronal expression, CCR4 has been suggested to be a modulator of neuronal functions [26,29]. However, the role of the CCR4-CCL17 axis in regulating behavior has yet to be resolved. In this study, we investigated the role of CCR4 and CCL17 in locomotor activity, anxiety-related behavior, social exploration, as well as object exploration and recognition. For this, we subjected CCR4-/- and CCL17 -/- mice and wildtype (WT) controls to various behavioral tests, including open-field, dark-light, elevated plus maze, social exploration and object recognition tests.

Material and methods Animals CCR4-/- and CCL17-/- mice have been generated as described before [14,22]. Both lines have been backcrossed onto the C57BL/6J background for at least 15 generations. In the present study, 8 female CCR4-/- mice and 10 female CCL17-/- mice have been investigated and compared to the same number of female C57BL/6J mice. All mice were bred in the SPF facility of the University of Münster Medical School. Mice were weaned at 28 days of age and housed in groups of 3–4 littermates in

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transparent standard polycarbonate cages (Makrolon, type II-L, 37 x 21 x 14 cm3) with sawdust bedding. All animals had free access to food (Altromin no. 1324, Altromin GmbH, Lage, Germany) and water. A 12 h light, 12 h dark cycle with lights on at 06:00 am, temperature of 211°C, and humidity of 5010% was maintained in the housing room. Behavioral testing started at 11 weeks of age in the same order as described below with at least three days between two subsequent tests. All experiments were performed by an experimenter blind for the genotype of the animals. Mice from one cage were tested successively before those from the next cage were tested. Cages of WT and knockout groups alternated in the order of testing.

Behavioral analysis Elevated Plus-Maze The elevated plus-maze (EPM) consists of four arms (30 x 7 cm2 each) each extending from the center (7 x 7 cm2) at an angle of 90°. Two opposing arms were enclosed by 15 cm high walls. The center of the EPM was dimly lit by a single light source from directly above (40 lx). At the beginning of the test, mice were placed in the center facing a closed arm and were then allowed to explore the maze for 5 min. Arm entries were defined as entering an arm with all four paws. Arm entries and time spent in arms were acquired by a blinded observer using ANY-maze software (Stoelting, Dublin, Ireland). Distance traveled was automatically tracked by the software. The relative number of entries into and the relative time spent in the open arms were calculated as ratio of open arm entries/time divided by total arm entries/time.

Open field test The open field test (OF) was conducted in a wooden box with a square area of 80 x 80 cm 2 surrounded by 40 cm high walls. The center area had a defined size of 40 x 40 cm 2 and was moderately illuminated (72 lx). Mice were placed close to the wall, in the middle between two adjacent corners and allowed to freely explore the arena for 8 min. Total distance traveled was recorded as measure of locomotor activity; the number of entries into and the time spent in the

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center were recorded as measure of anxiety-related behavior. These measures were automatically acquired by ANY-maze tracking software (Stoelting, Dublin, Ireland).

Dark-light test The dark-light test (DL) was performed in a modified Makrolon type III cage (1/3 dark, 2/3 light) as described before [30]. The bright zone was illuminated at 126 lx.

Social exploration test The social exploration test (SET) was performed in a wooden box with a square area of 42 x 42 cm2 with 40 cm high walls. A wire-mesh enclosure of 8 x 6 cm2 was located in the middle of one wall. The interaction zone was defined as the area surrounding this enclosure 8 cm to each side. At the opposite wall, corner zones of 9 x 9 cm2 were defined for each corner. The test comprised two trials of 150 s each. In trial 1 (T1) the enclosure was empty, in trial two (T2) an unfamiliar C57BL/6J mouse of the same sex was presented in the enclosure to measure social exploration. At the beginning of each trial the mouse was placed into one of the corner zones. The position of the animals and the duration of stay in the defined zones was automatically tracked by the ANY-maze software (Stoelting, Dublin, Ireland). The interaction ratio was calculated as the time spent in the interaction zone during the second trial in relation to the time in the interaction zone in the first trial.

Object recognition test The object recognition test (ORT) was conducted in a wooden box with a square area of 40 x 40 cm2 with 40 cm high walls. It consisted of two trials of 5 min each. In trial 1 (T1; acquisition trial) mice can explore two identical objects. In trial 2 (T2; test trial) animals can explore a novel object and a familiar one. Objects were placed left and right of the center on midline between the anterior and posterior wall of the box. Two types of bottles were used as objects, one made from glass, one from plastic also differing in size and shape. In preliminary investigations we showed that animals had no preference for either of the objects. Furthermore, the order of the objects (familiar/novel) and the location of the novel object (left/right) were systematically rotated to avoid any effects of object or place preference in this experiment. Exploration was defined as the nose directed towards the object 7

within one head-length and was analyzed from video files by a trained observer blind for the genotype of the animals. Distance traveled was automatically tracked by ANY-maze (Stoelting, Dublin, Ireland). As measure of object recognition memory, a recognition index was calculated as the exploration time of the novel object related to the total exploration time of the novel and familiar object in trial 2.

Nest building behavior Mice were habituated to cotton nestlets during group housing. One week after the social exploration test, CCR4-/- mice and wild type controls were separated and housed individually in Makrolon type II cages with a new cotton nestlet one hour before lights out. In addition, 8 CCL17-/- and 8 WT controls of the same sex and age, however without prior behavioral testing underwent this procedure. The next morning, all nests were scored from 1 (no nest) to 5 (perfect nest) according to [31] by two trained experimenters. In case of deviating scores between them the mean of the two ratings was used.

Statistics Data were checked for normal distribution by Shapiro-Wilk test. Normally distributed data of knockout and WT mice were analyzed by Student’s t test for independent data. In addition, Levene’s test for equality of variances was calculated. In the case of significantly different variances, corrected p-values as automatically calculated by SPSS were reported. Data deviating from normal distribution were analyzed by non-parametric Mann-Whitney U-test. For the analysis of locomotor activity in the open field per minute a repeated measures ANOVA with within subject factor time (T) and between subject factor genotype (G) was calculated. The level of significance was set at p < .05 for all tests. All analyzes were calculated with SPSS 23 (IBM).

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Results Altered behavior in the elevated plus-maze in CCR4-/- mice In order to assess the role of CCR4 in anxiety-related behavior 8 CCR4-/- and 8 WT mice were subjected to the elevated plus maze (EPM), open field (OF), and dark-light test (DL). In the EPM CCR4/-

mice showed increased entries into open arms relative to total arm entries (Fig. 1A, U = 9, p = .015).

CCR4-/- mice also spent more time in open arms relative to the total time spent on either arm (Fig. 1B, t = –2.075, p = .066). While these findings suggested that CCR4-/- mice exhibit a reduced anxietyrelated behavior our additional measures demonstrated reduced frequencies of total arm entries (enclosed and open) in these mice (Fig. 1C, t = 2.887, p = .012) pointing towards reduced locomotor activity. We also determined that CCR4-/- mice exhibited decreased absolute entries of closed arms (t = 6.779, p < .001) and time spent in closed arms (t = 4.190, p < .001) but no absolute increase in open arm entries and time spent therein (entries: t = –.153, p = .881; time: t = –1.478, p = .161). These data underscored the notion that CCR4 affects locomotor activity complicating the interpretation of the measures of anxiety-related behavior. In the OF no behavioral differences between the genotypes with regard to entries into the center (Fig. 1D, t =.995, p = .336) and time spent in the center (Fig. 1E, t = –.042, p = .967) were observed indicating that CCR4 does not affect anxiety-related behavior in this test. In the DL test CCR4-/- mice displayed equivalent entries into the light zone (Fig. 1G, t = 1.904, p = .078), spent equivalent time therein (Fig. 1H, t = 1.665, p = .118) and showed comparable latency to enter it (Fig. 1I, U = 17, p = .125). In this test, the mean numbers of light zone entries, time spent in the light zone and latency to enter it pointed into the opposite direction as in the EPM. These findings demonstrate that CCR4 deficiency reduced anxiety-related behavior only in the EPM, which was possibly affected by altered locomotor activity, but not in the OF and DL test indicating no prominent effect on anxiety-related behavior.

Decreased locomotor activity in CCR4-/- mice In order to assess locomotor activity in CCR4-/- mice, the total distance traveled by these animals was analyzed in the OF. We also included behavioral tests that are not classically used for the assessment 9

of locomotor activity such as the EPM, the social exploration test (SET), the object recognition test (ORT), and the DL test. In the OF CCR4-/- mice traveled a significantly shorter distance than WT mice demonstrating that CCR4-/- mice exhibited a reduced locomotor activity (Fig. 1F, U = 10, p = .021). Analysis per minute revealed that both genotypes habituated comparably to the unfamiliar open field as indicated by a decrease of locomotion over time (Suppl. Fig. 1 A, RM-ANOVA: T: F(4.3, 60.1) = 11.130, p < .001; TxG: F(4.3, 60.1) = 1.092, p = .371). They also covered less distances in the EPM (Suppl. Fig. 1B, U = 3, p = .001), the SET (Suppl. Fig. 1D,E, T1: t = 2.533, p = .024, T2: t = 2.318, p = .036), and the ORT (Suppl. Fig. 1F,G, T1: t = 3.876, p = .002, T2: t = 4.754, p < .001) while no significant differences were found in the DL test (Suppl. Fig. 1C, t = 1.395, p = .185). The results from these behavioral tests point towards a locomotor activity phenotype in CCR4-/- mice.

Decreased social exploration and object exploration but no alteration in recognition memory in CCR4-/- mice Social behavior was assessed in the social exploration test (SET) consisting of two trials. CCR4-/- mice exhibited reduced social exploration with the unfamiliar conspecific presented in an enclosure inside the test apparatus in the second trial (Fig. 2A, t = 3.660, p = .003). However, this effect was not specific for the social partner, since CCR4-/- mice also showed a reduced exploration time of the empty enclosure in the first trial thus resulting in an unchanged interaction ratio (Fig. 2B, t = –.695, p = .498). These data demonstrate that both, exploration of the empty enclosure and of the social partner were reduced in CCR4-/- mice. In addition, there was a tendency that CCR4-/- mice spent more time in the corners during the social exploration trial (Fig. 2C, t = –2.253, p = .055) likely the result of their reduced drive to approach and interact with the social partner. With regard to object exploration and object recognition memory, CCR4-/- mice showed reduced exploration of the objects (Fig. 2D, t = 4.059, p = .001). Additional data showing that the recognition ratio was not altered between both genotypes (Fig. 2E, t = –1.603, p = .131) indicated that object recognition memory was not impaired in the absence of CCR4.

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Differences in nest building in CCR4-/- mice In order to elucidate whether reduced exploration and activity levels in CCR4-/- mice also affects ethologically relevant behaviors, we analyzed nest building. As indicated in Figure 2F, CCR4-/- mice built their nests significantly worse compared to WT controls (Fig. 2F, U = 12, p = .034).

No effect of CCL17 on anxiety-related behavior and locomotor activity We next studied whether the CCR4 ligand CCL17 affects behavior and subjected 10 CCL17 deficient and 10 WT mice to various behavioral tests. In contrast to CCR4-/- mice, neither relative entries into open arms (Fig. 3A, t = –.306, p = .763) nor relative time spent on open arms of the EPM (Fig. 3B, t = 1.565, p = .135) differed significantly in CCL17-/- mice from WT mice. CCL17 deficiency did also not affect the number of total arm entries in the EPM (Fig. 3C, t = .108, p = .915). Furthermore, the number of center entries (Fig. 3D, t = –.410, p = .687) and the time spent in the center of the OF (Fig. 3E, U = 44, p = .684) were equivalent in CCL17-/- and WT mice. In the DL test, CCL17 deficient mice exhibited no differences in the number of entries into the light zone (Fig. 3G, t = –1.182, p = .254), the time spent therein (Fig. 3H, U = 37, p = .342), and the latency to enter it (Fig. 3I, U = 37, p = .328) when compared to WT controls. Thus, CCL17-/- mice did not exhibit altered anxiety-related behavior in the EPM, OF, and DL test. In contrast to CCR4-/- mice, CCL17 deficient and WT mice traveled an equivalent distance in the OF indicating that CCL17 does not affect locomotor activity in this test (Fig 3F, t = –1.283, p = .216). They also did not differ with regard to habituation to the open field over time (Suppl. Fig. 2A, RM-ANOVA: T: F(3.7, 66.3) = 32.217, p < .001; TxG: F(3.7, 66.3) = 1.311, p = .276). In the DL and in the ORT T2, however, CCL17-/- mice traveled a significantly longer distance when compared to WT mice (DL Suppl. Fig. 2C, t = –2.910, p = .010), (ORT T2 Suppl. Fig. 2G, t = –2.334, p = .031). These data demonstrate that in contrast to CCR4, CCL17 does not influence anxiety-related behavior activity. In the DL test and ORT however CCL17-/- mice exhibited an enhanced locomotor activity thus presenting an opposite behavior when compared to CCR4-/- mice.

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Increased exploration in CCL17-/- mice without affecting recognition memory We next investigated the effects of CCL17 on social behavior, object exploration, and recognition. There was no difference in social behavior between CCL17-/- and WT mice, neither in the time spent in the interaction zone in T2 (Fig. 4A, t =.407, p = .689), the interaction ratio (Fig. 4B, t = .337, p = .740), nor the time in the corner zone in T2 (Fig. 4C, t = .574, p = .573). However, CCL17-/- mice showed increased exploration of the objects in T2 in the ORT (Fig. 4D, t = –2.359, p = .036) indicating that CCL17 mediates opposing effects to CCR4 on object exploration. In analogy to CCR4 -/- mice, CCL17-/- mice displayed an equivalent recognition index when compared to controls (Fig. 4E, t = .502, p = .622) demonstrating that CCL17 and CCR4 do not affect recognition memory.

No differences in nest building in CCL17-/- mice With regard to nest building behavior, 8 CCL17-/- and 8 WT mice of same age and sex as the other animals investigated in this study were tested. There was no difference between CCL17-/- and WT mice (Fig. 4F, t = –1.234, p = .238).

Discussion Chemokines are small molecules that fulfill important messenger functions in immune responses [32,33]. However, their role in the regulation of behavior remains largely unknown. The aim of this study was to investigate the impact of CCR4 and its ligand, CCL17 on locomotor, emotional, and cognitive behaviors. We found that CCR4-/- mice displayed fewer anxiety-related behaviors in the elevated plus maze compared to WT controls, but not in additional anxiety-related tests. CCR4-/- mice further exhibited reduced locomotor activity and object exploration, diminished social exploration, and nest building compared to WT animals. Deficiency in CCL17 however resulted in a different behavioral phenotype. CCL17 deficiency did not affect locomotor activity, and even increased it in the dark-light test and the object recognition test. Object exploration was also enhanced in CCL17-/-

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mice, suggesting that CCL17 and the second CCR4 ligand, CCL22 may regulate CCR4 signaling differentially. Our findings from the elevated plus maze suggest that deficiency of CCR4 results in altered anxietyrelated responses. CCR4 deficiency resulted in increased relative open arm entries and enhanced time spent on open arms, which are generally interpreted as reduced anxiety-related behaviors [34,35]. Regulation of anxiety-related behavior by chemokine receptors has been reported before. For instance, CCR6 signaling modulates anxiety-related behavior under basal conditions [13], while CCR2 and CX3CR1 are involved in stress-induced anxiety-related behaviors that are mediated by inflammatory monocytes migrating to the brain [10]. In humans, the chemokines CCL2, CCL5, and CXCL12 have been reported as biomarkers of generalized anxiety disorder with comorbid personality disorder [36]. However, in additional anxiety-related tests such as open field and dark-light test anxiety-related behaviors were not altered in the absence of CCR4. Thus, additional factors may influence the performance of CCR4-/- mice in the elevated plus maze. It has been suggested that elevated plus maze behavior involves a cognitive component [37]. Cognitively impaired mice exhibit a reduced capability for learning and remembering the potential danger of open or the enhanced protection of closed arms [38]. The relatively increased open arm entries observed in CCR4-/- mice could therefore reflect potential cognitive impairments in these animals. However, CCR4-/- mice did not exhibit severe cognitive deficits at least with regard to recognition memory. Alternatively, it is possible that a reduced locomotor activity of CCR4-/- mice as shown by diminished total arm entries might interfere with anxiety-related measures in the elevated plus maze. It has been demonstrated before, that the forced exposure to the elevated plus maze induces open arm entries [39]. In combination with a reduced activity, this could explain the behavior of CCR4-/- mice in the elevated plus maze, characterized by unaltered frequency of open arm entries and time spent on open arms, but reduced frequencies and time of closed arm visits. These findings preclude final conclusions on the behavior of CCR4-/- mice in the elevated plus maze, but argue against a prominent effect of CCR4 on overall anxiety-related behavior.

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The behavioral phenotype observed in CCR4-/- mice was strongly characterized by decreased locomotor activity. CCR4-/- mice showed reduced distances traveled in nearly all tests, reduced social exploration, and diminished object exploration. It has been demonstrated before that mice under test situations develop mild stress in response to the novel environments [40]. To minimize a potential stress response in our experiments, testing conditions were chosen with low illumination levels. In addition, no differences were observed in the habituation over time to the open field in CCR4-/- and WT mice, speaking against a stress-induced reduction of locomotor activity in the absence of CCR4 (Suppl. Fig. 1A). Our findings support earlier studies demonstrating that CCRs are functionally involved in locomotor and exploratory activity. In analogy to CCR4-/- mice, CCR7-/- mice display reduced exploratory activity [13]. In contrast, elevated locomotor activity has been found in CCR6-/- and CXCR5-/- mice [11,13]. However, despite diminished object exploration, CCR4-/- mice did not exhibit deficits in object recognition memory. We further observed reduced nest building abilities in CCR4-/- mice. We cannot exclude that this behavior may result from a reduced general activity level in these mice. Nest building provides a shelter for mice from predators or harsh weather conditions allowing better heat conservation and thermoregulation [41]. It is therefore an ethologically relevant behavior especially for mice in the wild. We did not observe overt differences in nests of group housed CCR4-/- mice when compared to WT mice (data not shown) suggesting that CCR4-/- mice are able to build nests but may need more time than WT mice because of their overall reduced activity. So far, CCR4-/- mice showed no overt differences in offspring survival [14]. However, it is still possible that reduced nest building abilities or general activity levels might affect maternal care with profound influences on the offsprings’ behavioral development [42-44]. Therefore, future studies in CCR4-/- mice are needed to address specifically the potential impact of maternal behavior on the observed behavioral phenotype by cross-fostering studies or breeding of heterozygous animals in order to separate genetic and nongenetic effects.

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To analyze whether the CCR4 ligand CCL17 is required to modulate the observed behavioral alterations, we also characterized the behavior of CCL17-/- mice. In striking contrast to CCR4-/- mice, object exploration was enhanced in CCL17-/- mice. Thus, CCL17 appears unnecessary for, or inhibits the regulation of locomotor, exploratory, and social behavior. This points towards a prominent role for CCL22, the other ligand of CCR4, in this context. In immune responses, CCL17 and CCL22 have been demonstrated to mediate redundant as well as non-redundant functions. These differences have been explained by distinct in vivo expression patterns of the ligands, their differential capacity to induce CCR4 desensitization and internalization, and the fact that CCR4 exists in at least two conformations [45-49] that are differentially activated by each ligand. CCL17 and CCL22 may therefore represent selective ligands for the two conformations of the receptor. Alternatively, it is possible that CCL17 and CCL22 are not expressed and released in the same brain regions meaning that CCL22 is the critical regulator of these processes. It is also possible, that both ligands are redundantly released and CCL22 is able to compensate for the lack of CCL17. Mice deficient for CCL22 or deficient for both CCR4 ligands may provide valuable insights into the specific effects of each ligand in these behavioral domains. The mechanisms underlying CCR4 mediated regulation of behavior are still unexplained. In general, there are two possibilities: First, CCR4 could alter behavior by organizing effects during development, e.g. by affecting neural circuits or endocrine regulation mechanisms. Along this line, several reports favor a functional role for chemokines and their receptors in nervous system development, the most prominent example represents the CXCR4 receptor and its ligand CXCL12 [4]. Second, behavioral alterations in CCR4 deficient mice might be caused by immediate activating or regulating effects of CCR4 signaling such as direct actions on the nervous system or regulation of adult neurogenesis [11] or neural signaling [26,50,51]. Furthermore, it has been shown that neuroimmune circuitry regulates depression and anxiety-related behavioral responses. For example, exposing mice to lipopolysaccharide (LPS) reduces locomotor activity. An imbalance of kynurenine pathway metabolites causing reduced serotonin production has been suggested to underlie inflammationassociated depressive disorders and associated behavioral alterations [52]. Along this line, 15

chemokine receptors have been identified on serotonergic neurons and could thus affect serotonergic neurotransmission in brain regions involved in the regulation of depression. However, little is known about the mechanisms that mediate chemokine-dependent effects on behavior in the absence of an inflammatory response. In addition to CCR4 expression on immune cells, CCR4 has also been found on brain endothelial cells [53], microglia, and astrocytes [54], as well as on cultured hippocampal neurons [26]. Expression of CCR4 has also been identified in brain regions that control locomotor and exploratory behavior and social interaction, such as the striatal dopamine system [55,56] or the hippocampus which controls accumbal dopamine transmission and modulates locomotor activity [57] and social behavior [58,59]. CCR4 is also expressed in the anterior hypothalamus where its ligand CCL22 increases body temperature through cyclooxygenasedependent activation of brown adipose tissue [60]. With regard to neuronal function, it has been shown that CCR4 ligands affect neuronal calcium signaling in cultured hippocampal neurons [26]. Additional studies demonstrated that a subset of dorsal root ganglia neurons induced Ca2+ influx after binding of CCL17 [29]. CCR4 may affect hippocampal-dependent behaviors through regulation of hippocampal neural activity. Taken together, these data illustrate that CCR4 is widely expressed in the brain where its activation might control a variety of functions including the regulation of locomotor, exploratory, and social behaviors. Which cell types, brain regions, and pathways are involved in these processes need to be determined in future studies. All experiments in this study have been performed with adult female mice. Female mice exhibit an estrous cycle of 4-5 days shown before to affect behavior in some cases. However, open field behavior has been shown to be unaffected by different stages of the cycle in C57BL/6 mice [61]. Since all experiments in this study were performed with mice backcrossed for at least 15 generations to this C57BL/6 background and the observed phenotype was reproduced in various behavioral tests conducted on different test days, it is unlikely that differences in the phenotype observed were brought about by different cycle phases. Future studies will address the question, whether CCR4mediated effects on locomotor and exploratory behavior differ between males and females.

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Taken together, our study provides evidence that CCR4 is involved in the modulation of locomotor, exploratory, and social behavior as well as nest building. It remains open whether these effects are mediated via immediate activating/inhibiting or developmental organizing effects of CCR4. Deficiency in the CCR4 ligand CCL17 did not phenocopy the knockout of the receptor, suggesting that the second CCR4 ligand CCL22 is also or exclusively involved in CCR4-dependent regulation of this behavioral phenotype. Future studies will be needed to unravel the underlying mechanisms of CCR4dependent behavioral alterations and whether the CCL17/CCL22/CCR4 axis is involved in human behavior and the pathogenesis of psychiatric disorders.

Funding This study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG], DFG EXC 1003, Grant FF-2014-01 Cells in Motion–Cluster of Excellence, Münster, Germany) to JA. JA is a member of the DFG funded cluster of excellence “Cells in Motion”. The study was also supported by Grant Alf3/018/16 of the Interdisciplinary Centre for Clinical Research (IZKF, Münster, Medical Faculty) to JA, by the fund “Innovative Medical Research” of the University of Münster Medical School (IMF AM211515) to OA, by the DFG SCHE692/3-1 to SS; by the Strategic Research Fund of the Heinrich Heine University Düsseldorf (SFF-F2012/79-5-Scheu) to SS, by the DFG through SFB704 to IF. IF is a member of the DFG funded cluster of excellence “ImmunoSensation”. The funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Conflicts of interest V.A. is member of advisory boards and/or gave presentations for the following companies: AstraZeneca, Eli Lilly, Janssen-Organon, Lundbeck, Otsuka, Servier, and Trommsdorff. All other authors declare that they have no conflicts of interest.

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Acknowledgements We thank Christiane Schettler and Arezoo Fattahi-Mehr for excellent technical assistance and genotyping of mice. Furthermore, we greatly appreciate the assistance of the veterinarian Dr. Sandra Stöppeler from the Animal Facility of the University of Münster Medical School for providing photos of nests from mouse lines in SPF housing facilities.

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Figure Legends Figure 1 CCR4-/- mice show increased relative entries into open arms (A) and a tendency for increased relative time spent on the open arms (B) of the elevated plus-maze (EPM) when compared to WT controls. The total number of arm entries (C) was significantly lower in CCR4-/- mice. In the open field test (OF) CCR4-/- mice did not differ from wild type controls with regard to the anxiety-related measures center entries (D) and center time (E) while they ran significantly less during the 8 min test session (F). In the dark-light test (DL) a tendency for reduced entries into the light zone was observed in CCR4 knockout mice (G) while the time spent in the light zone (H) and the latency to enter it (I) did not differ from wild type mice. *: p < .05; for all variables and both groups n = 8. Figure 2 The time spent in the interaction zone in T2 (A) of the social exploration test (SET) was significantly lower in CCR4-/- mice compared to WT controls. There was no difference between the genotypes with regard to the interaction ratio (B), while the time spent in the opposite corners in T2 (C) was significantly higher in CCR4-/- mice. In the object recognition test (ORT) the total time exploring the objects in T2 (D) was significantly reduced in CCR4-/- mice while the recognition index (E) was not different between the genotypes. The nest building test (NBT) revealed significantly reduced nesting behavior (F) in CCR4-/- mice. *: p < .05, **: p < .01; for all variables and both groups n = 8. Figure 3 CCL17-/- mice did not differ from wild types in the elevated plus maze (EPM), neither in entries into open arms (A), time spent on open arms (B) nor the number of total arm entries (C). There was also no difference between the genotypes regarding center entries (D), time spent in the center (E) and the distance traveled (F) in the open field (OF). In the dark-light test (DL) the number of entries (G) and the time spent in the light zone (H) were not different, either. While the mean latency to enter the light zone (I) was markedly reduced in CCL17 deficient mice, it did not reach the level of statistical significance. For all variables and both groups n = 10. 22

Figure 4 CCL17-/- mice did not differ from wild types in the social exploration test, neither in the time in the interaction zone in T2 (A), the interaction ratio (B), nor the time spent in the opposite corner in T2 (C). In the object recognition test (ORT) there was a significant increase in the total time spent exploring the objects in CCL17-/- mice as compared to WT controls (D) while the recognition index was equal (E). The nest building test (NBT) revealed no differences in nesting behavior (F) between CCL17-/- and WT mice. *: p < .05¸ for all variables except nest building and both groups n = 10; for nest building, both groups n = 8.

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