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ackr2 in bone remodeling
Bone 101 (2017) 113–122
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Contribution of atypical chemokine receptor 2/ackr2 in bone remodeling Izabella Lucas de Abreu Lima a, Janine Mayra da Silva a, Letícia Fernanda Duffles Rodrigues b, Davidson Frois Madureira a, Angélica Cristina Fonseca c, Gustavo Pompermaier Garlet d, Mauro Martins Teixeira e, Remo Castro Russo f, Sandra Yasuyo Fukada g, Tarcília Aparecida da Silva h,⁎ a
Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil Department of Pediatric Dentistry and Orthodontics, Faculty of Dentistry, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil Department of Biological Sciences, School of Dentistry of Bauru, São Paulo University, FOB/USP, Al. Octávio Pinheiro Brisola 9-75, CEP 17012-901 Bauru, SP, Brazil d Department of Biological Sciences, School of Dentistry of Bauru, São Paulo University, FOB/USP, Al. Octávio Pinheiro Brisola 9-75, CEP 17012-901 Bauru, SP, Brazil e Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil f Laboratory of Pulmonary Immunology and Mechanics, Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil g Department of Physics and Chemistry, Faculty of Pharmaceutical Science of Ribeirão Preto, São Paulo University, Avenida do Café, s/n, Cidade Universitária, 14040-903 Ribeirao Preto, SP, Brazil h Department of Oral Pathology and Surgery, Faculty of Dentistry, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil b c
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Article history: Received 5 December 2016 Revised 29 April 2017 Accepted 2 May 2017 Available online 3 May 2017 Keywords: Chemokines Orthodontics Bone remodeling
a b s t r a c t Introduction: Bone remodeling is a tightly regulated process influenced by chemokines. ACKR2 is a decoy receptor for CC chemokines functioning as regulator of inflammatory response. In this study we investigated whether the absence of ACKR2 would affect bone phenotype and remodeling induced by mechanical loading. Methods: An orthodontic appliance was placed between incisors and first molar of ACKR2 deficient (ACKR2−/−) and C57BL6/J (wild-type/WT) mice. Microtomography, histology and qPCR were performed to evaluate bone parameters, orthodontic tooth movement (OTM), bone cells counts and the expression of ACKR2, bone remodeling markers, CC chemokines and chemokines receptors. Bone marrow cells (BMC) from WT and ACKR2−/− mice were differentiated in osteoclasts and osteoblasts for analysis of activity and expression of specific markers. Results: Mechanical stimulus induced ACKR2 production in periodontium. The expression of ACKR2 in vitro was mostly detected in mature osteoclasts and early-differentiated osteoblasts. Although ACKR2−/− mice exhibited regular phenotype in maxillary bone, the amount of OTM, osteoclasts counts and the expression of pro-resorptive markers were increased in this group. In contrast, the number of osteoblasts and related markers were decreased. OTM resulted in augmented expression of CC chemokines and receptors CCR5 and CCR1 in periodontium, which was higher in ACKR2−/− than WT mice. In vitro experiments demonstrated an augmented formation of osteoclasts and diminished differentiation of osteoblasts in ACKR2−/− mice. Conclusions: These data suggests that ACKR2 functions as a regulator of mechanically-induced bone remodeling by affecting the differentiation and activity of bone cells and the availability of CC chemokines at periodontal microenvironment. Therapeutic strategies based on increase of ACKR2 might be useful to hinder bone loss in inflammatory conditions. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Orthodontic tooth movement (OTM) is achieved by remodeling of periodontal ligament and alveolar bone [1–4]. Chemokines and their receptors are key molecules in this process by affecting osteoclasts
⁎ Corresponding author at: Departamento de Clínica, Patologia e Cirurgia Odontológicas, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Av. Presidente Antônio Carlos 6627, CEP 31.270-901 Belo Horizonte, Minas Gerais, Brazil. E-mail addresses: [email protected] (G.P. Garlet), [email protected] (S.Y. Fukada), [email protected] (T.A. Silva).
http://dx.doi.org/10.1016/j.bone.2017.05.003 8756-3282/© 2017 Elsevier Inc. All rights reserved.
recruitment and activity [1–3,5–8]. Studies in humans demonstrated a differential expression of CC chemokines at compression and tension sites of periodontal ligament submitted to mechanical force [5]. Furthermore, functional studies proved the relevance of CC chemokines as CCL2, CCL3, CCL5 and chemokines receptors (CCRs) CCR1, CCR2 and CCR5 in different models of alveolar bone remodeling, including orthodontic tooth movement [7–11]. Recently, the novel atypical chemokine receptor (ACKR) subfamily, comprising four molecules, was described [12,13]: ACKR1, previously named duffy antigen receptor for chemokines (DARC); ACKR2, previously known as D6 or CCBP2; ACKR3, also called CXC-chemokine
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receptor 7 (CXCR7) or RDC1; and ACKR4, previously called CC chemokine receptor-like 1 (CCRL1) or CCX-CKR [13]. Of these, ACKR2 was initially recognized as a CCL3 binding receptor [14]. Subsequently, it was demonstrated that ACKR2 is able to interact with most agonists of CC chemokine receptors, from CCR1 through CCR5, because of significant homology with these molecules [14,15]. Therefore, ACKR2 is known as a scavenger/decoy of CC chemokines in inflamed sites [16] functioning as a regulator of inflammatory response [14,17]. Considering that the expression of CC chemokines and respective receptors is augmented at periodontal tissues under mechanical loading [5,7–9], and that previous results implicated such molecules in bone remodeling [9–11,18,19], herein we hypothesized that ACKR2 could modify the bone resorptive/formation process by regulating the local availability of CC chemokines. 2. Materials and methods 2.1. Animals Ten-week-old wild-type (WT) (C57BL6/J) and ACKR2 deficient mice (ACKR2−/−) included in this study were obtained from Laboratory of Immunopharmacology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG). All animals were treated under Institutional Ethics Committee regulations for animal experiments (protocol #130/2012). For every set of experiments, 5 mice were used at each time-point. Each animal weight was recorded throughout the experimental period, in which no significant weight loss was observed. Mice were maintained under standard condition with a 12 h light/dark cycle, controlled temperature (24 ± 2 °C) and free access to commercial chow and drinking water.
(Tb.N), trabecular separation (Tb.Sp), bone volume (BV), bone surface (BS), tissue volume (TV), bone surface/volume ratio (BS/BV), bone surface density (BS/TV), percent bone volume (BV/TV), intersection surface (iS), trabecular bone pattern factor (Tb.Pf) and trabecular thickness (Tb.Th).
2.4. Histology Samples obtainment was performed as previously described [20]. Briefly, the anterior maxillae containing first, second and third molars were fixed in 10% buffered formalin, decalcified in 14% EDTA (pH 7.4) for 21 days and embedded in paraffin. The entire blocks were cut into sagittal sections of 4 μm thickness. The slides selected for bone cells counting presented the first and second molars, their mesial and distal-buccal root, the third molar, and adjacent structures, including the periodontal ligament and alveolar bone. At least five serial vertical sections containing the above mentioned structures were evaluated for each animal for each analysis.
2.5. Bone cells counting The distal-buccal root of the first molar, on its coronal two-thirds of the mesial periodontal site, was used for counting osteoclasts stained for tartrate resistant acid phosphatase (TRAP; Sigma-Aldrich, Saint Louis, MO, USA). The distal bone of the first molar distal-buccal root, on its mesio-coronal two-thirds, was used for osteoblast counting, stained with Masson's trichrome.
2.2. Orthodontic tooth movement 2.6. Osteoclast and osteoblast cultures The experimental protocol for OTM was based on a previous study [20]. Briefly, a nickel titanium coil (Lancer Orthodontics, Inc., Vista, San Marcos, CA, USA) was bonded by a light cored resin (Transbond, Unitek/3M, Monrova, CA, USA) between maxillary first molar and incisors at the right side of the maxilla. The force magnitude was set at 0.35 N applied in mesial direction; it was calibrated by a tension gauge (Shimpo Instruments, Itasca, IL, USA). The coil was not reactivated throughout the experimental period. The left side of the maxilla was used as control. The amount of OTM was measured through the difference between the distance of the cemento-enamel junction (CEJ) of first and second molar of the experimental side (right hemimaxilla) in relation to the control side (left hemi-maxilla) of the same animal. The measurements were obtained on microtomography (MicroCT) images. Three measurements were conducted for each evaluation and the variability was below 5%. For MicroCT and histological analysis, WT and ACKR2−/− groups were euthanatized with an overdose of anesthetic after 12 days of mechanical loading. For molecular examination, these groups were euthanatized at 0 and 72 h. 2.3. MicroCT imaging Maxillae, with the orthodontic appliance in situ, were scanned, using Skyscan 1176 (Bruker-MicroCT, Kontich, Belgium). Scanning trajectory was round and a filter of 0.5 mm was used. A 12.45 μm camera pixel size with camera XY Ratio of 0.9870, voltage of 50 kV and current of 500 μA was used. Images were analyzed using Dataviewer and CTAn software (Bruker-MicroCT). The threshold for analysis was set at 33. Maxillae were examined in fixed coronal and sagittal zones for analysis of bone density. The region of interest was standardized as circular in the alveolar bone region between the first molar roots. Bone phenotype was evaluated in maxillae on all three planes. The parameters evaluated were tissue surface (TS), trabecular number
Osteoclasts and osteoblasts were differentiated from bone marrow cells (BMCs). Tibias and femurs of WT and ACKR2−/− mice were flushed using α-MEM (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL of penicillin, and 100 mg/mL of streptomycin. For osteoclast formation, BMCs were plated in 10 mL of α-MEM with 30 ng/mL of M-CSF (R&D Systems, Minneapolis, MN, USA) and cultured for 3 d. The adherent cells (osteoclast precursors) were plated in 24-well plates at a density of 2 × 105 cells/ well and stimulated with M-CSF (10 ng/mL) and RANKL (30 ng/mL) for 48 and 72 h as previously described [20]. Then, cells were stained with TRAP (Sigma-Aldrich) and percentage of TRAP-positive cells was determined by counting the TRAP-positive cells with 3 or more nucleus in 10 fields per well at 40× magnification. For pit resorption assay, cells were plated on Corning® Osteo Assay Surface (Corning Life Sciences, NY, USA) and pits formation evaluated after 10 days. Data were expressed as number of pits/field. For osteoblast differentiation, subconfluent cells in primary culture were plated in 24 well plate at a density 5 × 104 cells/well and cultured in osteogenic medium (α-MEM supplemented with 10− 7 M dexamethasone (Sigma-Aldrich), 5 μg/mL ascorbic acid (GIBCO), and 7 mM β-glycerophosphate (Sigma-Aldrich). The calcium deposits formed at 14 and 21 days were quantified using alizarin solution (1%). After staining, alizarin deposit was eluted in 10% solution of acetic acid and methanol (4:1 v/v) and absorbance quantified by spectrophotometry at 490 nm. Bone cells viability was determined using 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyl- tetrazolium bromide (MTT) reduction assay (Vybrant® MTT Cell Proliferation Assay Kit, Invitrogen). Briefly, cells were incubated with MTT solution for 4 h at 37 °C. Formazan crystals were extracted by adding dimethyl sulfoxide (DMSO) (Sigma-Aldrich) according to the manufacturer's instructions. After 15 min, the absorbance was measured at 540 nm.
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Fig. 1. ACKR2 expression in vivo and in vitro. (A) ACKR2 expression in periodontium of C57BL6/J mice after 72 h of mechanical loading. (B) ACKR2 expression by bone marrow cells-derived osteoclasts and (C) osteoblasts. Data are expressed as mean ± SEM. *P b 0.05 comparing control to respective experimental group.
Table 1 Bone parameters in maxillae of wild-type and ACKR2 deficient mice. Parameters 2
TS μm Tb.N μm−1 Tb.Sp μm BV μm3 BS μm2 TV μm3 BS/BV μm−1 BS/TV μm−1 BV/TV % iS μm3 Tb.Pf μm−1 Tb.Th μm
WT (mean ± SEM) 2
2
11810 × 10 ± 564 × 10 3.79 × 10−3 ± 0.24 × 10−3 156.10 ± 5.87 3.98 ± 0.20 13560 × 102 ± 583 × 102 8.09 ± 0.56 3.41 × 10−2 ± 0.14 × 10−2 1.68 × 10−2 ± 0.06 × 10−2 49.39 ± 1.36 5626 × 102 ± 174 × 102 1.06 × 10−2 ± 0.10 × 10−2 131.40 ± 9.41
ACKR2−/− (mean ± SEM)
P values
10220 × 102 ± 381 × 102 4.62 × 10−3 ± 0.59 × 10−3 156.20 ± 15.34 3.93 ± 0.79 12590 × 102 ± 1765 × 102 6.67 ± 0.55 3.28 × 10−2 ± 0.30 × 10−2 1.86 × 10−2 ± 0.12 × 10−2 58.32 ± 8.44 6507 × 102 ± 1026 × 102 0.84 × 10−2 ± 0.34 × 10−2 126.50 ± 9.74
0.0800 0.2666 0.9972 0.9539 0.6289 0.1478 0.7228 0.2510 0.3558 0.4448 0.5791 0.7365
Parameters related to bone mineral density obtained by MicroCT of the alveolar bone region between the first molar roots. TS, tissue surface; Tb.N, trabecular number; Tb.Sp, trabecular separation; BV, bone volume; BS, bone surface; TV, tissue volume; BS/BV, specific bone surface; BS/TV, bone surface density; BV/TV, percent bone volume; iS, intersection surface; Tb.Pf, trabecular bone pattern actor; Tb.Th, trabecular thickness.
2.7. Real–time PCR Periodontal ligament and surrounding alveolar bone samples were obtained from upper first molars after 0 and 72 h of mechanical loading. Tissue samples and samples from osteoclasts and osteoblasts in vitro were submitted to the extraction of total RNA (RNeasyFFPE kit, Qiagen Inc., Valencia, CA, USA) according to the manufacturers' instructions. The integrity of RNA samples was checked by analyzing 1 mg of total RNA on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The
complementary DNA (cDNA) was synthesized using 3 μg of RNA through a reverse transcription reaction (Superscript III, Invitrogen Corporation, Carlsbad, CA, USA). The targets analyzed were: ACKR2, runt-related transcription factor 2 (RUNX2); dentin matrix acidic phosphoprotein (DMP); receptor activator of nuclear factor kappa B (RANK); receptor activator of nuclear factor kappa B ligand (RANKL); osteoprotegerin (OPG); metalloproteinase 13 (MMP13); interleukin-33 (IL-33); tumor necrosis factor alpha (TNF-α); interleukin-10 (IL-10), CCL3, CCL5, CCL2, CCR1 and CCR5. Real-Time PCR array was performed in a Viia7 instrument (Life Technologies) using TaqMan chemistry (Invitrogen) associated with inventoried optimized primers/probes sets (Invitrogen), with basic reaction conditions of 50 °C (20 s), 95 °C (10 min), (40 cycles) 95 °C (15 s) and 60 °C (1 min). For analysis of ACKR2 expression in samples from osteoclasts and osteoblasts culture, 18S was used as housekeeping reference gene, and the basic reaction conditions were (40 cycles) 95 °C (10 min), 94 °C (1 min), 56 °C (1 min) and 72 °C (2 min) for the other targets. The mean Ct values from duplicate measurements were used to calculate expression of the target gene, with normalization to an internal control (β-actin) using the 2−ΔΔCt method. Samples from osteoclasts and osteoblasts culture were also submitted to RNA extraction with the RNeasyFFPE kit (Qiagen Inc) according to the manufacturers' instructions. The integrity of RNA samples was checked by 2100 Bioanalyzer (Agilent Technologies), followed by cDNA synthesis (QuantiTect Reverse Transcription Kit, Qiagen). RealTimePCR was performed with Viia7 (Life Technologies) with TaqMan primers/probes sets according to the manufacturers' instructions, with standard amplification protocols. The results are expressed as target genes normalized by constitutive genes (GAPDH and ACTB), determined by the 2−ΔΔCt method.
Fig. 2. (A) Amount of orthodontic tooth movement in WT and ACKR2−/− mice after 12 days. MicroCT representative images of maxillae of WT mice at control side (B) and experimental side (C). MicroCT representative images of maxillae of ACKR2−/− mice at control side (D) and experimental side (E). White angulated arrows indicate the space between the crown of the first and second molars. White line indicated the space of periodontal ligament between the distal-buccal root and the mesial bone. White straight arrows indicate the direction of orthodontic OTM. Data were expressed as mean ± SEM. *P b 0.05. DB, distal alveolar bone; MB, mesial alveolar bone; R, root.
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2.8. Statistical analysis Results were expressed as mean ± SEM. As data sets presented normal distribution (Kolmogorov-Smirnov), one-way Analysis of variance (ANOVA) was used to analyze differences among
groups, followed by Newman-Keuls multiple comparison post hoc test. The data obtained from all evaluations were processed using GraphPad Prism (version 5.01, GraphPad Software, San Diego, CA, USA). The level of significance for all statistical tests was set at 5%.
Fig. 3. Number of TRAP-positive cells and osteoblasts at periodontal sites under mechanical loading. (A) Scheme showing the compression site (in gray) used for TRAP-positive cells count. (B) Quantification and (C) representative histological sections of TRAP-positive osteoclasts of WT and (D) ACKR2−/− mice after 12 days of mechanical loading. (E) Scheme showing the tension site (in gray) used for osteoblast count. (F) Quantification and (G) representative histological sections of osteoblasts of WT and (H) ACKR2−/− mice after 12 days of mechanical loading. Black angulated arrows indicate TRAP-positive cells (C–D) and white angulated arrows indicate osteoblasts (G–H). Black and white straight arrows indicate the direction of orthodontic tooth movement. Bar = 100 μm. Data are expressed as mean ± SEM. *P b 0.05 comparing control group to the respective experimental group. #P b 0.05 comparing WT to ACKR2−/− experimental groups. DB, distal alveolar bone; MB, mesial alveolar bone; R, root.
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3. Results 3.1. ACKR2 expression is induced by mechanical stimuli and osteoblasts and osteoclasts differentially express ACKR2 Initially, we assessed whether mechanical stimulus would interfere with ACKR2 expression in periodontium. It was observed an increased
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expression of ACKR2 in periodontal tissues at 72 h after force application (Fig. 1A). After that, in order to determine if cells involved with bone remodeling express ACKR2, we did analyze ACKR2 expression in bone marrow-derived osteoclasts and osteoblasts. Our results showed an increased expression of ACKR2 by mature osteoclasts (72 h) in relation to early-differentiated cells (48 h) (Fig. 1B). In contrast, early osteoblasts
Fig. 4. mRNA expression of bone cells markers (A) RUNX2, (B) DMP, (C) RANK, (D) RANKL/OPG, (E) MMP13, (F) IL-33, and inflammatory molecules (G) TNF-α and (H) IL-10 in alveolar bone and periodontium samples of WT and ACKR2−/− mice after 72 h of mechanical loading. Data are expressed as mean ± SEM. *P b 0.05 comparing control to the respective experimental group. #P b 0.05 comparing WT to ACKR2−/− experimental groups. One-way ANOVA and Newman–Keuls multiple comparison test.
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(14 days) exhibited higher expression of ACKR2 when compared with mature osteoblasts (21 days) (Fig. 1C).
Furthermore, the lack of ACKR2 resulted in a disruption of bone lining cells and decrease of osteoblast numbers at the distal bone of distalbuccal root in relation to WT mice (Fig. 3E–H).
3.2. ACKR2−/− mice exhibit regular parameters of maxillary bone To evaluate if deficiency of ACKR2 would affect maxillary bone phenotype, we first analyzed bone parameters in maxillae using MicroCT. The comparison of bone parameters of ACKR2−/− and WT animals under steady state conditions showed no significant differences (Table 1). 3.3. Bone resorption is increased in ACKR2−/− mice subjected to mechanical loading Once ACKR2 deficiency did not result in modification of maxillary bone phenotype, we next submitted ACKR2 deficient mice to OTM to test bone response during mechanical demands. The amount of OTM (Fig. 2A–E) and TRAP positive osteoclasts (Fig. 3A–D) were significantly increased after 12 days of mechanical loading, in ACKR2−/− mice when compared to WT mice. The increased OTM in ACKR2−/− mice was also evidenced by greater space between molar's crown and a decreased space of periodontal ligament between the distal-buccal root and the mesial bone (Fig. 2C, E). Collectively, the amount of bone resorption was increased in ACKR2−/− than WT mice.
3.4. ACKR2−/− mice exhibited decreased expression of osteoblastic markers and increased expression of osteoclastic and inflammatory markers in periodontium Considering the decreased osteoblast numbers at tension sites in ACKR2−/− mice, we decided to evaluate if the expression of osteoblast markers RUNX2 and DMP was consistently reduced in these mice. In fact, we observed a decreased expression of these markers after 72 h of mechanical loading in ACKR2−/− mice (Fig. 4A–B). Since ACKR2−/− mice also presented increased osteoclast numbers, we next investigated markers of osteoclast activity RANK, RANKL, OPG and MMP13. Consistently, after 72 h of mechanical loading, ACKR2−/− mice exhibited increased RANKL/OPG ratio and expression of RANK and MMP13 (Fig. 4C–E). The expression of the anti-osteoclastogenic cytokine IL-33 was also significantly increased in ACKR2−/− mice when compared to WT mice (Fig. 4F). As OTM depends on inflammatory mediators, which, in turn, are regulated by ACKR2, we investigated the expression of TNF-α and IL-10. ACKR2−/− mice presented an increased expression of TNF-α (Fig. 4G)
Fig. 5. mRNA expression of CC chemokines and receptors (A) CCL3, (B) CCL5, (C) CCL2, (D) CCR1 and (E) CCR5 in alveolar bone and periodontium samples of WT and ACKR2−/− mice after 72 h of mechanical loading. Data are expressed as mean ± SEM. *P b 0.05 comparing control to the respective experimental group. #P b 0.05 comparing WT to ACKR2−/− experimental groups. One-way ANOVA and Newman–Keuls multiple comparison test.
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after 72 h of mechanical loading, however, no significant difference in mRNA expression of IL-10 was observed between the groups (Fig. 4H). 3.5. Augmented expression of CC chemokines and receptors in ACKR2−/− mice periodontium Chemokines and their receptors have pivotal functions in strainmanaged bone remodeling and the ACKR2 acts as agonist of these molecules. Indeed, the expression of CCL2, CCL3, CCL5, CCR1 and CCR5 was increased at periodontal sites after 72 h of mechanical loading in WT mice. Noteworthy, the augment of these molecules was significantly higher in ACKR2−/− when compared to WT mice (Fig. 5A–E).
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3.6. Osteoclast differentiation from BMCs is increased in ACKR2−/− mice In vitro experiments were performed in order to analyze if bone cells differentiation is modified in ACKR2−/− mice. Comparing BMCs derived from WT and ACKR2−/−, we observed that BMCs from ACKR2−/− mice generated increased numbers of osteoclasts after 72 h (Fig. 6A, B, D, E, G, H) and resorptive activity (Fig. 6C, F, I). In contrast, BMCs from ACKR2−/− mice resulted in reduced numbers of differentiated osteoblasts and mineralized nodules formation at 14 and 21 days (Fig. 6J–O). Bone cells differentiation were not linked to viability of BMCs, since it was higher than 95% and no differences were observed between the groups (data not shown).
Fig. 6. In vitro differentiation of Bone marrow cells from WT and ACKR−/− mice. (A, B) Osteoclasts from BMCs of wild-type (WT; C57BL6/J) mice after 48 and 72 h of cell differentiation, respectively. (G, H) Quantification of TRAP-positive cells derived from WT and ACKR2−/− mice after 48 and 72 h of differentiation. (D, E) Osteoclasts from BMCs of ACKR2−/− mice after 48 and 72 h of cell differentiation, respectively. (C, F) Resorption pit formation after treatment with RANKL of BMCs from WT and ACKR2−/− mice, respectively. (I) Resorption field in culture of osteoclasts. (J, K) Osteoblasts from BMCs of WT mice after 14 and 21 days of cell differentiation, respectively. (L, O) Quantification of nodule area formation by osteoblasts derived from WT and ACKR2−/− mice after 14 and 21 days of cell differentiation. (M, N) Osteoblasts from BMCs of ACKR2−/− mice after 14 and 21 days of cell differentiation, respectively. Data were expressed as mean ± SEM. *P b 0.05 comparing WT to ACKR2−/− experimental groups.
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Fig. 7. mRNA expression of BMCs-derived osteoclasts (A–C) and osteoblasts (D–H) in WT and ACKR2−/− mice. #P b 0.05 comparing WT to ACKR2−/− at the same time point. One-way ANOVA and Newman–Keuls multiple comparison test.
3.7. Bone markers expression in BMCs from ACKR2−/− and WT mice BMCs-derived osteoclasts from ACKR2−/− and WT mice exhibited similar expression of TRAP, cathepsin K, RANK, alkaline phosphatase (ALP) and Osterix (OSX) (Fig. 7A–C, G, H). Concerning BMCs-derived osteoblasts, a slightly increase of RUNX2 and collagen, type I, alpha 1 (COL1A1) expression was observed in ACKR2−/− mice at 21 days. In contrast, a reduction of bone sialoprotein (BSP) expression was detected at day 14 (Fig. 7D–F).
4. Discussion Alveolar bone remodeling during mechanical loading is modulated by the interplay between bone cells and leukocytes [21,22]. CC chemokines and its receptors are important players of this process [7–
9]. In the view of the potential role of the ACKR2 in the regulation of CC chemokines and consequently in bone remodeling, we initially analyzed maxillary bone in steady state conditions. Interestingly, similar bone parameters were observed for ACKR2−/− and WT mice, demonstrating that ACKR2 does not modify physiological bone remodeling. However, when maxillary bone was submitted to mechanical force a pronounced alveolar bone resorption was observed in ACKR2−/− mice. Such phenotype was associated with increase of osteoclasts counts and augmented expression of osteoclastic markers and chemokines. Consistently, bone marrow-derived cells from ACKR2−/− mice exhibited greater osteoclast differentiation. Our data also confirmed that mechanical stimulus induces ACKR2 expression and that ACKR2 is expressed by osteoclasts and osteoblasts. It is the first demonstration linking ACKR2 and bone. ACKR2 is able to interact with several agonists of CC chemokine receptors [14,15], consequently affecting their availability [23]. As the
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production of these molecules is triggered by mechanical force [7,8], it seems reasonable that the absence of such receptor only impacts bone phenotype in a condition where there is an increase of chemokines, such as mechanical loading induced-bone remodeling. Indeed, we observed that ACKR2−/− mice presented increased OTM and augmented expression of CCL2, CCL3, CCL5, CCR1 and CCR5. This result is in line with greater osteoclasts numbers and resorption in this group, since CC chemokines and receptors are positive signals for osteoclast recruitment and activation in infectious [10,11] and non-infectious conditions, affecting periodontal tissues [7,18]. Bone resorption is regulated by RANKL binding to the receptor RANK [5], and this event is prevented by a decoy receptor OPG, consequently inhibiting resorption [5,24]. Consistent with the phenotype observed in ACKR2−/− mice submitted to OTM, we verified an increase of RANKL/OPG ratio and of RANK and MMP-13, a matrix metalloproteinase with a critical role in bone resorption [25]. These results point to that the effects of ACKR2 in bone are at least in part mediated via RANK/RANKL. Previous studies demonstrated that CC chemokines are related with higher RANKL expression and consequently greater osteoclast differentiation and activity [26,27]. Our in vitro data revealed that in the absence of ACKR2, BMCs ability to generate osteoclasts is augmented, indicating an inhibitory effect of this receptor in osteoclast differentiation. Unexpectedly, the expression of specific osteoclastic markers by these cultures was similar between the groups. It can be associated with the subtle difference observed in cells counts and the timing of BMCs differentiation. As bone remodeling is a result of coupled function of osteoclasts and osteoblasts, we evaluate if ACKR2 deficiency would also modify osteoblasts. We observed that ACKR2−/− mice presented a decreased number of osteoblasts when compared to WT mice. Consistently, osteoblast markers, RUNX2 and DMP, were also decreased in our analysis. In vitro experiments confirmed a reduced formation of osteoblasts from BMCs of ACKR2−/− mice. As expected, the expression of BSP from these cells was reduced, but intriguingly, COL1A1 and RUNX2 were slightly higher in this group at day 21. It can suggest a delay of osteoblast differentiation with a persistence of early-differentiated cells at late stages of cell cultures from ACKR2−/− mice. We also detected the expression of ACKR2 in osteoblasts, mostly early-differentiated than mature cells. In line with these findings, an increased expression of ACKR2 was detected in mature osteoclasts. Taken together, these findings indicate that osteoblasts and osteoclasts participate in controlling CC chemokines availability in bone milieu and that this process is important to coordinate the function of these cells. ACKRs also affect the production of inflammatory mediators [16]. In this study, the expression of inflammatory molecules involved in bone remodeling as TNF-α [28,29], IL-10 [9] and IL-33 [20] was evaluated. We did obtain an increased expression of TNF-α in ACKR2 deficient mice. This is in accordance with previous studies showing that TNF-α production is substantially abrogated by the presence of ACKR2, and increased by the absence of ACKR2 [30]. Despite the increased levels of TNF-α, the expression of IL-10 was similar in both groups. Regarding IL-33, an anti-osteoclastogenic cytokine [20], an increased expression was seen in ACKR2−/− mice. It could represent a compensatory mechanism in response to the augmented OTM, but this observation needs further investigation since no data in the literature is available about ACKR2 and IL-33 association. Our results clearly showed that ACKR2 is associated with anti-resorptive events. These findings provide rationale for development of novel therapeutics to reduce bone damage – centred on a reduction of availability of CC chemokines in bone resorption conditions. Acknowledgments Authors thank to Frederico Marianetti Soriani (UFMG) for help with qPCR analyses. We also would like to thank Carina Cristina Montalvany Antonucci and Ivana Márcia Alves Diniz (UFMG) for help with in vitro
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experiments. We are also grateful to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil – grant number 0027613) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil – grant number 306982/2013) for financial support. References [1] A. Maeda, K. Soejima, K. Bandow, K. Kuroe, K. Kakimoto, S. Miyawaki, et al., Force-induced IL-8 from periodontal ligament cells requires IL-1beta, J. Dent. Res. 86 (7) (2007) 629–634, http://dx.doi.org/10.1177/154405910708600709. [2] B.F. Boyce, L. Xing, Bruton and Tec: new links in osteoimmunology, Cell Metab. 7 (4) (2008) 283–285, http://dx.doi.org/10.1016/j.cmet.2008.03.013. [3] G.E1. Wise, G.J. King, Mechanisms of tooth eruption and orthodontic tooth movement, J. Dent. Res. 87 (5) (2008) 414–434, http://dx.doi.org/10.1177/154405910808700509. [4] L.J. Raggatt, N.C. Partridge, Cellular and molecular mechanisms of bone remodeling, J. Biol. 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Fra, et al., Differential recognition and scavenging of native and truncated macrophage-derived chemokine (MDC/CCL22) by the D6 decoy receptor, J. Immunol. 172 (8) (2004) 4972–4976, http://dx.doi.org/10.4049/jimmunol.172.8.4972. [16] K.M. Lee, C.S. McKimmie, D.S. Gilchrist, K.J. Pallas, R.J. Nibbs, P. Garside, et al., D6 facilitates cellular migration and fluid flow to lymph nodes by suppressing lymphatic congestion, Blood 118 (23) (2011) 6220–6229, http://dx.doi.org/10.1182/blood2011-03-344044. [17] R.J. Nibbs, S.M. Wylie, J. Yang, N.R. Landau, G.J. Graham, Cloning and characterization of a novel promiscuous human β-chemokine receptor, D6, J. Biol. Chem. 272 (51) (1997) 32078–32083, http://dx.doi.org/10.1074/jbc.272.51.32078. [18] P.D. Doodes, Y. Cao, K.M. Hamel, Y. Wang, R.L. Rodeghero, T. Kobezda, et al., CCR5 is involved in resolution of inflammation in proteoglycan-induced arthritis, Arthritis Rheum. 60 (10) (2009) 2945–2953, http://dx.doi.org/10.1002/art.24842. [19] S.Y. 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Full Length Article
Contribution of atypical chemokine receptor 2/ackr2 in bone remodeling Izabella Lucas de Abreu Lima a, Janine Mayra da Silva a, Letícia Fernanda Duffles Rodrigues b, Davidson Frois Madureira a, Angélica Cristina Fonseca c, Gustavo Pompermaier Garlet d, Mauro Martins Teixeira e, Remo Castro Russo f, Sandra Yasuyo Fukada g, Tarcília Aparecida da Silva h,⁎ a
Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil Department of Pediatric Dentistry and Orthodontics, Faculty of Dentistry, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil Department of Biological Sciences, School of Dentistry of Bauru, São Paulo University, FOB/USP, Al. Octávio Pinheiro Brisola 9-75, CEP 17012-901 Bauru, SP, Brazil d Department of Biological Sciences, School of Dentistry of Bauru, São Paulo University, FOB/USP, Al. Octávio Pinheiro Brisola 9-75, CEP 17012-901 Bauru, SP, Brazil e Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil f Laboratory of Pulmonary Immunology and Mechanics, Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil g Department of Physics and Chemistry, Faculty of Pharmaceutical Science of Ribeirão Preto, São Paulo University, Avenida do Café, s/n, Cidade Universitária, 14040-903 Ribeirao Preto, SP, Brazil h Department of Oral Pathology and Surgery, Faculty of Dentistry, Federal University of Minas Gerais, ICB/UFMG, Avenida Presidente Antônio Carlos 6627, 31.270-9010 Belo Horizonte, MG, Brazil b c
a r t i c l e
i n f o
Article history: Received 5 December 2016 Revised 29 April 2017 Accepted 2 May 2017 Available online 3 May 2017 Keywords: Chemokines Orthodontics Bone remodeling
a b s t r a c t Introduction: Bone remodeling is a tightly regulated process influenced by chemokines. ACKR2 is a decoy receptor for CC chemokines functioning as regulator of inflammatory response. In this study we investigated whether the absence of ACKR2 would affect bone phenotype and remodeling induced by mechanical loading. Methods: An orthodontic appliance was placed between incisors and first molar of ACKR2 deficient (ACKR2−/−) and C57BL6/J (wild-type/WT) mice. Microtomography, histology and qPCR were performed to evaluate bone parameters, orthodontic tooth movement (OTM), bone cells counts and the expression of ACKR2, bone remodeling markers, CC chemokines and chemokines receptors. Bone marrow cells (BMC) from WT and ACKR2−/− mice were differentiated in osteoclasts and osteoblasts for analysis of activity and expression of specific markers. Results: Mechanical stimulus induced ACKR2 production in periodontium. The expression of ACKR2 in vitro was mostly detected in mature osteoclasts and early-differentiated osteoblasts. Although ACKR2−/− mice exhibited regular phenotype in maxillary bone, the amount of OTM, osteoclasts counts and the expression of pro-resorptive markers were increased in this group. In contrast, the number of osteoblasts and related markers were decreased. OTM resulted in augmented expression of CC chemokines and receptors CCR5 and CCR1 in periodontium, which was higher in ACKR2−/− than WT mice. In vitro experiments demonstrated an augmented formation of osteoclasts and diminished differentiation of osteoblasts in ACKR2−/− mice. Conclusions: These data suggests that ACKR2 functions as a regulator of mechanically-induced bone remodeling by affecting the differentiation and activity of bone cells and the availability of CC chemokines at periodontal microenvironment. Therapeutic strategies based on increase of ACKR2 might be useful to hinder bone loss in inflammatory conditions. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Orthodontic tooth movement (OTM) is achieved by remodeling of periodontal ligament and alveolar bone [1–4]. Chemokines and their receptors are key molecules in this process by affecting osteoclasts
⁎ Corresponding author at: Departamento de Clínica, Patologia e Cirurgia Odontológicas, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Av. Presidente Antônio Carlos 6627, CEP 31.270-901 Belo Horizonte, Minas Gerais, Brazil. E-mail addresses: [email protected] (G.P. Garlet), [email protected] (S.Y. Fukada), [email protected] (T.A. Silva).
http://dx.doi.org/10.1016/j.bone.2017.05.003 8756-3282/© 2017 Elsevier Inc. All rights reserved.
recruitment and activity [1–3,5–8]. Studies in humans demonstrated a differential expression of CC chemokines at compression and tension sites of periodontal ligament submitted to mechanical force [5]. Furthermore, functional studies proved the relevance of CC chemokines as CCL2, CCL3, CCL5 and chemokines receptors (CCRs) CCR1, CCR2 and CCR5 in different models of alveolar bone remodeling, including orthodontic tooth movement [7–11]. Recently, the novel atypical chemokine receptor (ACKR) subfamily, comprising four molecules, was described [12,13]: ACKR1, previously named duffy antigen receptor for chemokines (DARC); ACKR2, previously known as D6 or CCBP2; ACKR3, also called CXC-chemokine
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receptor 7 (CXCR7) or RDC1; and ACKR4, previously called CC chemokine receptor-like 1 (CCRL1) or CCX-CKR [13]. Of these, ACKR2 was initially recognized as a CCL3 binding receptor [14]. Subsequently, it was demonstrated that ACKR2 is able to interact with most agonists of CC chemokine receptors, from CCR1 through CCR5, because of significant homology with these molecules [14,15]. Therefore, ACKR2 is known as a scavenger/decoy of CC chemokines in inflamed sites [16] functioning as a regulator of inflammatory response [14,17]. Considering that the expression of CC chemokines and respective receptors is augmented at periodontal tissues under mechanical loading [5,7–9], and that previous results implicated such molecules in bone remodeling [9–11,18,19], herein we hypothesized that ACKR2 could modify the bone resorptive/formation process by regulating the local availability of CC chemokines. 2. Materials and methods 2.1. Animals Ten-week-old wild-type (WT) (C57BL6/J) and ACKR2 deficient mice (ACKR2−/−) included in this study were obtained from Laboratory of Immunopharmacology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG). All animals were treated under Institutional Ethics Committee regulations for animal experiments (protocol #130/2012). For every set of experiments, 5 mice were used at each time-point. Each animal weight was recorded throughout the experimental period, in which no significant weight loss was observed. Mice were maintained under standard condition with a 12 h light/dark cycle, controlled temperature (24 ± 2 °C) and free access to commercial chow and drinking water.
(Tb.N), trabecular separation (Tb.Sp), bone volume (BV), bone surface (BS), tissue volume (TV), bone surface/volume ratio (BS/BV), bone surface density (BS/TV), percent bone volume (BV/TV), intersection surface (iS), trabecular bone pattern factor (Tb.Pf) and trabecular thickness (Tb.Th).
2.4. Histology Samples obtainment was performed as previously described [20]. Briefly, the anterior maxillae containing first, second and third molars were fixed in 10% buffered formalin, decalcified in 14% EDTA (pH 7.4) for 21 days and embedded in paraffin. The entire blocks were cut into sagittal sections of 4 μm thickness. The slides selected for bone cells counting presented the first and second molars, their mesial and distal-buccal root, the third molar, and adjacent structures, including the periodontal ligament and alveolar bone. At least five serial vertical sections containing the above mentioned structures were evaluated for each animal for each analysis.
2.5. Bone cells counting The distal-buccal root of the first molar, on its coronal two-thirds of the mesial periodontal site, was used for counting osteoclasts stained for tartrate resistant acid phosphatase (TRAP; Sigma-Aldrich, Saint Louis, MO, USA). The distal bone of the first molar distal-buccal root, on its mesio-coronal two-thirds, was used for osteoblast counting, stained with Masson's trichrome.
2.2. Orthodontic tooth movement 2.6. Osteoclast and osteoblast cultures The experimental protocol for OTM was based on a previous study [20]. Briefly, a nickel titanium coil (Lancer Orthodontics, Inc., Vista, San Marcos, CA, USA) was bonded by a light cored resin (Transbond, Unitek/3M, Monrova, CA, USA) between maxillary first molar and incisors at the right side of the maxilla. The force magnitude was set at 0.35 N applied in mesial direction; it was calibrated by a tension gauge (Shimpo Instruments, Itasca, IL, USA). The coil was not reactivated throughout the experimental period. The left side of the maxilla was used as control. The amount of OTM was measured through the difference between the distance of the cemento-enamel junction (CEJ) of first and second molar of the experimental side (right hemimaxilla) in relation to the control side (left hemi-maxilla) of the same animal. The measurements were obtained on microtomography (MicroCT) images. Three measurements were conducted for each evaluation and the variability was below 5%. For MicroCT and histological analysis, WT and ACKR2−/− groups were euthanatized with an overdose of anesthetic after 12 days of mechanical loading. For molecular examination, these groups were euthanatized at 0 and 72 h. 2.3. MicroCT imaging Maxillae, with the orthodontic appliance in situ, were scanned, using Skyscan 1176 (Bruker-MicroCT, Kontich, Belgium). Scanning trajectory was round and a filter of 0.5 mm was used. A 12.45 μm camera pixel size with camera XY Ratio of 0.9870, voltage of 50 kV and current of 500 μA was used. Images were analyzed using Dataviewer and CTAn software (Bruker-MicroCT). The threshold for analysis was set at 33. Maxillae were examined in fixed coronal and sagittal zones for analysis of bone density. The region of interest was standardized as circular in the alveolar bone region between the first molar roots. Bone phenotype was evaluated in maxillae on all three planes. The parameters evaluated were tissue surface (TS), trabecular number
Osteoclasts and osteoblasts were differentiated from bone marrow cells (BMCs). Tibias and femurs of WT and ACKR2−/− mice were flushed using α-MEM (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL of penicillin, and 100 mg/mL of streptomycin. For osteoclast formation, BMCs were plated in 10 mL of α-MEM with 30 ng/mL of M-CSF (R&D Systems, Minneapolis, MN, USA) and cultured for 3 d. The adherent cells (osteoclast precursors) were plated in 24-well plates at a density of 2 × 105 cells/ well and stimulated with M-CSF (10 ng/mL) and RANKL (30 ng/mL) for 48 and 72 h as previously described [20]. Then, cells were stained with TRAP (Sigma-Aldrich) and percentage of TRAP-positive cells was determined by counting the TRAP-positive cells with 3 or more nucleus in 10 fields per well at 40× magnification. For pit resorption assay, cells were plated on Corning® Osteo Assay Surface (Corning Life Sciences, NY, USA) and pits formation evaluated after 10 days. Data were expressed as number of pits/field. For osteoblast differentiation, subconfluent cells in primary culture were plated in 24 well plate at a density 5 × 104 cells/well and cultured in osteogenic medium (α-MEM supplemented with 10− 7 M dexamethasone (Sigma-Aldrich), 5 μg/mL ascorbic acid (GIBCO), and 7 mM β-glycerophosphate (Sigma-Aldrich). The calcium deposits formed at 14 and 21 days were quantified using alizarin solution (1%). After staining, alizarin deposit was eluted in 10% solution of acetic acid and methanol (4:1 v/v) and absorbance quantified by spectrophotometry at 490 nm. Bone cells viability was determined using 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyl- tetrazolium bromide (MTT) reduction assay (Vybrant® MTT Cell Proliferation Assay Kit, Invitrogen). Briefly, cells were incubated with MTT solution for 4 h at 37 °C. Formazan crystals were extracted by adding dimethyl sulfoxide (DMSO) (Sigma-Aldrich) according to the manufacturer's instructions. After 15 min, the absorbance was measured at 540 nm.
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Fig. 1. ACKR2 expression in vivo and in vitro. (A) ACKR2 expression in periodontium of C57BL6/J mice after 72 h of mechanical loading. (B) ACKR2 expression by bone marrow cells-derived osteoclasts and (C) osteoblasts. Data are expressed as mean ± SEM. *P b 0.05 comparing control to respective experimental group.
Table 1 Bone parameters in maxillae of wild-type and ACKR2 deficient mice. Parameters 2
TS μm Tb.N μm−1 Tb.Sp μm BV μm3 BS μm2 TV μm3 BS/BV μm−1 BS/TV μm−1 BV/TV % iS μm3 Tb.Pf μm−1 Tb.Th μm
WT (mean ± SEM) 2
2
11810 × 10 ± 564 × 10 3.79 × 10−3 ± 0.24 × 10−3 156.10 ± 5.87 3.98 ± 0.20 13560 × 102 ± 583 × 102 8.09 ± 0.56 3.41 × 10−2 ± 0.14 × 10−2 1.68 × 10−2 ± 0.06 × 10−2 49.39 ± 1.36 5626 × 102 ± 174 × 102 1.06 × 10−2 ± 0.10 × 10−2 131.40 ± 9.41
ACKR2−/− (mean ± SEM)
P values
10220 × 102 ± 381 × 102 4.62 × 10−3 ± 0.59 × 10−3 156.20 ± 15.34 3.93 ± 0.79 12590 × 102 ± 1765 × 102 6.67 ± 0.55 3.28 × 10−2 ± 0.30 × 10−2 1.86 × 10−2 ± 0.12 × 10−2 58.32 ± 8.44 6507 × 102 ± 1026 × 102 0.84 × 10−2 ± 0.34 × 10−2 126.50 ± 9.74
0.0800 0.2666 0.9972 0.9539 0.6289 0.1478 0.7228 0.2510 0.3558 0.4448 0.5791 0.7365
Parameters related to bone mineral density obtained by MicroCT of the alveolar bone region between the first molar roots. TS, tissue surface; Tb.N, trabecular number; Tb.Sp, trabecular separation; BV, bone volume; BS, bone surface; TV, tissue volume; BS/BV, specific bone surface; BS/TV, bone surface density; BV/TV, percent bone volume; iS, intersection surface; Tb.Pf, trabecular bone pattern actor; Tb.Th, trabecular thickness.
2.7. Real–time PCR Periodontal ligament and surrounding alveolar bone samples were obtained from upper first molars after 0 and 72 h of mechanical loading. Tissue samples and samples from osteoclasts and osteoblasts in vitro were submitted to the extraction of total RNA (RNeasyFFPE kit, Qiagen Inc., Valencia, CA, USA) according to the manufacturers' instructions. The integrity of RNA samples was checked by analyzing 1 mg of total RNA on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The
complementary DNA (cDNA) was synthesized using 3 μg of RNA through a reverse transcription reaction (Superscript III, Invitrogen Corporation, Carlsbad, CA, USA). The targets analyzed were: ACKR2, runt-related transcription factor 2 (RUNX2); dentin matrix acidic phosphoprotein (DMP); receptor activator of nuclear factor kappa B (RANK); receptor activator of nuclear factor kappa B ligand (RANKL); osteoprotegerin (OPG); metalloproteinase 13 (MMP13); interleukin-33 (IL-33); tumor necrosis factor alpha (TNF-α); interleukin-10 (IL-10), CCL3, CCL5, CCL2, CCR1 and CCR5. Real-Time PCR array was performed in a Viia7 instrument (Life Technologies) using TaqMan chemistry (Invitrogen) associated with inventoried optimized primers/probes sets (Invitrogen), with basic reaction conditions of 50 °C (20 s), 95 °C (10 min), (40 cycles) 95 °C (15 s) and 60 °C (1 min). For analysis of ACKR2 expression in samples from osteoclasts and osteoblasts culture, 18S was used as housekeeping reference gene, and the basic reaction conditions were (40 cycles) 95 °C (10 min), 94 °C (1 min), 56 °C (1 min) and 72 °C (2 min) for the other targets. The mean Ct values from duplicate measurements were used to calculate expression of the target gene, with normalization to an internal control (β-actin) using the 2−ΔΔCt method. Samples from osteoclasts and osteoblasts culture were also submitted to RNA extraction with the RNeasyFFPE kit (Qiagen Inc) according to the manufacturers' instructions. The integrity of RNA samples was checked by 2100 Bioanalyzer (Agilent Technologies), followed by cDNA synthesis (QuantiTect Reverse Transcription Kit, Qiagen). RealTimePCR was performed with Viia7 (Life Technologies) with TaqMan primers/probes sets according to the manufacturers' instructions, with standard amplification protocols. The results are expressed as target genes normalized by constitutive genes (GAPDH and ACTB), determined by the 2−ΔΔCt method.
Fig. 2. (A) Amount of orthodontic tooth movement in WT and ACKR2−/− mice after 12 days. MicroCT representative images of maxillae of WT mice at control side (B) and experimental side (C). MicroCT representative images of maxillae of ACKR2−/− mice at control side (D) and experimental side (E). White angulated arrows indicate the space between the crown of the first and second molars. White line indicated the space of periodontal ligament between the distal-buccal root and the mesial bone. White straight arrows indicate the direction of orthodontic OTM. Data were expressed as mean ± SEM. *P b 0.05. DB, distal alveolar bone; MB, mesial alveolar bone; R, root.
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2.8. Statistical analysis Results were expressed as mean ± SEM. As data sets presented normal distribution (Kolmogorov-Smirnov), one-way Analysis of variance (ANOVA) was used to analyze differences among
groups, followed by Newman-Keuls multiple comparison post hoc test. The data obtained from all evaluations were processed using GraphPad Prism (version 5.01, GraphPad Software, San Diego, CA, USA). The level of significance for all statistical tests was set at 5%.
Fig. 3. Number of TRAP-positive cells and osteoblasts at periodontal sites under mechanical loading. (A) Scheme showing the compression site (in gray) used for TRAP-positive cells count. (B) Quantification and (C) representative histological sections of TRAP-positive osteoclasts of WT and (D) ACKR2−/− mice after 12 days of mechanical loading. (E) Scheme showing the tension site (in gray) used for osteoblast count. (F) Quantification and (G) representative histological sections of osteoblasts of WT and (H) ACKR2−/− mice after 12 days of mechanical loading. Black angulated arrows indicate TRAP-positive cells (C–D) and white angulated arrows indicate osteoblasts (G–H). Black and white straight arrows indicate the direction of orthodontic tooth movement. Bar = 100 μm. Data are expressed as mean ± SEM. *P b 0.05 comparing control group to the respective experimental group. #P b 0.05 comparing WT to ACKR2−/− experimental groups. DB, distal alveolar bone; MB, mesial alveolar bone; R, root.
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3. Results 3.1. ACKR2 expression is induced by mechanical stimuli and osteoblasts and osteoclasts differentially express ACKR2 Initially, we assessed whether mechanical stimulus would interfere with ACKR2 expression in periodontium. It was observed an increased
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expression of ACKR2 in periodontal tissues at 72 h after force application (Fig. 1A). After that, in order to determine if cells involved with bone remodeling express ACKR2, we did analyze ACKR2 expression in bone marrow-derived osteoclasts and osteoblasts. Our results showed an increased expression of ACKR2 by mature osteoclasts (72 h) in relation to early-differentiated cells (48 h) (Fig. 1B). In contrast, early osteoblasts
Fig. 4. mRNA expression of bone cells markers (A) RUNX2, (B) DMP, (C) RANK, (D) RANKL/OPG, (E) MMP13, (F) IL-33, and inflammatory molecules (G) TNF-α and (H) IL-10 in alveolar bone and periodontium samples of WT and ACKR2−/− mice after 72 h of mechanical loading. Data are expressed as mean ± SEM. *P b 0.05 comparing control to the respective experimental group. #P b 0.05 comparing WT to ACKR2−/− experimental groups. One-way ANOVA and Newman–Keuls multiple comparison test.
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(14 days) exhibited higher expression of ACKR2 when compared with mature osteoblasts (21 days) (Fig. 1C).
Furthermore, the lack of ACKR2 resulted in a disruption of bone lining cells and decrease of osteoblast numbers at the distal bone of distalbuccal root in relation to WT mice (Fig. 3E–H).
3.2. ACKR2−/− mice exhibit regular parameters of maxillary bone To evaluate if deficiency of ACKR2 would affect maxillary bone phenotype, we first analyzed bone parameters in maxillae using MicroCT. The comparison of bone parameters of ACKR2−/− and WT animals under steady state conditions showed no significant differences (Table 1). 3.3. Bone resorption is increased in ACKR2−/− mice subjected to mechanical loading Once ACKR2 deficiency did not result in modification of maxillary bone phenotype, we next submitted ACKR2 deficient mice to OTM to test bone response during mechanical demands. The amount of OTM (Fig. 2A–E) and TRAP positive osteoclasts (Fig. 3A–D) were significantly increased after 12 days of mechanical loading, in ACKR2−/− mice when compared to WT mice. The increased OTM in ACKR2−/− mice was also evidenced by greater space between molar's crown and a decreased space of periodontal ligament between the distal-buccal root and the mesial bone (Fig. 2C, E). Collectively, the amount of bone resorption was increased in ACKR2−/− than WT mice.
3.4. ACKR2−/− mice exhibited decreased expression of osteoblastic markers and increased expression of osteoclastic and inflammatory markers in periodontium Considering the decreased osteoblast numbers at tension sites in ACKR2−/− mice, we decided to evaluate if the expression of osteoblast markers RUNX2 and DMP was consistently reduced in these mice. In fact, we observed a decreased expression of these markers after 72 h of mechanical loading in ACKR2−/− mice (Fig. 4A–B). Since ACKR2−/− mice also presented increased osteoclast numbers, we next investigated markers of osteoclast activity RANK, RANKL, OPG and MMP13. Consistently, after 72 h of mechanical loading, ACKR2−/− mice exhibited increased RANKL/OPG ratio and expression of RANK and MMP13 (Fig. 4C–E). The expression of the anti-osteoclastogenic cytokine IL-33 was also significantly increased in ACKR2−/− mice when compared to WT mice (Fig. 4F). As OTM depends on inflammatory mediators, which, in turn, are regulated by ACKR2, we investigated the expression of TNF-α and IL-10. ACKR2−/− mice presented an increased expression of TNF-α (Fig. 4G)
Fig. 5. mRNA expression of CC chemokines and receptors (A) CCL3, (B) CCL5, (C) CCL2, (D) CCR1 and (E) CCR5 in alveolar bone and periodontium samples of WT and ACKR2−/− mice after 72 h of mechanical loading. Data are expressed as mean ± SEM. *P b 0.05 comparing control to the respective experimental group. #P b 0.05 comparing WT to ACKR2−/− experimental groups. One-way ANOVA and Newman–Keuls multiple comparison test.
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after 72 h of mechanical loading, however, no significant difference in mRNA expression of IL-10 was observed between the groups (Fig. 4H). 3.5. Augmented expression of CC chemokines and receptors in ACKR2−/− mice periodontium Chemokines and their receptors have pivotal functions in strainmanaged bone remodeling and the ACKR2 acts as agonist of these molecules. Indeed, the expression of CCL2, CCL3, CCL5, CCR1 and CCR5 was increased at periodontal sites after 72 h of mechanical loading in WT mice. Noteworthy, the augment of these molecules was significantly higher in ACKR2−/− when compared to WT mice (Fig. 5A–E).
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3.6. Osteoclast differentiation from BMCs is increased in ACKR2−/− mice In vitro experiments were performed in order to analyze if bone cells differentiation is modified in ACKR2−/− mice. Comparing BMCs derived from WT and ACKR2−/−, we observed that BMCs from ACKR2−/− mice generated increased numbers of osteoclasts after 72 h (Fig. 6A, B, D, E, G, H) and resorptive activity (Fig. 6C, F, I). In contrast, BMCs from ACKR2−/− mice resulted in reduced numbers of differentiated osteoblasts and mineralized nodules formation at 14 and 21 days (Fig. 6J–O). Bone cells differentiation were not linked to viability of BMCs, since it was higher than 95% and no differences were observed between the groups (data not shown).
Fig. 6. In vitro differentiation of Bone marrow cells from WT and ACKR−/− mice. (A, B) Osteoclasts from BMCs of wild-type (WT; C57BL6/J) mice after 48 and 72 h of cell differentiation, respectively. (G, H) Quantification of TRAP-positive cells derived from WT and ACKR2−/− mice after 48 and 72 h of differentiation. (D, E) Osteoclasts from BMCs of ACKR2−/− mice after 48 and 72 h of cell differentiation, respectively. (C, F) Resorption pit formation after treatment with RANKL of BMCs from WT and ACKR2−/− mice, respectively. (I) Resorption field in culture of osteoclasts. (J, K) Osteoblasts from BMCs of WT mice after 14 and 21 days of cell differentiation, respectively. (L, O) Quantification of nodule area formation by osteoblasts derived from WT and ACKR2−/− mice after 14 and 21 days of cell differentiation. (M, N) Osteoblasts from BMCs of ACKR2−/− mice after 14 and 21 days of cell differentiation, respectively. Data were expressed as mean ± SEM. *P b 0.05 comparing WT to ACKR2−/− experimental groups.
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Fig. 7. mRNA expression of BMCs-derived osteoclasts (A–C) and osteoblasts (D–H) in WT and ACKR2−/− mice. #P b 0.05 comparing WT to ACKR2−/− at the same time point. One-way ANOVA and Newman–Keuls multiple comparison test.
3.7. Bone markers expression in BMCs from ACKR2−/− and WT mice BMCs-derived osteoclasts from ACKR2−/− and WT mice exhibited similar expression of TRAP, cathepsin K, RANK, alkaline phosphatase (ALP) and Osterix (OSX) (Fig. 7A–C, G, H). Concerning BMCs-derived osteoblasts, a slightly increase of RUNX2 and collagen, type I, alpha 1 (COL1A1) expression was observed in ACKR2−/− mice at 21 days. In contrast, a reduction of bone sialoprotein (BSP) expression was detected at day 14 (Fig. 7D–F).
4. Discussion Alveolar bone remodeling during mechanical loading is modulated by the interplay between bone cells and leukocytes [21,22]. CC chemokines and its receptors are important players of this process [7–
9]. In the view of the potential role of the ACKR2 in the regulation of CC chemokines and consequently in bone remodeling, we initially analyzed maxillary bone in steady state conditions. Interestingly, similar bone parameters were observed for ACKR2−/− and WT mice, demonstrating that ACKR2 does not modify physiological bone remodeling. However, when maxillary bone was submitted to mechanical force a pronounced alveolar bone resorption was observed in ACKR2−/− mice. Such phenotype was associated with increase of osteoclasts counts and augmented expression of osteoclastic markers and chemokines. Consistently, bone marrow-derived cells from ACKR2−/− mice exhibited greater osteoclast differentiation. Our data also confirmed that mechanical stimulus induces ACKR2 expression and that ACKR2 is expressed by osteoclasts and osteoblasts. It is the first demonstration linking ACKR2 and bone. ACKR2 is able to interact with several agonists of CC chemokine receptors [14,15], consequently affecting their availability [23]. As the
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production of these molecules is triggered by mechanical force [7,8], it seems reasonable that the absence of such receptor only impacts bone phenotype in a condition where there is an increase of chemokines, such as mechanical loading induced-bone remodeling. Indeed, we observed that ACKR2−/− mice presented increased OTM and augmented expression of CCL2, CCL3, CCL5, CCR1 and CCR5. This result is in line with greater osteoclasts numbers and resorption in this group, since CC chemokines and receptors are positive signals for osteoclast recruitment and activation in infectious [10,11] and non-infectious conditions, affecting periodontal tissues [7,18]. Bone resorption is regulated by RANKL binding to the receptor RANK [5], and this event is prevented by a decoy receptor OPG, consequently inhibiting resorption [5,24]. Consistent with the phenotype observed in ACKR2−/− mice submitted to OTM, we verified an increase of RANKL/OPG ratio and of RANK and MMP-13, a matrix metalloproteinase with a critical role in bone resorption [25]. These results point to that the effects of ACKR2 in bone are at least in part mediated via RANK/RANKL. Previous studies demonstrated that CC chemokines are related with higher RANKL expression and consequently greater osteoclast differentiation and activity [26,27]. Our in vitro data revealed that in the absence of ACKR2, BMCs ability to generate osteoclasts is augmented, indicating an inhibitory effect of this receptor in osteoclast differentiation. Unexpectedly, the expression of specific osteoclastic markers by these cultures was similar between the groups. It can be associated with the subtle difference observed in cells counts and the timing of BMCs differentiation. As bone remodeling is a result of coupled function of osteoclasts and osteoblasts, we evaluate if ACKR2 deficiency would also modify osteoblasts. We observed that ACKR2−/− mice presented a decreased number of osteoblasts when compared to WT mice. Consistently, osteoblast markers, RUNX2 and DMP, were also decreased in our analysis. In vitro experiments confirmed a reduced formation of osteoblasts from BMCs of ACKR2−/− mice. As expected, the expression of BSP from these cells was reduced, but intriguingly, COL1A1 and RUNX2 were slightly higher in this group at day 21. It can suggest a delay of osteoblast differentiation with a persistence of early-differentiated cells at late stages of cell cultures from ACKR2−/− mice. We also detected the expression of ACKR2 in osteoblasts, mostly early-differentiated than mature cells. In line with these findings, an increased expression of ACKR2 was detected in mature osteoclasts. Taken together, these findings indicate that osteoblasts and osteoclasts participate in controlling CC chemokines availability in bone milieu and that this process is important to coordinate the function of these cells. ACKRs also affect the production of inflammatory mediators [16]. In this study, the expression of inflammatory molecules involved in bone remodeling as TNF-α [28,29], IL-10 [9] and IL-33 [20] was evaluated. We did obtain an increased expression of TNF-α in ACKR2 deficient mice. This is in accordance with previous studies showing that TNF-α production is substantially abrogated by the presence of ACKR2, and increased by the absence of ACKR2 [30]. Despite the increased levels of TNF-α, the expression of IL-10 was similar in both groups. Regarding IL-33, an anti-osteoclastogenic cytokine [20], an increased expression was seen in ACKR2−/− mice. It could represent a compensatory mechanism in response to the augmented OTM, but this observation needs further investigation since no data in the literature is available about ACKR2 and IL-33 association. Our results clearly showed that ACKR2 is associated with anti-resorptive events. These findings provide rationale for development of novel therapeutics to reduce bone damage – centred on a reduction of availability of CC chemokines in bone resorption conditions. Acknowledgments Authors thank to Frederico Marianetti Soriani (UFMG) for help with qPCR analyses. We also would like to thank Carina Cristina Montalvany Antonucci and Ivana Márcia Alves Diniz (UFMG) for help with in vitro
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experiments. We are also grateful to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil – grant number 0027613) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil – grant number 306982/2013) for financial support. References [1] A. Maeda, K. Soejima, K. Bandow, K. Kuroe, K. Kakimoto, S. Miyawaki, et al., Force-induced IL-8 from periodontal ligament cells requires IL-1beta, J. Dent. Res. 86 (7) (2007) 629–634, http://dx.doi.org/10.1177/154405910708600709. [2] B.F. Boyce, L. Xing, Bruton and Tec: new links in osteoimmunology, Cell Metab. 7 (4) (2008) 283–285, http://dx.doi.org/10.1016/j.cmet.2008.03.013. [3] G.E1. Wise, G.J. King, Mechanisms of tooth eruption and orthodontic tooth movement, J. Dent. Res. 87 (5) (2008) 414–434, http://dx.doi.org/10.1177/154405910808700509. [4] L.J. Raggatt, N.C. Partridge, Cellular and molecular mechanisms of bone remodeling, J. Biol. 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