臺灣博碩士論文加值系統

Syk脾酪胺酸激酶為一非受體型酪胺酸激酶，主要高表現於造血細胞 (hematopoietic cells)中，在免疫系統則扮演調控發炎反應的角色，舉凡T細胞、B細胞受體以及某些抗體恆定區接受器(Fc receptor)的訊息傳遞路徑，皆須靠Syk傳遞激活訊號以誘使發炎反應之產生。樹突細胞 (dendritic cell, DC)為免疫系統中重要的抗原呈現細胞並能活化後天性免疫，亦有一群調節性樹突細胞(regulatory dendritic cell, regDC)負責維持免疫耐受性。腸道中即存在特殊之CD103+ regDC，此regDC主要分布於腸道固有層 (lamina propria, LP)以及腸繫膜淋巴結 (mesenteric lymph node, MLN)，其高表現的第二型視黃酸合成酶 (ALDH1a2)能將維生素A代謝物轉化成視黃酸 (retinoic acid, RA)，可促使T細胞分化為具有腸道歸巢受器 (gut-homing receptor) α4β7及CCR9的Foxp3+調節性T 細胞 (regulatory T cell, Treg)。迄今為止，Syk在DC的研究皆聚焦在免疫活化上，對於其在regDC的角色則尚未被探討，因此我的研究主要探討Syk對於regDC之功能與發育上所扮演的調控角色。研究結果顯示，在3% DSS小鼠腸炎模式中，CD11cΔSyk老鼠相較於Sykfl/fl老鼠有更顯著的發炎情況。CD11cΔSyk老鼠MLN中的CD103+ regDC比例及數量相對Sykfl/fl 老鼠也有明顯下降，其ALDH1a2酵素活性亦較低。在體外培養RA誘導的CD103+ regDCs中，我們發現CD11cΔSyk CD103+ regDCs表達較低之ALDH1a2活性、Il-10、Tgf-b1及Ccr9。給予RA誘導的CD103+ regDCs熱凝膠多糖(curdlan)刺激後，CD11cΔSyk CD103+ regDCs相較於Sykfl/fl CD103+ regDCs亦無增強ALDH1a2活性、Il-10、Tgf-b1及Arg-1。這些結果顯示Syk在DC中對於維持腸道恆定相當重要，同時在RA誘導的CD103+ regDC之發育及功能上扮演重要的調控角色。

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase which mainly expresses in hematopoietic cells. Syk plays a crucial role in regulating immune responses such as triggering downstream signal pathway of T cell receptors (TCRs), B cell receptors (BCRs) and some Fc receptors (FcRs). Dendritic cells (DCs) are professional antigen-presenting cells (APCs) and can trigger adaptive immune responses. There is also a distinct subset of DCs, called regulatory dendritic cells (regDCs), responsible for immune tolerance. In gut lamina propria (LP) and mesenteric lymph nodes (MLN), CD103+ regDCs highly express retinal aldehyde dehydrogenase family 1, subfamily A2 (ALDH1a2), which transforms the metabolite of vitamin A into retinoic acid (RA). In the presence of RA, CD103+ regDCs can drive the differentiation of Foxp3+ regulatory T cells (Tregs) and induce their expression of gut homing receptors CCR9 and α4β7. Up to date, the majority of Syk studies focus on inflammatory DCs. However, there is no report to describe the role of Syk in regDCs. Thus, my thesis is to study the role of Syk in the development and function of regDCs. The results showed that in 3% DSS colitis mouse model, inflammatory phenotype was more severe in CD11cΔSyk mice than that in Sykfl/fl mice. In addition, the relative frequency and absolute number of CD103+ regDC in MLN was significantly decrease in CD11cΔSyk mice, and the activity of ALDH1a2 among these CD103+ regDCs was also much lower. Furthermore, we found that RA-induced CD103+ regDCs from CD11cΔSyk mice in vitro expressed low level of ALDH1a2 activity, Il-10, Tgf-b, and Ccr9 compared to those from Sykfl/fl mice. After treating with curdlan (dectin-1 ligand), Syk-deficient RA-induced CD103+ regDCs also failed to enhance ALDH1a2 activity, gene expression of Il-10, Tgf-b, and Arg-1. These data suggested that Syk in DCs is important for gut homeostasis and Syk plays a critical role in regulating development and function of RA-induced CD103+ regDCs.

誌謝 I
中文摘要 II
Abstract III
List of figures XI
List of tables XV
1. Introduction 1
1.1 Inflammatory Bowel Disease (IBD) 2
1.2 Dendritic cell (DC) 3
1.3 DCs in gut immune system 4
1.4 Spleen Tyrosine Kinase (Syk) 5
1.5 Syk in DCs 6
2. Rationale and Specific aims 8
2.1 Rationale 9
2.2 Specific aims 9
3. Materials and Methods 10
3.1 Mice 11
3.2 Bone marrow-derived DCs culture 11
3.3 Bone marrow-derived regDCs culture 12
3.4 Dextran Sulfate Sodium (DSS)-induced colitis mouse model 12
3.5 Isolation of intestinal lamina propria lymphocytes 13
3.6 Isolation of CD4+ splenic T cells 14
3.7 Co-culture of T cells and DCs 14
3.8 Western Blot 15
3.9 Aldehyde dehydrogenase activity assay 15
3.10 Flow Cytometry 16
3.11 Quantitative real-time PCR (qPCR) 16
3.12 Statistical analysis 17
4. Results 18
4.1 The effect of Syk on gut regDCs 19
4.1.1 Loss of Syk in DCs resulted in increasing the susceptibility of DSS-induced colitis. 19
4.1.2 There is no difference in Il-6, Il-17a, Tnf-a and Il-10 gene expression in colon between control and colitis mice. 19
4.2 The role of Syk in the development and function of gut regDCs. 20
4.2.1 DC-specific Syk deficiency had no effect on the ratio and ALDH1a2 activity of CD11c+ DCs in peripheral LNs and spleen. 20
4.2.2 DC-specific Syk deficiency had no effect on the ratio and number of CD103+ ALDH1a2+ CD11c+ DCs in peripheral LNs and spleen. 21
4.2.3 DC-specific Syk deficiency had no effect on the ratio and number of CD103+ CD11c+ DCs and CD103- CD11c+ DCs in peripheral LNs and spleen. 21
4.2.4 ALDH1a2 activity of CD11c+ DCs was reduced in MLN from CD11cΔSyk mice. 22
4.2.5 The population of CD103+ALDH1a2+ DCs was decreased in MLN from CD11cΔSyk mice. 23
4.2.6 Both population and ALDH1a2 activity of CD103+CD11b+ CD11c+ and CD103+ CD11b- CD11c+ DCs were diminished in MLN from CD11cΔSyk mice. 23
4.2.7 The population but not ALDH1a2 activity of CD103+ CD11c+ DCs was reduced in intestinal LP from CD11cΔSyk mice. 24
4.2.8 The Aldh1a2 gene expression was downregulated in small intestine (SI) and colon from CD11cΔSyk mice. 25
4.2.9 DC-specific Syk deficiency had no effect on the population of CD4+ Foxp3+ Treg cells in MLN. 25
4.3 Establishment of regDC culture system in vitro. 26
4.3.1 Establishment of RA+IL-4-induced CD11c+ regDCs in vitro. 26
4.3.2 Syk-deficient RA+IL-4-induced CD11c+ regDCs augmented regulation-associated genes except for Aldh1a2. 27
4.3.3 RA+IL-4-induced CD11c+ regDCs responded to LPS stimulation. 28
4.3.4 Syk-deficient RA+IL-4-induced CD11c+ regDCs promoted CD4+ Foxp3+ Treg differentiation robustly in vitro. 29
4.3.5 Establishment of low-dose IL-4-induced CD11c+ regDCs in vitro. 29
4.3.6 Establishment of TGF-β-induced CD11c+ regDCs in vitro. 30
4.3.7 Syk-deficient TGF-β-induced CD11c+ regDCs augmented the expression of TIM-3. 31
4.3.8 Establishment of RA-induced CD11c+ regDCs in vitro. 32
4.4 The role of Syk in the development and function of RA-induced CD11c+ regDCs. 33
4.4.1 RA-induced CD11c+ regDCs was CD11b+ and highly expressed dectin-1. 33
4.4.2 RA-induced CD11c+ regDCs expressed low cDC2-related gene but high cDC1-related gene compared to GM-CSF-cultured CD11c+ DCs. 34
4.4.3 Syk-deficient RA-induced CD11c+ regDCs expressed reduced level of regulation-associated genes such as Il10 and Tgf-b1. 34
4.4.4 Syk-deficient RA-induced CD11c+ regDCs expressed reduced level of gut-homing receptor gene Ccr9. 35
4.4.5 RA-induced CD11c+ regDCs did not respond to LPS stimulation. 36
4.4.6 Zymosan and Pam3CSK4 failed to enhance ALDH1a2 activity in RA-induced CD11c+ regDCs. 37
4.4.7 Curdlan failed to enhance ALDH1a2 activity in RA-induced Syk-deficient CD11c+ regDCs. 38
4.4.8 Syk-deficient RA-induced CD11c+ regDCs expressed reduced level of regulation-associated genes after treatment with curdlan. 39
4.4.9 CD103 but not CCR7 expression was downregulated by curdlan stimulation in RA-induced CD11c+ regDCs. 39
4.4.10 β-catenin was not detectable in RA-induced CD11c+ regDCs even after treating with curdlan. 40
5. Discussion 42
6. Figures 49
7. Tables 81
8. Reference 83

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