Background Systemic lupus erythematosus (SLE) is a severe systemic autoimmune disease characterised by immune-complexes which cause systemic inflammation and damage. Neutrophil extracellular traps (NETs) are an important source of autoantigens in SLE patients leading to the production of autoantibodies. Functionally, SLE-specific autoantibodies as immune-complexes are important triggers of excessive NET formation. As such, effective targeting of pathogenic autoantibodies in SLE is subject to several promising experimental treatment strategies. Recently, the combination of Rituximab (RTX) and Belimumab (BLM) in patients with severe SLE led to a strong decrease of autoantibodies and diminished excessive NET formation as well as improvement of clinical disease. A consortium was formed to study different experimental treatment strategies that target the humoral autoimmune system, including RTX, Bortezomib (BTZ) or combination treatment with RTX and BLM.
Objectives The present study aimed to investigate the effects of B cell targeted therapies on relevant autoantibody levels and excessive NET formation in severe SLE.
Methods This study involved three cohorts of anti-dsDNA positive, severe SLE patients that were eligible to experimental treatment with RTX (n=16), BTZ (n=6) or RTX +BLM (n=16). A cross-sectional cohort of 35 anti-dsDNA positive SLE patients served as a control cohort. A panel of SLE relevant autoantibodies against dsDNA, histones, nucleosomes and C1q were measured by ELISA. As a functional result of autoantibody levels, NET formation was quantified by our novel highly-sensitive NET quantification assay using 3D confocal microscopy1.
Results Comparing three regimens, RTX +BLM resulted in the strongest reduction of anti-dsDNA (median ratio of baseline; 0.32 vs 0.78 vs 0.65; p=0.08), anti-histone (0.36 vs 0.51 vs 0.53; p=0.45), anti-nucleosome (0.38 vs 0.61 vs 0.58; p=0.15), and significantly the strongest reduction of anti-C1q antibodies (0.55 vs 0.91 vs 1.00; p=0.016) compared to RTX and BTZ. Excessive NET formation diminished significantly with a ratio of 0.66 [0.49–0.93] after RTX (p=0.005) and 0.25 [0.15–0.47] after RTX +BLM (p=0.0002), however it was not reduced after BTZ with 1.37 [0.90–1.61]. As such, excessive NET formation correlated with disease activity (p=0.004), except for the BTZ cohort. Importantly, regression of excessive NET formation was associated with reduction of anti-C1q antibodies. In an independent cohort of SLE patients, we confirmed that the presence of anti-C1q antibodies correlated with excessive NET formation (p=0.03). We further observed that the presence of three or more autoantibody specificities associated with excessive NET formation (p=0.02).
Conclusions This study demonstrates a synergetic effect of RTX +BLM compared to RTX or BTZ on the reduction of relevant autoantibodies in SLE patients which associated with significant regression of NET formation. The reducing effects of RTX +BLM, RTX and BTZ on anti-C1q antibodies underpinned the observed, immunological effects on humoral autoimmunity.
Reference  Kraaij, et al. Autoimmunity reviews2016.
Disclosure of Interest None declared
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