BackgroundThe peripheral blood (PB) monocyte pool contains osteoclast progenitors (OCPs), which contribute to osteoresorption in inflammatory arthritides and are influenced by the cytokine and chemokine milieu. We aimed to define the importance of chemokine signals for migration and activation of OCPs in rheumatoid arthritis (RA) and psoriatic arthritis (PsA).MethodsPB and, when applicable, synovial fluid (SF) samples were collected from 129 patients with RA, 53 patients with PsA, and 110 control patients in parallel to clinical parameters of disease activity, autoantibody levels, and applied therapy. Receptors for osteoclastogenic factors (CD115 and receptor activator of nuclear factor-κB [RANK]) and selected chemokines (CC chemokine receptor 1 [CCR1], CCR2, CCR4, CXC chemokine receptor 3 [CXCR3], CXCR4) were determined in an OCP-rich subpopulation (CD3−CD19−CD56−CD11b+CD14+) by flow cytometry. In parallel, levels of CC chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CXC chemokine ligand 9 (CXCL9), CXCL10, and CXCL12 were measured using cytometric bead array or enzyme-linked immunosorbent assay. Sorted OCPs were stimulated in culture by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, and they were differentiated into mature osteoclasts that resorb bone. Selected chemokines (CCL2, CCL5, CXCL10, and CXCL12) were tested for their osteoclastogenic and chemotactic effects on circulatory OCPs in vitro.ResultsThe OCP population was moderately enlarged among PB cells in RA and correlated with levels of tumor necrosis factor-α (TNF-α), rheumatoid factor, CCL2, and CCL5. Compared with PB, the RANK+ subpopulation was expanded in SF and correlated with the number of tender joints. Patients with PsA could be distinguished by increased RANK expression rather than total OCP population. OCPs from patients with arthritis had higher expression of CCR1, CCR2, CCR4, CXCR3, and CXCR4. In parallel, patients with RA had increased levels of CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10, with significant elevation in SF vs PB for CXCL10. The subset expressing CXCR4 positively correlated with TNF-α, bone resorption marker, and rheumatoid factor, and it was reduced in patients treated with disease-modifying antirheumatic drugs. The CCR4+ subset showed a significant negative trend during anti-TNF treatment. CCL2, CCL5, and CXCL10 had similar osteoclastogenic effects, with CCL5 showing the greatest chemotactic action on OCPs.ConclusionsIn our study, we identified distinct effects of selected chemokines on stimulation of OCP mobilization, tissue homing, and maturation. Novel insights into migratory behaviors and functional properties of circulatory OCPs in response to chemotactic signals could open ways to new therapeutic targets in RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-017-1337-6) contains supplementary material, which is available to authorized users.

Detailed characterization of medullary and extramedullary reservoirs of osteoclast progenitors (OCPs) is required to understand the pathophysiology of increased periarticular and systemic bone resorption in arthritis. In this study, we focused on identifying the OCP population specifically induced by arthritis and the role of circulatory OCPs in inflammatory bone loss. In addition, we determined the relevant chemokine axis responsible for their migration, and targeted the attraction signal to reduce bone resorption in murine collagen-induced arthritis (CIA). OCPs were expanded in periarticular as well as circulatory compartment of arthritic mice, particularly the CCR2hi subset. This subset demonstrated enhanced osteoclastogenic activity in arthritis, whereas its migratory potential was susceptible to CCR2 blockade in vitro. Intravascular compartment of the periarticular area contained increased frequency of OCPs with the ability to home to the arthritic bone, as demonstrated in vivo by intravascular staining and adoptive transfer of splenic LysMcre/Ai9 tdTomato-expressing cells. Simultaneously, CCL2 levels were increased locally and systemically in arthritic mice. Mouse cohorts were treated with the small-molecule inhibitor (SMI) of CCR2 alone or in combination with methotrexate (MTX). Preventive CCR2/CCL2 axis blockade in vivo reduced bone resorption and OCP frequency, whereas combining with MTX treatment also decreased disease clinical score, number of active osteoclasts, and OCP differentiation potential. In conclusion, our study characterized the functional properties of two distinct OCP subsets in CIA, based on their CCR2 expression levels, implying that the CCR2hi circulatory-like subset is specifically induced by arthritis. Signaling through the CCL2/CCR2 axis contributes to OCP homing in the inflamed joints and to their increased osteoclastogenic potential. Therefore, addition of CCL2/CCR2 blockade early in the course of arthritis is a promising approach to reduce bone pathology.

Our study aimed to determine the functional activity of different osteoclast progenitor (OCP) subpopulations and signals important for their migration to bone lesions, causing local and systemic bone resorption during the course of collagen-induced arthritis in C57BL/6 mice. Arthritis was induced with chicken type II collagen (CII), and assessed by clinical scoring and detection of anti-CII antibodies. We observed decreased trabecular bone volume of axial and appendicular skeleton by histomorphometry and micro-computed tomography as well as decreased bone formation and increased bone resorption rate in arthritic mice in vivo. In the affected joints, bone loss was accompanied with severe osteitis and bone marrow hypercellularity, coinciding with the areas of active osteoclasts and bone erosions. Flow cytometry analysis showed increased frequency of putative OCP cells (CD3 B220 NK1.1 CD11b CD117 CD115 for bone marrow and CD3 B220 NK1.1 CD11b CD115 Gr-1 for peripheral haematopoietic tissues), which exhibited enhanced differentiation potential in vitro. Moreover, the total CD11b population was expanded in arthritic mice as well as CD11b F4/80 macrophage, CD11b NK1.1 natural killer cell and CD11b CD11c myeloid dendritic cell populations in both bone marrow and peripheral blood. In addition, arthritic mice had increased expression of tumour necrosis factor-α, interleukin-6, CC chemokine ligand-2 (Ccl2) and Ccl5, with increased migration and differentiation of circulatory OCPs in response to CCL2 and, particularly, CCL5 signals. Our study characterized the frequency and functional properties of OCPs under inflammatory conditions associated with arthritis, which may help to clarify crucial molecular signals provided by immune cells to mediate systemically enhanced osteoresorption.

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BackgroundPeripheral blood (PB) monocyte pool contains cells capable of differentiating into osteoclasts (OCs). These osteoclast progenitors (OCPs) contribute to osteoresorption in inflammatory arthritides under influence of the cytokine milieu and chemokine mediated trafficking.ObjectivesOur study aimed to define chemokine receptor profile of peripheral OCPs in rheumatoid arthritis (RA), with comparison to psoriatic arthritis (PSA), as well as their susceptibility to chemotactic signals.Methods129 RA, 53 PSA and 110 control patients were enrolled after Ethical approval. PB samples and synovial fluid (SF) samples, with clinical data of disease activity, inflammation and autoantibody levels were collected. Patients starting anti-TNF therapy were followed up 6 months. TNF-α and CTX serum levels were measured by ELISA. Frequency of OCP-rich subpopulation (CD3-CD19-CD56-CD11b+CD14+), expression of OC differentiation (CD115, RANK) and chemokine (CCR1, CCR2, CCR4, CXCR4) receptors was assessed by flow cytometry. OCPs were sorted using FACS, cultured with M-CSF and RANKL, stained for TRAP enzyme and mature OCs counted. Levels of CCL2, CCL3, CCL4, CCL5, CXCL9 and CXCL10 were measured using cytometric bead array, and of CXCL12 by ELISA. Osteoclastogenic effects of CCL2, CCL5 and CXCL10 were analyzed in cell culture, and chemotactic effects on OCPs were studied by cell migration assay using Transwell, with count of number of migrated cells and subsequently differentiated mature osteoclasts.ResultsOCP population was moderately enlarged in PB, further expanded in SF and correlated with TNF-α and rheumatoid factor (RF) levels in patients with RA. However, sorted OCPs generated similar number of mature OCs as control. RANK+ subpopulation was enlarged in SF vs PB and correlated with number of tender joints. In PSA, the OCP population was not enlarged, but had a higher RANK expression. OCPs in RA and PSA had higher expression of CCR1, CCR2, CCR4, CXCR4, and all except CCR4 showed positive PB-to-SF gradient. RA had higher levels of CCL2, CCL3, CCL4, CCL5, CXCL9 and CXCL10, with a positive PB-to-SF gradient for all except CCL5 and CXCL9. OCP frequency correlated with levels of CCL2 and CCL5. Subset expressing CXCR4 was associated with TNF-α, CTX and RF levels and was lower in patients treated with DMARD, who at the same time had lower osteoresorption (CTX). Subset expressing CCR4 showed significant negative trend during anti-TNF treatment. CCL2, CCL5 and CXCL10 showed significant osteoclastogenic effect. CCL5 showed greatest chemotactic effect, attracting the highest number of cells in the migration assay. At the same time, attracted cells possessed greater osteoclastogenic potential.ConclusionsOur study provides evidence of the specific importance of certain chemokine signals for stimulation of OCP mobilization, subsequent tissue homing, and maturation, explaining local as well as generalized bone loss seen in RA. Novel insights in regards to migratory behavior and functional properties of PB OCPs in response to chemotactic signal...