Mechanisms of bone loss in inflammatory arthritis: diagnosis and therapeutic implications

Rheumatoid arthritis represents an excellent model in which to gain insights into the local and systemic effects of joint inflammation on skeletal tissues. Three forms of bone disease have been described in rheumatoid arthritis. These include: focal bone loss affecting the immediate subchondral bone and bone at the joint margins; periarticular osteopenia adjacent to inflamed joints; and generalized osteoporosis involving the axial and appendicular skeleton. Although these three forms of bone loss have several features in common, careful histomorphometric and histopathological analysis of bone tissues from different skeletal sites, as well as the use of urinary and serum biochemical markers of bone remodeling, provide compelling evidence that different mechanisms are involved in their pathogenesis. An understanding of these distinct pathological forms of bone loss has relevance not only with respect to gaining insights into the different pathological mechanisms, but also for developing specific and effective strategies for preventing the different forms of bone loss in rheumatoid arthritis

Breathing New Life into Interstitial Lung Disease in Rheumatoid Arthritis

Clinical heterogeneity is a hallmark of many autoimmune disorders, and clinical or subclinical pulmonary involvement is a common extraarticular feature of the rheumatoid arthritis (RA) phenotype. High-resolution computed tomography reveals evidence of pulmonary abnormalities in more than half of patients with RA, and clinically significant interstitial lung disease (ILD) will develop in approximately 10% of patients.It is currently difficult to identify these patients and to intervene early in the clinical course of their lung disease

IL-17A deficiency promotes periosteal bone formation in a model of inflammatory arthritis

BACKGROUND: Interleukin-17A (IL-17A) plays a pathogenic role in several rheumatic diseases including spondyloarthritis and, paradoxically, has been described to both promote and protect from bone formation. We therefore examined the effects of IL-17A on osteoblast differentiation in vitro and on periosteal bone formation in an in vivo model of inflammatory arthritis. METHODS: K/BxN serum transfer arthritis was induced in IL-17A-deficient and wild-type mice. Clinical and histologic inflammation was assessed and periosteal bone formation was quantitated. Murine calvarial osteoblasts were differentiated in the continuous presence of IL-17A with or without blockade of secreted frizzled related protein (sFRP)1 and effects on differentiation were determined by qRT-PCR and mineralization assays. The impact of IL-17A on expression of Wnt signaling pathway antagonists was also assessed by qRT-PCR. Finally, regulation of Dickkopf (DKK)1 expression in murine synovial fibroblasts was evaluated after treatment with IL-17A, TNF, or IL-17A plus TNF. RESULTS: IL-17A-deficient mice develop significantly more periosteal bone than wild-type mice at peak inflammation, despite comparable severity of inflammation and bone erosion. IL-17A inhibits calvarial osteoblast differentiation in vitro, inducing mRNA expression of the Wnt antagonist sFRP1 in osteoblasts, and suppressing sFRP3 expression, both potentially contributing to inhibition of osteoblast differentiation. Furthermore, a blocking antibody to sFRP1 reduced the inhibitory effect of IL-17A on differentiation. Although treatment with IL-17A suppresses DKK1 mRNA expression in osteoblasts, IL-17A plus TNF synergistically upregulate DKK1 mRNA expression in synovial fibroblasts. CONCLUSIONS: IL-17A may limit the extent of bone formation at inflamed periosteal sites in spondyloarthritis. IL-17A inhibits calvarial osteoblast differentiation, in part by regulating expression of Wnt signaling pathway components. These results demonstrate that additional studies focusing on the role of IL-17A in bone formation in spondyloarthritis are indicated

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 mug/ml of Liberase TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4(+) and CD8(+) T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

Towards an arthritis flare-responsive drug delivery system

Local delivery of therapeutics for the treatment of inflammatory arthritis (IA) is limited by short intra-articular half-lives. Since IA severity often fluctuates over time, a local drug delivery method that titrates drug release to arthritis activity would represent an attractive paradigm in IA therapy. Here we report the development of a hydrogel platform that exhibits disassembly and drug release controlled by the concentration of enzymes expressed during arthritis flares. In vitro, hydrogel loaded with triamcinolone acetonide (TA) releases drug on-demand upon exposure to enzymes or synovial fluid from patients with rheumatoid arthritis. In arthritic mice, hydrogel loaded with a fluorescent dye demonstrates flare-dependent disassembly measured as loss of fluorescence. Moreover, a single dose of TA-loaded hydrogel but not the equivalent dose of locally injected free TA reduces arthritis activity in the injected paw. Together, our data suggest flare-responsive hydrogel as a promising next-generation drug delivery approach for the treatment of IA

A lipopolysaccharide-induced DNA-binding protein for a class II gene in B cells is distinct from NF-kappa B

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant decrease in mRNA encoding the Ia proteins which persists for at least 18 h. Within the upstream regulatory region of A alpha k, an NF-kappa B-like binding site is present. We have identified an LPS-induced DNA-binding protein in extracts from athymic mice whose spleens consist predominantly of B cells. Binding activity is present in low levels in unstimulated spleen cells and is increased by LPS treatment. This protein binds to two sites in a regulatory region of the Ia A alpha k gene, one of which contains the NF-kappa B-like binding site. DNA fragments containing these sites cross-compete for protein binding. Analysis by DNase I footprinting identified a target binding sequence, named the LPS-responsive element. Although this target sequence contains an NF-kappa B-like binding site, competition with a mutant oligonucleotide demonstrated that bases critical for NF-kappa B binding are not required for binding of the LPS-inducible protein. Therefore, we hypothesized that this inducible protein represents a new mediator of LPS action, distinct from NF-kappa B, and may be one mechanism to account for the decrease in mRNA encoding the Ia proteins

The role of TNF-receptor family members and other TRAF-dependent receptors in bone resorption

The contribution of osteoclasts to the process of bone loss in inflammatory arthritis has recently been demonstrated. Studies in osteoclast biology have led to the identification of factors responsible for the differentiation and activation of osteoclasts, the most important of which is the receptor activator of NF-κB ligand/osteoclast differentiation factor (RANKL/ODF), a tumor necrosis factor (TNF)-like protein. The RANKL/ODF receptor, receptor activator of NF-κB (RANK), is a TNF-receptor family member present on both osteoclast precursors and mature osteoclasts. Like other TNF-family receptors and the IL-1 receptor, RANK mediates its signal transduction via TNF receptor-associated factor (TRAF) proteins, suggesting that the signaling pathways activated by RANK and other inflammatory cytokines involved in osteoclast differentiation and activation are interconnected

Detection of IgG4-Specific Autoantibodies in Rheumatoid Arthritis Serum Samples

Background: Rheumatoid arthritis (RA) is a chronic multi-system autoimmune disease characterized by inflammatory synovitis. Autoantibodies such as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) have been implicated in the pathogenesis of RA, and are currently important criteria for diagnosis within the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria.1 Yet, many patients diagnosed with RA do not have measurable circulating ACPA or RF which may result in delayed diagnosis and treatment. After IgG1, IgG4 is the second most predominant isotype among ACPA and RF; however it is not detected in currently available diagnostic assays. Recent data have demonstrated that patients deemed “sero-negative” based on standard assays may have high titers of IgG4-specific ACPA and/or RF. Objectives: The purpose of this study is to quantitate and compare IgG1- to IgG4-specific anti-CCP antibodies and rheumatoid factor in patients with rheumatoid arthritis. We will determine the frequency of IgG4 autoantibodies, and examine whether they have a differential expression among RA patients. We will also correlate their presence with disease activity, anti-rheumatic drug therapy, and levels of serum cytokines. Ultimately, this work may help to determine if a diagnostic test that detects IgG4 isotype of ACPA and RF will aid in earlier diagnosis and better characterization of rheumatoid arthritis. Methods: To explore our objectives, we have initiated a cross-sectional study with the goal of enrolling 1,000 patients with a confirmed diagnosis of rheumatoid arthritis, based on the 2010 ACR/EULAR classification criteria.1 We are collecting clinical information about each patient including demographics, current treatments, clinical disease activity, laboratory values, and radiographic results. Concurrently, we are collecting serum samples from each patient that will be analyzed for 1) total levels of IgG4 and IgG1; 2) total ACPA and RF; 3) levels of IgG1-specific and IgG4-specific ACPA and RF; and 4) cytokine levels (IL-6, TNF, IL-1, IL-17, IFNy, IL-21, and G-CSF). Results: To date, we have recruited 102 RA patients including 70 females (68.6%) and 32 males (31.4%). Their ages range from 24 to 85 years (mean 58.4 ± 12.4 years). Acute phase reactant levels were available for 98 of the 102 patients, allowing calculation of the disease activity score using 28 joints (DAS28). The mean DAS28 was 3.67 ± 1.0, which is within the moderate disease activity range. The proportion of patients in each disease category was: remission (12.2%), low disease activity (21.4%), moderate disease activity (61.2%), and high disease activity (6.1%). Based on their medical records, at any point in time, 46.1% (n=47) of the recruited subjects had positive RF titers vs. 39.2% (n=40) without RF; 58.8% (n=60) had ACPA vs 26.5% (n=27) without ACPA. For 14.7% (n=15) of the subjects, RF and/or ACPA were either unknown or untested. Of patients with RF, 91.4% (n=43) also had ACPA; of patients with ACPA, 71.7% % (n=43) also had RF. Of the patients tested for both, 27.9% (n=24) were negative for both RF and ACPA. Conclusion: Subject recruitment and data collection are well underway for this large cross-sectional study that will shed light to the role of IgG4- specific autoantibodies in the pathogenesis and diagnosis of rheumatoid arthritis. Reference: 1Aletaha D Neogi T, Silman A, et al. 2010 Rheumatoid arthritis classification criteria: An American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum 2010; 62(9):2569-81/Ann Rheum Dis. 2010; 69:1580-8

Physical activity and attitudes towards exercise in people with axial and peripheral spondyloarthritis

OBJECTIVE: To evaluate physical activity and attitudes towards exercise among people with axial and peripheral spondyloarthritis (SpA). METHODS: Using baseline information from an on-going, longitudinal, prospective SpA cohort study (n=264), self-reported attitudes and beliefs towards exercise were assessed using questionnaires. Total metabolic equivalent (MET) hours of self-reported physical activity per week, time spent in activities, and activity levels were calculated from the Nurses\u27 Health Study Physical Activity Questionnaire II (NHSPAQ II). Adjusted multivariable linear models estimated the relationship between physical activity and disease status (axial versus peripheral). RESULTS: Regardless of predominant anatomic distribution of disease, most participants were well-educated, non-Hispanic white men. Approximately 40% met the United States Department of Health and Human Services physical activity recommendations. Positive attitudes, beliefs, and perceived benefits towards exercise were similar by anatomic distribution of disease. Despite similar MET-hours per week, participants with axial disease had greater concerns regarding discomfort and joint injuries than those with peripheral disease. Compared to those with peripheral SpA (n=201), participants with axial SpA (n=63) spent less time engaging in light and moderate activities (adjusted beta in light activity: -1.94 minutes/week, 95% Confidence Interval (CI): -2.96 to -0.93; adjusted beta in moderate activity: -1.05 minutes/week, 95% CI: -2.12 to 0.02). CONCLUSION: Participants with axial SpA had greater concerns regarding discomfort and injuries from exercise than those with peripheral SpA. Although no differences in time spent in vigorous activities were observed, participants with axial SpA spent less time than those with peripheral SpA in light to moderate activities

The role played by cell-substrate interactions in the pathogenesis of osteoclast-mediated peri-implant osteolysis

Prosthetic wear debris-induced peri-implant osteolysis is a major cause of aseptic loosening after total joint replacement. In this condition, wear particles released from the implant components induce a granulomatous inflammatory reaction at the interface between implant and adjacent bone, leading to progressive bone resorption and loss of fixation. The present study was undertaken to characterize definitively the phenotype of osteoclast-like cells associated with regions of peri-implant focal bone resorption and to compare the phenotypic features of these cells with those of mononucleated and multinucleated cells associated with polyethylene wear particles. Peri-implant tissues were obtained from patients undergoing hip revision surgery for aseptic loosening after total joint replacement. Cells were examined for the expression of several markers associated with the osteoclast phenotype using immunohistochemistry, histochemistry, and/or in situ hybridization. CD68 protein, a marker expressed by multiple macrophage lineage cell types, was detected in mononucleated and multinucleated cells associated with polyethylene particles and the bone surface. Cathepsin K and tartrate-resistant acid phosphatase were expressed highly in both mononucleated and multinucleated cells associated with the bone surface. Levels of expression were much lower in cells associated with polyethylene particles. High levels of β(3 )integrin protein were detected in cells in contact with bone. Multinucleated cells associated with polyethylene particles exhibited faint positive staining. Calcitonin receptor mRNA expression was detected solely in multinucleated cells present in resorption lacunae on the bone surface and was absent in cells associated with polyethylene particles. Our findings provide further evidence that cells expressing the full repertoire of osteoclast phenotypic markers are involved in the pathogenesis of peri-implant osteolysis after total joint replacement. They also demonstrate that foreign body giant cells, although believed to be phenotypically and functionally distinct from osteoclasts, express many osteoclast-associated genes and gene products. However, the levels and patterns of expression of these genes in the two cell types differ. We speculate that, in addition to the role of cytokines and growth factors, the substrate with which these cells interact plays a critical role in their differential phenotypic and functional properties