- 學位論文
發育過程中的第七型類鐸受體訊號抑制樹突細胞的活化和功能
TLR7 signaling during dendritic cell development suppresses their activation and function
摘要
樹突狀細胞（DC）包括傳統樹突細胞（conventional DCs , cDC）和槳狀樹突細胞（plasmacytoid DCs , pDC），這兩種細胞在先天和適應性免疫中扮演重要角色。我們先前發現類鐸受體（Toll like receptor, TLR）誘導的發炎反應會減少pDC的發育，但會增加cDC的產生。然而，由發炎反應誘導出的DC的功能仍不清楚。在我們的研究中證實在DC發育過程中接受了TLR7信號會削弱分化出的DC的活化和功能，這包括表現在細胞表面的活化標誌物比如CD40，PD-L1，CD86，MHCII等訊號在pDC和cDC中的表現量均顯著降低。DCs也產生較少的發炎細胞素，例如IL-6，IL-12和TNF-α在RNA和蛋白質的量都減少，然而，IL-10（一種抑制發炎細胞素）則略有增加。此外，用DQ-OVA熒光裂解的方式測試抗原分解能力的實驗中，發現這種能力也被抑制，這些結果顯示發炎反應所誘導的DC具有更大的耐受引發能力。此外，DC發育中的TLR7信號傳導也會降低粒線體膜電位和質量。與對照組相比，事先用R848處理的DC中的粒線體的ROS降低了。這些結果顯示，代謝能力變差有可能與DC功能受損有關。為了研究R848如何改變DC活化及功能，我們使用了STAT3，AKT，mTOR和p38的抑制劑，我們發現mTOR抑製劑（Rapamycin）可部分恢復發炎反應所誘導的DCs活化標誌的表現。p38抑制劑（SB203580）則可以通過上調活化標記和促炎細胞素的產生，明顯逆轉了R848所改變的DC的活化。此外，p38抑制劑也逆轉了TLR7所介導的粒線體ROS，粒線體膜電位和質量的降低。因此，在我們的研究中，已經證明在DC發育過程中TLR7訊號通過活化p38去造成所分化出的DCs的功能的嚴重損害，粒線體動力學和ROS。這些結果或許可以解釋慢性發炎造成的免疫缺陷，並提供治療慢性發炎或自體免疫疾病（例如，發炎性腸炎（Inflammatory bowel disease, IBD）及類風濕性關節炎）的治療方法。
並列摘要
Dendritic cells (DCs), including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) play important roles in innate and adaptive immunity. Previously, we found that TLR-induced inflammation impairs the development of pDC but increases cDC generation. However, the functions of inflammation-induced DCs remain unclear. Here we showed that TLR7-signaling during DC development impaired the activation and functions of DCs. CpG-induced upregulation of activation markers, including CD40, PD-L1, CD86, MHCII was significantly lower in both pDC and cDC derived from TLR priming. DCs also produce less inflammatory cytokines, such as IL-6, IL-12 and TNF-α, both at RNA and protein levels. However, IL-10, a suppressive cytokine, on the contrary, was slightly increased. Moreover, the antigen processing ability was revealed by DQ-OVA cleavage, which was also suppressed, suggesting that inflammation-induced DCs are more tolerogenic. Moreover, TLR7 signaling in DC development also impaired mitochondrial membrane potential and mass. As a result, mitochondrial ROS in R848-pretreated DC was decreased as compared to control. These results indicate that metabolic insufficiency may associate with impaired function of DC. To examine the signaling pathways altered DC function by R848, we use pharmacological inhibitor，including inhibitor to STAT3, AKT, mTOR and p38. The activation markers of DCs induced by inflammation were partially restored by mTOR inhibitor. However, SB203580, a p38 inhibitor significantly reversed R848-altered activation of DC by CpG, inducing upregulation activation markers and production of cytokine. In addition, TLR7-mediated reduction of mitochondrial ROS, mitochondrial membrane potential and mass were blocked by p38 inhibitors. Together, we have demonstrated that TLR7 signaling during DC development severely impairs their functions, and mitochondrial dynamics and ROS. One of the signaling pathways that induing the phenotype is p38. These results may shed light into chronic inflammation-triggered immune-deficiency and provide a therapeutic approach for the treatment of diseases, including IBD and rheumatoid arthritis.