Dietary Omega-3 Fatty Acid Supplementation Improves Sickle Cell Bone Disease By Affecting Osteoblastogenesis and Adipogenesis

Sickle bone disease (SBD) is a severe and invalidating complication related to recurrent bone vaso-occlusive events affecting patients with sickle cell disease (SCD). In a humanized mouse model for SCD, we previously showed that SBD is sustained by impaired osteoblast function and increased osteoclast activity associated with local up-regulation of pro-inflammatory cytokines and anti-oxidant systems (Dalle Carbonare L., Blood 2015;126:2320-2328). Growing evidence supports a role of ω-3 fatty acid supplementation in improving bone homeostasis (Fallon EM, et al J Surg Res 2014;191:148-155.). A diet enriched with ω-3 fatty acids beneficially impacts SCD inflammatory vasculopathy, and blunts the acute and chronic SCD-related organ damage in humanized SCD mice (Kalish BT et al Haematologica 2015;100:870-880). Here, we sought to compare the dietary effects of ω-6 (soybean oil-based, SD)- vs ω-3 (fish oil-based, FD)-enriched diets on SBD in SCD mice (Hba^tm1(HBA)Tow Hbb^{tm2(HBG1,HBB*)Tow}). Treated SCD and control healthy mice (AA, Hba^tm1(HBA)Tow Hbb^{tm3(HBG1,HBB)Tow}) (n=6 animals in each group) were exposed to recurrent hypoxia/reoxygenation (rH/R) stress, which closely mimics SBD natural history (Dalle Carbonare L., Blood 2015). In SCD mice, FD prevented rH/R-induced bone loss compared to animals exposed to SD by decreasing osteoclast and increasing osteoblast activities. Up-regulation of molecular osteogenic markers such as Runx2 and Sparc and down-regulation of Rank and RankL, molecular markers of osteoclast recruitment and activity were observed in FD SCD mice exposed to rH/R compared to SD SCD animals. Similar changes, but to a lesser extent, were also observed in AA control mice exposed to rH/R stress. Expression of IL-6 (Il6) and matrix-metalloproteinase-9 (Mmp9) regulators which interact with RankL on osteoclastic activity and bone resorption were studied. In SCD mice, FD markedly reduced the up-regulation of both genes compared to SD SCD animals in conjunction with down-regulation of peroxiredoxin-2 (Prx2) gene expression, an important cytoprotective and antioxidant system. We also evaluated bone adipogenesis, which is believed to be an important contributor to bone impairment in SBD. Bone immunohistochemistry for Peripilin-1 which coats storage lipid droplets revealed increased adipogenesis in SD SCD mice compared to either FD SCD animals or AA controls, in association with downregulation of miR205, which decreases adipogenesis and enhances osteogenic activity. Our data thus indicate that in SCD exposed to rH/R, FD (i) improves osteoblastogenesis; (ii) decreases osteoclast activity; (iii) modulates the bone inflammatory response; iv) reduces adipogenesis. These findings provide new insights on the mechanism of action of ω-3 fatty acid supplementation on the pathogenesis of SBD and strengthen the rationale for ω-3 fatty acid dietary supplementation in SCD as a complementary therapeutic intervention targeting an amplified inflammatory response and sickle cell-related bone impairment.