Method: DDOC are characterized using our established in-vitro co-cultures of purified CD11c+DC with RANKL or CD4+T cells (or Foxp3+T-reg) & sonicated-Aa (or RAW264.7+M-CSF/RANKL), which are correlated with mRNA/protein expressions by RT-PCR & Western blots, respectively, for the markers of TRAP-mediated osteoclastogenesis via paired t-test (p<0.05) as reported (Infect & Immun 2009). For physiological relevance, CFSE-labeled CD11c+DC are adoptively-transferred onto NOD/SCID calvarias with Aa+IFA injections (n=3).
Result: Our resulting data showed that: i) neutralization of TGF-β activity by mAb abolishes DDOC development in co-cultures, based on TRAP & resorptive-pit assays in-vitro (p<0.03) & in-vivo (p<0.02); ii) transfection of dominant-negative SOCS-3 molecules into CD11c+DDOC via adenoviral vector and subsequent adoptive-transfer study yield a significant inhibition of osteoclatogenesis and bone resorption in vitro & in vivo (p<0.05); iii) addition of Foxp3+CD4+T-reg cells into CD11c+DC with sonicated-Aa in co-cultures result in significantly reduced SOCS-3 & TRAP expressions and osteoclastogenesis on bone resorption assays in vitro & the calvarias in NOD/SCID mice, respectively (p<0.05).
Conclusion: These findings suggest that: i) TGF-β/SOCS-3 signaling is involved in modulating the development of DDOC function, and ii) Foxp3+T-reg cells may be a potentially integral player underlying the inflammation-induced bone loss & osteoclastogenesis through DC/T-cell interactions at the osteo-immune interface. Project supported by NIH-DE018356 & -015786, USA; University of Rochester, National Health Research Institute EX100-S34199N & National Science Committee NSC-100 -2314-B037-050-MY3, Taiwan.
Keywords: Bone, Immune response, Inflammatory mediators, Osteoblasts/osteoclasts and Resorption