New BBB Model Reveals That IL-6 Blockade Suppressed the BBB Disorder, Preventing Onset of NMOSD

(A) 3D flow chamber and 3D flow membrane. The triple-cultured membranes were transferred in a 3D chamber. (B) Whole setup of migration assay under shear forces. Normal human PBMCs flowed onto luminal side with physiologic shear force by peristaltic pomp. (C) Schema of the 3D flow chamber in transmigration assay. Total migrated cells were recovered from the bottom chamber and enumerated. (D) Schema of leukocyte transmigration assay with NMO-IgG and/or satralizumab. (E) Flow-based leukocyte transmigration assays using a 3D flow chamber showed that application of NMO-IgG increased the numbers of total migrating PBMCs and CD4⁺, CD8⁺, and CD19⁺ cells relative to numbers migrating with control IgG and that the application of NMO-IgG plus satralizumab significantly suppressed that increase. *p < 0.05 by unpaired t test (n = 6 per group). All data are expressed as mean and standard error of mean. PBMC = peripheral blood mononuclear cells.

(A) Anti–IL-6 receptor antibody (MR16-1) was administered on day 7 after immunization. Anti–IL-6 receptor antibody significantly prevented the onset of clinical signs in EAE mice. *p < 0.05 by 2-way analysis of variance (n = 3–6 per group). (B) Anti–IL-6 receptor antibody suppressed lymphocyte migration into the spinal cords of EAE mice. Representative images showing immunohistochemical staining for CD4⁺ cells in the spinal cord on day 15 after immunization. The number of CD4⁺ T cells was markedly increased in the spinal cord of EAE mice. Anti–IL-6 receptor antibody administered on day 7 after immunization significantly prevented this increase. *p < 0.05 by Tukey multiple comparison test (n = 3–6 per group). Scale bar = 100 µm. (C) The induction of Th1 cells and (E) FoxP3-positive regulatory T cells was significantly upregulated on day 16 after immunization. There was a tendency, but not significantly, for Th17 cells to increase in EAE mice (D). Administration of anti–IL-6 receptor antibody on day 7 after immunization did not change the induction of these in EAE mice. *p < 0.05 by Tukey multiple comparison test (n = 4–8 per group). All data are expressed as mean and standard error of mean. EAE = experimental autoimmune encephalomyelitis; IL-6 = interleukin-6.

(A and B) Representative images showing immunohistochemical staining for albumin (A) and IgG (B) in the spinal cord on day 15 after immunization. Leakage of albumin and IgG into the spinal cord was higher in EAE mice than in control mice and was significantly reduced by treatment with anti–IL-6 receptor antibody (MR16-1). *p < 0.05 by Tukey multiple comparison test (n = 3–6 per group). Scale bars = 100 µm. All data are expressed as mean and standard error of mean. EAE = experimental autoimmune encephalomyelitis.

(A) Application of NMO-IgG to either the vascular side or the brain parenchymal side or both sides of the static BBB model significantly decreased the TEER value relative to that of control IgG at 72 hours. *p < 0.05 by unpaired t test (n = 3 per group). (B) Real-time TEER measurement by cellZscope showed that the TEER values had started to decrease within 24 hours of application of NMO-IgG in all groups, and the declining trend continued for 48 hours. (C–E, left panels) After addition of satralizumab and NMO-IgG to either the vascular side or the brain parenchymal side or both sides, the TEER values under conditions of satralizumab plus NMO-IgG were significantly higher than under conditions of NMO-IgG alone at 72 hours. *p < 0.05 by unpaired t test (n = 3 per group). All data are expressed as mean and standard error of mean. (C–E, right panels) Real-time TEER measurement by cellZscope showed a declining trend in TEER values under conditions of satralizumab plus NMO-IgG in all groups, but the decline remained less than that for NMO-IgG alone for 96 hours.

(A) Analysis of microvolume IgG translocation through the BBB by the Odyssey Infrared Imaging System revealed that the BBB Papp for satralizumab was almost 3 times that for control IgG. *p < 0.05 by unpaired t test (n = 6 per group). (B) Analysis of microvolume IgG translocation through the BBB by the spectrophotometer revealed that the relative accumulation of IgG for NMO-IgG was almost 1.5 times that for control IgG. *p < 0.05 by unpaired t test (n = 6 per group). (C) ELISA with anti-satralizumab antibody showed that the relative accumulation of satralizumab for satralizumab + NMO-IgG was significantly increased to almost 3 times that for satralizumab + control IgG. *p < 0.05 by unpaired t test (n = 8 per group). All data are expressed as mean and standard error of mean.