自己免疫性脳脊髄炎(EAE)モデル

実験的自己免疫脳脊髄炎(EAE)モデルは多発性硬化症(MS)に関連する前臨床実験モデルです。

MSは中枢神経系の慢性的炎症性疾患であり、脳炎あるいは脱髄疾患を引き起こします。MSは自己免疫性疾患の一つであり、自己反応性T細胞により引き起こされ、筋肉硬直や麻痺、視覚障害や失明、感覚喪失や運動失調など症状、繰り返し再発する特徴があります。今は様々なMS動物モデルがありますが、そのなかにはEAEモデルはMSに似てる炎症及び脱髄疾患の病理特性を持ち、MSの実モデルとして広く使われています。MSにはIL-23/IL-17の経路、EAE及びMS1-3の発病メカニズムが絡んでいます。

EAEモデルは髄鞘のタンパク質、例えばMOG(ミエリンオリゴデンドロサイト糖タンパク質、Myelin/oligodendrocyte glycoprotein)及びCFA(完全フロイントアジュバント Complete Freund's adjuvant)により誘導されます。フロイントのアジュバント(CFA)は免疫の日と2日後に、百日咳毒素(PTX)の腹腔内に注射して作れます。ミエリン特異的T細胞は末梢で活性化され、血液脳関門を通過して中枢神経系に入って再活性化され、一連の炎症反応を誘発して脱髄と軸索細胞のアポトーシスを引き起こし、最終的には神経の損傷と機能が失なわれます。標準化スコアリングシステムによって疾患誘発の程度を検定し、臨床症状を評価します。病理組織染色では局在の脱髄と炎症性白血球浸潤が観察されます。バイオサイトジェンは自社で開発したB-h17Aヒト化マウス(ノックアウトIL-17A遺伝子及びノックインIL-17A遺伝子)をMS治療ためのヒトIL-17Aターゲットのに対する薬効評価の為に、信頼性高いモデルとして提供しています。

B-hIL17Aマウスの作製と表現型解析

1.基本情報

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2. タンパク質発現の解析

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Serum from wild type (WT) C57BL/6 and homozygous B-hIL17A mice were analyzed by ELISA.

Mouse IL17A was detectable in the WT mice, while human IL17A was detectable in the homozygous B-hIL17A mice.



3. B-hIL17Aマウス白血球サブタイプの解析(脾臓)

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Analysis of leukocytes subpopulation in spleen.Splenocytes were isolated from C57BL/6 and B-hIL17A mice (n=3) and profiled by flow cytometry. As shown in the panels above, leukocytes subpopulations in homozygous B-hIL17A mice are similar to those in the C57BL/6 mice, indicating that development of T, NK, Monocyte, DC and macrophage cells are not affected by the knock-in of hIL17A.



4. B-hIL17AマウスT細胞サブタイプの解析(脾臓)

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Analysis of T cell subpopulation in spleen.Lymphocytes were isolated from spleen in C57BL/6 and B-hIL17A mice (n=3). T cells subpopulations were profiled by flow cytometry. As shown above, the T cell subpopulations in homozygous B-hIL17A mice are similar to those in the C57BL/6 mice, indicating that T cell development is not affected by the introduction of hIL17A.

MS/EAEモデルの作製びその表現型解析

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Experimental Autoimmune Encephalomyelitis (EAE) is an induced demyelinating disease model that closely resembles the progression and symptoms of the human neurological disease Multiple Sclerosis (MS).


EAE(実験的自己免疫脳脊髄炎)の評価

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EAE induction by MOG35–55/CFA Emulsion PTX in 10-week-old B-hIL17A mice(female). Data are expressed as mean ± SEM from a typical experiment (n=5). MOG: myelin-oligodendrocyte glycoprotein; PTX: pertussis toxin. 


EAEマウスモデルのH&EIHC染色


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HE staining showing lumbar enlargement, 4x. Inset 20x.

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Immunofluorescence (IF) staining of lumbar. Green, MBP; Blue, DAPI. Sections: 4x. Inset: 20x.

Local inflammation of the CNS in B-hIL17A mice (female, n=5) during EAE. On day 45 after MOG/CFA and PTx immunization, spinal cords were removed. The tissue sections were stained with H&E(A,B) and IHC (C,D)(Green, MBP; Blue, DAPI). The sections at the lumbar level are shown. The results showed that the infiltration of inflammatory cells in the MOG group was significantly increased, and the myelin protein was greatly reduced.



EAEモデルのリンパ節内IL-17A+細胞の増殖

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IL-17 is primarily produced by CD4 Th17 cells during the development of EAE. To detect IL-17 production, cells from lymph node of B-hIL17A mice (female, n=5) immunized with MOG/CFA were stimulated for 6 hours by PMA and ionomycin in the presence of brefeldin A. IL-17-producing cells were analyzed by FACS, along with IFN-gamma. The percentage of IL-17+CD3+CD4+ T cells in CD3+CD4+ T cells was increased in response to MOG immunization in B-hIL17A mice (left panel).  So was the percentage of IFN-gamma T cells.

EAEモデルCNSのIL-17A+細胞の増殖


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CNS (brain) cells of B-hIL17A mice (female, n=5)  immunized with MOG/CFA were stimulated for 6 hours by PMA and ionomycin in the presence of brefeldin A. IL-17-producing cells were analyzed by FACS, along with IFN-gamma. The percentage of IL-17+CD3+CD4+ T cells in CD3+CD4+ T cells was increased in response to MOG immunization in B-hIL17A mice (left panel).  So was percentage of IFN-gamma T cells.

Reference:

[1] Iwakura Y, Ishigame H. The IL-23/IL-17 axis in inflammation.J Clin Invest. 2006 May;116(5):1218-22.

[2] Gaffen SL,et al., The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing. Nat Rev Immunol. 2014 Sep;14(9):585-600. doi: 10.1038/nri3707.

[3] Kuwabara T,et al., The Role of IL-17 and Related Cytokines in Inflammatory Autoimmune Diseases. Mediators Inflamm. 2017;2017:3908061.


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