TGF-β Regulates the Development of Dendritic cell-derived Osteoclasts

Background: Bone-loss is a major health problem attending several inflammatory-bone disorders including periodontitis, due to elevated osteoclasts (OC) frequency & activity in response to osteogenic cytokines. Dendritic cells (DC) are professional antigen presenting cells that infiltrate periosseous tissues during inflammation, & whose subset(s) have been recently proposed to act as OC precursors. Previously, we reported the development of functional OC from murine CD11c⁺DC in response to RANKL & Aggregatibacter actinomycetemcomitans sonicated-antigens (Aa) [called; DC-derived OC or DDOC]. Objectives: Here, we aimed to identify factors involved in regulating DDOC development. Methods: DDOC were generated using our established in-vitro co-culture system of purified murine bone marrow-derived CD11c⁺DC with 100ng/ml RANKL or CD4⁺T-cells & 10ug/ml Aa. A genome-wide approach was employed via microarray-analysis to search for regulator(s) of RANKL-dependent DDOC development compared to DC+Aa as a negative control. Results: Focusing on cytokines & their receptors, we found that DDOC-development consistently correlates with ~1.5 fold-increase in TGF-β-receptor-II (TGF-βRII) expression. Real-time PCR & flow-cytometric analyses validated microarray results, & therefore we hypothesized that TGF-β promotes DDOC development. We neutralized TGF-β activity in our in-vitro co-cultures & found DDOC development to be completely abolished based on TRAP & resorptive-pit assays (p<0.03). Compelled to explore the in-vivo physiological relevance of our findings, we injected freshly purified live murine CD11c⁺carboxyfluoroscein succinimidyl ester-labeled DC onto the calvarias of NOD/SCID mice that received Aa+IFA 24hrs earlier. Dual-color quantitative-immunohistochemistry demonstrated the presence of CFSE⁺ CD11c⁺ TRAP⁺ OC-like cells associated with high local bone loss in experimental but not control animals (namely; sham; Aa+IFA only or with heat-killed DC), suggesting that CD11c⁺DC can become OC under inflammatory conditions in-vivo. TGF-β neutralization, blocked DDOC-development & its associated bone-loss in-vivo (p<0.02). Conclusion: Our findings strongly suggest that TGF-β is essential for regulating RANKL-dependent DDOC development, an osteo-immune mechanism that could be part of inflammation-induced bone loss. NIH/NIDCR-DE018356.