茯苓免疫調節蛋白活化樹突細胞及調節 TH1 免疫反應

Abstract

茯苓蛋白 PCP (Poria cocos protein) 為由茯苓菌核萃取出的蛋白二聚體，分子量為 35.6 kDa，包含 14.3 kDa 單元及 21.3 kDa 含醣基單元。PCP 具有活化 RAW264.7 巨噬細胞及小鼠腹腔巨噬細胞的能力，在 anti-CD3/CD28 mAbs 存在的環境與 T 細胞共同培養可活化 T 細胞並分泌 TH1 細胞素 IFN-γ (interferon-gamma)，且於異位性皮膚炎之貼膚試驗中可抑制 TH2 免疫反應。因為 PCP 無法直接活化 CD90.2+ T 細胞，故本研究推測 PCP 必須利用脾細胞中的抗原呈現細胞 (antigen-presenting cells, APCs)，如樹突細胞 (dendritic cells, DCs) 使輔助型 T 細胞分化成 TH1 型，並設計試驗驗證之。首先，在探討 PCP 是否能活化 DCs 的研究中，PCP (100 μg/mL) 可增加小鼠 DCs 之 CD40、CD80 及 CD86 表現及 IL-6 及 IL-12p70 分泌量，證明 PCP 可活化 DCs。由 TLR (toll-like receptor) 2-/- 及 TLR4-/- 基因剔除鼠之 DCs 表面分子表現及 IL-6 細胞素分泌試驗結果推測 PCP 不完全透過 TLR2 或 TLR4 路徑活化 DCs。接著，在探討樹突細胞調節 TH 免疫反應之試驗中，預先與 PCP 及 OVA 抗原培養過的 DCs 可增加對 OVA 有特異性之 DO11.10 CD90.2+ T 細胞增生及分泌 IFN-γ，並減少 IL-4 分泌量，顯示 PCP 有助於 DCs 呈現 OVA 並使免疫反應導向 TH1。最後，於 OVA 誘導之小鼠氣喘試驗中，餵食 PCP (100 μg/day) 可增加支氣管肺泡灌洗液 (bronchoalveolar lavage fluid, BALF) IFN-γ 濃度及血清 OVA-specific IgG2a 含量，並降低 BALF 嗜酸性球 (eosinophils) 數、TH2 細胞素 (IL-4、IL-5、IL-13) 濃度、血清 OVA-specific IgE 含量及肺部組織的發炎細胞浸潤情形。由以上試驗結果證明 PCP 可透過活化樹突細胞使 TH1 型細胞分化，並抑制 TH2 型細胞分化，減少動物氣喘模式之小鼠 TH2 免疫反應。

Poria cocos protein (PCP), which is purified from dried sclerotium of Poria cocos, is a 35.6 kDa heterodimer protein including 14.3 and 21.3 kDa glycosyl subunits. In our previous results, PCP could active RAW264.7 cells and mouse peritoneal macrophages. This protein could also up-regulate murine T cells to secrete IFN-γ in the presence of anti-CD3/CD28 mAbs and inhibit TH2 immune-response in patch test for atopic dermatitis. However, PCP could not directly activate the differentiation of CD90.2+ T cells to TH1 cells, suggesting antigen-presenting cells as dendritic cells (DCs) could be responsible for the activation of PCP on TH1 cells. In this study, we first found that PCP could up-regulate the expression of CD40, CD80, and CD86 and induce secretion of IL-6 and IL-12p70 on murine DCs. In the results of PCP incubated with DCs of TLR2-/- and TLR4-/- mice, we found that PCP might induce DCs activation through neither TLR2 nor TLR4 pathway. Second, PCP significantly increased the antigen-presentimg ability of murine DCs to promote cell proliferation and IFN-γ secretion of OVA-specific DO11.10 CD90.2+ T cells. Meanwhile, these DCs could inhibit the OVA-specific IL-4 secretion by DO11.10 CD90.2+ T cells. According to the results, we suggested that PCP can help DCs presenting antigen and skewing immune-response to TH1. Finally, in OVA-induced asthma mouse model, oral administration of PCP (100 μg/day) could increase the production of IFN-γ in bronchoalveolar lavage fluid (BALF) and OVA-specific IgG2a in serum. Oral administration of PCP could also down-regulate the amount of eosinophils and TH2 cytokines (IL-4, IL-5, and IL-13) in BALF, OVA-specific IgE in serum, and cell infiltration in mouse lung sections. In conclusion, PCP can activate DCs to promote differentiation of TH1 cells and suppress TH2 immune-response in mouse model of asthma.