前期的研究，發現由金針菇有效成分中的免疫調節蛋白(FIP-fve)，具有免疫調節的 功能，蛋白上的醣類結合模組(CBM)為重要結構，來刺激人類周邊血液的淋巴球細胞 產生 IFN-。本計畫由 ”衛生假說” 的學理基礎，來說明 Th1/Th2免疫細胞間相互的拮 抗作用來達成平衡，由 FIP-fve 所誘發的 IFN-可作為導向 Th1 細胞分化的應用，具 有減緩 IgE所引起的慢性氣喘及呼吸道發炎等過敏反應的效果。對於 FIP-fve透過何種 方式或活化路徑? 來啟動傾向於 Th1細胞的分化仍需釐清。本研究將分為三年來執行: 第一年，建立 FIP-fve 與受體上多醣類的交互作用機制，1) 藉由醣蛋白合成抑制劑與 去醣酵素等實驗， 2) 醣類 ligand 之免疫染色與 3) 專一性競爭的方法以及 4) 質譜 的應用與分析，來說明 FIP-fve與 TLR4/MD-2上 glycans的交互作用。第二年，探討 TLR4/MD-2 的內移作用與訊息傳遞路徑之關聯，與誘發 T-bet 與 IFN-活化的過程， 1) 利用免疫染色的方法來測定兩條路徑分子磷酸化的狀態、2) 對於各個路徑之下游基 因改變情形，3) 利用 TLR4/MD-2 抑制劑 ResatorvidTM、Dynamin 抑制劑 Dynasore 的處理及 MyD88 inhibitor peptide 分別來抑制 TLR4/MD-2 兩條訊息傳遞的路徑 TRIF-dependent 及 MyD88-dependent pathways，探討 FIP-fve 的內移作用在誘發 IFN-之訊息傳遞路徑與調控機制。第三年，研究不同 Notch ligands的選擇與 Th1/Th2 分化機制的轉換與免疫平衡之關係。1) 利用次世代定序技術，選殖微角家塵蟎致敏基 因，並鑑定具有誘發產生 IgE 抗體，並產生 Th2 細胞的反應傾向的過敏特質，2) 選 擇合理性的 Notch 受體抑制劑抗體OMP21M18及MSB6521，分別阻斷 DLL1/Notch3 及 Jagged1/Notch 接受器與受體的結合，3) DAPT 及 RO4929097等 Notch 下游關鍵 活化基因 -secretase 抑制劑，及 4) Bay-11-7082 NFB 的抑制劑，來建立 FIP-fve 的作用與 Notch ligands選擇的訊息傳遞路徑與 Th1/Th2分化傾向的機制。預期的研究 成果，將對於臨床上 80%以上由家塵蟎所誘發的氣喘患者，在此類患者體內 IgE 濃度 高、同時有 IL-4 細胞白介素上升、及噬酸性白血球偏多的現象。本計畫目標將藉由 FIP-fve對體內 Th1/Th2 免疫調節機制的探討，提供金針菇在過敏疾病上的食療應用、 及作為健康食品的學理性基礎。 In previous study, we demonstrated FIP-fve is a lectin isolated from Flammulina velutipes which carbohydrate binding module in response to the production of type II interferon associated with Th1-dominant immune responses from human peripheral mononuclear cells (hPBMCs). Explanations of “hygiene hypothesis” indicated the mechanisms of Th1/Th2 polarized might operate synergistically. But, which targeted pathways mediated to Th1 responses by the FIP-fve induced immune modulatory pathways remain to be elucidated. In this study, we proposed three possible mechanisms by which selections of proteoglycan ligands on hPBMCs, TLR4 internalization initiates different sets of adapters and Notch ligands alternative signals to be achieved the Th1/Th2 skewing. First, we removed the carbohydrate moieties from cell surface by tunicamycin and deglycosylation enzymes treatments reveal that reduce the secretion level of IFN-γ induction from hPBMCs. Then, we tried to illuminate the internalization mechanisms and downstream signal pathways involved the production of IFN-γ elicited by FIP-fve in human peripheral blood lymphocytes. Proof-to-concept we specific designed the immune staining to detect the status of phosphorylated and expression of downstream genes. Combined treated the Resatorvid TM , dynamin inhibitor, Dynasore, and MyD88 inhibit peptides to block the two major TLR4-signalling including TRIF-dependent and MyD88-depedent pathways, respectively. Furthermore, we used next generation sequencing (NGS) technologies to identify especially the Dermatophagoides Microceras (Der m 2, MW 14,000) allergen which can estimate of the major specificities of allergens which are potent inducers of IgE synthesis favoring Th2 response involved in type I hypersensitivity. Rationale use of pharmacological Notch inhibitors including OMP-21M18 and MBS605931 antibodies (ligands blocking), DAPT and RO4929097 (-secretase inhibitors) in addition to Bay-111-7082 (NFB inhibitor) to inducible inactivation of Notch signaling involved in Th1/Th2 cell polarization reflected FIP-fve and auto-adjuvant responses, respectively. In conclusion, this investigation provides a greater rationale of the mechanisms that development of associated FIP-fve to the clinically relevant allergen HDMs provided scientific evidences supported immune modulatory effects in the prevention of allergic diseases and may be as health food products.
