指導教授：李建國臺灣大學：免疫學研究所趙穎吟Chao, Ying-YinYing-YinChao2014-11-262018-07-092014-11-262018-07-092014http://ntur.lib.ntu.edu.tw//handle/246246/262032類鐸受體（Toll-like receptor）是先天性免疫中辨識細菌及病毒感染很重要的成員，而類鐸受體主要是表現在一些先天性免疫細胞上，像是巨噬細胞及樹突細胞。除此之外，在非免疫細胞上，像是上皮細胞也會表現此種受體。有趣的是造血前驅細胞上也表現這些類鐸受體，但是它表現在前驅細胞的功能仍舊未明。在過去的研究中，我們使用體外培養的方法，將分離共同淋巴前驅細胞（common lymphoid progenitor; CLP）和 Fms-like tyrosine-3 (Flt3) 配體 （Flt3 ligand, FL） 一起培養後，可以使 CLP 主要發育為漿狀樹突細胞（pDC），然而受到類鐸受體 9配體（TLR9 ligand） CpG ODN 刺激後， CLP 卻轉而發育為傳統樹突細胞 (conventional DC, cDC)。值得注意的是，單獨加入 CpG ODN 或是同時添加 FL 與 CpG ODN 會誘導一群特殊的樹突細胞，這群細胞會同時表現 CD11b 和 B220 和 CD11c 表面分子。這群 DPDC 的形態與特徵比較像是 cDC 而不像 pDC，且這群細胞在缺少 STAT1 或第一型干擾素受體後並不影響其生成，顯示第一型干擾素並沒有參與其發育。當把野生型 CLP 與 Tlr9-/- CLP 一起培養時，Tlr9-/- CLP 並無法發育成 cDC 與 DPDC，因此它們的發育應該是經由 TLR 9 訊息傳遞過程直接調控而非其誘發因子。此外，這群細胞在缺少 STAT3 後也不會形成，表示 STAT3 在 TLR 9 訊號傳遞過程會正向調控。綜合以上的結果，我們發現感染或發炎反應會改變原來樹突細胞發育過程，使得原本應該是促進 pDC 生成的，都變成促進 cDC 及 DPDC 的生成，或許這是提供先天免疫系統一個可能快速對抗感染的方式。Toll-like receptors (TLRs) are one family of the pattern recognition receptors in the innate immunity to sense and response to microbial or viral infection. TLRs are expressed in innate immune cells, including macrophage and dendritic cells (DC) and non-immune cells such as epithelium in the gut and lung. Interestingly, TLRs also expressed in hematopoietic stem and progenitor cells (HSPCs). However, the roles of TLR response in HSPCs are largely unknown. We showed here that Flt3 ligand (FL) preferentially promoted pDC formation from common lymphoid progenitor (CLP), while TLR9 signaling promoted cDC differentiation. CpG ODN alone or CpG ODN plus FL, and not FL alone, also induced an unique DC population, which expressed both CD11b and B220 (double positive) other than CD11c. Interestingly, the morphology and phenotype of these DPDCs were more closer to those of cDCs and not to those of pDCs. The development of CpG-induced cDCs and DPDCs were not altered in the absence of STAT1, or IFNAR1, the type I interferon (IFN-I) receptor, suggesting that the process is IFN-I-independent. However, the effect of CpG ODN on DC development was dependent on direct TLR9 signaling pathway and not on TLR9-induced genes, as biased cDC development was impaired in Tlr9-/- CLPs when co-cultured with WT CLPs. Moreover, this effect was abolished in the absence of STAT3, suggesting that STAT3 might positively regulate TLR9-induced development of cDC and DPDC. These results suggest that inflammation-induced remodeling of DC population may provide a unique way for a rapid reaction of the innate immune system upon infection or inflammation.誌謝 i 中文摘要 iii Abstract iv Abbreviation vi Table of contents viii Chapter I Introduction 1 1.1 DC subsets 2 1.2 DC development 3 1.3 Cytokines in DC development 4 1.4 The role of TLR expression on HSPCs 6 1.5 Circulation of HSPC. 7 1.6 Hematopoiesis in inflammatory conditions 7 1.7 Specific aims 8 Chapter II Materials and Methods 10 2.1 Mice 11 2.2 Isolation of cell populations and flow cytometry 11 2.3 In vitro DC culture 12 2.4 Flt3L preparation 12 2.5 Adoptive transfer and analysis of DC subtypes 13 2.6 Cytospin and staining 14 2.7 Statistical Analysis 14 2.8 Fluorescent antibodies 14 Chapter III Results 16 3.1 TLR9 response enhances cDC development and induces the generation of an unique CD11c+B220+ DCs from CLPs. 17 3.2 CpG is necessary for cDC and B220+CD11b+ cells developmental process at early stage. 17 3.3 To characterize the DC subsets from FL- or FL plus CpG-derived cells 18 3.4 CpG-stimulated development of DPDCs is IL-15-independent. 19 3.5 The effects of CpG on DC development from CLPs are IFN-I independent. 19 3.6 The effects of CpG on DC development are dependent on TLR9 signaling 20 3.7 CpG-triggerd cDC and DPDC developmental is STAT3-dependent. 21 3.8 CpG regulate DC development from CLP in vivo. 22 Chapter IV Discussion 24 4.1 HSPCs respond to TLR ligand and develop into cDC 25 4.2 The natures of TLR-induced cDC is dissimilar from FL-derived cDC 26 4.3 Impaired DCs development in Stat1-/- mice with upon FL stimulation 27 4.4 The detailed mechanisms of STAT3 involved in TLR9-dependent DC differentiation remain to be clarified 28 4.5 The functions of TLR-induced DCs need to be determined 29 Figures 30 References 553767714 bytesapplication/pdf論文使用權限：同意有償授權(權利金給回饋學校)樹突細胞發育發炎反應淋巴共同前驅細胞類鐸受體活化訊號傳導與轉錄子三thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262032/1/ntu-103-R01449011-1.pdf