茯苓免疫調節蛋白活化小鼠腹腔巨噬細胞之訊息路徑及促進 T 細胞活化與第一型 T 輔助細胞免疫反應

A Novel Fungal Immunomodulatory Protein (PCP) Isolated from Poria cocos Induces Toll-like Receptor 4-dependent Activation in Mouse Peritoneal Macrophages and Promotes T Cell Activation and Th1 Immune Response

張慧欣(Hui-Hsin Chang)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.10346

摘要

茯苓 (Poria cocos) 為重要的藥用真菌，具有多種藥理療效，惟其活性物質的結構及作用機轉尚未釐清，故本論文之研究目的為闡明茯苓活性成分，並針對其活化巨噬細胞及淋巴細胞之分子機制做進一步的研究。本研究自茯苓乾燥菌核中經由 DE-52 陰離子交換樹脂與膠體過濾層析純化所得之新免疫調節蛋白 PCP，層析與電泳結果顯示天然之 PCP 為異質雙體的醣蛋白，其分子量為 35.6 kDa，分別由 21.3 kDa 與 14.3 kDa 兩次單元蛋白以雙硫鍵鍵結所構成，其中 21.3 kDa 之次單元蛋白具有 N 型與 O 型之醣化修飾。
第一部份試驗主要偵測茯苓蛋白 PCP 對小鼠巨噬細胞之影響，於體外試驗顯示茯苓蛋白 PCP 能刺激 RAW 264.7 巨噬細胞產生 TNF-α 與 IL-1β，同時亦能調控 NF-κB 相關基因的表現量。於初代小鼠腹腔巨噬細胞共同培養發現，茯苓蛋白 PCP 能使其細胞增加 MHC class II 與 CD86 的表現，同時經由 TLR4 受器的辨識，透過 MyD88 依賴型信號傳導途徑，以達到活化巨噬細胞的效果。試驗中證明茯苓蛋白 PCP 能與 TLR4 受器結合，並使其細胞產生酪氨酸磷酸化；進一步利用 TLR4 免疫缺陷鼠 (C57BL/10ScN) 進行試驗，結果發現茯苓蛋白 PCP 活化巨噬細胞及與細胞結合的能力顯著降低。茯苓蛋白 PCP 經由酵素去醣基後會降低其引發的巨噬細胞反應，因此茯苓蛋白 PCP 上的醣基為 TLR4 調控腹腔巨噬細胞活化作用的主要因子。
第二部份探討茯苓蛋白 PCP 活化淋巴細胞的作用與機制，結果指出茯苓蛋白 PCP 能直接活化小鼠脾細胞，增加其細胞增生作用及細胞激素 IFN-γ 的產量，然而在 IL-5 分泌量則無提升之效果；定量即時聚合酶連鎖反應分析結果顯示茯苓蛋白 PCP 亦能有效提高 IL-2 與 IFN-γ 基因表現；經由流式細胞儀分析結果顯示，茯苓蛋白 PCP 雖無法增加小鼠脾細胞中 CD4+ 與 CD8+ T 細胞的細胞族群比例，卻能顯著增加 T 細胞活化標記 CD69 的表現量。進一步利用磁珠分離系統分選特定細胞，結果指出於 anti-CD3/CD28 抗體存在之下，茯苓蛋白 PCP 能增加純化後 CD90+ 細胞的增生能力，並呈劑量效應。藉由偵測細胞增生作用、細胞激素分泌量與 CD44 及 CD69 活化標記的表現，證實茯苓蛋白 PCP 具有活化 CD4+ 與 CD8+ T 細胞的效果。同時茯苓蛋白 PCP 能增加 CD4+ T 細胞 T-bet 轉錄因子的基因表現，促進其 STAT4 轉錄因子酪氨酸磷酸化，及 IL-2 與 IFN-γ 產量的增加。動物試驗結果顯示，口服茯苓蛋白 PCP 能抑制小鼠血清中 IgG1 的含量，並增加其血清 IgG2a 含量與脾細胞 IFN-γ 的分泌，同時茯苓蛋白 PCP 亦能提升 OVA 致敏小鼠體內 OVA 特異性 IgG2a 含量，推測茯苓蛋白 PCP 能促進第一型 T 輔助細胞 (Th1) 反應。此外，茯苓蛋白 PCP 經由酵素水解蛋白後，則顯著降低其原有活化 T 細胞的能力，此結果說明茯苓蛋白 PCP 主要藉由其蛋白分子誘發 T 細胞的活化。
綜合上述研究結果顯示，茯苓蛋白 PCP 為具開發潛力的免疫活化物質，可經由 TLR4 活化巨噬細胞，同時促進 Th1 的免疫反應。本研究結果有助於瞭解茯苓及其活性物質的功效，亦能促進其醫藥潛力與產業應用。

關鍵字

免疫調節蛋白 ； 茯苓 ； 巨噬細胞活化 ； TLR4 ； T 細胞分化 ； 醣基化

並列摘要

Poria cocos (Schw.) Wolf is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and mechanisms involved have not been fully characterized. The objective of the present study was to investigate the bioactive substance from P. cocos and its molecular mechanism involved in immune modulation focused on macrophage and lymphocyte activation. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-linked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) as well as the regulation of nuclear factor-kappa B (NF-κB)-related gene expression. In primary mouse macrophages, we observed an increase in the expression of major histocompatibility complex (MHC) class II and CD86 molecules on peritoneal cavity macrophages. PCP directly activated macrophages to induce Toll-like receptor (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. We demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages.
Further investigation on lymphocyte activation indicated that PCP directly activated mouse splenocytes, markedly increased cell proliferation and the levels of interferon-gamma (IFN-γ) secretion but not IL-5 production. Similarly, the selectively enhanced transcriptional expression of IL-2 and IFN-γ by PCP was demonstrated using quantitative real-time PCR. Although there were slight increases in the total cell population of CD4+ and CD8+ T cells in PCP-stimulated splenocytes, PCP significantly increased expression of the activation marker CD69 on both splenic CD4+ and CD8+ T cells. The potent CD4+ and CD8+ T cell-activating capability of PCP was demonstrated by the enhancement of cell proliferation, cytokine secretion, activation marker CD44 and CD69 expression upon anti-CD3/CD28 costimulation. The expression of T-bet, tyrosine phosphorylation of STAT4, IFN-γ and IL-2 secretion during PCP-induced CD4+ T cell activation were upregulated. In contrast to the functional deficiency of deglycosylated PCP on macrophage activation, the core protein of PCP was shown to be involved strongly in induction of T cell activation, as demonstrated by inhibition of T cell response using deproteinized PCP. In vivo experiments indicated that oral administration of PCP (50 mg/kg body weight) suppressed the level of serum IgG1, and enhanced amounts of serum IgG2a and T helper 1 (Th1)-associated cytokine secretion in BALB/c mouse spleen cell cultures. Oral administration of PCP upon immunization with ovalbumin (OVA) exhibited that OVA specific IgG2a levels were also significantly increased compared with those of PBS-treated mice, suggesting that PCP could suppressed OVA-induced Th2 response to drive Th1 development.
Taken together, these studies characterize a new potential immune stimulator, PCP, which induces TLR4-dependent activation within murine macrophages and triggers a Th1-dominant immune response. These observations provide strong support for further studies of PCP and P. cocos to explore their overall modulatory nature toward mammalian cells and reveal their pharmaceutical potential and industrial value.

並列關鍵字

immunomodulatory protein ； Poria cocos ； macrophage activation ； TLR4 ； T cell development ； glycosylation

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