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- Effect of tumor necrosis factor inhibitor therapy on osteoclasts precursors in rheumatoid arthritisPublication . Perpétuo, Inês Pedro; Caetano-Lopes, Joana; Rodrigues, Ana Maria; Campanilho-Marques, Raquel; Ponte, Cristina; Canhao, Helena; Ainola, Mari; Fonseca, João EuricoObjective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16- monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6.
- Methotrexate and low-dose prednisolone downregulate osteoclast function by decreasing receptor activator of nuclear factor-κβ expression in monocytes from patients with early rheumatoid arthritisPublication . Perpétuo, Inês Pedro; Caetano-Lopes, joana; Rodrigues, Ana Maria; Campanilho-marques, Raquel; Ponte, Cristina; Canhao, Helena; Ainola, Mari-Mia; Fonseca, João EuricoObjective: Rheumatoid arthritis (RA) is a systemic, immune-mediated inflammatory disease that ultimately leads to bone erosions and joint destruction. Methotrexate (MTX) slows bone damage but the mechanism by which it acts is still unknown. In this study, we aimed to assess the effect of MTX and low-dose prednisolone (PDN) on circulating osteoclast (OC) precursors and OC differentiation in patients with RA. Methods: Patients with RA before and at least 6 months after MTX therapy were analysed and compared with healthy donors. A blood sample was collected in order to assess receptor activator of NF-κβ (RANK) ligand surface expression on circulating leucocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines and OC differentiation assays were performed. Results: Classical activation markers of monocytes and RANK increased in patients with RA at baseline, compared with control healthy donors, and after MTX and low-dose PDN (MTX+PDN) exposure they decreased to control levels. Although the number of OC was not different between groups, the percentage of resorbed area and the resorbed area per pit reduced after treatment. Serum soluble receptor activator of nuclear factor-kappa (RANKL) levels increased at baseline compared with healthy donors and normalised after therapy. Conclusion: Our results suggest that MTX+PDN play an important role in downregulating OC function, which we believe occurs through the decrease in RANK surface expression in monocytes.
- Ankylosing Spondylitis patients have impaired osteoclast gene expression in circulating osteoclast precursorsPublication . Perpétuo, Inês Pedro; Caetano-Lopes, Joana; Vieira De Sousa, Elsa Cristina; Campanilho-Marques, Raquel; Ponte, Cristina; Canhao, Helena; Ainola, Mari-Mia; Fonseca, João EuricoIntroduction: Ankylosing spondylitis (AS) is typically characterized by focal bone overgrowth 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-κβ (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.
- Effect of tumor necrosis factor inhibitor therapy on osteoclasts precursors in ankylosing spondylitisPublication . Perpétuo, Inês Pedro; Raposeiro, Rita; Caetano-Lopes, Joana; Vieira De Sousa, Elsa Cristina; Campanilho-Marques, Raquel; Ponte, Cristina; Canhao, Helena; Ainola, Mari; Fonseca, João EuricoIntroduction: Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. Methods: 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. Results: RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-β levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. Conclusion: In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli.