探討益生菌經由類鐸受器誘發 Notch 訊息傳遞以調控樹突細胞免疫反應的能力

1. 探討益生菌經由類鐸受器誘發Notch訊息傳遞以調控樹突細胞免疫反應的能力探討益生菌經由類鐸受器誘發Notch訊息傳遞以調控樹突細胞免疫反應的能力 • 樹突細胞對後天免疫系統的貢獻主要在於對T細胞的調控，並且被認為可藉由細胞表面的Notch ligands去活化T細胞上的Notch訊息傳遞以改變T細胞亞群的分化而有不同的免疫反應。已知益生菌能影響樹突細胞的成熟和免疫調節功能，並且可經由與樹突細胞表面不同的類鐸受器(TLRs)結合來影響，但是益生菌是否會藉由TLRs訊息傳遞來影響到樹突細胞Notch或Notch ligands的表現目前仍不清楚。實驗中先利用人類周邊血液單核球及樹突細胞之體外培養，與加熱失去活性的益生菌作用後觀察細胞激素的變化以了解各種Lactobacilli益生菌對於誘發人體免疫反應之效能，接著以小鼠骨髓衍生樹突細胞篩選出最具潛力的A菌，觀察此菌對於樹突細胞Notch、Notch ligands、Hes1、IL-10和IL-12等基因表現之影響，並在IL-10存在環境下觀察基因的表現是否有細胞激素的參與，再以gamma secretase inhibitor (GSI) 抑制Notch訊息傳遞後觀察樹突細胞成熟分子的表現是否受到影響，以及觀察餵食A菌之OVA致敏小鼠是否改善氣喘的症狀。結果顯示A菌能大幅提升小鼠骨髓衍生樹突細胞Delta1、Delta4、Jagged1、IL-12和Hes1 在24小時前期增加表現量，而Notch3和IL-10基因則在24小時後期能持續增加表現量，此一現象可能是藉由菌之細胞壁的peptidoglycan與樹突細胞上的TLR2以及菌之CpG DNA與TLR9作用而造成，TLR4也可能參與不同程度各基因的變化，同時IL-10則是降低Notch和Notch ligands基因的表現。另外，A菌刺激之樹突細胞能增加下游T細胞IFN-γ的分泌，同時藉由TLR2、TLR4、TLR9影響下游T細胞的增生，並且從GSI不改變樹突細胞成熟分子的表現可看出Notch基因的變化不影響樹突細胞本身之功能，而餵食A菌的小鼠能有效減緩以methacholine誘發之呼吸道阻力增加情形。從結果中推論益生菌能藉由TLR2、4、9改變樹突細胞早期Notch ligands表現量，引起Notch訊息傳遞再啟動Notch3基因的表現，輔以細胞激素之調節，並且可能參與樹突細胞對T細胞之影響走向Th1分化，以緩和過敏性氣喘的反應。

2. Studies for the immunomodulatory effect of probiotics via toll-like receptors on dendritic cells by Notch pathway induction • Notch ligands on dendritic cells initiate Notch signals on T cells to drive T cell differentiation and result in different immune responses in adaptive immunity. Probiotics are recognized by dendritic cells via Toll-like Receptor (TLR) 2, TLR4 or TLR9. Activating TLR signals effect on maturation and immune modulation on dendritic cells. Though there is studies on Nocth signals and TLRs recently, the relationship between probiotics and Notch signals is still unknown. Thus we used heat-inactivated probiotics to stimulate human peripheral blood mononuclear cells and monocyte-derived dendritic cells and investigated what kinds of immune responses were induced. Furthermore, we selected probiotic A as the most potential strain and investigated the gene expression of Notch, Notch ligands, Hes1, IL-10 and IL-12 on probiotic A–stimulated bone marrow-derived murine dendritic cells in vitro. We observed that probiotic A up-regulated Delta1, Delta4, Jagged1, Hes1 and IL-12 gene expression on dendritic cells in early phases between 3 to 24 hours while Notch3 and IL-10 gene were continuous increase after 24 hours stimulation. Moreover, IL-10 might reverse the changes. Our data suggest that probiotic A induced Notch/Notch ligands expression by TLR2 and TLR9 signaling in murine dendritic cells. However, TLR4 also involved these gene expression. In conclusion, it is possible that Notch, TLR and cytokine signals are integrated to modulate specific effector functions in dendritic cells and, therefore, induce the development of Th1 cells to ease the airway resistance in murine asthma model.