臺灣博碩士論文加值系統

組織常駐巨噬細胞會依據其居住的微環境不同而具有不同的功能和特徵。在胸腺中，90%的胸腺細胞無法通過篩選過程而走向細胞凋亡。這些凋亡的胸腺細胞需要及時被清除，以避免組織壞死和自體發炎反應的產生，而胸腺巨噬細胞則是負責清除胸腺中凋亡細胞的專業吞噬細胞。我們推測胸腺巨噬細胞在胸腺微環境中可能具有獨特的代謝程序來滿足其特定的需求，例如在缺氧的微環境下，胸腺巨噬細胞需要責處理大量的凋亡胸腺細胞的吞噬和降解。通過離體胸腺巨噬細胞轉錄體學分析，我們發現了一基因Solute Carrier Family 1 Member 3 （Slc1a3）其編碼出麩胺酸運輸蛋白是一種在胸腺巨噬細胞中高度表達的代謝物運輸蛋白。
在這項研究中，我們證實了與腹腔巨噬細胞和骨髓衍生巨噬細胞（BMDM）相比，胸腺巨噬細胞中有較高的Slc1a3 mRNA表現量，且在胸腺中，Slc1a3蛋白只在胸腺巨噬細胞中特異性表達。而我們在胸腺間質液中檢測到較高含量的麩胺酸，證實了胸腺微環境中的受質和胸腺巨噬細胞胞的轉運蛋白是相互對應的，這個結果和胸腺巨噬細胞的表現圖譜是一致的。
為了找出哪些因子調節了胸腺巨噬細胞中Slc1a3的表現，我們進一步剖析了胸腺環境線索中的不同成分。我們發現不論是只有麩胺酸或是介白素4（IL-4）的存在都不足以誘導BMDM中Slc1a3的轉錄，而大量的凋亡胸腺細胞的存在會向上調控BMDM中Slc1a3 mRNA的表現。由於麩胺酸是產生穀胱甘肽的關鍵成分，而穀胱甘肽是細胞內主要抵禦氧化壓力的抗氧化劑，因此我們利用 Slc1a3 抑製劑 TFB-TBOA 處理離體胸腺切片，測試 Slc1a3 是否參與胸腺巨噬細胞中氧化壓力恆定的維持。儘管我們的實驗結果顯示胸腺巨噬細胞在此實驗系統中的存活度較差，但我們也確實觀察到了 TFB-TBOA 處理的胸腺切片中有凋亡胸腺細胞的累積，這暗示了Slc1a3在維持清除凋亡細胞上具有潛在的作用。
總之，我們的研究證實了胸腺微環境和胸腺巨噬細胞中獨特的麩胺酸-麩胺酸轉運蛋白配對，我們也發現吞噬凋亡胸腺細胞的需求可能是驅使 Slc1a3 表現的原因之一。此外，我們的實驗結果也意味著Slc1a3可能在支持胸腺巨噬細胞功能上的需求扮演重要的角色。

Tissue-resident macrophages have distinct functions and features according to their residing microenvironment. In the thymus, 90% of the thymocytes fail the selection process and undergo apoptosis. These apoptotic thymocytes need to be removed in a timely fashion to prevent necrosis and autoinflammation. Thymic resident macrophages are the professional phagocytes responsible for the clearance of these apoptotic cells in the thymus. We reasoned that thymic resident macrophages likely have a unique metabolic program to meet the particular demands in the thymus microenvironment, such as the heavy load of phagocytosing and degradation of apoptotic thymocytes under the hypoxia microenvironment. By ex vivo thymic macrophage RNAseq analysis, we identified the gene Solute Carrier Family 1 Member 3 (Slc1a3), which encodes glutamate transporter, is a highly expressed metabolite transporter in the thymic macrophages.
In this study, we confirmed Slc1a3 mRNA was highly expressed in thymic macrophages compared with peritoneal cavity and bone marrow-derived macrophages. In the thymus, the Slc1a3 protein is specifically expressed in thymic macrophages. In the agreement of the expression profile, we detected higher glutamate contents in the thymic interstitial fluid, confirming the substrate and the transporter pair in the thymic microenvironment.
We further dissected different components in the thymic environmental cues to find out which factors regulated Slc1a3 expression in thymic macrophages. We found that the presence of neither glutamate nor IL-4 alone was sufficient to induce the transcriptional level of Slc1a3 in BMDMs, while the presence of a large number of apoptotic thymocytes upregulated the expression of Slc1a3 mRNA in BMDMs. As glutamate is the key component to produce glutathione, which is the major intracellular antioxidant to defend against oxidative stress, we tested if Slc1a3 is involved in oxidative stress homeostasis in thymic macrophages, by treating ex vivo thymic slices with TFB-TBOA, a Slc1a3 inhibitor. Although the results showed found that thymic macrophages had poor viability in this experimental system, we did observe the accumulation of apoptotic thymocytes in the TFB-TBOA treated thymic slices, suggesting the potential role of Slc1a3 in supporting apoptotic cell clearance.
In summary, our study identified the unique glutamate-glutamate transporter pair in the thymic microenvironment and thymic macrophages. We found that the efferocytosis demand of apoptotic thymocytes may be one of the drivers for the expression of Slc1a3. Moreover, our data suggest Slc1a3 may have an important role in supporting thymic macrophage for their functional demands.

誌謝 ii
中文摘要 iv
Abstract vi
Table of Content viii
List of Tables x
List of Figures xi
List of the Appendix Figures xii
1. Introduction 1
1.1 Thymic resident macrophages maintain thymus homeostasis 1
1.2 The relationship between efferocytosis and metabolic programming in macrophages 2
1.3 Glutamate metabolism and Glutamate transporters 2
1.4 The glutamate high-affinity transporter Slc1a3 3
1.5 Oxidative stress and Glutathione system 7
1.6 Rationale and Hypothesis 8
2. Material and Methods 9
2.1 Animals 9
2.2 Cell culture 9
2.3 Tissue-resident macrophages isolation 10
2.4 Immunofluorescent staining 11
2.5 Apoptotic thymocytes preparation 12
2.6 Sample preparation for glutamate measurement 13
2.7 Glutamate measurement 15
2.8 Glutamate treatment 16
2.9 Il-4 and apoptotic thymocytes treatment 16
2.10 Thymic slice preparation and TFB-TBOA treatment 16
2.11 TFB-TBOA treatment of thymocytes 17
2.12 Construction of Slc1a3 over-expression plasmids 17
2.13 Polyethyleneimine (PEI) transfection 20
2.14 Lentivirus production 21
2.15 Virus titration 22
2.16 Lentivirus transduction and puromycin selection 22
2.17 MSCV-based retroviral transduction 22
2.18 Cell lysate preparation and protein extraction 23
2.19 Western blot 24
2.20 Flow cytometry and antibodies 25
2.21 RNA extraction and reverse transcription 26
2.22 Quantitative Real-time PCR (qPCR) 27
2.23 Software and statistics analysis 28
3. Results 29
3.1 Thymic macrophages express Slc1a3 mRNA and protein 29
3.2 Glutamate is abundant in the thymic microenvironment 30
3.3 The exogenous glutamate and IL-4 does not induce Slc1a3 transcriptional level but efferocytosis enhances Slc1a3 mRNA expression in BMDMs 31
3.4 It is hard to use an ex vivo system to explore the function of Slc1a3 in thymic macrophages due to its low viability 32
3.5 Slc1a3 inhibition in the thymus impacted the viability of thymocytes via thymic macrophages 34
3.6 Slc1a3 shRNA-3 had better knockdown efficiency of Slc1a3 mRNA in NIH-3T3 cell lines 36
3.7 Generation of Slc1a3 over-expression plasmid 37
3.8 Studying if Slc1a3 is involved in maintaining redox homeostasis in BMDMs through retroviral MSCV-Slc1a3-GFP over-expression system 38
4. Discussion 40
5. Reference 44
6. Figures 49
7. Appendix Figure 88

List of Tables
Table 1. The list of primers used in the construction of SLC1A3 over-expression plasmid 19
Table 2. PCR program used in the construction of SLC1A3 over-expression plasmid 20
Table 3. The list of antibodies used for flow cytometry 26

List of Figures
Figure 1. Thymic macrophages uniquely express high levels of Slc1a3 transcripts and the transporter protein on the plasma membrane. 50
Figure 2. Glutamate is an abundant metabolite found in the thymic microenvironment. 51
Figure 3. The presence of exogenous glutamate cannot regulate Slc1a3 mRNA expression in BMDMs. 53
Figure 4. Efferocytosis of apoptosis thymocytes can regulate Slc1a3 expression in BMDMs, but not IL-4. 54
Figure 5. The viability and oxidative stress analysis of thymic macrophages in short-term TFB-TBOA treated thymic slices. 59
Figure 6. The viability and oxidative stress analysis of thymic macrophages in long-term TFB-TBOA treated thymic slices. 62
Figure 7. The viability of thymocytes in short-term TFB-TBOA treated thymic slices. 65
Figure 8. The cellularity of thymocytes in short-term TFB-TBOA treated thymic slices. 67
Figure 9. The viability of thymocytes in long-term TFB-TBOA treated thymic slices. 69
Figure 10. The cellularity of thymocytes in long-term TFB-TBOA treated thymic slices. 71
Figure 11. The accumulation of apoptotic double-positive thymocytes in long-term TFB-TBOA treated thymic slices. 74
Figure 12. The TFB-TBOA treatment did not directly impact thymocytes survival. 76
Figure 13. The TFB-TBOA treatment did not directly impact the cellularity of thymocytes. 78
Figure 14. The viability in different thymocytes subsets upon TFB-TBOA 81
Figure 15. Identification of Slc1a3 knockdown efficiency using three different shRNA constructs using NIH-3T3 cell line. 82
Figure 16. Validation of Slc1a3 over-expression constructs. 86
Figure 17. The transduction efficiency of MSCV-Slc1a3 over-expressing construct in BMDMs. 87

List of the Appendix Figures
Appendix Figure 1. Glutamate metabolism is highly activated in the thymic microenvironment. 88
Appendix Figure 2. The viability analysis of CD64+ subsets in thymic slices treated with or without DMSO for 12 h and 24 h 89
Appendix Figure 3. The 3 different Slc1a3 shRNA from the RNAi core is ready to use. 91
Appendix Figure 4. The Slc1a3 expression of murine cell lines is obtained from the Geneinvestigator Database. 92
Appendix Figure 5. The plasmid map of pcDNA3.1(+) based SLC1A3 over-expression constructs 94
Appendix Figure 6. The plasmid map of MSCV-mSLC1A3-IRES-GFP SLC1A3 over-expression construct 95
Appendix Figure 7 The Schematic of our findings and our hypothesis 96
Appendix Figure 8 The transcriptional expression levels of glutamate transporters and cysteine/glutamate antiporters in different tissue-resident macrophages through RNA-seq 98

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