The 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). PB 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. The 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. In 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.

The available treatments for cholestatic liver fibrosis are limited, and the disease often progresses to liver cirrhosis. Tamoxifen is a selective modulator of estrogen receptors, commonly used in breast cancer therapy. A recent in vitro study showed that tamoxifen deactivates hepatic stellate cells, suggesting its potential as an antifibrotic therapeutic, but its effects in vivo remain poorly investigated. In the present study, we show that tamoxifen protects against the cholestatic fibrosis induced by a diet supplemented with 0.025% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Mice fed with a DDC-supplemented diet for four weeks and treated with tamoxifen developed a significantly milder degree of liver fibrosis than vehicle-treated mice, as evidenced by a lower percentage of Sirius red-stained area (60.4% decrease in stained area in male and 42% decrease in female mice, p < 0.001 and p < 0.01, respectively) and by lower hydroxyproline content. The finding was further confirmed by qPCR analysis, which showed a lower expression of genes for Col1a1, Acta2, Sox9, Pdgf, and Krt19, indicating the inhibitory effect on hepatic stellate cells, collagen production, and biliary duct proliferation. The degree of protection was similar in male and female mice. Tamoxifen per se, injected into standard-diet-fed mice, increased the expression of genes for Il6 (p < 0.01 and p < 0.001 in male and female mice, respectively) and Tgfβ (p < 0.01 for both sexes), and had no adverse effects. We showed that tamoxifen sex-independently protects against cholestatic DDC-induced liver fibrosis. The increased expression of Il6 and Tgfβ seems to be a plausible protective mechanism that should be the primary focus of further research.

Epstein–Barr virus (EBV) infection and various chemokines, including CCL20, CXCL8 and CXCL10 are considered to participate in the pathogenesis of multiple sclerosis (MS), and several studies point to a direct regulatory effect of EBV on the expression of these chemokines. In our study we hypothesized that serum concentrations of CCL20, CXCL8 and CXCL0 are induced in patients with relapsing-remitting MS (RRMS) in comparison to healthy individuals, and that they are associated with EBV infection. Serum concentrations of CXCL8 and CXCL10 were lower in RRMS patients in relapse in comparison to healthy controls. Although potential effects of corticosteroid therapy introduced in a subgroup of RRMS patients prior to sampling were excluded by subgroup comparison, this possibility has to be considered while interpreting the results. We found an inverse association between serum concentrations of CXCL8 and anti-Epstein–Barr Virus Nuclear Antigen (EBNA) IgG and decreased expression of CXCL8 in peripheral blood mononuclear cells (PBMC) in relapse compared to remission. Lower serum concentrations of CXCL8 and CXCL10 in RRMS patients and decreased peripheral production of CXCL8 in relapse may indicate compensatory anti-inflammatory counter-regulation in MS.

Introduction: The existence of different osteoclast progenitor (OCP) subsets has been confirmed by numerous studies. However, pathological inflammation-induced osteoclastogenesis remains incompletely understood. Detailed characterization of OCP subsets may elucidate the pathophysiology of increased osteoclast activity causing periarticular and systemic bone resorption in arthritis. In our study, we rely on previously defined OCP subsets categorized by the level of CCR2 expression as circulatory-like committed CCR2^hi OCPs, which are substantially expanded in arthritis, and marrow-resident CCR2^lo OCPs of immature phenotype and behavior.Methods: In order to perform transcriptome characterization of those subsets in the context of collagen-induced arthritis (CIA), we sorted CCR2^hi and CCR2^lo periarticular bone marrow OCPs of control and arthritic mice, and performed next-generation RNA sequencing (n=4 for each group) to evaluate the differential gene expression profile using gene set enrichment analysis with further validation.Results: A disparity between CCR2^hi and CCR2^lo subset transcriptomes (863 genes) was detected, with the enrichment of pathways for osteoclast differentiation, chemokine and NOD-like receptor signaling in the CCR2^hi OCP subset, and ribosome biogenesis in eukaryotes and ribosome pathways in the CCR2^lo OCP subset. The effect of intervention (CIA) within each subset was greater in CCR2^hi (92 genes) than in CCR2^lo (43 genes) OCPs. Genes associated with the osteoclastogenic pathway (Fcgr1, Socs3), and several genes involved in cell adhesion and migration (F11r, Cd38, Lrg1) identified the CCR2^hi subset and distinguish CIA from control group, as validated by qPCR (n=6 for control mice, n=9 for CIA mice). The latter gene set showed a significant positive correlation with arthritis clinical score and frequency of CCR2^hi OCPs. Protein-level validation by flow cytometry showed increased proportion of OCPs expressing F11r/CD321, CD38 and Lrg1 in CIA, indicating that they could be used as disease markers. Moreover, osteoclast pathway-identifying genes remained similarly expressed (Fcgr1) or even induced by several fold (Socs3) in preosteoclasts differentiated in vitro from CIA mice compared to pre-cultured levels, suggesting their importance for enhanced osteoclastogenesis of the CCR2^hi OCPs in arthritis.Conclusion: Our approach detected differentially expressed genes that could identify distinct subset of OCPs associated with arthritis as well as indicate possible therapeutic targets aimed to modulate osteoclast activity.

Rheumatoid arthritis (RA) is chronic, autoimmune joint inflammation characterized by irreversible joint destruction. Besides increased resorption, destruction is a result of decreased bone formation, due to suppressed differentiation and function of the mesenchymal lineage-derived osteoblasts in inflammatory milieu. In this study, we analyzed the cellular composition of synovial tissue from 11 RA and 10 control patients harvested during planned surgeries in order to characterize resident synovial progenitor populations. Synovial cells were released by collagenase, and labeled for flow cytometry by two antibody panels: 1. CD3-FITC, CD14-PE, 7-AAD, CD11b-PECy7, CD235a-APC, CD19-APCeF780; and 2. 7-AAD, CD105-PECy7, CD45/CD31/CD235a-APC, and CD200-APCeF780. The proportions of lymphocytes (CD3⁺, CD19⁺) and myeloid (CD11b⁺, CD14⁺) cells were higher in synovial tissue from the patients with RA than in the controls. Among non-hematopoietic (CD45⁻CD31⁻CD235a⁻) cells, there was a decrease in the proportion of CD200⁺CD105⁻ and increase in the proportion of CD200⁻CD105⁺ cells in synovial tissue from the patients with RA in comparison to the control patients. The proportions of both populations were associated with inflammatory activity and could discriminate between the RA and the controls.

Antinuclear antibodies (ANA) are currently considered as an epiphenomenon of apoptotic processes, possibly in control of autoreactivity in patients with multiple sclerosis (MS). Apoptosis of reactive lymphocytes by the Fas/FasL system is described as an effective control mechanism for autoreactivity in MS. We aimed to provide a context to the potential link between ANA and peripheral lymphocyte apoptosis in MS. The presence of ANA was detected in sera by immunofluorescence assay, and concentrations of sFas and sFasL were determined in the sera of 44 and cerebrospinal fluid (CSF) of 11 relapsing-remitting (RR) MS patients using cytometric bead-based array, and their association with the disease characteristics was determined. ANA were detected in the sera of 43.2% of RRMS patients, and their frequency was the highest in patients with disease duration of less than one year (88,89%). In addition, the number of experienced relapses was lower in ANA-positive patients. Concentrations of sFasL were inversely associated with patients’ expanded disability status scale (EDSS) scores. Low concentrations of both soluble factors strongly discriminated patients with moderate to severe disability, from patients with mild or absent disability only in a group of patients with prolonged disease duration (>10 years). Both soluble mediators were significantly higher in ANA-positive patients. FasL concentrations were inversely associated with the number of relapses. There is a potential link between the presence of ANA and peripheral lymphocyte apoptosis mediated by Fas/FasL system in MS, whose precise role and significance needs to be determined by future mechanistic studies.