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- Differences in osteoclast activity between rheumatoid arthritis and ankylosing spondylitisPublication . Perpétuo, Inês Pedro; Fonseca, João Eurico, 1969-; Ainola, Mari-MiaIn many inflammatory diseases, such as rheumatoid arthritis (RA) and ankylosing spondylitis (AS), bone is a target for immune cells unbalancing bone remodeling. RA typically presents as a symmetric polyarthritis, affecting more women than men and is linked with the presence of autoantibodies in the serum such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF). Human leukocyte antigen (HLA)-DR genes are strongly associated with the disease. Chronic inflammation in RA leads to cartilage and bone destruction, which is typically recognized as erosive disease on x-rays. In contrast, AS is characterized by axial disease involving the sacroiliac joints and the spine. AS affects more men than women and is strongly associated with HLA-B27 haplotypes. The long-term outcome is characterized by ankylosis of the spine and sacroiliac joints. While RA is a disease characterized by destruction of bone and cartilage, the predominant finding in AS is bone formation rather than its destruction. In this work, we hypothesize that the inability of osteoclasts (OC) or its precursors to respond to osteoclastogenic stimuli in AS patients contributes to the excessive bone formation characteristic of this disease. Therefore, the aim of this thesis was the characterization of OC circulating precursors (monocytes) in RA and AS, as well as their ability to differentiate in resorbing OC when cultured in vitro. Moreover, we also aimed to understand the effect of therapies, such as methotrexate (MTX) and tumor necrosis factor inhibitors (TNFi), in the OC precursors in RA and AS patients. We first investigated whether cytokines were dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration (VERA) before and after treatment with corticosteroids and MTX. Pro-inflammatory cytokines were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Patients were also analyzed after therapy. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1β and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to promote the chronicity of inflammation. These cytokines are also associated with the promotion of osteoclastogenesis. In established RA this pattern was more evident within the SF. Early treatment with MTX or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. VERA patients already display increased levels of cytokines related with Th17 polarization and osteoclastogenesis, a deregulation also found in SF of established RA, suggesting that a cytokine-milieu favoring Th17 and OC activity is an early event in RA pathogenesis. We then aimed to assess the effect of MTX on circulating OC precursors and OC differentiation in RA patients. RA patients were assessed before therapy and at least 6 months after the introduction of MTX therapy and results controlled with healthy donors. We determined receptor activator of NF-κB (RANK) ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines and in vitro OC differentiation were also performed. We found that serum RANKL, classical activation monocytes markers (C-C chemokine receptor 2 - CCR2, CD86, HLA-DR) and RANK were increased in RA patients with active disease compared to healthy donors, and after MTX exposure these parameters normalized to control levels. Although we found no differences in OC number, cells differentiated from RA patients showed higher resorption activity than from healthy donors. Again, after MTX treatment, osteoclasts resorption activity was normalized. The results of this work suggested that MTX plays an important role in downregulating OC function through the decrease in RANK surface expression in monocytes. We then proceeded to study the effect of TNFi in osteoclastogenesis in RA patients. RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Results were controlled with healthy donors. After TNFi therapy, RANKL surface expression was downregulated in B lymphocytes and the frequency of circulating OC precursors was also decreased. Cells from TNFi treated patients had decreased osteoclast numbers and resorption activity as well as decreased expression of specific genes important for osteoclastogenesis, like tumour necrosis factor receptorassociated factor (TRAF6), fos-related antigen 2 (FRA-2) and for bone resorption like cathepsin K. Therefore, we suggest that in RA TNFi decreases bone resorption through the direct reduction of the number of circulating precursors and the inhibition of intracellular signalling pathways acting through TRAF6. After exploring OC precursor behavior in untreated and treated RA patients we aimed to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes phenotype and in vitro OC differentiation in AS patients. xli Patients with active AS without any ongoing therapy and age and gender matched healthy donors were recruited. We observed that pro-inflammatory cytokine levels were higher in a cohort of untreated AS patients when compared to healthy donors, but CD51/CD61 expression (integrin αvβ3 or vitronectin receptor, important for osteoclast attachment to the bone matrix) was downregulated in the classical OC precursors. No differences in the in vitro osteoclastogenesis or bone resorption was observed when compared to healthy donors, however we found low expression of colony stimulating factor 1 receptor (CSF1R), RANK and nuclear factor of activated T cell c1 (NFATc1) in AS osteoclast precursors that consequently led to a decreased expression of important resorption genes such as cathepsin K. These findings showed us that despite the high levels of proinflammatory cytokines present in AS patients, circulating monocytes have low OC specific gene expression supporting our hypothesis of an impaired response of OC precursors to pro-osteoclastogenic stimuli in AS patients. In an effort to understand the effects of TNFi therapy in circulating OC precursors and their differentiation ability from AS patients, we followed up a cohort of patients before and after therapy. Results were controlled with healthy donors. We found that IL-17A and IL-23 circulating levels decreased after TNFi treatment. OC number was decreased in AS patients before treatment when compared to control. However, no differences in OC precursor frequency or in the number OC in culture were found after treatment. RANK, CSF1R and NFATc1 expression was downregulated in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiated from AS TNFi-treated patients showed higher resorption activity than cells from patients before treatment. These results showed us that in AS patients, TNFi treatment reduces systemic proosteoclastogenic stimuli but when OC precursors are exposed to TNFi therapy they have increased in vitro activity in response to osteoclastogenic stimuli. From this work we were able to understand some of the mechanisms by which MTX and TNFi therapies act on circulating OC precursors in RA and AS patients. Although RA and AS are two chronic immune mediated diseases their effect on bone metabolism is different. The work here discussed shows that RA and AS OC precursors have a different behaviour in vitro, even coming from a similar pro-inflammatory milieu. Our findings support the hypothesis that OC from AS patients have impairment in their activity when compared both to RA patients or healthy controls. This difference can be partially explained by an intrinsic inability to respond to osteoclastogenic stimuli and by downregulation of key OC differentiation and activity genes in AS patients.
- 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.