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November 2021; 8 (6) ArticleOpen Access

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

View ORCID ProfileYukio Takeshita, Susumu Fujikawa, Kenichi Serizawa, Miwako Fujisawa, Kinya Matsuo, Joe Nemoto, Fumitaka Shimizu, Yasuteru Sano, Haruna Tomizawa-Shinohara, View ORCID ProfileShota Miyake, Richard M. Ransohoff, Takashi Kanda
First published October 19, 2021, DOI: https://doi.org/10.1212/NXI.0000000000001076
Yukio Takeshita
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • ORCID record for Yukio Takeshita
  • For correspondence: takeshy@yamaguchi-u.ac.jp
Susumu Fujikawa
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: smfujika@yamaguchi-u.ac.jp
Kenichi Serizawa
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: serizawakni@chugai-pharm.co.jp
Miwako Fujisawa
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: fuji0418@yamaguchi-u.ac.jp
Kinya Matsuo
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: matsuok@yamaguchi-u.ac.jp
Joe Nemoto
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: nemoto@yamaguchi-u.ac.jp
Fumitaka Shimizu
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: fshimizu@yamaguchi-u.ac.jp
Yasuteru Sano
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: yasuteru@yamaguchi-u.ac.jp
Haruna Tomizawa-Shinohara
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: shinohara.haruna89@chugai-pharm.co.jp
Shota Miyake
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • ORCID record for Shota Miyake
  • For correspondence: miyake.shota34@chugai-pharm.co.jp
Richard M. Ransohoff
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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  • For correspondence: rransohoff@thirdrockventures.com
Takashi Kanda
From the Department of Neurology and Clinical Neuroscience (Y.T., S.F., M.F., K.M., J.N., F.S., Y.S., T.K.), Yamaguchi University Graduate School of Medicine; Kenichi Serizawa (K.S., H.T.-S., S.M.), Haruna Tomizawa-Shinohara and Shota Miyake, Product Research Department, Chugai Pharmaceutical Co., Ltd, Kanagawa, Japan; and Richard M Ransohoff (R.M.R.), Third Rock Ventures, Boston, MA.
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Citation
New BBB Model Reveals That IL-6 Blockade Suppressed the BBB Disorder, Preventing Onset of NMOSD
Yukio Takeshita, Susumu Fujikawa, Kenichi Serizawa, Miwako Fujisawa, Kinya Matsuo, Joe Nemoto, Fumitaka Shimizu, Yasuteru Sano, Haruna Tomizawa-Shinohara, Shota Miyake, Richard M. Ransohoff, Takashi Kanda
Neurol Neuroimmunol Neuroinflamm Nov 2021, 8 (6) e1076; DOI: 10.1212/NXI.0000000000001076

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    Figure 1 Construction of Newly In Vitro BBB Model With Triple Coculture System of Temperature-Sensitive Conditionally Immortalized Human BBB Cell Lines

    (A) Establishment of the BBB cell lines that maintain the BBB properties. Morphology of hEC is spindle-shape. hEC expressed von Willebrand factor (vWF) as a lineage marker of endothelium. Morphology of hPCT is cobblestone-shape. hPCT expressed PDGFß as a lineage marker of pericyte. Morphology of hAST is star-shape. hAST expressed glial fibrillary acidic protein (GFAP) as a lineage marker of hAST. (B) Three conditionally immortalized human cell lines were transfected with temperature-sensitive SV40 large T antigen (Ts-SV40 LT). At 33°C, activated Ts-SV40 LT binds and inhibits p53 and Rb, which are strong tumor suppressors, leading to continuous cell proliferation. At 37°C, inactivated Ts-SV40 LT exhibits growth arrest, leading to differentiation into mature cells. BBB = blood-brain barrier; hAST = human astrocyte; hEC = human brain microvascular EC; hPCT = human pericyte.

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    Figure 2 Construction of Ex Vivo BBB Model With Triple Coculture System for Leukocyte Transmigration and Effect of Satralizumab on NMO-IgG–Induced Transmigration of Leukocytes in Ex Vivo

    (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.

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    Figure 3 Effects of IL-6 Receptor Blockade on Lymphocyte Migration Into the Spinal Cord In Vivo

    (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.

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    Figure 4 Effects of IL-6 Receptor Blockade on BBB Permeability In Vivo

    (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.

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    Figure 5 Effects of Satralizumab on the Barrier Function of the BBB In Vitro

    (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.

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    Figure 6 Intracerebral Transferability of Satralizumab in the Presence of NMO-IgG In Vitro

    (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.

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    Figure 7 The Pathophysiology of NMOSD at the BBB and the Action Mechanism of Satralizumab at the BBB

    (A) There are several steps on both sides of the BBB involved in the onset of NMOSD. First, NMO-IgG (anti-GRP78 or an unknown antibody or antibodies) activates nuclear factor-kappa B (NF-κB) signals in ECs. Second, NMO-IgG decreases the barrier function on the vascular side. Third, NMO-IgG increases the intracerebral transferability of NMO-IgG itself. Fourth, NMO-IgG attacks the AQP4 of astrocytes (ASTs) and induces IL-6 expression in ASTs. Fifth, IL-6 signaling affects endothelial cells on the CNS side. Sixth, IL-6 signaling more strongly decreases the barrier function on the CNS side than on the vascular side. Seventh, IL-6 signaling induces the expression of several chemokines (CCL2 and CXCL8) in endothelial cells. Eight, the induced chemokines enhance infiltration of inflammatory cells. Finally, NMOSD develops. (B) First, NMO-IgG (anti-GRP78 or an unknown antibody or antibodies) activates NF-κB signals in endothelial cells. Second, NMO-IgG decreases the barrier function on the vascular side. Third, NMO-IgG elevates the intracerebral transferability of satralizumab more than NMO-IgG. Fourth, NMO-IgG attacks the AQP4 of ASTs and induces IL-6 expression in ASTs. Fifth, satralizumab blocks IL-6 signaling on the CNS side. Sixth, satralizumab inhibits the reduction in barrier function by blockade of IL-6 signaling. Seventh, blockade of IL-6 signaling by satralizumab suppresses the expression of several chemokines (CCL2 and CXCL8) in endothelial cells. Eight, satralizumab inhibits infiltration of inflammatory cells. Finally, satralizumab prevents the onset of NMOSD. AQP4 = aquaporin-4; BBB = blood-brain barrier; EC = endothelial cell; IL-6 = interleukin-6; NMOSD = neuromyelitis optica spectrum disorder.

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