Background/Purpose: Immature dendritic cells (DCs) can develop into osteoclasts (OCs), however, the mechanisms regulating this differentiation switch are not yet understood. We have shown that protein citrullination can play important roles in OC differentiation, both at steady state and in rheumatoid arthritis (RA) when anti-citrulline protein antibodies (ACPAs) induce bone erosion through an IL-8 mediated induction of OC development. In the present study we analyzed how protein citrullination and ACPAs regulate differentiation plasticity towards the OC lineage in DCs.

Methods: We have previously shown that the endogenously produced lactic acid (LA), a glycolysis side product, can increase the ability of developing DCs to differentiate into osteoclasts in dense cell cultures, whereas sparse cultures, associated with low LA levels, promoted the development of immunostimulatory DCs with little ability to form OCs. By exploiting this mechanism we generated different monocyte-derived DC types in parallel with macrophages and cultured these cells in presence of M-CSF and RANKL to induce OC development. Mass spectrometry was applied to analyze protein expression and citrullination at different stages of OC differentiation. Activity of peptidylarginine deiminases, the enzymes responsible for protein citrullination, was measured by ELISA, cytokine levels were analyzed using CBA technology. We studied in detail the autocrine effects of IL-8 in OC cultures.

Results: Citrullinated-actin peptides were identified in all stages DC-OC transdifferentiation and citrullinated-vimentin peptides were identified in mature OCs. Expression of PAD2 and PAD4 increased during DC differentiation and PAD activity was detected in both DCs and OCs. Interestingly, the efficiency of DC-OC transdifferentiation correlated with the level of PAD activity and with higher detection of citrullinated peptides in DCs. The PAD inhibitor Cl-Amidine efficiently interfered with OC development form DC precursors. Polyclonal and certain monoclonal ACPAs increased osteoclastogenesis from DCs and the intensity bone resorption. DC-derived OC differentiation was inhibited by Cl-Amidine in the presence of ACPAs. The IL-8 receptors CXCR1 and CXCR2 were present on immature DCs and the increased osteoclastogenesis was associated with elevated IL-8 levels in ACPA-treated cultures. IL-8 neutralization blocked the ACPA-mediated increase of OC development.

Conclusion: Our results indicated that DCs are heterogenic in their ability to form OCs and the differentiation plasticity towards the OC lineage might be influenced by protein citrullination. The increase of LA at sites of immune activation might play an important role in the reprogramming DCs and increasing OC development. Antibodies that target citrullinated proteins and have been suggested to play an improtant pathogenic role in RA-associated bone destruction ACPAs can further increase osteoclastogenesis from DCs through an IL-8 dependent mechanism, which further support OC development in an autocrine manner. Blocking IL-8 and PAD activities represent important therapeutic possibilities that might interfere with DC transdifferentiation into OC and ACPA-induced bone damage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/protein-citrullinations-by-pad-enzymes-promote-dendritic-cell-transdifferentiation-into-osteoclast-and-generate-targets-for-ra-specific-antibodies/