Differences in Bone Cell Activity Between Rheumatoid Arthritis and Ankylosing Spondylitis
Condition: Rheumatoid Arthritis; Bone Resorption; Ankylosing Spondylitis
MeSH terms: Spondylitis; Arthritis; Arthritis, Rheumatoid; Spondylarthritis; Spondylitis, Ankylosing; Bone Resorption
Study type: Observational

Summary: 
      Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are characterized by chronic
      systemic inflammation and share common pathogenic pathways. In both diseases, cytokines like
      TNF (tumor necrosis factor) and interleukin (IL)-17, known for their pro-inflammatory and
      osteoclastogenic effects, are relevant players, however, while RA is characterized by bone
      erosions, AS favors bone overgrowth. Understanding this paradox may hold the key for a better
      management of both diseases. Our hypothesis is that there are differences in the cellular
      environment and intracellular signaling between AS and RA. To test this hypothesis we will
      evaluate the cytokine milieu, the kinetics of bone cells differentiation and their activity
      in untreated and immunosuppressed RA and AS patients. We will also perform the same
      observations in patients exposed to targeted treatments.
    

Detailed description: 
      Objectives:

        1. Assess the influence of immune system cells in the modulation of bone turnover in
           patients with RA, AS and healthy donors (Task 2).

        2. Analyze the osteoclast precursors and their differentiation into fully functional
           osteoclasts in a subgroup of patients with RA, AS and healthy donors (Task 3, 4 and 6).

        3. Study osteoblast differentiation potential and activity in a subgroup of patients with
           RA and AS (Task 5 and 6)

        4. Assess the effect of the therapy on bone cell differentiation and function in a subgroup
           of patients with RA and AS treated with TNF-blockers (Task 2, 3, 4 and 5).

      Work program and timetable Starting date: July 2011. Finishing date: July 2013 Task 1 -
      Patients Patients with RA diagnosis (according to the revised American Rheumatism Association
      criteria, 1988) and AS diagnosis (according to the European Spondyloarthropathy Study Group
      criteria, 1991) followed up in the Rheumatology and Bone and Metabolic Diseases Department of
      Hospital de Santa Maria (HSM) will be recruited for this study. Twenty-five patients with
      active RA (Disease Activity Score 28 (DAS28)>3.2) and 25 patients with active AS (Bath
      Ankylosing Spondylitis Disease Activity Index (BASDAI)>4) will be included in the study.
      Fifteen healthy donors, sex and age matched, will be recruited and used as a control group.

      Patients will be submitted to a clinical protocol that includes information on sex, age,
      disease duration, previous therapies, presence of rheumatoid factor and anti
      cyclic-citrullinated peptide antibodies, Human Leukocyte Antigen (HLA)-B27 status, DAS28
      score and Health Assessment Questionnaire - HAQ (Health Assessment Questionnaire, for RA
      patients), BASDAI, Maastricht Ankylosing Spondylitis Enthesitis Score (MASES), Bath
      Ankylosing Spondylitis Functional Index - BASFI (for AS patients) and Ankylosing Spondylitis
      Disease Activity Score (ASDAS). Blood will be collected from patients before starting
      treatment with glucocorticoids and disease-modifying anti-rheumatic drugs (DMARDs:
      methotrexate, sulphasalazine and leflunomide) and 3 months after reaching a stable dose of
      these drugs. For those that latter on start TNF-blockers blood samples will be collected
      after 6 months of starting therapy. Because it is expected that only a minority of this
      initial cohort will need to be treated with TNF antagonists, a second group of 25 RA and 25
      AS patients, selected for starting TNF antagonists, will be assessed before starting the drug
      and 6 months later.

      Written informed consent will be obtained from all patients prior to any protocol-specific
      procedure and this study will be conducted in accordance with the regulations governing
      clinical trials such as the Declaration of Helsinki, as amended in Tokyo (2004). This study
      was approved by HSM Ethics Committee.

      All the procedures in the following tasks will be performed in RA and AS patients (serum and
      blood) and healthy donors (serum), unless stated differently.

      Task 2 - Study of the inflammatory stimuli and bone turnover markers Sub-populations of
      immune system cells (neutrophils, B and T cell subpopulations, including Th17 cells) will be
      analysed for surface RANKL expression by flow cytometry.

      Pro-inflammatory cytokines like IL-1β, IL-6, IL-17A, IL-20, IL-23, TNF and bone modulating
      proteins (OPG, sRANKL, sclerostin and dickkopf-1) will be quantified by ELISA. Bone
      degradation enzymes like tartrate-resistant acid phosphatase (TRAP)5b and bone turnover
      markers like type I collagen carboxyterminal cross-linked telopeptide (ICTP) and procollagen
      type 1 amino-terminal propeptide (P1NP) will also be studied.

      The data obtained from flow cytometry experiments and protein quantification will be compared
      between diseases before and after treatment and with healthy donors. This will allow us to
      understand the disease-specific differences in the systemic and local inflammatory
      environment in untreated patients and the effect of the different therapies over this same
      environment.

      Task 3 - Analysis of osteoclast precursors and their differentiation into functional
      osteoclasts Circulating CD14+ cells will be characterized by analysing markers like HLA-DR,
      cluster of differentiation (CD)16, CD86, CD11b and CD62L, as well as osteoclast
      differentiation-associated proteins, such as the surface integrin CD51/CD61 (αVβ3 integrin),
      RANK (receptor activator of nf-kappa beta) and the calcitonin receptor by flow cytometry.

      In order to fully characterize the osteoclasts precursors, the expression of
      osteoclast-related genes like DC-STAMP, MITF (microphthalmia-associated transcription
      factor), CTSK (cathepsin K), TRACP and ATP6v0d2 in CD14+ cells will be assessed by real time
      quantitative polymerase chain reaction (RT-qPCR). The results obtained will be normalized
      with the housekeeping genes beta glucuronidase (GUSB) and phosphomannomutase-1 (PMM1).

      Data obtained from flow cytometry and gene expression experiments will be compared between
      treated and untreated patients and with healthy donors. This task will provide us
      quantitative information on the disease-specific changes of the CD14+ monocytes subpopulation
      and it will allow us to understand the effect of therapy in the circulating precursor cells.

      Task 4 - Function and cellular dynamics of the differentiated osteoclasts The isolated CD14+
      monocytes will be cultured for 21 days in the presence of M-CSF (macrophage colony
      stimulating factor) and hrRANKL (human recombinant receptor activator of nf-kappa beta
      ligand) at 37º, 5% CO2 in multiwell culture plates.

      Osteoclasts will be analyzed at days 7, 14 and 21 of culture to assess cellular dynamics.
      Expression of osteoclast-specific genes will be analyzed at these time-points by RT-qPCR (see
      Task 3). Flow cytometry analysis will be performed to assess the CD51/CD61 and RANK surface
      expression. Data obtained during time points (days 7, 14 and 21) from patients and healthy
      controls will be compared with the same data obtained from the precursor cells.

      At the 21st culture day cells will be used in two functional assays: TRAP staining and
      resorption assay. In the TRAP staining assay we will assess the number of osteoclasts formed
      in culture by counting TRAP-positive multinucleated cells with more than 3 nuclei
      (osteoclasts). Comparison of this value with nuclei count in CD14+ monocytes in culture
      plates will allow us to determine the fusion index of these precursors. The resorption assay
      is carried by culturing monocytes over bone slices and allows us to measure the resorption
      activity of functional osteoclasts. This will be measured by image analysis of the bone
      slices stained with toluidine-blue where the resorbed area acquires a blue to purple color.
      The analysis of the fusion rate of precursors and of the osteoclast resorbed area will allow
      us to identify differences in osteoclast function between RA and AS patients and healthy
      donors.

      Both monocytes and osteoclasts will be studied regarding the RANK/RANKL and the αVβ3 integrin
      and c-fms signalling pathways that lead to the recruitment and activation of TRAF6, tyrosine
      kinase Src and extracellular signal-regulated kinase (ERK)1/2 activating NF-kB and other
      transcription factors. These signalling pathways will be studied using the Luminex xMAP
      platform with EpiQuant Cell Signalling Assays (Millipore). Comparison of the results from
      untreated patients and controls will allow us to understand if there is an impairment in
      osteoclast differentiation/activation classical pathways in AS as compared to RA.

      The results obtained from treated and untreated patients will provide insight on the action
      of the therapies on osteoclast differentiation and function. Comparing overall results from
      untreated patients and healthy donors will allow us to understand key differences between
      osteoclast differentiation and activation in these pathologies.

      Task 5 - Function and cellular dynamics of osteoblasts In a subgroup of RA and AS patients
      submitted either to hip replacement or cervical surgery osteoblast differentiation will be
      studied. Immediately after surgery, a small piece of trabecular bone (1cm3) will be extracted
      from the sample in order to isolate the osteoblasts. After isolation, osteoblasts will be
      cultured in Dulbecco's minimal essential medium (DMEM) supplemented with vitaminD3 (10-8M)
      and cells will be studied at 80% confluence. Osteoblasts' function will be studied in vitro
      by methylthiazole tetrazolium (MTT) assay (cell proliferation), alkaline phosphatase staining
      and mineralization nodules formation. Cells will be also used for RNA extraction and gene
      expression analysis Specific genes that encode proteins such as osteopontin, collagen type I,
      osteocalcin, alkaline phosphatase, RANKL, osteoprotegerin, runx2 and osterix will be analyzed
      to characterize the isolated cells. These results will allow us to understand the differences
      in osteoblast differentiation and activity between diseases.

      Task 6 - Bone gene expression assays In a subgroup of RA and AS patients submitted either to
      hip replacement or cervical surgery we will frozen bone samples at -80ºC. With the bone still
      frozen, a small sample of trabecular bone will be reduced to fine powder in a cryogenic mill
      and the RNA extraction will be performed using TRIzol reagent. The resulting RNA will be
      assessed for its integrity and quantified in a Bioanalyser. Therefore, the RNA extracted will
      be the total of RNA present in bone, from blood, osteoblasts, osteoclasts, osteocytes,
      adipocytes and bone marrow cells.

      The gene expression both in the bone microenvironment and in the cells from the in vitro
      studies will be evaluated by RT-PCR. Specific genes that encode proteins both from
      osteoblasts and osteoclasts such as osteopontin, collagen type I, osteocalcin, alkaline
      phosphatase, RANKL, osteoprotegerin, runx2, osterix, cathepsin K, beta3 integrin subunit,
      calcitonin receptor, ATPase subunit d2, TRAF6, RANK, TRAP, the co-receptors OSCAR and TREM-2,
      among others, will be studied. Osteocytes' activity will also be assessed by the expression
      of specific genes, SOST, Dkk1, DMP-1, Phex, E11 antigen, MEPE and CD44. The genes that code
      the inflammatory cytokines IL-1, IL-6, IL-17 and TNF will also be studied.

      In order to assess the PCR efficiency, standard curves will be built from RNA extracted from
      frozen bone samples from individuals with normal bone mineral density and with no other
      disease that could have influence in bone metabolism. The osteoclast, osteoblast and
      osteocyte genes of interest will be quantified by the standard curve method.

      The RNA expression study on the bone samples will allow us to understand the local cell
      activity and it characterize bone cells and their function in a disease context.
    

Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Patients with RA diagnosis (according to the revised American Rheumatism Association
        criteria, 1988) and AS diagnosis (according to the European Spondyloarthropathy Study Group
        criteria, 1991)
      
Biospecimen description: 
      Blood and bone samples
    
Keywords: Rheumatoid arthritis; Ankylosing spondylitis; Osteoclasts; TNF antagonists