Chondroitin sulfate (CS) is a member of glycosaminoglycans (GAGs) which are an important family of highly functionalized, linear, and negatively charged bioactive polysaccharides that are ubiquitous components of animal connective tissue. CS is firstly isolated from cartilage and consists of repeating (1->4)-linked disaccharide units of β-D-glucuronic acid linked (1->3) to N-acetyl-β-D-galactosamine. The two most common isomers, CS-A and CS-C, are sulfated at C-4 and C-6 of the galactosamine residue, respectively (Fig. 1). Several reports have shown that CS has anti-inflammation characteristics. CS is therefore commonly used as an ingredient for dietary supplements taken as an alternative medicine to treat osteoarthritis. CS is also approved and regulated as a symptomatic slow-acting drug for this disease (SYSADOA) in Europe and some other countries.

The recently research about immune functions of CS has been focused on its anti-inflammatory effects. But the signaling pathways and mechanism associated with the action of CS in cells still haven't been clearly defined. The pro-inflammatory responses induced by the microbe compounds via toll-like receptors (TLRs) in macrophages are characterized by secretion of cytokines such as tumour necrosis factor (TNF)-a, interleukin (IL)-1 and IL-6, etc. Recent studies have documented a series of IL-6 activities that are critical for resolving innate immunity and promoting adaptive immune responses. Overproduction of IL-6 leads to inflammation and diseases such as rheumatoid arthritis and Crohn's disease. In this study, we focused on the suppressive effects of CS and its oligosaccharides on the IL-6 secretion induced by TLR ligands in macrophage-like cells. And also the permeation mechanism of disaccharides derived from CS (Di-CSs) (Fig. 1) has been investigated.

Chapter 1. The permeation study of Di-CSs across human intestinal Caco-2 cell monolavers

Although CS has caused widespread interest in food and pharmaceutical industries, the intestinal absorption of CS has been controversial due to its high molecular weight and charge density. So far, a variety of analytical methods have demonstrated that CS can be absorbed by oral route. Furthermore, lots of studies showed that the structure and molecular size of CSs strongly influence their absorption and bioavailability, but the detailed absorption mechanism of CS still has not been revealed. In this study, CS was enzymatically hydrolyzed to prepare disaccharides (Di-CSs), and their intestinal permeability was investigated by using monolayers of intestinal Caco-2 cells which have been widely used to study the transport mechanisms of drugs. The amount of permeated disaccharides was determined by strong anion-exchange high-performance liquid chromatography (SAX-HPLC). Treatment of Caco-2 cell monolayers with sodium azide (NaN3), a metabolic inhibitor, did not affect the transepithelial transport of Di-4S and Di-6S through Caco-2 cell monolayers, suggesting that active transport is not involved in the transport of Di-CSs.

Interferon-gamma (IFN-y) and Cytochalasin (Cyto) B are known to increase the paracellular passive diffusion across the intestinal epithelial cell monolayers by altering the cytoskeletal structure which results in opening tight junctions. The permeabilities of Di-4S and Di-6S were remarkably increased when the transepithelial electrical resistance (TER) value of Caco-2 cell monolayers was dropped markedly by Cyto B or IFN-y treatment. These results suggested that the most plausible mechanism for Di-CSs permeation through Caco-2 cell monolayers is paracellular diffusion.

The three different types of authentic Di-CSs, Di-4S, Di-6S and Di-OS (Fig. 1), were also introduced in this study to investigate whether the sulfur group would affect the transepithelial transport of CS disaccharides. Our data indicated that the permeability of the three different disaccharides was not significantly different. This may suggest that the sulfur group is not the key factor determining the transepithelial transport of Di-CSs.

Chapter 2. The effects of CS and its oliaosaccharides on IL-6 secretion induced by TLR liaands in macrophage-like cells

The immune-modulating effects of CS and its oligosaccharides were concentrated on TLR-mediated inflammation in a macrophage-like cell line J774.1. Secretion of IL-6 from J774.1 cells stimulated by Pam3CSK4, Poly (I:C), LPS and CpG, which is the ligand of TLR1/TLR2, TLR3, TLR4 and TLR9 respectively, was investigated by co-treatment with CS or its oligosaccharides.

The disaccharide of CS-A, Di-4S, suppressed the IL-6 secretion induced by all four TLR ligands. Di-6S also significantly suppressed the IL-6 secretion induced by Pam3CSK4, Ploy I:C and CpQ The strongest suppressive effect on IL-6 secretion was observed when cells were stimulated by CpG; except CS-A, all other molecular types and sizes of CS can markedly suppress IL-6 secretion. Di-4S and Di-6S also significantly suppressed CpG-induced IL-6 secretion in another macrophage-like cell line RAW 264.

Furthermore, our experimental data suggested that CpG-induced IL-6 suppression in J774.1 caused by CS-A was molecular size-dependent; the smaller sized CS-A exerted more significant IL-6 suppression. However, similar size-dependency was not observed by CS-C treatment. Our results also pronounced the structure-dependent IL-6 suppression of CS and Di-CSs while J774.1 cells were stimulated by CpG. Between the two intact CSs utilized in our study, CS-C presented significantly stronger IL-6 suppression than CS-A. Interestingly, among the three types of Di-CSs with different sulfation (Fig. 1), Di-4S showed markedly stronger IL-6 suppressive effect than Di-6S and Di-OS. Similar structure-dependent IL-6 suppression caused by Di-CSs was also observed while J774.1 cells were induced by LPS, Pam3CSK4 and Poly:IC. Although the sulfur group of CS was thought to play an important role in its immune-modulating behavior, non-sulfated Di-OS showed close IL-6 suppression compared with Di-6S. This suggested that the sulfate residue is not always essential for the activity, but position of the sulfate residues may be important.

The results presented in this chapter indicated that characteristics like molecular size and structure are likely to be the determinants which govern the immune-modulating functions of CS and its oligosaccharides, especially on the TLR-related inflammatory responses.

Chapter 3. The mechanism study of CSs/Di-CSs on CpG-induced IL-6 suppressive effects in marcophage-like J774.1 cells

CS-C and Di-CSs also suppressed CpG-induced IL-6 mRNA expression, suggesting that the action point of CS-C and Di-CSs in suppressing CpG-induced IL-6 secretion exists at a stage earlier than the IL-6 gene transcription (Fig. 2B). CpG stimulated the activation of TLR9 to trigger the molecule myeloid differentiation primary response gene (88) (MyD88)-dependent inflammatory signaling pathway. The effect of CSs/Di-CSs on the CpG-induced degradation of IL-1 receptor-associated kinase 1 (IRAK1), which was involved in the early stage of MyD88-dependent inflammatory signaling pathway, was therefore examined. IRAK1 degradation was clearly inhibited by Di-4S, Di-6S and CS-C, indicating this inhibition to be a possible mechanism point of the IL-6 suppression by CS (Fig. 2C). However, some steps that precede the signaling pathway triggered by TLR9 can also switch off the downstream signaling activation. Several other action points of CSs/Di-CSs preceding the signaling pathway by TLR9 activation were therefore checked in this study (Fig. 2D-G). Results of size exclusion chromatography (SEC) indicated that CSs/Di-CSs couldn't directly bind with CpG (Fig. 2D). Flow cytometry results of fluorescein isothiocyanate (FITC)-labelled CpG demonstrated that CSs/Di-CSs couldn't affect the phagocytosis of CpG by J774.1 cells (Fig. 2E) which was necessary for CpG recognition by TLR9 and triggered the IL-6 inflammatory signaling pathway. Immune fluorescence results suggested that oligosaccharides derived from CS did not affect CpG traffic to TLR9 (Fig. 2F), although CSs appeared at the same location of CpG and TLR9. Finally, results of immunoprecipitation suggested that Di-4S interfered with the CpG-TLR9 interaction (Fig. 3G), which would be one of the additional mechanisms.

CS has been reported to directly bind with mannose receptor in a structure-dependent manner. The similar CS binding domain of mannose receptor also exists in TLR9. Thus, it is probable that CSs/Di-CSs directly bind with TLR9. The competition of CpG and CSs/Di-CSs in binding with TLR9 may affect the interaction of TLR9 and CpG, and therefore weaken the stimulation of IL-6 signaling pathway by CpG. It would be also possible that CSs/Di-CSs play as antagonistic ligands of TLR9, thereby suppressing the IL-6 signaling pathway activation.

Understanding the possible permeation mechanism of Di-CSs would be beneficial to improve their bioavailability and also be helpful to exert their immune functions. With enough evidence and knowledge that Di-CSs can be actually absorbed in the oral route, further interesting immune research on CS would be worthy to be expected.

Our results have suggested that CS-C and Di-CSs would be effective suppressing agents for TLR9-mediated inflammation in macrophages. Until now, there was no report describing the natural substances which suppress the CpG-induced inflammatory reaction. The immune-modulating effects of CS-C and Di-CSs presented in this study will be helpful to understand the anti-inflammatory mechanism of CS and its oligosaccharides and other sulfated polysaccharides, especially in terms of the TLR-mediated inflammatory responses.

Fig. 1. Structures of CS disaccharides (Di-OS, Di-4S and Di-6S). Di-4S, the disaccharide unit of CS A, is sulfated on C-4 of the galactosamine while Di-6S, the disaccharide unit of CS-C, is sulfated on C-6 of the galactosamine. Whereas Di-OS carries no sulfated group.

Fig. 2. Schematic diagram of possible action points of CSs/Di-CSs which suppressed the CpG-induced IL-6 secretion in macrophage J774.1 cells. Part A: IL-6 translation, Part B: IL-6 transcription, Part C: signaling pathway after TLR9 activation, Part D: direct binding of CSs/Di-CS with CpG, Part E: phagocytosis of CpG by J774.1 cells, Part F: CpG traffic to lysosome, Part G: CpG recognized by TLR9.

コンドロイチン硫酸(CS)は、動物の結合組織に広く存在する直鎖状多糖類グリコサミノグリカンの一種である。β-D-グルクロン酸(61→3)N-アセチル-β-D-ガラクトサミンの2糖単位が1→4結合により繰り返し配列した構造を持つ。もっとも一般的な2つのisomerはCS-AとCS-Cで、それぞれガラクトサミン残基のC-4あるいはC-6の位置が硫酸化されている。CSは抗炎症作用を持つと言われており、関節炎治療のための経ロサプリメントの原料としても用いられているが、その抗炎症作用の分子メカニズムはまだ明確にされていない。本研究は、微生物成分がマクロファージのToll様受容体(TLR)を刺激することによって誘導される炎症性サイトカインの分泌が関節炎などの炎症の一反応であることに着目し、TLR刺激によって誘導されるマクロファージ様細胞のIL6分泌に対するCSの抑制効果について解析を進めたもので、3章からなる。

経口的に摂取されたCSの構造や分子サイズがその吸収性に大きく影響することは報告されている。しかし、CSや低分子化したCSの腸管吸収機構の詳細については明らかにされていない。第1章では、csを酵素分解して2糖(Di-CS)を調製し、その腸管透過性をヒト腸管Caco-2細胞の単層培養系により解析した結果が述べられている。未分解CSはほとんど細胞層を透過しないが、Di-CSは透過することが示された。その透過性はエネルギー代謝阻害剤で細胞層を処理しても変化がなかったことから、Di-CSsの透過には能動輸送系は寄与していないことが示唆された。細胞間受動拡散の速度を高めることが知られているサイトカインなどで処理して上皮細胞層の電気抵抗を顕著に低下させた細胞層では、Di-CSの透過量が顕著に増加した。これらの結果から、Di-CSの透過経路として最も可能性が高いのは細胞間拡散であると考えられた。透過性に硫酸基がどのように関わるかを、3種の異なるタイプのDi-CS、すなわちDi・4S、Di-6SおよびDi-OSの標品を用いて解析した結果、透過性に関して、CSの硫酸基の有無やその結合部位は重要な要因ではないことが示唆された。

第2章では、TLRのリガンドによって誘導される、マクロファージ様細胞からの炎症性サイトカインIL6分泌元進に及ぼすCSやそのオリゴ糖の影響を調べている。マクロファージ様細胞J774.1を各種のTLRリガンドで刺激した時、TLR1/TLR2,TLR3,TLR4およびTLRに対する刺激は、細胞からのIL-6分泌をft進した。一方、CSやそのオリゴ糖は、このIL・6分泌充進を抑制する作用を示した。CpGによって誘導されるIL6分泌に対し、未分解のCSではCS・Cが強い抑制活性を持つこと、低分子CSではCS・A由来のものが強い抑制作用を持つこと、2糖類ではDi-6SやDi・OSに比べてDi-4Sが顕著に高いIL-6分泌抑制作用を示すことなど、明確な構造依存性が認められた。なお、硫酸基の有無は活性に必須ではないことも示唆された。

第3章では、CpGで誘導されるIL-6分泌に及ぼすCSの抑制効果を、主にDi-CSに注目してその作用機構の面から解析している。Di-CSはIL-6の分泌だけでなく、IL-6mRNA発現も抑制し、その抑制作用点がIL-6遺伝子の転写段階よりも上流に存在することが示唆された。IL-6転写の上流にあるMyD88一依存的な炎症反応のシグナル伝達経路にDi-CSが影響を及ぼすかどうかを、その初期段階に関わる分子IL-1receptor・associated kinase 1(IRAK1)の分解への影響という視点から検討したところ、IRAK1の分解はDi-CSによって顕著に抑制され,これがCSによるIL-6分泌抑制の分子機構の一つと考えられた。一方、TLR9リガンドであるCpGにDi-CSが直接結合してしまう可能性、CpGが誘導するJ774.1細胞の食細胞活性にDi-CSが影響を及ぼす可能性、Di-CSsがCpGの細胞内移行に影響を与える可能性はいずれも否定されたが、Di-4Sを用いた免疫沈降試験によって、Di-4Sが細胞内でのCpGとTLR9の相互作用を妨害していることが示され、これが作用点の一つとなっていることが示唆された。マクロファージなどの表面に存在するマンノース受容体に存在するcs結合ドメインがTLR9分子にも存在することから、Di-CSは、これを介してTLR9と直接結合し、CpGのTLR9への結合を競合的に抑制するという仮説が最後に述べられている

以上要するに、本研究は、炎症抑制作用が期待されている食品成分コンドロイチン硫酸がマクロファージの炎症性サイトカイン分泌元進を抑制する現象を見出し、その作用機構の一端を分子レベルで解析したもので、学術上、応用上寄与するところが少なくない。よって、審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。