Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids

ObjectivesTo explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis.MethodsLatent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines.ResultsAll MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort.ConclusionSynovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.

Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

Evaluation of the MRSA/SA ELITe MGB assay for the detection of Staphylococcus aureus in bone and joint infections

Bone and joints infections represent a potentially devastating complication of prosthetic orthopaedic joint replacement, thus requiring both rapid and appropriate antibiotic treatment. Staphylococcus aureus is one of the most frequent pathogens involved in this pathology. Being able to assert its presence is the first step of patients’ efficient management. This monocenter study was aimed at evaluating the MRSA/SA ELITe MGB assay for the molecular detection of S. aureus and methicillin-resistant S. aureus (MRSAin bone and joint biopsies and synovial fluids. This test together with conventional techniques including standard cultures and 16S rRNA amplification assay were performed on 208 successive perioperative samples collected prospectively for one year from 129 patients. Using conventional techniques, a microbial pathogen was detected in 76 samples from 58 patients, out of which 40 were identified as S. aureus . The limit of detection (LODof the MRSA/SA ELITe MGB assay was experimentally determined for bone and joint biopsies and synovial fluids using negative samples spiked with S. aureus ATCC43300. The sensitivity of S. aureus detection with the MRSA/SA ELITe MGB assay was 82.5% (33/40 samplesand 97.5% (39/40 samplesusing manufacturer’s LOD and experimentally determined LOD respectively. Interestingly using the osteo-articular specific LOD, 15 additional samples were detected positive for S. aureus DNA with the MRSA/SA ELITe MGB assay; in all cases, those samples were taken from patients considered to be infected with S. aureus according to their clinical and microbiological records. The results were available within 24h, which could help to shorten the therapeutic decisions.

Synovial Fluid of Patient With Rheumatoid Arthritis Enhanced Osmotic Sensitivity Through the Cytotoxic Edema Module in Synoviocytes

Rheumatoid arthritis (RA) is an autoimmune disease that causes inflammation of the synovial membrane ultimately leading to permanent damage in the affected joints. For this study, synovial fluids from 16 patients diagnosed with either RA or osteoarthritis (OA) were used to examine volume regulation and cooperative water channels, both of which are involved in the cytotoxic edema identified in RA-fibroblast-like synoviocytes (FLS). The osmolarity and inflammatory cytokine interleukin (IL)-6 of synovial fluids from RA patients were mildly enhanced compared to that from OA patients. RA-FLS demonstrated the enhanced property of regulatory volume increase in response to IL-6 and synovial fluids from RA patients. Although there was no difference in the protein expression of the volume-associated protein sodium–potassium–chloride cotransporter1 (NKCC1), its activity was increased by treatment with IL-6. Membrane localization of NKCC1 was also increased by IL-6 treatment. Additionally, both the protein and membrane expressions of aquaporin-1 were increased in RA-FLS by IL-6 stimulation. The IL-6-mediated enhanced osmotic sensitivity of RA-FLS likely involves NKCC1 and aquaporin-1, which mainly constitute the volume-associated ion transporter and water channel elements. These results suggest that RA-FLS provide enhanced electrolytes and concomitant water movement through NKCC1 and aquaporin-1, thereby inducing cellular swelling ultimately resulting in cytotoxic edema. Attenuation of cytotoxic edema and verification of its related mechanism will provide novel therapeutic approaches to RA treatment within the scope of cytotoxic edema.

The effect of deactivated phospholipids on joints lubrication: Osteoarthritis and lubricating properties

PLs bilayers coating the major synovial joints such as knees and hips as the lubricant are responsible for the lubrication of articular cartilage. Lamellar-repulsive effect has been considered as a lubrication mechanism but it is likely that lubricin and hyaluronan with PLs participate in the lubrication process. The molecules of lubricin and hyaluronan adsorbed by PLs have a supportive role and provide the efficient lubrication of synovial joints via the hydration mechanism (~ 80% water content). Lipid profiles of injured and healthy knees’ synovial fluids show significant differences. The phospholipid content in synovial fluid (SF) during joint inflammation, osteoarthritis is significantly higher (2 to 3 times) above the normal concentration of PL, and has a poor boundary-lubricating ability because of deactivated PL molecules. Deactivated PL molecule has no ability to form bilayers, lamellar phases, and liposomes.

Analysis of Chemisorbed Tribo-Film for Ceramic-on-Ceramic Hip Joint Prostheses by Raman Spectroscopy

To understand the possible lubricant mechanism in ceramic-on-ceramic hip joint prostheses, biochemical reactions of the synovial fluid and the corresponding frictional coefficients were studied. The experiments were performed in a hip joint simulator using the ball-on-cup configuration with balls and cups made from two types of ceramics, BIOLOX®forte and BIOLOX®delta. Different lubricants, namely albumin, γ-globulin, hyaluronic acid and three model synovial fluids, were studied in the experiments and Raman spectroscopy was used to analyze the biochemical responses of these lubricants at the interface. BIOLOX®delta surface was found less reactive to proteins and model fluid lubricants. In contrast, BIOLOX®forte ball surface has shown chemisorption with both proteins, hyaluronic acid and model fluids imitating total joint replacement and osteoarthritic joint. There was no direct correlation between the measured frictional coefficient and the observed chemical reactions. In summary, the study reveals chemistry of lubricant film formation on ceramic hip implant surfaces with various model synovial fluids and their components.