Citrullination Controls Dendritic Cell Transdifferentiation into Osteoclasts

Abstract

An increased repertoire of potential osteoclast (OC) precursors could accelerate the development of bone-erosive OCs and the consequent bone damage in rheumatoid arthritis (RA). Immature dendritic cells (DCs) can develop into OCs, however, the mechanisms underlying this differentiation switch are poorly understood. We investigated whether protein citrullination and RA-specific anti-citrullinated protein Abs (ACPAs) could regulate human blood-derived DC-OC transdifferentiation. We show that plasticity toward the OC lineage correlated with peptidyl arginine deiminase (PAD) activity and protein citrullination in DCs. Citrullinated actin and vimentin were present in DCs and DC-derived OCs, and both proteins were deposited on the cell surface, colocalizing with ACPAs binding to the cells. ACPAs enhanced OC differentiation from monocyte-derived or circulating CD1c⁺ DCs by increasing the release of IL-8. Blocking IL-8 binding or the PAD enzymes completely abolished the stimulatory effect of ACPAs, whereas PAD inhibition reduced steady-state OC development, as well, suggesting an essential role for protein citrullination in DC-OC transdifferentiation. Protein citrullination and ACPA binding to immature DCs might thus promote differentiation plasticity toward the OC lineage, which can facilitate bone erosion in ACPA-positive RA.

FIGURE 1.

OC development from MΦ and DC precursors. (A) Schematic representation of OC development from MΦ and DCs. CD14 and CD1a expression was measured by flow cytomtery. (B) Protein abundances were determined in samples from three different stages of DC- and MΦ-derived OC development by mass spectrometry and label-free quantification and analyzed by PCA. Principal components 1 and 2 of triplicate samples from day 0 (iDC and MΦ), day 3 (intermediate stage), and day 12 (mature OCs) are plotted. (C) Expression of characteristic MΦ, DC, and OC markers were quantified using mass spectrometry, and the values in the table represent protein abundances that are log₁₀-transformed. Mean ± SD values are calculated from triplicate samples.

FIGURE 2.

Protein citrullination and high PAD activity levels are associated with differentiation plasticity toward OCs in iDCs. (A) PCA indicates clearly distinct protein profiles in DCs obtained from sparse or dense cultures (principal components 1 and 2 are shown). Triplicate samples were analyzed from three independent experiments. (B) Images showing resorption activity on calcium phosphate–coated surface representing OCs that were generated from sparse or dense culture–derived iDCs (original magnification ×40). (C) Higher PAD activity was observed in the lysates of DCs that were obtained from dense cultures. Activity levels are normalized to sparse cultures; the graph represents four individual experiments, each with three replicates. *p < 0.05. (D) Normalized PAD activity levels are shown in cell lysates of iDCs and mature OCs. (E) Immunohistochemistry images indicate 3,3-diaminobenzidene staining of PAD4 and PAD2 enzymes with their respective irrelevant controls during OC development from DC precursors. Slides were counterstained with Mayer’s hemotoxylin and analyzed in light microscopy (original magnification ×100). (F) Citrullinated actin was identified using mass spectrometry in lysates of DCs developing in sparse or dense cultures. The graph shows Mascot score values obtained for the identified peptide; symbols represent three different DC cultures. Mean ± SD values are shown. (G) Reduced DC–OC transdifferentiation in the presence of the PAD inhibitor Cl-amidine.

FIGURE 3.

Citrullinated actin and vimentin are potential ACPA targets on the surface of iDC-derived OCs. Confocal microscopy images displaying vimentin (A) or actin (B) localization in green and ACPA (clone 1325:04C03) binding in red on the surface of iDCs or DC-derived OCs (original magnification ×400). (C) Staining with the control IgG for ACPA and vimentin or actin. Nuclear stainings using DAPI are indicated in blue. In the last panels, the images respresent the ACPA staining merge with actin or vimentin; yellow coloring indicates colocalization of the tested proteins with monoclonal ACPA.

FIGURE 4.

Targeting citrullinated cell surface proteins by ACPAs induces DC–OC transdifferentiation. TRAP staining (upper panels) and bone resporption assays (lower panels) representing OC cultures generated from monocyte-derived iDCs derived from the PB of healthy individuals (A) and RA patients (B). The iDCs were cultured in the presence of 0.1 or 1 μg/ml ACPAs or control IgG. The images were obtained using light microscopy [(A and B) original magnification ×200 (top row), ×40 (bottom row)]. The graphs represent the fold difference (normalized using controls M-CSF/RANKL but no Abs) in OC numbers and in resorption areas. Mean ± SD values were calculated from at least three independent experiments. (C) ACPAs or control IgGs were applied at different concentrations during DC-derived OC development; OC activity was measured in bone erosion assay (normalized to controls receiving M-CSF/RANKL but no Abs). (D) F(ab')₂ ACPAs induced an increase in OC development. The graph represents the OC numbers developing in the presence of F(ab')₂ ACPA and ACPA Ab with their respective control IgGs. Mean ± SD values were calculated from at least three independent experiments. (E) CD1c⁺ DCs were isolated from PB of healthy individuals, and the cells were cultured in the presence of M-CSF, RANKL, and ACPAs or control IgG. OCs were visualized using TRAP staining (original magnification ×100); the graph represents normalized values obtained from three independent experiments. (F) The PAD inhibitor Cl-amidine prevented the ACPA-induced increase in OC development. Cl-amidine was used in concentrations of 0.2, 2, 20, and 200 μM, and IgG and ACPA concentrations were 1 μg/ml. The graph represents OC numbers developing in the presence of IgG or ACPA. Mean ± SD values were calculated in triplicate wells; representative results from three independent experiments are shown. *p < 0.05.

FIGURE 5.

ACPAs regulate osteoclastogenesis through induction of IL-8. IL-8 (A) and IL-6 (B) levels were measured in the supernatants of DC-derived OC cultures at different time points using cytometric bead array. ACPAs and control IgG were added to culture at a final concentration of 1 μg/ml. Mean ± SD values were calculated from triplicate samples; representative data from three independent experiments are shown. (C) F(ab')₂ ACPAs induced increased IL-8 release. The graph shows the increase in the IL-8 levels mediated by F(ab')₂ ACPAs and ACPA Ab but not F(ab')₂ IgG or control IgG. (D) Expression of the IL-8Rs CXCR1 and CXCR2 was analyzed on iDCs using flow cytometry. Histograms in gray show stainings with isotype control Abs. (E) IL-8 neutralizing Abs abolished the stimulatory effects of ACPAs on OC differentiation. The graphs show OC numbers; mean ± SD values were calculated from triplicate samples. *p < 0.05.