Juvenile idiopathic arthritis (JIA) is a common rheumatological condition. While past research suggests JIA is an autoimmune disease, the autoantigens driving this supposed T cell–mediated immune response are still unknown. Clues to answering this question may lie in proteins present in the synovial fluid. Past researchers have seen an increase in transthyretin (TTR) in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis.1 Also giving weight to this hypothesis is some well-established research indicating a connection with B cells and autoantibodies in JIA pathogenesis; however, these mechanisms are unknown. Recently, Clement and colleagues sought to shed light on this by characterizing the TTR immune response in patients with JIA.2
The researchers first used global proteomic profiling to look for possible antigens present in synovial fluid that could be important in JIA. After enrolling 50 JIA patients and patients with active knee arthritis, the team collected between 6 mL and 15 mL of synovial fluid from each patient. As a control group, they also obtained samples from 24 subjects with knee injuries requiring surgery.
Looking at seven JIA patients, the team digested samples of synovial fluid using hyaluronidase and then depleted albumin and IgG proteins. Using 50 µg of each sample, they performed 4% to 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The team stained gels and then performed an in-gel trypsin digest. For the analysis, they relied on nanospray-liquid chromatography-linear trap quadrupole-tandem mass spectrometry (nano LC LTQ MS/MS) on an UltiMate Plus Nano HPLC system, and an LTQ Orbitrap Velos HR mass spectrometer (both from Thermo Scientific). This system was also interfaced with a TriVersa NanoMate nanoelectrospray ionization source.
From this, the team identified 391 proteins. Gene ontology helped determine that these proteins are involved in acute-phase response, complement activation, coagulation and immunoglobulin production. It also revealed several metalloproteases and other proteases, including plasmin kallikrein and cathepsins, in addition to tissue inhibitors of metalloproteinase.
Next, analyzing the JIA peptidome of those seven JIA patients, the researchers identified 788 unique naturally processed endogenous peptides derived from 108 proteins.
To profile immune complex–associated antigens in JIA, the team purified synovial fluid immunoglobulins from controls and JIA patients using protein A/G gel beads. They eluted self antigens at low pH and analyzed them using nanoLC MS/ MS. This resulted in the researchers identifying 32 proteins in the JIA group that were absent from the control group and thus were putative disease-associated markers.
To investigate TRR as a target antigen, the researchers used enzyme-linked immunosorbent assay (ELISA) to detect anti-TTR antibodies on both synovial fluid and sera of JIA patients and controls. Not only was there a statistically significant antibody response to TTR in both synovial fluid and sera from JIA patients as compared with healthy controls, there was also a significant increase in TTR protein levels.
After further examination of the synovial fluid, the researchers saw misfolding, aggregation and oxidation of TTR, which is known to stimulate a signaling cascade to induce B cell differentiation and IgG secretion in a T cell–dependent and/or –independent manner. Despite this, they saw a T cell response to 4 cryptic TTR peptides in only 3 out of 17 analyzed samples. The team notes that 4 peptides shared a common binding site for HLA-DR1, a cell surface receptor, and 2 peptides shared the same primary amino acid sequence, with the exception of post-translational modifications.
Clement et al. recommend that future experiments should focus on mapping additional TTR peptides with different HLA restriction and on finding an immunodominant peptide driving the T cell immune response in different HLA-susceptible haplotypes.
1. Akasaki, Y., et al. (2015) “Transthyretin deposition in articular cartilage: A novel mechanism in the pathogenesis of osteoarthritis,” Arthritis Rheumatology, 67(8) (pp. 2097–2107). doi: 10.1002/art.39178.
2. Clement, C.C., et al. (2016) “Autoimmune response to transthyretin in juvenile idiopathic arthritis,” JCI Insight, 1(2): e85633.
Post Author: Emily Humphreys. Emily has previous research experience in eye development, infectious diseases, and aging. Emily has been a regular contributor to Accelerating Science since 2012.