藉由 TLR2 活化劑刺激單核球細胞之共同刺激分子上升的機制
在抗原呈現細胞表面所表現之共同刺激分子如 CD80 及 CD86 等可與 T 細胞 上的 CD28 或 CTLA-4 辨認，進而調節 T 細胞的活化、分化等免疫反應，而 影響這些共同刺激分子的表現會調節免疫反應的作用。在先天免疫系統中，
Toll-like receptors 是模式識別受體家族成員中的一個主要份子，其中 TLR2 可 被格蘭氏陽性菌如金黃色葡萄球菌其細胞壁所組成的肽聚醣或 Pam3CSK4 所 活化。已知 TLR2 活化會促進抗原呈現細胞表面共同刺激分子的表現，但其 調控機制及訊息傳遞路徑仍不是非常清楚。本實驗計畫，以人類單核球細胞 與 THP-1 細胞為模式，給予 TLR2 活化劑刺激可增加共同刺激分子 CD40 、 CD80 與黏附分子 CD54 的表現。而使用不同 MAPK 抑制劑來抑制 TLR2 誘 導的訊息傳遞，發現 JNK 活化路徑較為重要。利用各種方式如抑制劑、 domi nant negative 突變質體轉染，各種方法均可得到此結果。特別是 CD80 其 mR NA 表現與蛋白質表現均被抑制，表示 JNK 訊息傳遞為 TLR2 活化 CD80 表 現的重要途徑。 AP-1 蛋白為 TLR2/JNK 訊息傳遞中 JNK 下游之轉錄因子。
我們利用核染色質免疫沉澱分析發現刺激 TLR2/JNK 路徑進而活化 AP-1 會結 合至 CD80 promoter 區域促進 CD80 的表現。此種共同刺激分子表現上升的作 用，實際上有其功能上意義，我們發現經由 TLR2 誘導 CD80 表現上升的單 核球，可促使 T 細胞的增生。因此在我們的實驗中發現， TLR2 活化劑刺激 共同刺激分子中的 CD80 的表現 MAPK 訊息傳導中 JNK 及其下游轉錄因子 A P-1 ，扮演一個關鍵調控的角色。
Upregulation of costimulatory molecules on monocytes stimulated by T LR2 agonists
The costimulatory molecules, such as CD80, CD86 on the antigen presenting cell have the abil ity to recognize the ligand (CD28 or CTLA-4) on T cell. The function of costimulatory molec ule on APC differentiation, and provides a signal necessary for T cell regulate the immune sys tem. In the innate immune system, Toll-like receptor is one of the major members in pattern re cognition receptor family. TLR2 can be activated by peptidoglycan (PGN), found in the cell w alls of Gram (+) bacteria and synthetic bacteria lipopeptide Pam3CSK4. TLR2 is reported to e nhance the expression of costimulatory molecules on the antigen presenting cell, however the regulatory mechanisms are still unclear. In the study, we used PGN and Pam3CSK4. to activat e TLR2 on monocyte and THP-1 cell and found the costimulatory molecules such as CD40, C D80, and adhesion molecule CD54 can be upregulated. By using MAPK inhibitors inhibit spe cific MAPK activation, downsterm of TLR2. We identify JNK pathway is more important tha n others. In addition to inhibitors, we also use other stratagie such as dominant negative mutan t DNA trasfectionto confirm the significance of JNK pathway, and got the similar evidence. Ei ther of CD80 mRNA and protein expression were inhibited, and this indicates that the JNK pat hway downsterm of TLR2 may regulate CD80 induce transcription level. Since AP-1 is one of stimulation of TLR2/JNK pathway of the transcription factor downsterm of JNK, we also foun d that AP-1 can binds to CD80 promoter by chromatin IP assay. To evaluate whether the upre gulation of costimulatory molecule have effects o immunomodulation, we use TLR2-activated monocytes to serve on APC to stimulate T cells, our result indicats that TLR2-activated mono cytes can stimulate T cell proliferation. Therefore, our results indicate that TLR2 agonist can s timulate CD80 upregulation mediated by JNK-AP-1 signaling pathway.
