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樹状細胞を活性化する食品由来の素材探索と用途開発に関する研究

辻, 亮平 東京大学 DOI:10.15083/0002002744

2021.10.27

概要

第1章 -序論-
 免疫とは外敵からの侵入に対して、速やかに排除する高等生物に広く備わった高度なシステムである。近年の研究で多くの免疫細胞が多岐に渡った機能を有していることが分かってきているが、これらの細胞の機能が何らかの原因で弱くなると、外敵の排除ができなくなり、感染症に罹患する。これまで人類は非常に多くの感染症と闘ってきている。抗生物質の発見やワクチン技術の開発により、感染症を克服してきた。しかしながら、抗生物質の多用によるMRSAやC. difficileなどの多剤耐性細菌の出現や、人の移動を含めたグローバルな物流の進化によるデング熱やジカ熱・エボラ出血熱といった新型感染症の発生や蔓延が起きており、感染症の完全な克服はできていない。
 哺乳動物の免疫は自然免疫系と獲得免疫系に大別される。獲得免疫系は特定の抗原に対して速やかに強い反応を示す一方で、新しい外敵に対しては自然免疫を介した学習が必要である。そのため、多剤耐性細菌や新型感染症の対策・予防には自然免疫からのアプローチが有効である。そこで本研究では、普遍的に存在する植物組織からの新規免疫賦活素材を探索し、その作用メカニズムと新しい切り口でのワクチンアジュバントとしての応用に向けた詳細な検討を行った。さらにIFN-αの産生誘導ができる乳酸菌を用いて、新規用途検討として獲得免疫系も含めた免疫老化やその他の老化表現型に対する抗老化効果の詳細な検討を行った。

第2章 -植物由来免疫賦活物質の探索と作用メカニズム解明-
 リグニンは植物の細胞壁を強固にし、外敵からの侵入を防ぐ構造を構成しているものであり、セルロースに次いで2番目に多いバイオマスである。しかしながら、生理活性はあまり知られておらず、また生理機能に着目した商業化の例も乏しい。我々は、発泡酒の製造のときに副産物として生じる大麦穀皮にリグニンが豊富に含まれることに着目し、自然免疫系を活性化するものの探索を行った。過去の知見を参考に、大麦穀皮を4つの画分に分画し、マウス骨髄細胞由来樹状細胞(以下、BM-DC)に添加したところ、Lignin-Rich Enzyme Lignin画分(以下、LREL)とPure Enzyme Lignin画分(以下、PEL)において活性化が起こり、さらにLRELのほうがPELよりもその活性が強かった。大麦穀皮以外の食品として食される植物組織より抽出したLREL画分も同様の活性があることを明らかにしたが、大麦穀皮由来のLRELが最も強い生理活性を有していた。
 次にLRELおよびPEL画分の化学的な特徴を調べることを目的として、リグニン構造、糖組成の分析を行った。さらにリグニンと糖の結合と免疫賦活活性との関係を、アルカリ加水分解したサンプルを用いて評価した。その結果、リグニンに特有なβ-O-4型構造を構成するモノマー化合物が検出でき、さらにLRELのほうがPELよりもリグニンの含有量が多い、という結果が得られ、分析的にリグニン構造が含まれる免疫賦活物質であることを世界で初めて見出した。また、糖組成分析の結果、通常の大麦穀皮と比較して、LRELおよびPELはガラクトース及びマンノースが濃縮されていることを明らかにした。さらにリグニンと多糖のエステル結合を分解したところ、BM-DCに対する免疫賦活作用が消失した。これらの結果から、LRELおよびPELは、ガラクトースとマンノースが濃縮された特徴的な組成の多糖とリグニンがエステル結合していることで生理活性が生じるリグニン・多糖結合体である化学的特徴を見出した。
 最後にLRELの免疫賦活活性の作用メカニズムをTLRファミリーのノックアウトマウスを用いて作用機構の解析を行った。その結果、LPSが天然リガンドとして知られるTLR4のノックアウトマウス由来のBM-DCで活性化が完全に消失した。また、多糖類の受容体のブロッキング抗体などを使用し、TLR4以外の受容体がLRELの免疫賦活作用に関わっていないこと、LPS阻害剤であるポリミキシンDにより、微生物由来LPSのコンタミではないことを明らかにした。これらの結果より、LRELは新規のTLR4リガンドであることが示された。

第3章 -LRELのin vivoでの作用と応用開発-
 LRELのin vivoにおける免疫賦活能を検証することを目的として、C57BL/6JマウスにLRELを腹腔内投与した。すると、LREL投与により、一過的に血中IL-12p40濃度の上昇が確認された。また、投与24時間後の脾臓細胞において、ミエロイド樹状細胞(mDC)およびNK細胞の活性化が認められた。これらの結果から、LRELはin vivoにおいても、免疫賦活作用を有することを明らかにした。
 次に経口投与でも免疫賦活作用が見られるのかを検証した。LRELの単回経口投与試験において、腸間膜リンパ節中のmDCの活性化が確認された。また、LRELの反復経口投与試験において、腸間膜リンパ節のmDCの有意傾向の活性化に加え、IFN-γ陽性のCD4+T細胞の比率が上昇することを見出した。これらの結果から、LRELは経口投与でも免疫賦活能があることに加え、2週間投与し続けることで、自然免疫系だけではなく獲得免疫にも影響を与えることができる素材であることが示された。
 最後にLRELの経口ワクチンのアジュバントとしての可能性を検証した。ワクチンアジュバントは抗原提示細胞に抗原を取り込ませて抗体産生等に繋げるために、非常に重要な役割を持っており、既存のワクチンにも使用される不可欠なものである。BALB/CマウスにOVAとLRELを週に1度、5回反復して経口で投与したところ、血液中の総IgGおよび抗OVA IgGの量がLREL群で有意に増加した。また、小腸洗浄液において、総IgAの濃度はLREL群で有意に増加したが、抗OVA IgAの濃度は増加しなかった。IgAは一般的にIgGよりも抗原に対する特異性が低く、広範な抗原に反応することが知られていることから、これらのデータにより、LRELには経口ワクチンのアジュバントとして、一定の効果があることが示唆された。

第4章 -Lactococcus lactis strain Plasmaの抗老化機能の検証-
 生体内に存在する樹状細胞にはmDCのほかにプラズマサイトイド樹状細胞(pDC)がある。pDCは非常にレアなサブセットではあるが、生体内に広く存在し、ウイルスの感染を感知してIFN-αを産生する。Lactococcus lactis strain Plasma(以下、LC-Plasma)はpDCを活性化し、TLR9を介してIFN-α産生を誘導する乳酸菌として単離された。様々な非臨床試験および臨床試験において、ウイルス感染症に対する効果が見出されている。
 免疫系は他の臓器と同じく、経年的に老化することが知られている。免疫系が老化するとpDCのIFN-α誘導能が落ちることに加え、NK細胞やT細胞の機能も低下し、感染症やガンへの罹患率が高くなることが知られている。本章ではLC-Plasmaの新たな機能を見出すことを目的として、免疫老化や個体の老化に対する影響を、老化促進マウス(SAM)を使用して検証した。
 免疫系の老化が促進することが知られているSAMP1マウスにおいて、LC-Plasmaの免疫老化に対する抗老化効果を検証した。8週齢のSAMP1マウスにLC-Plasmaを1mg/headとなるように15週間混餌投与したところ、脾臓リンパ球のTLR9リガンド(CpG)に対するIFN-α産生促進能がLC-Plasma群で有意に増加していた。また、CD4+T細胞およびCD8+T細胞において、ナイーブマーカーであるCD62Lの陽性細胞の割合が有意に増加していた。これらの結果から、LC-Plasmaを経口摂取することで、pDCの活性低下が抑制され、T細胞の老化が抑制される可能性が示唆された。
 そして、免疫系の老化が報告されていない系統であるSAMP10マウスを使用して、個体レベルでの老化に対する効果を検証した。老年初期にあたる28週齢のSAMP10マウスを2群に分け、LC-Plasmaを1mg/headとなるように20週間混餌投与した。すると、脾臓リンパ球のCD4+T細胞において、SAMP1マウスと同様にCD62L陽性CD4+T細胞の比率がLC-Plasma群で高くなる、免疫老化抑制効果を再現できた。また、皮膚老化に対する影響を評価したところ、LC-Plasma群で老化時に見られる表皮の薄化が抑制されていることが確認された。さらに筋肉の老化に対する影響を評価したところ、ヒラメ筋の全体重に対する比重量がLC-Plasma群で増加していた。最後に個体レベルでの老化度を評価したところ、LC-Plasma群で老化度が統計的に有意に減少していた。
 これらの結果から、LC-Plasmaの長期経口摂取によって、免疫老化のみならず、個体レベルでの老化を抑制することができるという、抗老化効果があることが示唆された。

第5章–総括-
 本研究では自然免疫と獲得免疫系の懸け橋となっている樹状細胞の活性化に着目し、TLRシグナルを活性化する素材の探索とその応用開発に関わる検討を実施してきた。穀物穀皮を用いた検討において、TLR4を活性化する画分を抽出することに成功し、経口ワクチンのアジュバントとしての可能性を見出した。またTLR9リガンドを有するLC-Plasmaの検討において、新規に抗老化効果を見出した。
 これらの新たな樹状細胞活性化素材が生体防御作用、さらには老化制御などの機能性食品素材として活用され、多くの人々の健康維持に貢献できることを今後に期待したい。

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