- 學位論文
發炎反應以及感染對樹突細胞從共同淋巴前驅細胞發育之調控
Inflammation- and Infection-mediated Modulation of Dendritic Cell Development from Common Lymphoid Progenitors
摘要
樹突狀細胞(Dendritic cell, DC)對先天性及後天性免疫的調控反應都很重要。樹突狀細胞主要分為漿狀樹突細胞(plasmacytoid DC, pDC)及傳統樹突細胞 (conventional DC, cDC)，兩者的生命週期都很短。因此，它們需要持續地從骨髓系統及淋巴系統的造血前驅細胞更新補充。我們先前的研究指出在穩定狀態下, Flt3配體(Flt3 ligand, FL)主要是促進共同淋巴前驅細胞(common lymphoid progenitors, CLP)分化成pDC而不是cDC。CLP會表現不同的類鐸受體 (Toll-like receptor, TLR)包含TLR7及TLR9，因此在發炎及感染的情況下, 從CLP分化成DC的調控過程仍然不是十分清楚。在這裡我們的實驗證據顯示，經過TLR7和TLR9配體R848及CpG分別刺激後，從CLP生成的pDC會大幅減少，相反的cDC的產生則會增加。除此之外，經過TLR刺激，cDC的亞群會從偏向生成CD24+cDC轉變成CD24-cDC。這樣的現象主要是經由TLR訊息傳導途徑的直接調控，另外有一部分則是間接受到TLR下游分子，像是第一型干擾素(type I interferon, IFN) 的影響。有趣的是，TLR及第一型干擾素下游訊息傳遞蛋白分子STAT1是促進cDC生成不可缺少的因子。然而第一型干擾素也有參與TLR刺激造成的cDC的增加。流行性感冒病毒在體外感染CLP後也和類鐸受體的刺激有類似的反應；也會造成cDC生成的增加及pDC生成的減少。流感病毒可以被不同的模式辨識受體 (pattern recognition receptor, PRR)辨認包含TLR7、RIG-I及NLRP3，但是沒有任何一個單獨的受體會造成這樣的改變。此外，不管是STAT1還是第一型干擾素也都不會影響流感病毒對DC分化的作用，因此調控TLR及流感病毒感染的分子機制差異性非常大。為了進一步了解DC發育的分子訊息調控的研究，我們使用造血前驅及幹細胞的細胞株 (immortalized hematopoietic stem and progenitor cell line, iHSPC)。iHSPC中的AKT及STAT3的活化都會因為FL加R848的刺激比只加FL的組別增加。此外，Stat3及Akt3 (AKT的一種異構物)在iHSPC中基因靜默表現(knockdown)後，cDC生成提高但是pDC生成卻減少了，表示STAT3及AKT3會正向調控pDC的發育。總結來說，我們發現發炎以及感染會改變CLP發育成pDC及cDC的平衡，從原本偏向生成pDC變成偏向生成cDC。這結果也暗示DC的發育是非常多變的，而且會受到環境因子的影響。
並列摘要
Dendritic cells (DCs) are critical for the innate and the adaptive immunity. DCs, including plasmacytoid DCs (pDCs) and conventional DC (cDCs) are short-lived. Therefore, they are constantly replenished from progenitor cells of myeloid and lymphoid lineages. We have previously shown that Flt3 ligand (FL) predominantly promotes the development of pDC, and not cDC, from common lymphoid progenitors (CLPs) during steady-state. While CLPs express various TLRs, including TLR7 and TLR9, DC development from CLPs during inflammation and infection is still incompletely understood. Here, we showed that, R848 and CpG, a TLR7 and TLR9 ligand, respectively, promoted cDC production while decreased pDC generation from CLPs. Moreover, development of cDC subsets was also shifted from CD24+cDC to CD24-cDC following the TLR stimulation. The effect mainly depended on direct TLR signaling pathway and, in part, on indirect downstream molecules of TLR, such as type I interferon (IFN). Interestingly, STAT1, a direct signals of TLR and type I IFN, was indispensable for TLR-mediated enhancement of cDC development. However, type I IFN contributed partially to the TLR-mediated enhancement of cDC production. Infection of influenza virus (IAV) in vitro also resulted in increased production of cDCs but reduced generation of pDCs. While several PRRs, including TLR7, RIG-I and NLRP3, have been shown to sense pathogen-associated molecular patterns of IAV, none of the individual molecule was involved. Moreover, neither STAT1 nor type I IFN was dispensable for the phenotype, suggesting that the molecular mechanisms for TLR- and IAV infection-mediated are distinct. To facilitate the study of the signaling molecules dictate the programming of DC development, an immortalized hematopoietic stem and progenitor cell line (iHSPC) was used. Activation of AKT and STAT3 was enhanced upon FL plus R848 stimulation as compared to FL alone in iHSPC. Knockdown of Stat3 or Akt3, a isoform of AKT, in iHSPC enhanced cDC development but inhibited pDC development, suggesting that STAT3 and AKT3 may positively regulate pDC development. In conclusion, we demonstrate that inflammation and infection reprogram DC homeostasis from CLPs by shifting pro pDC to pro cDC development. Therefore, DC development is highly dynamic, depending on environmental cues.