探討第四型腺苷酸激酶在第一型輔助T細胞中扮演的角色

Abstract

輔助型Ｔ細胞（helper T cell; TH cell）可依據其所表現的轉錄因子（transcription factor）與所分泌的細胞激素（cytokines）分為以下幾類：第一型（TH1）, 第二型（TH2），與第十七型（TH17）輔助T細胞。當中，TH1細胞參與在當個體遇到病毒或是細菌的感染時，可以產生伽瑪干擾素（IFN-γ）來幫助巨噬細胞（Macrophage）清除病原體。當TH1接受到IFN-γ的訊號後會刺激TH1的轉錄因子：T-bet的表現。近年來許多的研究開始著重在代謝對於細胞（包含免疫細胞）之影響。目前已經發現不同的免疫細胞對於代謝路徑的使用上有不同的偏好，另外，一些特定的代謝中間產物也已經被證實能夠影響到免疫細胞的功能。然而，在代謝當中一個常見的物質：ATP在免疫細胞中的角色尚未完全釐清。第四型腺苷酸激脢（adenylate kinase 4）能夠藉由在ATP, ADP, AMP之中轉移磷酸基來改變ATP的平衡。本研究利用慢病毒轉導（lentivirus transduction）之方式將小髮夾RNA（shRNA）送入T細胞中抑制第四型腺苷酸激酶 mRNA的表現來探討第四型腺苷酸激酶在TH1細胞中扮演之角色，藉由此方式第四型腺苷酸激酶 mRNA的表現可以被抑制近90%。先前研究證實，當第四型腺苷酸激酶的表現被抑制時細胞當中ATP的含量有顯著提升的現象，而在TH1當中也可以發現到一樣的現象。另外，由於TH1主要的特徵與其功能為表現T-bet與伽瑪干擾素，本研究利用這兩項指標來觀察其功能是否有受影響，結果發現在抑制第四型腺苷酸激酶表現的組別中，產生伽瑪干擾素的TH1細胞比例相較對照組有顯著的增加。另外利用酵素結合免疫吸附分析方法（ELISA）也可偵測到抑制第四型腺苷酸激酶的第一型輔助T細胞培養液中含有較高濃度的伽瑪干擾素。然而在兩組當中T-bet的表現量卻沒有顯著差異，顯示第四型腺苷酸激酶對伽瑪干擾素的影響不是藉由調控T-bet而導致的現象。根據以上實驗，本研究發現了第四型腺苷酸激酶在TH1細胞當中扮演一個抑制伽瑪干擾素生成的角色。

Effector CD4+ T cells can be classified into different subsets, TH1, TH2, TH17, and TREG, by expressing specific transcription factors and cytokines. TH1 cells participate in fighting against viral and mycobacterial infection by secreting IFN-γ which can enhance the pathogen clearance by macrophage. IFN-γ signaling induces the expression of T-bet, the master transcription factor of TH1. Recent studies have raised the importance of metabolism to immune cell function. Many researches demonstrated that the preference of metabolic pathway or the intermediate product in metabolic reaction affect the function of immune cells. However, the role of ATP, a common product in metabolic pathway, in immune cells remains elusive. Adenylate kinase 4 (ΑΚ4), an enzyme that transfer phosphate group amount ATP, ADP, and AMP, is a modulator of ATP. Here, we aim to investigate the role of ΑΚ4 in TH1 cells. To facilitate this study, the ΑΚ4 expression was knockdown by lentiviral transducing AK4 shRNA into TH1 cell. After knockdown treatment, the expression of AK4 mRNA decreases up to 90%. As expected, there is higher amount of ATP in ΑΚ4 knockdown TH1 cells. The function of TH1 cells, represented by the expression of IFN- γ, was investigated comparing ΑΚ4 knockdown and scramble groups. ΑΚ4 knockdown elevated the percentage of IFN-γ producing cells. Furthermore, elevated IFN-γ level was detected from the supernatant of re-stimulated ΑΚ4 knockdown TH1 cells by ELISA. In this study, we demonstrated that AK4 negatively regulates the IFN-γ production in TH1 cells.