Darja Flegar

Chemokine signals are crucial for enhanced homing and differentiation of circulating osteoclast progenitor cells.

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.

Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis.

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–/loCD117+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.

AB0026 Chemokine signals are critical for homing and enhanced differentiation of circulating osteoclast progenitor cells

Background Peripheral 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. Objectives Our 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. Methods 129 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. Results OCP 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. Conclusions Our 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 signals could open way to new therapeutic targets in RA. Acknowledgements This work was supported by a grant from the Croatian Science Foundation (project number 5699). Disclosure of Interest None declared

AB0085 Osteoclast Progenitors Are Attracted by CCl2/CCR2 and CCl5/CCR5 Chemotactic Signals To The Sites of Osteitis Associated with Collagen Induced Arthritis

Background Osteitis, replacement of bone marrow adipocytes with inflammatory cells, is an early sign of inflammation in rheumatoid arthritis (RA), seen as bone marrow edema by magnetic resonance imaging. Osteoclast progenitors (OCPs), normally present within circulating monocytes, are presumed to be increasingly attracted to inflamed joints in RA, where their enhanced activation and differentiation leads to excessive osteoresorption. Objectives Our goal was to identify attraction signals important for homing of OCPs to the sites of osteitis. Therefore, we analyzed changes in immune cell populations as well as OCP chemokine receptor profile and migration potential in circulation, affected joints and periarticular bone in collagen induced arthritis (CIA) as a mouse model of RA. Methods After obtaining Ethical approval, C57BL/6 mice were immunized with chicken type II collagen in complete Freunds adjuvant and evaluated for CIA development by clinical and histological scoring and anti-collagen antibody detection. Subchondral bone loss and osteitis were assessed by histology and histomorphometry. Distal tibial bone marrow, peripheral blood (PBL) and collagenase-digested tarsometatarseal joints were analyzed by flow-cytometry for the expression of hematopoietic markers and chemokine receptors (CD3, B220, NK1.1, CD11b, CD115, Gr1, CCR2, CCR5, CCR9). Transwell system was used to assess migration potential of M-CSF/RANKL stimulated PBL cells toward CCL2 or CCL5 gradient in vitro. For migration tracking, labeled PBL cells were transferred to recipient CIA mice and assessed by in vivo fluorescence imaging. Results Immunized mice developed CIA with the maximum incidence of 60%. Arthritic areas showed histological presence of osteitis and significant subchondral bone loss (BV/TV 46.8±13.4% in control vs 32.1±8.6% in CIA) with increased number of mature osteoclasts (1.4±1.1 in control vs 5.4±4.7 in CIA). Flow-cytometry analysis revealed significant expansion of several populations in CIA, including lymphoid-negative CD11b+/Gr-1+ OCP subset within affected joints (24±4.1% in control vs 49±12.7% in CIA); lymphoid-negative CD11b+CD115+ OCP subset in PBL (4.6±0.7% in control vs 8.8±3.3% in CIA) and periarticular bone marrow (9.6±1.1% in control vs 21.9±3.4% in CIA). PBL OCP populations expressing chemokine receptors were reduced in arthritis (CCR2+ 32.1±4.3% in control vs 24.7±5.1% in CIA; CCR5+ 23.3±4.5% in control vs 18.1±3.7% in CIA; CCR9+ 5.9±3.7% in control vs 0.4±0.1% in CIA). In vitro migration of PBL cells toward chemotactic gradients was significantly enhanced in CIA (11.5 (IQR 11–13.25) in control vs 20.5 (IQR 20–25) in CIA for CCL2; 12.5 (IQR 9–18.5) in control vs 33.5 (IQR 23–35.75) in CIA for CCL5). Fluorescently-labeled PBL cells were efficiently attracted to tarsometatarseal joints 48 hours after in vivo transfer in mice with CIA. Conclusions In fully developed CIA OCP populations are highly induced, with the reduction of circulating OCP subpopulations expressing chemokine receptors, possibly indicating their increased migration and homing to bone surfaces of the inflamed joints. Therapeutic blocking of chemokine signals may therefore be a promising approach to reduce osteoresorption in RA. Acknowledgement This work was fully supported by Croatian Science Foundation (project nr. 5699) Disclosure of Interest None declared

THU0011 CD32+ B Lymphocytes and IL21R+ T Lymphocytes Are Associated with Disease Activity and Increased Levels of Proinflammatory Cytokines in Patients with Rheumatoid and Psoriatic Arthritis

Background Autoimmunity is presumed to be a major driving force in the pathogenesis of chronic rheumatic diseases, including rheumatoid arthritis (RA) and psoriatic arthritis (PSA). Although these forms of arthritis differ in their clinical features, both are marked by persistent inflammation and osteoresorption underpinned by aberrant lymphocyte populations and disturbed cytokine network. Objectives Various subpopulations of both T and B lymphocytes have been implied in RA and PSA, but their relevance to disease onset and progression remains largely unclear. The aim of our study was to define the association of T, B and NK cell subpopulations with cytokine levels and clinical parameters in RA and PSA patients. Methods Mononuclear cells were isolated from peripheral blood of healthy controls (n=35), RA (n=36) and PSA (n=13) patients, after obtaining Ethical approval and informed consent. Flow cytometry was applied to discriminate between T lymphocyte subpopulations: Th1/2 (CD3+CD4+CCR6-), Th17 (CD3+CD4+CCR4+CCR6+), Tfh (CD3+CD4+CXCR5+), Tc (CD3+CD8+) and memory Tc (CD3+CD8+CCR4+); B lymphocyte subpopulations: naïve (CD19+IgD+CD27-), unswitched memory (CD19+IgD+CD27+), class-switched memory (CD19+IgD-CD27-) and plasmablasts (CD19+IgD+CD27hiCD38+); and NK cells (CD3-CD19-CD56+). Markers of lymphocyte maturation (CD32), activation (CD86, IL21R, CD25) and migration (CD11b) were also analyzed. Frequencies of lymphocyte subpopulations were correlated with clinical parameters, including DAS28 (for RA and PSA) and BASDAI (for PSA with spondylitis). Finally, serum levels of various cytokines (TNF, IL4, IL6, IL10, IL17, CCL2, CCL3, CCL4, CCL5) were measured by flow cytometry bead based assay. Results Several subpopulations were found to be significantly expanded: CD32+ naïve (Ctrl 1.8%, RA 5.8%, PSA 6.0%) and memory B lymphocytes, both class-switched (Ctrl 1.8%, RA 5.3%, PSA 3.8%) and unswitched (Ctrl 4.0%, RA 16.8%, PSA 13.7%); memory Tc lymphocytes (Ctrl 4.8%, RA 6.8%, PSA 9.1%); and CD11b+ B lymphocytes (Ctrl 15.1%, RA 20.9%, PSA 20.2%). Significant correlations between lymphocyte subpopulations and clinical parameters included: positive association of IL21R+ T lymphocytes with DAS28 in RA (ρ=0.45) and negative with BASDAI in PSA (ρ=-0.86); negative association of class-switched memory B lymphocytes with DAS28 in RA (ρ=-0.52), but positive with BASDAI in PSA (ρ=0.71). Correlation of lymphocyte subpopulations with cytokine levels showed: significant positive association of CD32+ naïve B lymphocytes with TNF and CCL5 (ρ=0.55 and 0.54); CD32+ class-switched and unswitched memory B cells with TNF (ρ=0.47 and 0.55) and CCL4 (ρ=0.47 and 0.54); CD11b+ B lymphocytes with CCL4 (ρ=0.47); and IL21R+ T lymphocytes with TNF, CCL3 and CCL4 (ρ=0.42, 0.47 and 0.49). Conclusions Our results indicate novel T and B lymphocyte subpopulations induced in both RA and PSA. CD32+ B lymphocytes, as well as IL21R+ T lymphocytes, may be of particular interest as possible therapeutic targets, since their frequency is associated with disease activity and increased levels of proinflammatory and proresorptive cytokines. Acknowledgement The work has been fully supported by Croatian Science Foundation (project no. 5699). Disclosure of Interest None declared

Expression of Chemokines and Chemokine Receptors on Peripheral Blood Mononuclear Cells of Patients with Rheumatoid Arthritis

Background Rheumatoid arthritis (RA) is characterized by chronic inflammatory response as well as enhanced bone destruction. Development of bone erosions is critically dependent on osteoclasts, which are highly specialized bone cells capable of resorbing the mineralized matrix. Osteoclast progenitors (OCPs), contained among myeloid hematopoietic lineage, could be found in the peripheral blood and synovial tissue of patients with RA, mediating bone loss locally, in the form of bone erosions and joint osteolysis, and systemically, with loss of skeletal bone density. Objectives To define these chemotactic signals by analyzing expression of several chemokines and chemokine receptors on T lymphocytes and OCPs in the peripheral blood of RA patients, to measure the levels of their respective ligands in serum and synovial fluid of RA patients, and to assess the osteoclastogenic potential of OCPs. Methods Mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. The phenotype of isolated mononuclear cells was determined using flow cytometry. OCPs (CD3-CD19-CD56-CD11b+CD14+) were analyzed for the expression of the following chemokine receptors: C5AR1, CCR1, CCR2, CCR4, CXCR4. T lymphocytes (CD3+CD4+ or CD3+CD8+) were analyzed for expression of CXCR5, CCR4, CCR6 chemokine receptors. Chemokine ligand concentrations (MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5) were measured in serum and synovial fluid of RA patients using flow cytometry bead based array. OCPs were sorted and plated into cell culture with M-CSF and RANKL. After two weeks, the cells were stained for TRAP enzyme and positive, mature, osteoclasts were counted. Results Human peripheral blood OCPs similarly expressed chemokine receptors CCR1, CCR2, CCR4 and CXCR4 in RA and healthy subjects. However, MCP1/CCL2, MIP1a/CCL3 and MIP1b/CCL4 concentrations were significantly higher in synovial fluid (and blood for CCL2 and CCL4). Cell culture revealed no significant differences in mature osteoclast count between RA and control group. The proportion of T lymphocytes expressing CCR4 was two-fold higher in RA patients compared to controls. T lymphocyte expression of CXCR5 and CCR6 was similar between RA and control group. Conclusions Although OCPs in RA have a differentiation potential similar to controls, levels of several chemokines are upregulated, indicating a possible chemotactic mechanism of OCP migration to affected joints. These results may help to reveal migration mechanism of T lymphocytes and OCPs specifically associated with RA in order to develop more efficient therapeutic approaches. Acknowledgements This work has been supported by Croatian Science Foundation (project number 5699). Disclosure of Interest None declared