mRNA Profiling of Inflammatory Stress Responses after Aquaporin-4 Antibody and Human Complement Treatment Reveals Upregulation of NF-κB and IL6 Pathways

Neuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune disease affecting the central nervous system via autoantibodies that target the water channel aquaporin-4 (AQP4) on astrocytes. Binding to AQP4 initiates activation of innate immune components, especially the complement system. Both in vivo and in vitro models have been developed to investigate the molecular pathomechanisms of NMOSD. The goal of our study was to characterize the molecular response of four different human cell lines to a treatment with AQP4 antibody E5415A and human complement. We aimed to identify overlapping transcriptomic changes seen in the in vivo pathophysiology of NMOSD. Tested cell lines were AQP4-ECFP overexpressing U-87MG glioblastoma cells, U-87MG expressing only ECFP, HEK293 cells transiently transfected with AQP4-EmGFP, and human primary astrocytes. Complement-dependent cytotoxicity was induced after E5415A and active human complement treatment in AQP4-expressing cells, primarily by the classical complement pathway, but also with a contribution of the alternative pathway. Transcriptomic analysis revealed that both the in vitro U-87MG-AQP4-ECFP model and an in vivo rat model share genes primarily involved in nuclear factor K-light-chain-enhancer of activated B cells (NF-κB) and interleukin-6 (IL6) pathways. These findings were confirmed on the mRNA and protein levels in the in vitro model. As further validation, serum samples from AQP4 antibody seropositive and seronegative NMOSD patients were applied instead of E5415A on U-87MG-AQP4-ECFP cells and showed the same outcome. Additionally, NF-κB upregulation was shown by immunohistochemistry in medulla oblongata lesions of AQP4 antibody seropositive NMOSD patients. To conclude, our findings demonstrate IL6 and NF-κB pathways as major contributors to inflammation caused by complement activation in AQP4 antibody-positive NMOSD. We observed U-87MG-AQP4-ECFP cells to be a suitable model to study NMOSD pathomechanisms, as they show a gene expression profile towards NF-κB and IL6 pathway upregulation comparable with an in vivo model.