TGF-β/SOCS-3 signaling and Foxp3+T-reg modulate DC/T-cell interactions during DDOC-associated osteoclastogenesis

Objective: Inflammation-induced bone loss is associated with elevated osteoclast (OC) activity. Dendritic cells (DC) are professional antigen-presenting-cells involved in T-cell immunity, whose subsets have been shown to act like OC-precursors [OCp; JBMR 2007]. We reported the development of functional OC derived from CD11c⁺DC (called: DDOC) in response to RANKL plus inflammatory stimuli (i.e., Aggregatibacter actinomycetemcomitans-Aa; J. Immunol. 2006 & Blood 2009]. In this study, we proposed to study how the signal interactions work between DC/T-cells and osteotropic cytokines’ that modulate DDOC-mediated osteoclastogenesis.

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.