Study Of Novel Autoantibodies In Rheumatoid Arthritis And The Pathogenesis Of Related Autoantigen

Study objectivesRheumatoid arthritis(RA)is an aggressive,symmetric polyarticular inflammatory disease that involves mainly the small joints and may also present with multiple extraarticular manifestations.Persisting synovitis could lead to joint damage and functional decline,resulting in disability and deformity.Prompt diagnosis and intervention at early stage are fundamental for preventing irreversible joint destruction.Anticitrullinated protein antibody(ACPA)is one of the most specific autoantibodies for RA diagnosis,but there is still nearly one-third of patients ACPA-negative.This study aims to identify novel autoantibodies in RA,and further verify their diagnostic value in ACPA-negative RA,improving the early diagnosis and intervention of patients with ACPA-negative RA.Study methodsA total of 1011 sera,including 559 RA(276 ACPA-positive RA and 283 ACPAnegative RA),239 autoimmune disease controls and 213 healthy controls,were collected in the study.The study involved three stages:the high-density protein microarray was first employed to screen candidate autoantigens.The focusedmicroarray composed of candidate autoantigens were then used for validation by hybridizing with a large number of serum samples.Enzyme linked immunosorbent assay(ELISA)/Western blot(WB)detection were followed to verify the presence of the most promising candidates in ACPA-negative RA.Study resultNine novel autoantibodies were identified by two sequential microarrays with positivity 17.93%-27.59%and specificities>90%in ACPA-negative RA.Among these,anti-PTX3 and anti-DUSP11 autoantibodies presented with the highest RA sensitivity and were consistently verified by ELISA and WB detection.Pooling samples of all cohorts,the positivities of anti-PTX3 and anti-DUSP11 autoantibodies in ACPA-negative RA were 27.56%and 31.80%,respectively,similar to those in ACPA-positive RA,and significantly higher than in healthy controls(anti-PTX3 antibody:4.69%;anti-DUSP11 antibody:2.35%)and disease controls(anti-PTX3 antibody:10.04%;anti-DUSP11 antibody:8.49%)(p<0.0001).The combination of anti-PTX3 with anti-DUSP11 autoantibodies significantly increased the diagnostic sensitivity(38.00%)with specificity of 88.72%.Study ConclusionNewly identified anti-PTX3 autoantibody and anti-DUSP11 autoantibody are potential biomarkers for diagnosis of ACPA-negative RA.Study ObjectiveThe expression level of the autoantigen penetratin-3(PTX3)has been previously reported to be upregulated in both serum and synovial tissue of rheumatoid arthritis(RA).And there is a strongly positive correlation between PTX3 levels and disease activity.However,the specific role of PTX3 in RA remains unclear.The aim of this study was to investigate the involvement of PTX3 in the pathogenesis of RA and reveal the underlying mechanism.Study methodsA total of 115 RA patients(including 90 anti-citrullinated protein antibody(ACPA)positive RA and 25 ACPA-negative RA),52 autoimmune disease controls and 95 healthy controls(HC)were included in the study.First,we employed the enzyme linked immunosorbent assay to measure the expression levels of PTX3 as well as PTX3’s classical ligand C1q in the peripheral blood of RA subsets and controls.Subsequently,we stimulated monocytes from RA patients and HC with PTX3 and C1q individually or jointly,examining cell death and the secretion of inflammatory factors.Further,we mimicked the internal environment with the sera from RA or HC.We stimulated HC sera or RA sera pre-incubated monocytes with the combination of PTX3 and C1q,evaluating the cell morphology,inflammatory factor secretion,NODlike receptor thermal protein domain associated protein 3(NLRP3)inflammasome activation and GSDMD-mediated pyroptosis.Study resultPTX3 as well as its ligand C1q was found to be significantly upregulated in the peripheral blood of both ACPA-positive and negative RA patients.Stimulated with the combination of PTX3 and C1q,NLRP3 inflammasome and GSDMD-mediated pyroptosis were activated.The monocytes disrupted to release large amounts of inflammatory cytokines such as IL-1β,IL-18,IL-6 and TNFα.Besides,the synergistic effect of PTX3 and C1q was most pronounced in the internal environment of RA.Study conclusionsUpregulated PTX3 as well as its ligand Clq in RA sera could act synergistically to accelerate the inflammatory storms of RA,by enhancing NLRP3 inflammasome overactivation and inducing GSDMD cleavage,accompanied by the rupture of monocyte membrane and subsequent release of large amounts of inflammatory cytokines.This study reveals the novel role of PTX3 in the pathogenesis of RA,providing the potential therapeutic target for RA.