Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors

Introduction: Ankylosing spondylitis (AS) is typically characterized by focal bone over-growth and also by systemic bone loss. We hypothesize that the increased osteoproliferation found in AS might be partially due to reduced ability of osteoclast precursors (OCPs) to differentiate into osteoclasts (OCs). Therefore, our aim was to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes' phenotype, and in vitro osteoclast differentiation in AS patients. Methods: Patients with active AS without any ongoing therapy and age-and gender matched healthy donors were recruited. Receptor activator of nuclear factor-K13 (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations were assessed. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and quantitative reverse transcription real-time PCR for OC-specific genes were performed. Results: Pro-and anti-inflammatory cytokine serum levels were higher in AS patients than in controls. RANKL neutrophil expression was higher in AS patients when compared to healthy donors, but CD51/CD61 expression was lower in the classical monocyte subpopulation. Concerning osteoclastogenesis, we found no differences in the in vitro osteoclast differentiating potential of these cells when compared to healthy donors. However, we observed low expression of CSF1R, RANK, and NFATc1 in AS OCPs. Conclusion: Despite the high levels of pro-inflammatory cytokines present in AS patients, no differences in the number of OC or resorbed area were found between AS patients and healthy donors. Moreover, we observed that OCPs have low OC specific gene expression. These findings support our hypothesis of an impaired response of OCPs to pro-osteoclastogenic stimuli in vivo in AS patients.Peer reviewe

Upregulation of Inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing

Peer reviewe

Identification of a cytokine network sustaining neutrophil and Th17 activation in untreated early rheumatoid arthritis

© 2010 Cascão et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustained synovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset and perpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophil and Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration and before treatment (VERA). Methods: Cytokines related to neutrophil and Th17 activation were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. Results: VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1b and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. In established RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. Conclusions: VERA patients already display increased levels of cytokines related with Th17 polarization and neutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggest that a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis.This work was supported by a grant from Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. RAM and RC were funded by Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 and SFRH/BD/40513/2007, respectively. MMS-C was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and a EULAR Young Investigator Award

Toezicht op de toezichthouders: Hoe kunnen non-gouvernementele organisaties ter verantwoording worden geroepen?

Abstract Background Our understanding of the biology of osteoblasts is important as they underpin bone remodelling, fracture healing and processes such as osseointegration. Osteoblasts isolated from human humeral samples display distinctive biological activity in vitro, which relates to the samples’ bone types (subchondral (S), trabecular (T), cortical (C)). Our aim was to isolate primary osteoblast cultures from different bone types from the proximal femur of a clinical population of dogs presented for total hip replacement and compare the behaviour of the osteoblasts derived from different bone types, to identify a preferred bone type for isolation. Results No differences were found for osteoblast doubling time (median for S = 2.9, T = 3.1 and C = 2.71 days, respectively; p = 0.33), final cell number (median for S = 54,849, T = 49,733, C = 61,390 cells/cm2; p = 0.34) or basal tissue non-specific alkaline phosphatase (TNAP) activity (median for S = 0.02, T = 0.02, C = 0.03 U/min/mg protein; p = 0.81) between bone types after 6 days of culture in basal media. There were no differences in mineralizing TNAP activity (S = 0.02, T = 0.02, C = 0.03 U/min/mg protein, p = 0.84) or in mineralized area (S = 0.05, T = 0.04, C = 0.04%, p = 0.92) among cells from different bone types. Conclusions There is no significant difference in mean doubling time, basal or mineralizing TNAP activity or mineralized area in osteoblasts derived from subchondral, cortical, or trabecular bone types from the canine femoral head. However, there appears to be a high level of inter-animal variability in the studied parameters, which was independent of age, body mass, and sex. Trabecular isolate osteoblasts have the least variation of the bone types studied, and therefore should be considered a preferred source for primary osteoblast cultures. The work here provides baselines for canine osteoblast function, which has utility for future comparative studies

Serum levels of bone turnover markers, bone metabolism proteins and cytokines.

<p>CTX-I levels are decreased in patients at 6 months follow-up when compared to healthy donors (p = 0.0268). DKK-1, IL-1 β, IL-6, IL-17A, IL-12p70, IL-23, TNF, MCP-1 and TGF-β are increased in patients at baseline when compared to healthy donors. After 6 months of therapy, follow-up patients had decreased levels of IL-17A, IL-23 and TGF-β when compared to their baseline. We have also observed that after therapy the levels of IL-1 β, IL-6, IL-12p70 and TNF were still significantly higher than healthy donors levels’. Each dot represents a sample. Line represents median. * vs Baseline, § vs Follow-up. DKK—dickkopf-related protein, CTX—carboxy-terminal collagen crosslinks, IL—interleukin, TNF—tumor necrosis factor, MCP—monocyte chemmotractant protein, TGF—transforming growth factor.</p

Gene expression profile of stimulated cells in culture for 21 days.

<p>All genes except CTSK are significantly decreased at day 1 in patients at follow-up when compared to healthy donors. At day 1 RANK expression in patients at baseline is also significantly reduced when compared to healthy donors (p = 0.0268). We found no differences between groups throughout the culture time except for Atp6v0d2 expression at day 21 in patients at baseline which is significantly reduced when compared both to healthy donors (p = 0.039) and patients at follow-up (p = 0.0234). Relative gene expression shown in Log scale. Dots in graphs represent median gene expression for each group at each time-point. d—day; CSF1R—gene encoding macrophage-colony stimulating factor (c-fms), RANK—gene encoding for receptor activator of nuclear factor-κβ, NFATc1—gene encoding for nuclear factor of activated T-cells, Atp6v0d2—gene encoding ATPase, H⁺ transporting, lysosomal V0 subunit D2, CTSK—gene encoding cathepsin K. p<0.05 is considered significant.</p

Osteoclast number is reduced in baseline patients, but bone resorption activity is increased after TNF-blocker exposure.

<p>A) Representative images of culture day 21 of cells stimulated with M-CSF, RANKL, dexamethasone and TGF-β and stained for TRAP. OC number increased throughout time and at culture day 21 baseline patients have significantly less osteoclasts than healthy donors (p = 0.0038). No differences were found in the number of nuclei per OC in any studied time of culture. B) Representative images of pit assay at culture day 21. Patients at follow-up had significantly higher number of pits and resorption area at culture day 21 when compared to their baseline (p = 0.0469 for both resorption pit number and percentage of resorbed area). Dots represent median counts for each group at each time-point and bars represent interquartile range [10–90]. d—day; OC—osteoclast; Scale bars 100μm, red arrows—osteoclasts, black arrows—resorption pits.</p