Introduction

Allergy is defined as an inappropriate, overactive and adverse immune reaction to exogenous harmless antigens including those derived from food. Although various therapies have been developed, using anti-allergic dietary factors to prevent allergies would be a promising strategy. Flavonoids are the most common polyphenolic compounds and the anti-allergic functions of dietary flavonoids have been reported. Aryl hydrocarbon receptor (AhR) is a transcription factor that plays an important physiological role in toxicological system-mediated regulation. Recent evidence also suggests that oral administration of AhR-ligands activates AhR and induces regulatory T cells (Tregs). Tregs are important in immunological tolerance, which is associated with food allergy and oral tolerance. Taken together, considering that some flavonoids are ligands of AhR, AhR-agonistic flavonoids must be potent to modulate the immunological function via Tregs induction, and therefore, to regulate different types of allergies including foodborne allergies. The aim of this study is (i) to screen potential AhR-agonistic dietary phytochemicals for use as Treg-inducible dietary factors, (ii) to characterize Tregs induced by dietary factors and to investigate their immune-regulatory activities, and (iii) to evaluate in vivo effect of Treg-inducible dietary factors administered perorally.

Chapter 1. Screening and evaluation of phytochemicals on the induction of Tregs in vitro

In this chapter, we attempted to find new anti-allergic dietary factors by screening dietary phytochemicals with AhR-agonistic activity in order to identify compounds inducing functional Tregs in vitro. NIH3T3 cells transfected with a xenobiotic responsive element (XRE)-luciferase reporter plasmid were used to monitor the effect of dietary phytochemicals on AhR-dependent transcriptional activity. Among 25 phytochemical samples tested, apigenin, (+)-catechin, fisetin, genistein, hesperetin, naringenin, quercetin hydrate, and rutin showed markedly high luciferase activities, and we found that only naringenin possesses the activity of inducing Tregs in vitro via a TGF-β-independent pathway. This result indicates that not all AhR ligands can induce Tregs and suggests that the specific structure of naringenin may contribute to its immune-regulatory activity.

To further confirm whether naringenin affects the induction of Tregs via AhR-ligand interaction, we next investigated the effect of AhR antagonists-resveratrol, TMF, and CH-223191-on the induction of Tregs. We found that the immunosuppressive effect induced by naringenin was attenuated upon the co-treatment with AhR antagonists. These AhR antagonists are reported to have different characteristics and action mechanisms one another, suggesting that the immunosuppressive activity of naringenin is regulated by its direct interaction with the ligand-binding site of the AhR molecule. Taken together, naringenin was shown to have the immue-regulatory activity on in vitro Tregs induction via an AhR-dependent pathway.

Chapter 2. Characterization and investigation of the function of Tregs induced by naringenin

In Chapter 2, we investigated the characteristics and immunomodulatory function of the Tregs induced by naringenin in vitro. We found that (i) the populations of both CD4+Foxp3+ T cells and CD4+CD25+Foxp3+ T cells were dose-dependently increased in CD4+ T cells activated and differentiated in vitro in the presence of naringenin, and that (ii) CD4+Foxp3+ T cells induced by naringenin expressed higher levels of CTLA4, GITR, LAP, and FR4 compared to the control. These results suggest that naringenin induced CD4+CD25+Foxp3+ Tregs which express typical Treg-associated surface molecules. We found that Foxp3 mRNA transcript levels were dose-dependently up-regulated by the induction of naringenin. NCABS2 is the functional non-evolutionarily conserved AhR binding site in the Foxp3 promoter and might participate in controlling Foxp3 expression. Naringenin exhibited higher NCABS2-driven luciferase activity by the reporter gene expression assay. Interestingly, AhR mRNA expression was also up-regulated in CD4+ T cells activated in the presence of naringenin in a naringenin-dose dependent manner.

We next investigated the expression of immune-suppressive cytokines by the naringenin-induced Tregs. Our data demonstrated that CD4+ T cells induced by naringenin at relatively low concentrations (6-12.5 μM) secreted significantly higher amount of IL-10 than the control CD4+ T cells and the CD4+ T cells induced by relatively high concentrations (25-50 μM) of naringenin. Moreover, we found that anti-IL-10 and anti-TGF-β antibodies attenuated the suppressive function of Treg cells induced by naringenin at low and high concentrations, respectively. We further determined that the suppressive effect of T cells induced by naringenin at high concentrations was mediated by membrane-bound TGF-β in a cell-cell contact mechanism.

Taken together, it was suggested that naringenin induced IL-10 producing Tr1-like cells and Foxp3+ Treg cells depending on the doses. We assume that naringenin up-regulated the AhR expression in CD4+ T cells dose-dependently and naringenin-activated AhR might bind Foxp3 promoter to regulate the induction of CD4+Foxp3+ Tregs expressing typical Treg-associated markers. In addition, in a specific range of concentration, naringenin induced IL-10 producing T cells via an AhR-dependent and/or unknown mechanisms.

Chapter 3. Evaluation of the immune-regulatory effect in vivo by oral administration of naringenin

AhR-ligands have been reported to induce Tregs in vivo that control and regulate the immunological tolerance, and we have found in this study that naringenin was a potent AhR agonistic dietary flavonoid and induced Tregs in vitro. Hence, we attempted to investigate whether naringenin possesses the immune-regulatory effect in vivo in terms of the induction of Tregs in BALB/c mice and DO11.10 mice. First we found that the populations of both CD4+Foxp3+ T cells and CD4+CD25+Foxp3+ T cells in spleen (SPL) and mesenteric lymph node (MLN) were up-regulated by feeding BALB/c mice with naringenin. The percentages of T cells expressing Treg-associated molecular markers in whole CD4+ SPL, CD4+ MLN, and CD4+ Peryer's patch (PP) T cells were mostly significantly up-regulated by naringenin administration. Furthermore, IL-2 production by CD4+ SPL T cells stimulated with anti-CD3 and anti-CD28 antibodies in vitro was significantly down-regulated by naringenin administration, suggesting that the CD4+Foxp3+ T cells, which do not secrete IL-2, included in the CD4+ SPL T cells exerted the suppressive effect against other normal CD4+ T cells unaffected by naringenin administration. These results suggested that oral administration of naringenin to BALB/c mice is potent to expand CD4+Foxp3+ Tregs in vivo. The percentages of CD4+IL-10+ T cells and CD4+TGF-β+ T cells among PP CD4+ T cells were also increased by oral administration of naringenin.

The up-regulation activity on expanding CD4+Foxp3+ Tregs of oral naringenin administration suggested that naringenin might enhance the induction of oral tolerance. Hence, we subsequently examined the immune-regulation effect of oral naringenin administration in the oral tolerance induction model using DO11.10 mice, which express rearranged T-cell receptor gene specific to 323-339 residues of ovalbumin and have been used for the investigation of T-cell development and immune-regulation. Although the generation of OVA-specific Foxp3+ Tregs was observed after oral administration of OVA, oral administration of naringenin showed no enhancing effect on OVA-specific CD4+Foxp3+ Tregs. In contrast, IL-10 production by SPL and PP CD4+ T cells from DO11.10 mice fed naringenin was significantly higher than by those from the control mice. However, no promoting effect of naringenin administration on oral tolerance induction was observed in the conditions applied in this study.

In our in vitro experiments, naringenin directly affected CD4+ T cells which were stimulated with anti-CD3 and anti-CD28 antibodies. However, in the case of oral administration, naringenin might also affect other immune cells, such as dendritic cells playing an important role in T cell activation. Therefore, further investigations on the effects of naringenin on other immune cells and its mechanisms of action are required to clarify the immune-regulatory effect of naringenin in vivo.

In conclusion, both the in vitro and in vivo results demonstrated that naringenin is potent to induce CD4+Foxp3+ Tregs and to up-regulate IL-10 producing CD4+ T cells in some conditions. These promising results, we believe, would provide us a new approach for developing anti-allergy food via the potential immunomodulatory activity of naringenin, and have wide range potential applications such as health supplements, quasi drug, as well as functional foods.

食品中に多く含まれる植物ポリフェノールであるフラボノイドには、肥満細胞の脱顆粒阻害作用を介した抗アレルギー作用があることが知られている。一方、フラボノイドには、解毒代謝の調節に関わる重要な転写因子であるaryl hydrocarbon receptor(AhR)のリガンド活性があることも知られている。最近になって、リガンドによるAhRの活性化が、アレルギーなどの免疫応答を抑制的に制御する制御性T細胞(Treg)を誘導することが報告された。これらの知見を総合すると、食品中のフラボノイドがAhRの活性化を介してTregを誘導し、その結果アレルギーを抑制している可能性も考えられる。本研究は、AhRアゴニスト活性をもつ植物由来食品成分のTreg誘導についてin vitro、in vivoの両面から詳細に検討したもので、3章からなる。

第1章では、Treg誘導活性を示す成分の探索を行っている。AhR依存的な異物応答配列(XRE)の下流にルシフェラーゼ遺伝子を結合したレポータープラスミドをトランスフェクトした細胞を用いて25種類の植物由来食品成分からAhRアゴニスト活性をもつものをスクリーニングした結果、アピゲニン、カテキン、ナリンゲニンなど8種類が選抜された。次に、これらをマウス脾臓CD4+T細胞に作用させ、Tregを誘導するかどうかを調べたところ、ナリンゲニンだけがその活性を有すること、このTreg誘導活性はTGF-β非依存的に起こることが示された。次に、ナリンゲニンによるTreg誘導がAhRを介した反応であるかどうかを確認するため、AhRアンタゴニストの存在下でナリンゲニンのTreg誘導活性を調べた。その結果、AhRアンタゴニストはナリンゲニンによって誘導されるT細胞増殖抑制効果を解除することが明らかとなり、このTreg誘導活性はAhR分子のリガンド結合部位との直接の相互作用を介したものであることが示唆された。

第2章では、ナリンゲニンにより誘導されたTregの免疫調節機能をin vitroで解析している。抗CD3および抗CD28抗体による刺激下、ナリンゲニンとともに培養したマウス脾臓CD4+T細胞を解析した結果、CD4+Foxp3+T細胞およびCD4+CD25+Foxp3+T細胞の集団が増加することが明らかとなった。また、CTLA4などの分子を発現するCD4+T細胞集団も増加していたことから、ナリンゲニンが典型的なTregを誘導していることが示唆された。Foxp3遺伝子のmRNA発現もナリンゲニンにより増加し、Foxp3遺伝子プロモーターに存在する非進化保存的なAhR結合部位であるNCABSを用いたレポーターアッセイから、ナリンゲニンはFoxp3遺伝子の転写活性化を介してTregの分化を誘導することが示唆された。さらにナリンゲニンはAhR遺伝子のmRNA発現も亢進した。サイトカイン量の測定から、低濃度のナリンゲニンによるTregの誘導にはIL-10が関わること、高濃度のナリンゲニンによる誘導には膜結合型TGF-βが関わっていることが示唆された。以上の結果から、ナリンゲニンはAhRの発現を増強するとともにAhRを活性化し、それがFoxp3遺伝子の発現を上昇させFoxp3+Tregを誘導した可能性が示唆された。

第3章ではナリンゲニンのin vivoにおける効果について評価している。卵白アルブミン(OVA)特異的T細胞レセプター遺伝子のトランスジェニックマウスであるDO11.10を用いたマウス経口免疫寛容誘導モデルでは、OVA経口投与で誘導される経口免疫寛容をナリンゲニンがさらに促進する効果は観察されなかったが、BALB/cマウスにナリンゲニンを2週間経口投与することにより、脾臓(SPL)と腸間膜リンパ節(MLN)において有意にCD4+Foxp3+T細胞とCD4+CD25+Foxp3+T細胞の集団が増加した。また、SPL、MLNおよびパイエル板(PP)のCD4+T細胞においてTreg関連分子マーカーを発現する細胞の割合がナリンゲニンの投与によって増加する現象が認められた。PPにおけるCD4+IL-10+T細胞とCD4+TGF-β+T細胞の割合もナリンゲニンの経口投与によって増加したことから、BALB/cマウスにおいてナリンゲニンの経口投与はCD4+Foxp3+Tregの集団を拡大することが示唆された。

以上、本研究は、日常食品から摂取しているフラボノイドの中に、アレルギー等の抑制にかかわる制御性T細胞を誘導する活性を持つものがあることを見出し、その誘導が、本来は解毒代謝の調節に関わると考えられてきた転写因子AhRを介して起こっていることをin vitroおよびin vivoの実験を用いて明らかにしたもので、学術上、応用上貢献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。