Background/Purpose: Juvenile idiopathic arthritis (JIA) is the most common chronic condition seen by pediatric rheumatologists.  The pathogenesis of disease is poorly understood, but is believed to be mediated by an adaptive immune response involving auto-reactive B-cells and T-cells.  There are high concentrations of immune complexes in the joint but specificities of these immune complexes are unknown.  We hypothesize that joint destruction is partly the result of an adaptive immune response mediated by antibodies, and that examination of peptides and proteins in the synovial fluid (SF) of children with JIA combined with the identification of antigens targeted by antibodies found in arthritic SF will help clarify the pathogenesis of JIA.

Methods: Subjects with JIA with active arthritis of the knee requiring intra-articular corticosteroid injection were recruited from our pediatric rheumatology clinic.  Subjects seen by pediatric orthopedics requiring arthroscopic knee surgery for traumatic injuries were recruited as controls.  SF was collected from the knee of 4 children with JIA and 2 orthopedic controls.  Specimens were diluted, centrifuged, and filtered.  Peptidome and proteome were separated  with ultrafiltration spin columns and advanced MS/MS was carried out. Protein A&G was used to isolate IgG inclusive immune-complexes. Antigens were eluted with glycine and analyzed using  trypsin restriction with MS/MS.  Ingenuity pathway analysis was used to analyze the proteome. 

Results: High concentrations of immune complexes were found in the joints of JIA patients, nearing 50% of the total sample.  Pathway analysis of the proteomes revealed statistically significant overlap with 5 relevant canonical pathways in all patients: acute phase response signaling, complement activation, coagulation system, intrinsic prothrombin activation, and extrinsic prothrombin activation.  Peptidome analysis revealed the breakdown products of structural components including multiple collagen subtypes.  Antibody-targeted antigens present in three out of the 4 patients but absent in controls were alpha-1-antitrypsin precursor, hyaluronan binding protein, haptoglobin, and protein S100-A9. 

Conclusion: Proteome analysis confirmed the validity of our methods, revealing significant overlap with 5 pathways already known to be important in the pathogenesis of JIA.  Given the role of short peptides as antigenic targets of T cell responses, the composition of the peptidome in the synovial tissues and fluids of JIA patients is likely to contribute to disease associated adaptive immune responses.  The preliminary finding of the breakdown products of structural components suggests a mechanism for amplification of the autoimmune process during inflammation.  While the role of the identified antibody-targeted antigens in pathogenesis remains unclear, it is of interest that some of these proteins, including alpha-1-antitrypsin and protein S100-A9 have been implicated as biological markers in both adult RA and JIA.  Future research will validate the presence of these antibodies in a larger sample.  These antibodies could have potential roles as biomarkers for disease and as targets for treatment.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immunoprecipitation-and-advanced-proteomics-for-the-discovery-of-novel-antigenic-targets-in-juvenile-idiopathic-arthritis/