FM - Teses de Doutoramento
Permanent URI for this collection
Browse
Browsing FM - Teses de Doutoramento by advisor "Ainola, Mari-Mia"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- 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.