TRIM21 autoantibodies are associated with blood-brain barrier dysfunction in a subgroup of neuromyelitis optica spectrum disorder

We previously reported that glucose-regulated protein 78 autoantibodies (GRP78 Ab) cause breakdown of the blood-brain barrier (BBB) in neuromyelitis optica spectrum disorder (NMOSD) patients. Objective of the present study is to identify novel antibodies associated with the breakdown of the BBB in NMOSD patients negative for anti-GRP78 Ab. Human brain microvascular endothelial cells (BMECs) were incubated with purified NMOSD-IgG from 4 patients. RNA-seq, a high-content imaging, living cell-based antibody binding assay, and mass spectrometry were used to evaluate molecular changes and identify the target protein of BMECs after exposure to NMOSD-IgG. The anti-tripartite motif-containing protein 21 (TRIM21) autoantibody was detected using an enzyme-linked immunosorbent assay. The clinical data of patients with NMOSD with anti-TRIM21 antibodies were verified. Changes in permeability were evaluated using three in vitro BBB models, including monolayer, spheroid and microfluidic tri-culture models. TRIM21, TROVE2 and SS-B were identified as the target antigens of NMOSD-IgG in GRP78 Ab-negative NMOSD patients using proteomics. NF-κB and tight junctions (claudin-5) in BMECs were identified as important signaling pathways after stimulation with TRIM21 Ab-positive NMOSD-IgG by RNA-seq and high-content imaging at the mRNA and protein levels. TRIM21 Abs from NMOSD patients reacted with TRIM21 in the cytoplasm of BMECs using double immunostaining. Stimulation with some commercial TRIM21 Abs, but not TROVE2 Abs or SS-B Abs, caused the nuclear translocation of NF-κB and an increase in 10- and 150-kDa dextran permeability. Removal of anti-TRIM21 Abs from NMOSD-IgG leads to a significant decrease in the biological effect of NMOSD-IgG on the increased permeability of 10-kDa dextran in BMECs. The positive rate of anti-TRIM21 Abs was 27% (13 of 48) in NMOSD patients, 27% (4 of 15) in myelin oligodendrocyte glycoprotein-antibody associated disease patients, 0% (0 of 43) in multiple sclerosis patients, 5% (2 of 43) in disease controls, and 0% (0 of 16) in healthy controls. Pooled NMOSD-IgG with anti-TRIM21 Abs negative for anti-GRP78 Abs decreased the barrier function more than HC-IgG using three in vitro BBB tri-culture models. In conclusion, anti-TRIM21 Abs in NMOSD may contribute to the breakdown of the BBB in patients with NMOSD via the activation of NF-κB and reduction in claudin-5 in BMECs.