Resolvin E1 and Chemokine-like Receptor 1 Mediate Bone Preservation

Li Gao, Dan Faibish, Gabrielle Fredman, Bruno S. Herrera, Nan Chiang, Charles N. Serhan, Thomas E. Van Dyke and Robert Gyurko
J Immunol January 15, 2013, 190 (2) 689-694; DOI: https://doi.org/10.4049/jimmunol.1103688

Abstract

The polyunsaturated ω-3 fatty acid eicosapentaenoic acid–derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss, and induces bone regeneration. Although the inflammation-resolving actions of RvE1 are characterized, the molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice overexpressing the RvE1 receptor chemokine-like receptor 1 (chemR23) on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in receptor activator of NF-κB ligand levels, whereas the osteogenic markers alkaline phosphatase, bone sialoprotein, and Runt-related transcription factor 2 remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable receptor activator of NF-κB ligand/OPG ratio, in addition to known anti-inflammatory and proresolving actions.

Introduction

It is becoming increasingly evident that future treatment of infectious and inflammatory diseases will rely on a detailed understanding and molecular appreciation of the resolution programs for inflammation and tissue injury (1, 2). The health benefits of ω-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid present in fish oil have been long known, but the cellular and molecular mediators responsible for their actions have only recently been uncovered (3). Resolvin E1, (5S,12R,18R-trihydroxy-eicosapentaenoic acid; RvE1) is an enzymatically oxygenated product of EPA, one of the main dietary essential ω-3 PUFAs (4). RvE1 has potent immunoregulatory actions stimulating the resolution of inflammation by blocking and counterregulating the production of proinflammatory mediators, controlling cell–cell interactions, regulating leukocyte infiltration and transmigration in vivo, and stimulating macrophages to enhance phagocytosis and clearance of apoptotic polymorphonuclear leukocytes (PMN) as well as bacteria (2). RvE1 is produced in vivo during the resolution phase of acute inflammation by endothelial–leukocyte cell interactions (2, 5).

Periodontal disease is a common bacterially induced chronic inflammatory condition resulting in leukocyte infiltration, osteoclast activation, and alveolar bone resorption (6). In a combined ligature-induced and Porphyromonas gingivalis–induced experimental model of periodontal disease in rabbits, RvE1 prevents leukocyte infiltration, significantly decreases osteoclast counts, and stops alveolar bone loss (6). Recent evidence shows that RvE1 also restores lost periodontal tissue, including bone (7). Specific binding sites for RvE1 have been identified on monocytes and identified as the human chemokine-like receptor 1 (chemR23 or CMKLR) (4), which also serves as a receptor for chemerin (4, 8). ChemR23 is expressed in the cardiovascular system, kidney, brain, gastrointestinal tissues, and bone marrow (8–10). ChemR23 specifically binds 3H-labeled RvE1, resulting in inhibition of TNF-α–stimulated NF-κB activation. Functional interactions between RvE1 and chemR23 have been further confirmed (4). Inflammatory cytokines such as TNF-α and IFN-γ upregulate monocyte chemR23 and cyclooxygenase-2 transcripts (4).

To date, limited information is available on the potential action of RvE1 in bone metabolism. ChemR23 is expressed in developing bone tissue (11). RvE1 was identified in the bone marrow of rats supplemented with dietary EPA (12), and RvE1 inhibits osteoclast differentiation and bone resorption in vitro (13). To investigate agonist actions of RvE1 in vivo, we engineered transgenic mice overexpressing the human chemR23 transgene to test the hypothesis that chemR23-mediated events impact changes in bone metabolism through direct actions on bone in addition to known anti-inflammatory and proresolving actions. This gain-of-function approach demonstrated a protection against bone destruction without osteogenic actions that was further enhanced with the administration of RvE1. The results indicate that RvE1 and chemR23 serve a protective role in acute inflammatory responses and bone homeostasis in vivo.

Materials and Methods

Animals

FVB mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mouse experiments were in conformity with the standards of the Public Health Service Policy on Human Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Boston University.

Resolvin synthesis

RvE1 was prepared by total organic synthesis, as in Ref. 4. The structural integrity of RvE1 was monitored using UV tandem LC-MS/MS (4). Immediately before use, RvE1 was diluted in PBS to final ethanol concentration of <1%.

Preparation of chemR23 transgenic mice

The full-length of hCD11b promoter cDNA was cloned upstream of the full-length chemR23 cDNA (GenBank accession: www.ncbi.nlm.nih.gov/nuccore/NM_004072). Transgenic mice were generated at Boston University School of Medicine Transgenic/Knockout Mouse Core Facility. Hemizygous colonies were amplified by out-breeding chemR23 transgenic males with wild-type FVB females.

Genotyping of mice

Genomic DNA was isolated from tail biopsies of mice and screened by PCR using primers directed to the mouse chemR23: forward primer, 5′-CTCGGTCTCCTAGGCAAC-3′; human chemR23 forward primer, 5′-GTCTTCCTCCCAATCCAT-3′. The mouse and human chemR23 amplicons shared the same reverse primer: 5′-TAGAAAGCCAGGACCCAG-3′.

Gene expression analysis

RNA was extracted using RNeasy Mini Kit (Qiagen). Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). ChemR23-specific probes (forward primer, 5′-CTGTCCACACCTGGGTCTTC-3′; reverse primer, 5′-CCCCACAGGGTCCATTTGG-3′) were designed using File Builder Software (Applied Biosystems) (4). The expression of bone sialoprotein (BSP) and Runt-related transcription factor 2 (RunX2) were measured by real-time PCR (7000 Sequence Detection System; ABI). The expression was normalized to hypoxanthine–guanine phosphoribosyltransferase-1.

Flow cytometry assay

Peritoneal exudates were collected after i.p. injection of zymosan-A (0.2 mg/ml in PBS) at time points indicated. Peritoneal cells were stained with monoclonal anti-human chemR23 Ab (50 μg/ml; R&D Systems, Minneapolis, MN). For leukocyte typing, peritoneal cells were labeled with FITC-conjugated anti-mouse Ly6G Ab (PMN) or PE-conjugated anti-mouse F4/80 Ab (macrophages). Cells were analyzed on a FACScan instrument using CellQuest software (BD Biosciences).

Ligature-induced alveolar bone loss

Mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). A 9-0 silk suture was placed into the gingival sulcus of the upper left second molar and tied around the tooth. One week later, mice were sacrificed, and the maxilla was cleaned and stained with methylene blue (1% in water). The distance between the cementoenamel junction and the alveolar bone crest was determined to assess alveolar bone loss (14).

Craniotomy

Mice were anesthetized as described earlier, and a 1-mm craniotomy defect was created in the parietal bone through a scalp incision using a round carbide burr (15). RvE1 (100 ng in 20 μl PBS) or vehicle was injected s.c. over the craniotomy every 2 d. Mice were sacrificed after 14 d, and histological sections of the parietal bone were stained with H&E and examined by light microscopy. The healing rate was quantified as the new bone formed relative to the initial defect in three sections in the center of the defect.

Receptor activator of NF-κB ligand and osteoprotegerin measurements

Primary bone cell cultures were isolated from neonatal mouse calvaria by collagenase/dispase digestion. Cells were cultured the presence of ascorbic acid, β-glycerophosphate, and vitamin D3 (16). After 10 d of differentiation, cells were stimulated with IL-6 and IL-6 receptor (both at 10 ng/μl) to stimulate proinflammatory conditions and simultaneously incubated with RvE1 (1–100 nM). Supernatants were collected after 48 h, and the expression of soluble receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) was measured by ELISA (R&D Systems).

MC3T3 cells

MC3T3-E1 Subclone 4 (American Type Culture Collection, Manassas, VA) cells were cultured in α-MEM medium (Life Technologies, Grand Island, NY), 10,000 IU penicillin, 10,000 μg/ml streptomycin, and 10% FBS in 6-well plates at a density of 105 cells/well. To support osteoblast differentiation, media were supplemented with ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mM) and changed every other day.

Immunohistochemistry

Calvarial osteoblasts were grown on glass coverslips, fixed in 4% paraformaldehyde, and stained with anti-chemR23 (R&D Systems) or isotype-matched nonspecific Ab (mouse IgG3; R&D Systems). Coverslips were mounted using the ProLong Gold anti-fade reagent (Invitrogen), which labels nuclei with DAPI.

Alkaline phosphatase activity

MC3T3 cells were cultured in media supplemented with ascorbic acid and β-glycerophosphate as described earlier for 14 or 21 d. Cytoplasmic fractions were prepared using an extraction kit (Pierce) and incubated with p-nitrophenyl phosphate. Reactions were stopped using 0.5 N sodium hydroxide. OD of the products at 405 nm was measured by spectrophotometry (Spectramax 340PC 384 with a SoftMax Pro software 4.3LS; Molecular Devices). Enzyme activity was calculated as the OD of the reaction product multiplied by the reaction volume and normalized to the reaction time. These values were normalized to protein content as measured using the Bradford protein assay.

Statistical analysis

In vitro and in vivo experiments were analyzed with Student t test and ANOVA, with p < 0.05 considered statistically significant.

Results

Characterization of human chemR23 transgenic mouse

The full length of hCD11b promoter cDNA was cloned upstream of the full-length human chemR23 cDNA and injected into the male pronucleus (Fig. 1A). The male transgenic founder was mated with wild-type (WT) FVB females to propagate the transgenic line. Offspring were genotyped using PCR. Both native murine chemR23 (316 bp, upper band) and the transgenic human chemR23 (253 bp, lower band) were amplified in the genomic DNA of transgenic mice, as both human- and murine-specific 5′ PCR primers along with a common 3′ primer were included in each PCR reaction (Fig. 1B). In WT DNA, only the murine chemR23 (316 bp) was identified. ChemR23tg mice are viable, fertile, and show no gross developmental or behavioral abnormalities. Transgenic chemR23 RNA expression levels were determined on leukocyte RNA extracts after reverse transcription and real-time PCR. chemR23tg leukocytes (Tg) expressed markedly higher amounts of chemR23 RNA than WT leukocytes (Fig. 1C, WT). Flow cytometry using anti-human chemR23 Ab showed increased chemR23 cell surface expression on peritoneal exudate leukocytes (Fig. 1D). WT littermate leukocytes showed some positive signal with chemR23 Ab, suggesting some background cross-reactivity of the anti-human Ab with the mouse chemR23.

FIGURE 1.
FIGURE 1.

Characterization of chemR23tg mouse. (A) ChemR23 transgene construction. The full-length human chemR23 cDNA was cloned downstream of the hCD11b promoter in pCDNA3 plasmid. The 2.9 kb KpnI–NotI fragment was used for pronuclear injections. (B) Genotyping of chemR23tg × FVB offspring with PCR. Genomic DNA was amplified with primers for mouse chemR23 (316 bp, upper band) and human transgenic chemR23 (253 bp, lower band) in the same reaction. Double bands in lanes 2, 3, 4, and 6 indicate transgenic mice (Tg). Single bands correspond to WT mice. Std, size standards. (C) Leukocyte RNA was reverse-transcribed and amplified with quantitative real-time PCR using human chemR23-specific primers. CT values represent the PCR cycle number at which an arbitrary threshold of PCR product accumulation is achieved. Lower CT values indicate higher gene expression. Transgenic lines Tg#3 and Tg#33 express chemR23 mRNA abundantly in comparison with WT samples. Positive control: purified transgene DNA. (D) Flow cytometry analysis of peritoneal leukocyte expression of chemR23 in transgenic and WT mice. Leukocytes were isolated from chemR23 transgenic mice (dark line) and WT controls (gray line) and incubated with PE-conjugated Ab for chemR23. Nonpermeabilized chemR23tg leukocytes show increased cell surface chemR23 immunoreactivity compared with WT leukocytes.

ChemR23 overexpression enhances leukocyte clearance in the peritoneal cavity induced by RvE1

Acute peritoneal inflammation was initiated in WT and chemR23tg mice by injecting 0.2 mg zymosan in 1 ml PBS i.p. Mice were sacrificed at 2, 4, 12, 24, and 72 h after zymosan injection, and peritoneal leukocytes were collected by abdominal lavage. Leukocyte counts were determined with Wright–Giemsa staining, and differential leukocyte analysis of the exudate was performed with flow cytometry using Ly6G and F4/80 Abs for PMN and macrophages, respectively. PMN recruitment peaked at 4 h and declined by 24 h, whereas macrophage recruitment increased gradually over the 3-d observation period in both WT and chemR23tg mice (data not shown). In a separate set of experiments, RvE1 (0, 1, 10, or 100 ng) was i.p. injected simultaneously with zymosan. At the 24-h time point, representing the resolution phase (17), peritoneal leukocytes were collected and quantified using flow cytometry after labeling neutrophils with FITC-conjugated anti-mouse Ly6G Ab and macrophages with PE-conjugated anti-mouse F4/80 Ab. Leukocyte counts were lower in the peritoneal exudate of chemR23tg mice regardless of RvE1 dose. RvE1 at 1 and 10 ng dose decreased total leukocyte (Fig. 2A) and neutrophil (Fig. 2B) recruitment in chemR23tg mice, whereas in WT mice, only the 10 ng dose was effective. In chemR23tg mice at the 100 ng RvE1 dose, total leukocyte numbers are higher than those at 10 ng RvE1, due to increased recruitment of macrophages in transgenic animals at 100 ng RvE1 (Fig. 2C). Expression of neutrophil recruitment as percent inhibition reveals that chemR23 overexpression shifts the RvE1 dose response to the left with actions in the chemR23tg evident at 1 ng/mouse of RvE1 compared with WT (Fig. 2D). Together, these results indicate an enhanced response to RvE1 in leukocyte trafficking in chemR23tg mice.

FIGURE 2.
FIGURE 2.

Leukocyte infiltration in zymosan-induced peritoneal inflammation with RvE1 in WT and chemR23tg mice. RvE1 (0–100 ng) was injected i.p. simultaneously with 0.2 mg zymosan in 1 ml PBS. Twenty-four hours later, leukocytes were harvested from the peritoneum and quantified with immunocytochemistry and flow cytometry. RvE1 (1 and 10 ng) decreased total leukocyte (A) and neutrophil (B) recruitment in chemR23tg mice, whereas in WT mice only the 10 ng dose was effective. Macrophage recruitment is increased with 100 ng RvE1 (C). In chemR23tg mice, the RvE1 inhibitory dose-response curve for PMN is shifted to the left (D). Mean ± SEM, n = 3 each data point. †p < 0.05 (between WT and chemR23tg, two-way ANOVA), *p < 0.05 (for doses indicated in pairwise comparisons).

ChemR23tg mice show diminished alveolar bone loss in experimental periodontitis

Induction of periodontal disease by molar ligation caused significant alveolar bone loss measured as the cementoenamel junction–alveolar bone crest (CEJ–ABC) distance in both WT (Fig. 3A) and chemR23 transgenic mice (Fig. 3B) compared with the corresponding contralateral nonligated molar. Bone loss surrounding the ligated molar was significantly lower in chemR23 transgenic mice compared with WT (p < 0.05), indicating a bone-preserving action of chemR23 (Fig. 3C).

FIGURE 3.
FIGURE 3.

Periodontal disease induced by molar ligation. A 9-0 silk suture was tied around the second maxillary molar to induce periodontal disease and alveolar bone loss. White arrow points to the CEJ–ABC distance on WT (A) and chemR23tg (B) samples. CEJ–ABC distance is similar in the nonligated (control) side of WT and chemR23tg mice; however, ligation induces significantly less bone loss in chemR23tg mice (C). Mean ± SD, n = 8 each group. *p < 0.05 (t test).

Healing of uniform craniotomy is accelerated by RvE1

RvE1 (100 ng in 20 μl PBS) was injected over a 1-mm craniotomy defect in the parietal bone every other day for 2 wk in WT and chemR23tg mice. Bone healing was quantified on histological sections of the parietal bone. New bone formation was significantly increased by RvE1 treatment in both WT and chemR23tg mice (Fig. 4A). There was no significant difference between WT and chemR23tg mice at baseline or after RvE1 treatment. Histological sections (Fig. 4B) indicated new bone formation at the edges of the defect as well as adjacent to it. Tartrate-resistant acid phosphatase staining was performed to identify osteoclasts on the histological sections. Osteoclast numbers were not significantly different in the vehicle-injected group compared with the RvE1-treated group (data not shown).

FIGURE 4.
FIGURE 4.

RvE1 significantly enhances bone healing in vivo. (A) A 1-mm-wide circular bone defect was created in the parietal bone of WT and chemR23tg mice and treated with subperiosteal injections of RvE1 (100 ng in 20 μl) every other day for 2 wk. Bone healing is expressed as percentage of original defect. RvE1 significantly enhanced bone healing in both WT and chemR23tg (Tg) mice. Mean ± SEM, n = 16 for each group. No significant difference between WT and Tg was found. *p < 0.05 (t test). (B) Histological section across a healing calvarial bone defect (Masson’s trichrome staining).

ChemR23 is expressed in neonatal calvarial cultures

Primary osteoblast cultures were isolated from WT neonatal calvarial cultures. RNA was extracted from 1- and 10-d-old cultures. After reverse transcription of RNA samples, a single 357-bp fragment corresponding to the murine chemR23 gene was amplified (Fig. 5A). Immunohistochemistry in 1- and 10-d-old calvarial cultures showed specific chemR23 staining (Fig. 5B, 10-d-old culture shown).

FIGURE 5.
FIGURE 5.

ChemR23 is expressed in calvarial osteoblast cultures. (A) RT-PCR of a 357-bp fragment of the chemR23 transcript (Std., 100-bp size standards; d1, 1-d-old culture; d10, 10-d-old culture). (B) Immunohistochemistry of chemR23 expression in 10-d-old calvarial cultures. Cell nuclei were labeled with DAPI. Isotype ctr: Negative control staining with a nonspecific Ab. Original magnification ×400.

Rescue of OPG expression by RvE1

To investigate the molecular mechanism of RvE1 on inflammatory bone loss, expression of RANKL and OPG were measured in neonatal calvarial cultures and stimulated with the inflammatory mediator IL-6 and its soluble receptor, IL-6R, for 48 h. RANKL expression was not significantly altered by RvE1 (10 ng) when administered with IL-6 (10 ng) and IL-6R (10 ng) as determined with ELISA (Fig. 6A). In contrast, RvE1 (1–100 nM) rescued OPG expression that was suppressed by administration of IL-6 and IL-6R (Fig. 6B). To characterize further the specificity of RvE1-induced OPG rescue, primary neonatal calvarial cell cultures were treated with the RvE1 precursor EPA (100 nM) or the chemR23 peptide ligand chemerin (100 nM) along with IL-6 and IL-6R. EPA and chemerin did not impact OPG levels under these conditions (Fig. 6B).

FIGURE 6.
FIGURE 6.

RANKL and OPG measurements in primary osteoblast cultures. Inflammatory phenotype was induced with administration of IL-6 (10 nM) and IL-6 receptor (IL6R, 10 nM) for 48 h. Culture supernatants were assayed for RANKL and OPG with ELISA. RANKL expression was not significantly altered by RvE1 (10 ng) when administered with IL-6 (10 ng) and IL-6R (10 ng) (A). RvE1 (1–100 nM) rescued OPG expression that was suppressed by administration of IL-6 and IL-6R (B). EPA and chemerin did not impact OPG levels under these conditions (B). Mean ± SEM, n = 4. *p < 0.05 (ANOVA).

RvE1 in osteogenesis

The impact of RvE1 on different cell types in the transgenic animals is consistent with cell specificity imparted by the CD11b promoter-driven transgene expression, as inflammatory (myeloid) cells demonstrate exaggerated response to RvE1, whereas bone formation appears similar in WT and transgenic mice. As osteoblasts express their native chemR23 receptor, we have examined whether modulation of bone formation by RvE1 contributes to bone anabolic actions in addition to OPG regulation. Cell cultures of the mouse calvarial osteoblast cell line MC3T3 was treated with RvE1 (100 nM) for 2 wk, and RNA was extracted for measuring BSP and the transcription factor RunX2. For alkaline phosphatase (ALP) measurements, cells were treated with RvE1 for 21 d, the cells were lysed, and ALP enzymatic activity in the cell lysates was determined. No significant changes in ALP in response to RvE1 were found (control, 1.53 ± 0.09; RvE1, 1.19 ± 0.17 enzyme activity units; p = 0.13). BSP and RunX2 expression were determined with real-time PCR. After 14 d of differentiation and RvE1 treatment, no significant alterations were found in BSP mRNA level (control, 3.71 ± 0.98; RvE1, 2.96 ± 0.46 relative value; p = 0.76) and RunX2 mRNA level (control, 1.883 ± 0.19; RvE1, 1.46 ± 0.23 relative value; p = 0.29).

Discussion

The results indicate that in addition to anti-inflammatory and proresolution actions mediated via the chemR23 receptor, RvE1 has a direct bone-preserving function in osteoblasts. ChemR23 overexpression limited to myeloid cells in the transgenic mice provides unique insight into the function of RvE1 in bone regeneration in periodontal disease as it is consistent with a two-pronged contribution of the resolution agonist. The enhanced reduction of inflammation through chemR23 in the transgenic animals is clear in both the peritonitis experiments and the resistance to periodontitis-induced bone loss. This is anticipated based on the distribution of the receptor to inflammatory cells and strongly implicates inflammation as a major determinant of disease. Also, consistent with the lack of overexpression of human chemR23 transgene on nonmyeloid osteoblasts, the impact of RvE1 on calvarial bone healing was not significantly higher in transgenic mice compared with WT. Importantly, we confirm the expression of chemR23 on osteoblasts and demonstrate a direct contribution of RvE1 in coupling bone formation and limiting bone resorption through modulation of OPG production by osteoblasts. Taken together with our earlier observations of direct actions on osteoclasts via BLT1, a clearer picture of RvE1 actions in resolution of inflammation and regeneration of bone emerges with resolution agonists having pleiotropic actions on inflammatory as well as mesenchymal cells.

RvE1 is a member of the E series resolvins, biosynthesized from the ω-3 PUFA EPA (2). RvE1 was first identified in vivo during the resolution phase of inflammation from peritoneal exudates (3). RvE1 is produced by hypoxia-activated human endothelial cells that convert EPA to an 18R-hydroxy–containing intermediate via aspirin-acetylated cyclooxygenase-2 as well as via a second route involving p450 like reactions (3). Once formed, 18R-HEPE is rapidly transformed by activated leukocytes to a 5(6)-epoxide-18R-hydroxy–containing intermediate where 5-LOX carries out consecutive steps. Physiologically relevant concentrations of resolvins are detectable in the blood after ω-3 supplementation (18). RvE1 complete stereochemistry is established (5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid); it is a stereoselective agonist that interacts with at least two identified G-protein–coupled receptors, chemR23 (4) and BLT1 (19). ChemR23 is expressed on monocytes, macrophages, dendritic cells, and, to a lesser extent, in PMN and CD4+ T lymphocytes (4, 8). Among bone cells, bone marrow stromal cells and osteoblasts express chemR23 (10), whereas osteoclasts express the BLT1 receptor (13).

Transgenic mice overexpressing the RvE1 receptor chemR23 in myeloid cells are viable and fertile. To characterize the inflammatory phenotype, zymosan-induced acute peritonitis was assessed in the presence and absence of RvE1. RvE1 has been shown to regulate leukocyte infiltration in the peritoneal cavity in 10–100 ng amounts in the mouse (5, 20, 21). In chemR23tg mice, the leukocyte response to RvE1 is heightened, as indicated by a shift in the dose-response curve for neutrophil inhibition, thus validating the expected transgenic phenotype. Notably, leukocyte recruitment appears lower in the chemR23tg mice even in the absence of exogenous RvE1. This observation may hint at an action by an endogenous ligand such as chemerin; however, chemerin is chemotactic, and it targets dendritic cells and NK cells, but not neutrophils (22). Transgenic macrophages display increased recruitment at the highest RvE1 dose (100 ng) leading to elevated total leukocyte numbers. RvE1 was shown to increase monocyte recruitment in the murine peritoneal cavity in response to zymosan (23), thus in situ differentiation of monocytes in chemR23tg mice may account for the increased macrophage yield.

Downstream signaling of the chemR23 receptor is not well understood at present. In chemR23-transfected Chinese hamster ovary cells, chemR23 mediates RvE1 actions via Akt phosphorylation leading to phosphorylation of ribosomal protein S6, a translational regulator (24). In human cartilage chondrocytes, stimulation of chemR23 with the peptide agonist chemerin resulted in Akt phosphorylation as well as p44/p42 MAPK phosphorylation and increased levels of proinflammatory cytokines and matrix metalloproteinases (25).

Periodontal disease is a bacterially induced chronic inflammatory disease leading to bone resorption (26). Leukocytes have been shown to play multiple roles in the progression of periodontal disease, including phagocytosis and killing of bacteria, secretion of inflammatory cytokines, mounting of specific immune response, and activation of osteoclasts (27). Leukocytes are essential in the host defense against oral pathogens; however, unresolved chronic inflammation in the periodontium is shown to cause periodontal bone loss (28). In this context, RvE1 was shown to resolve periodontal inflammation, inhibit periodontal bone loss (6), and enhance periodontal regeneration (7). Our observation that chemR23 gene overexpression protects against experimental periodontal disease confirms these findings and indicates that RvE1 actions in the periodontium are at least partially mediated by chemR23.

To elucidate whether leukocyte chemR23 regulates the inflammatory or bone-resorption modulating pathways, a noninfectious bone loss model, calvarial craniotomy was tested. Whereas RvE1 enhanced bone remodeling in all mice tested, no significant enhancement was conferred by chemR23 overexpression, indicating that in addition to bacterial clearance and resolution of inflammation, RvE1 may also directly regulate bone cells.

To assess direct RvE1 actions on bone cells, primary neonatal calvarial cultures from WT mice were tested. Neonatal calvarial cells represent a mixed population of osteogenic cells, including osteoblasts and bone marrow stromal cells (29). These two cell types are the chief sources of the cardinal regulators of osteoclast differentiation and hence bone resorption, RANKL and OPG. RANKL is the chief signal for osteoclast differentiation, whereas OPG antagonizes it as a decoy receptor (30). In periodontal disease, RANKL/OPG ratio is elevated (31). Osteoblasts express chemR23 (10), a finding we have confirmed in this study. In neonatal calvarial cultures, RvE1 rescues OPG expression that is suppressed by IL-6, whereas RANKL expression remains unchanged, thus inhibiting osteoclast differentiation and bone resorption. These observations provide molecular evidence for ligand-receptor interaction–mediated regulation of bone metabolism and complement our earlier findings showing that RvE1 treatment of bone marrow–derived primary osteoclast cultures results in a decrease of mature multinuclear osteoclasts and diminished bone resorption in vitro (13).

As RvE1 enhanced the healing of the craniotomy defect, we have examined whether RvE1 also stimulates bone formation. Osteogenic potential of RvE1 was examined in a mouse calvarial osteoblast cell line that upon differentiation produces extracellular collagen matrix, calcification nodules, and express key osteoblast markers such as ALP, BSP, and RunX2 (32). Multiple markers were examined to monitor early (RunX2, BSP) as well as late (ALP) osteoblast maturation (32, 33). RvE1 did not significantly alter osteoblast differentiation markers ALP, BSP, and RunX2 in calvarial osteoblasts, arguing against a direct action of RvE1 on bone formation. Indeed, in bone marrow stromal cells, knockdown of chemR23 (also referred to as CMKLR1) or the peptide ligand chemerin with small interfering RNA resulted in an increase of osteoblast differentiation markers, indicating that chemerin/chemR23 signaling inhibits osteoblast differentiation (10). RvE1 may indeed act on chemR23 receptors on osteoblasts; however, its main action is stimulation of OPG expression, and not osteoblast differentiation.

Inflammation and bone resorption occur together not only in periodontal disease but also in rheumatoid arthritis and systemic lupus erythematosus (34). Lipid mediators such as PGE2 and leukotriene B4 (LTB4) have been shown to play regulatory roles in both inflammation and bone resorption (35). However, the actions of inflammation-resolving lipid mediators such as RvE1 are in sharp contrast to those of PGE2 and LTB4. PGE2 stimulates both bone resorption and bone formation, thus in effect accelerating bone turnover (36). Elevated PGE2 production is associated with chronic osteomyelitis, failing joint prostheses, and periodontal disease (37). Leukotrienes such as LTB4, leukotriene D4, and 5-HETE stimulate bone resorption, inhibit bone formation, and contribute to inflammation-induced bone loss (38). RvE1 in contrast is protective in several inflammatory disease models including peritonitis, retinopathy, dermatitis, and colitis (17, 39, 40). In periodontal bone, RvE1 prevents alveolar bone loss induced by experimental periodontitis (6). In vitro, RvE1 inhibits osteoclast differentiation and in vitro bone resorption (13). Thus, resolvins are emerging as the first family of lipid mediators that simultaneously inhibit inflammation and block bone resorption.

In conclusion, findings in the chemR23 transgenic mice indicate that in addition to anti-inflammatory actions, RvE1 directly impacts bone remodeling by suppressing bone resorption.

Disclosures

C.N.S. and T.E.V.D. are inventors on patents (resolvins) assigned to Brigham and Women’s Hospital and Boston University and licensed to Resolvyx Pharmaceuticals. C.N.S. and T.E.V.D. are scientific founders of Resolvyx Pharmaceuticals and own equity in the company. C.N.S.’s interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. T.E.V.D.’s interests were reviewed and are managed by Boston University and now the Forsyth Institute in accordance with their conflict of interest policies. The other authors have no financial conflicts of interest.

Acknowledgments

We thank Makoto Arita for cDNA construct design and insightful comments.

Footnotes

  • ↵1 L.G. and D.F. share first authorship.

  • ↵5 T.E.V.D. and R.G. share senior authorship.

  • This work was supported in part by U.S. Public Health Service Grants DE19938, DE16933, and DE16191.

  • Abbreviations used in this article:

    ALP
    alkaline phosphatase
    BSP
    bone sialoprotein
    CEJ–ABC
    cementoenamel junction–alveolar bone crest
    chemR23
    chemokine-like receptor 1
    EPA
    eicosapentaenoic acid
    LTB4
    leukotriene B4
    OPG
    osteoprotegerin
    PMN
    polymorphonuclear leukocyte
    PUFA
    polyunsaturated fatty acid
    RANKL
    receptor activator of NF-κB ligand
    RunX2
    Runt-related transcription factor 2
    RvE1
    resolvin E1
    WT
    wild-type.

  • Received December 22, 2011.
  • Accepted November 6, 2012.

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