Background and purpose: The inflammation‐resolving lipid mediator resolvin E1 (RvE1) effectively stops inflammation‐induced bone loss in vivo in experimental periodontitis. It was of interest to determine whether RvE1 has direct actions on osteoclast (OC) development and bone resorption.Experimental approach: Primary OC cultures derived from mouse bone marrow were treated with RvE1 and analysed for OC differentiation, cell survival and bone substrate resorption. Receptor binding was measured using radiolabelled RvE1. Nuclear factor (NF)‐κB activation and Akt phosphorylation were determined with western blotting. Lipid mediator production was assessed with liquid chromatography tandem mass spectrometry.Key results: OC growth and resorption pit formation were markedly decreased in the presence of RvE1. OC differentiation was inhibited by RvE1 as demonstrated by decreased number of multinuclear OC, a delay in the time course of OC development and attenuation of receptor activator of NF‐κB ligand‐induced nuclear translocation of the p50 subunit of NF‐κB. OC survival and apoptosis were not altered by RvE1. Messenger RNA for both receptors of RvE1, ChemR23 and BLT₁ is expressed in OC cultures. Leukotriene B₄ (LTB₄) competed with [³H]RvE1 binding on OC cell membrane preparations, and the LTB₄ antagonist U75302 prevented RvE1 inhibition of OC growth, indicating that BLT₁ mediates RvE1 actions on OC. Primary OC synthesized the RvE1 precursor 18R‐hydroxy‐eicosapentaenoic acid and LTB₄. Co‐incubation of OC with peripheral blood neutrophils resulted in transcellular RvE1 biosynthesis.Conclusions and implications: These results indicate that RvE1 inhibits OC growth and bone resorption by interfering with OC differentiation. The bone‐sparing actions of RvE1 are in addition to inflammation resolution, a direct action in bone remodelling.British Journal of Pharmacology (2008) 155, 1214–1223; doi:10.1038/bjp.2008.367; published online 22 September 2008

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.

Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice ( db / db ), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor ( ERV1 ), and a newly bred strain of db / ERV1 mice, were used to determine the impact of RvE1 on the phagocytosis of Porphyromonas gingivalis in T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeled P. gingivalis , and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate the in vivo impact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis of P. gingivalis in WT animals but had no impact in db / db animals. In ERV1 -transgenic and ERV1 -transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals. In vivo dorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis of P. gingivalis in the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressing ERV1 .

Background: Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long‐term iNOS inhibition, and the differentiation and activity of OC from iNOS‐knockout (KO) mice in vitro.Methods: Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild‐type C57BL/6 mice. OC were counted 6 days later after tartrate‐resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks.Results: Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature‐induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild‐type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts.Conclusion: Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature‐induced periodontitis, thus confirming that iNOS‐derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.

This study evaluated the alveolar bone response to testosterone and the impact of Resolvin D2 (RvD2) on testosterone-induced osteoblast function. For the in vivo characterization, 60 male adult rats were used. Treatments established sub-physiologic (L), normal (N), or supra-physiologic (H) concentrations of testosterone. Forty rats were subjected to orchiectomy; 20 rats received periodical testosterone injections while 20 rats received testicular sham-operation. Four weeks after the surgeries, 10 rats in each group received a subgingival ligature around the lower first molars to induce experimental periodontal inflammation and bone loss. In parallel, osteoblasts were differentiated from neonatal mice calvariae and treated with various doses of testosterone for 48 h. Cell lysates and conditioned media were used for the determination of alkaline phosphatase, osteocalcin, RANKL, and osteoprotegerin. Micro-computed tomography linear analysis demonstrated that bone loss was significantly increased for both L and H groups compared to animals with normal levels of testosterone. Gingival IL-1β expression was increased in the L group (p<0.05). Ten nM testosterone significantly decreased osteocalcin, RANKL, and OPG levels in osteoblasts; 100 nM significantly increased the RANKL:OPG ratio. RvD2 partially reversed the impact of 10 nM testosterone on osteocalcin, RANKL, and OPG. These findings suggest that both L and H testosterone levels increase inflammatory bone loss in male rats. While low testosterone predominantly increases the inflammatory response, high testosterone promotes a higher osteoblast-derived RANKL:OPG ratio. The proresolving mediator RvD2 ameliorates testosterone-derived downregulation of osteocalcin, RANKL, and OPG in primary murine osteoblasts suggesting a direct role of inflammation in osteoblast function.