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研究生：	謝雨彤 Hsieh, Yu-Tung
論文名稱：	微小核醣核酸146a調控嗜中性球胞外誘捕功能於自體免疫性肺出血 Neutrophil extracellular traps formation regulated by microRNA 146a in autoimmune-mediated diffuse alveolar hemorrhage
指導教授：	王崇任 Wang, Chrong-Reen
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	40
中文關鍵詞：	嗜中性球胞外誘捕、微小核糖核酸146a 、瀰漫性肺出血、全身性紅斑狼瘡
外文關鍵詞：	Neutrophil extracellular traps, MiR-146a, Diffuse alveolar hemorrhage, Systemic lupus erythematosus
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瀰漫性肺出血是一種臨床上高致死性的肺部急症，而因自體免疫病態機轉所產生之肺臟出血表現，最常見於全身性紅斑狼瘡患者。它是一種慢性自體免疫疾病，以喪失免疫反應耐受性而著名。此類病人由於體內清除細胞凋亡及細胞胞外補網產生之細胞殘骸的功能障礙，導致體內累積過多自體抗原。由於病人的異常免疫反應耐受性，使體內對於自體抗原產生各種自體抗體，自體抗原與自體抗體形成了免疫複合體，沉積在許多身體組織及器官，進一步吸引免疫細胞前來攻擊而引發器官的損害。當免疫複合體沉積在肺部微血管，會導致肺部產生瀰漫性肺出血現象。先前研究已闡述細胞胞外補網在紅斑性狼瘡病人的病程發展中扮演重要角色。也有其他研究指出在人類及小鼠細胞中，微小核醣核酸146a的低表現量可增強細胞胞外部網的形成。在此碩士論文中，吾人首先發現紅斑狼瘡表現瀰漫性肺出血病人的單核球、多核球細胞及肺臟組織中，微小核糖核酸146a的表現量皆較對照組低。隨後發現在過量表現微小核糖核酸146a之分化的HL-60人類細胞中，可降低細胞胞外補網的形成。以腹腔注射pristane產生的狼瘡小鼠肺出血模型中，發現不論是肺組織或是以thioglycolate誘導分離出的嗜中性球，微小核醣核酸146a的表現量皆低於生理食鹽水注射的對照組。最後利用氣管灌注的方式將攜帶微小核醣核酸146a的慢病毒載體送入小鼠的肺部來過量表現此微小核糖核酸，結果顯示完全肺出血的頻率減少且血液中血紅素增加，此外細胞凋亡及細胞胞外補網的現象也降低。這些發現顯示在紅斑性狼瘡之病程發展中，不論是試管內、活體外或活體內的研究，微小核糖核酸146a在抑制細胞胞外補網的形成可能扮演重要的角色，具有成為臨床治療標靶之潛在性。

Diffuse alveolar hemorrhage (DAH) is a respiratory emergency characterized by acute-onset diffuse alveolar bleeding, leading to acute respiratory failure with a high mortality. Most patients of autoimmune-mediated DAH are caused by pulmonary capillaritis associated with systemic lupus erythematosus (SLE). It is a chronic autoimmune disease characterized by overproduction of autoantibodies due to a loss of immune tolerance with the activation of innate and adaptive immunity. In SLE patients, ineffective clearance of apoptotic cell debris and neutrophil extracellular traps (NETs) results in the accumulation of nuclear autoantigens, reacting with autoantibodies to form immune complexes (ICs) depositing in various organs, causing DAH and lupus nephritis. Neutrophil overactivation with NETs formation and impaired removal, can contribute to the disease development and progression by providing the autoantigen source and increasing pro-inflammatory responses in SLE. Nevertheless, the pathogenic role of NETs remains to be elucidated in autoimmune-mediated DAH. Previous studies have shown that deficient expression of miR-146a can enhance the NETs formation in both human and mouse cells. In this study, we found decreased miR-146a levels in peripheral blood mononuclear cells (PBMCs), neutrophils (PBNs) and lung tissues from SLE-associated DAH patients. Furthermore, miR-146a-overexpressed differentiated HL-60 cells had reduced formation of NETosis. The pristane-induced C57BL/6 mouse model could exhibit clinicopathological features similar to the SLE-associated DAH manifestation. By using this model, we demonstrated decreased miR-146a levels in lung tissues and neutrophils purified from thioglycolate-induced peritoneal exudates. Intra-pulmonary overexpressed miR-146a levels through intratracheal administration of LV-miR-146a vectors could reduce hemorrhagic frequencies and improved anemia status in the pristane-induced DAH mice. In conclusion, our in vitro, ex vivo and in vivo results implicate that down-regulated miR-146a expression plays a pathogenic role in the autoimmune-mediated DAH by increasing the NETs formation, and a therapeutic potential of intra-pulmonary miR-146a delivery to treat such patients.

Contents

中文摘要	I
Abstract	II
Acknowledgements	III
Abbreviation	IV
Introduction	1
Autoimmune-mediated diffuse alveolar hemorrhage (DAH) and systemic lupus erythematosus (SLE)	1
Neutrophil extracellular traps (NETs)	1
MicroRNA	2
MicroRNAs and NETs	2
MiR-146a in SLE	3
Pristane-induced DAH mouse model	4
Specific aim	5
Materials	6
Reagents	6
Plasmid	6
Cell line	6
Primers	6
TaqMan	7
Primary antibodies	7
Secondary antibodies	8
Immunofluorescent	8
Enzyme-linked immunosorbent assay (ELISA)	8
Flow cytometry	8
RNA isolation kit	8
Methods	9
SLE patients and age/sex-matched healthy control subjects	9
Isolation of human and murine cells	9
In vitro NETs experiments	10
Hydrophilic pristane preparation	11
Overexpression of miR-146a in HL-60 cell line	11
Doxorubicin (Dox)-induced cell apoptosis in MLE-12 cells	11
Overexpression of miR-146a in MLE-12 cells	12
Generation of Lenti-virus	12
Real-time quantitative reverse transcription-PCR (RT-qPCR)	13
Pristane-Induced DAH mice model	13
Hemogram and proteinuria determination in mice	13
Immunoblotting analysis	14
Lv-miR-146a IT administration and therapeutic evaluation	14
H&E, TUNEL and IF histopathological staining	14
Statistical Analyses	15
Results	16
Down-regulated miR-146a levels in PBMCs and PBNs from patients with SLE-associated DAH	16
Down-regulated miR-146a with NETs and apoptosis formation in the lung tissues of SLE patients with DAH	17
Increased NETs formation in SLE-associated DAH patients in vitro	17
Reduced NETs formation in miR-146a-overexpressed dHL-60 cells in vitro	18
Down-regulated miR-146a with in situ NETs formation in pristane-induced DAH mice model	19
Down-regulated miR-146a in pristane-induced apoptosis in murine alveolar epithelial cells in vitro	20
Reduced cell apoptosis in miR-146a-overexpressed murine alveolar epithelial cells	21
Pristane-induced NETs generation in vitro	21
Intra-pulmonary delivery of Lv-miR-146a improve DAH in pristane-induced mouse model through reducing NETs and apoptosis formation	22
Discussion	23
Conclusion	25
Figure legends	26
Figure 1. Down-regulated miR-146a levels in PBMCs and PBNs from patients with SLE-associated DAH.	26
Figure 2. Down-regulated miR-146a with NETs and apoptosis formation in the lung tissues of patients with SLE-associated DAH.	27
Figure 3. Increased NETs formation in PBNs from SLE-associated DAH patients in vitro.	28
Figure 4. Reduced NETs formation in miR-146a-overexpressed dHL-60 cells in vitro.	29
Figure 5. Down-regulated miR-146a with in situ NETs formation in pristane-induced DAH mice model.	30
Figure 6. Down-regulated miR-146a in pristane-induced apoptosis in murine alveolar epithelial cells in vitro.	31
Figure 7. Reduced cell apoptosis in miR-146a-overexpressed murine alveolar epithelial cells.	32
Figure 8. Pristane-induced NETs generation in vitro.	33
Figure 9. A working model of HMGB1 released from apoptotic alveolar cells to induce NETs formation in TLR-4 expressed neutrophils.	34
Down-regulated miR-146a expression in alveolar cells can induce cell apoptosis with the release of HMGB1, functioning as DAMP to engage TLR4 in neutrophils, followed by PKC activation to mobilize intracellular Ca2+ and promote PAD4 activation, leading to NETs formation. Furthermore, by down-regulating miR-146a expression, pristane can induce NETs formation in neutrophils, and up-regulate the expression of IL-8, a NETosis inducer, in alveolar cells.	34
Figure 10. Intra-pulmonary delivery of Lv-miR-146a improve DAH in pristane-induced mice model through reducing NETs and apoptosis formation.	35
References	37

Figure Contents

Figure 1. Down-regulated miR-146a levels in PBMCs and PBNs from patients with SLE-associated DAH.	26
Figure 2. Down-regulated miR-146a with NETs formation in the lung tissues of patients with SLE-associated DAH.	27
Figure 3. Increased NETs formation in PBNs from SLE-associated DAH patients in vitro.	28
Figure 4. Reduced NETs formation in miR-146a-overexpressed dHL-60 cells in vitro.	29
Figure 5. Down-regulated miR-146a with in situ NETs formation in pristane-induced DAH mice model.	30
Figure 6. Down-regulated miR-146a in pristane-induced apoptosis in murine alveolar epithelial cells in vitro.	31
Figure 7. Reduced cell apoptosis in miR-146a-overexpressed murine alveolar epithelial cells.	32
Figure 8. Pristane-induced NETs generation in vitro.	33
Figure 9. Intra-pulmonary delivery of Lv-miR-146a improve DAH in pristane-induced mice model through reducing NETs and apoptosis formation.	35
                                    

Águila, S., de Los Reyes-García, A. M., Fernández-Pérez, M. P., Reguilón-Gallego, L., Zapata-Martínez, L., Ruiz-Lorente, I., . . . Martínez, C. (2021). MicroRNAs as New Regulators of Neutrophil Extracellular Trap Formation. Int J Mol Sci, 22(4). doi:10.3390/ijms22042116
Angeletti, A., Volpi, S., Bruschi, M., Lugani, F., Vaglio, A., Prunotto, M., . . . Ghiggeri, G. M. (2021). Neutrophil Extracellular Traps-DNase Balance and Autoimmunity. Cells, 10(10). doi:10.3390/cells10102667
Arroyo, A. B., Águila, S., Fernández-Pérez, M. P., Reyes-García, A. M. L., Reguilón-Gallego, L., Zapata-Martínez, L., . . . González-Conejero, R. (2021). miR-146a in Cardiovascular Diseases and Sepsis: An Additional Burden in the Inflammatory Balance? Thromb Haemost, 121(9), 1138-1150. doi:10.1055/a-1342-3648
Arroyo, A. B., de Los Reyes-García, A. M., Rivera-Caravaca, J. M., Valledor, P., García-Barberá, N., Roldán, V., . . . González-Conejero, R. (2018). MiR-146a Regulates Neutrophil Extracellular Trap Formation That Predicts Adverse Cardiovascular Events in Patients With Atrial Fibrillation. Arterioscler Thromb Vasc Biol, 38(4), 892-902. doi:10.1161/atvbaha.117.310597
Ban, T., Sato, G. R., & Tamura, T. (2018). Regulation and role of the transcription factor IRF5 in innate immune responses and systemic lupus erythematosus. Int Immunol, 30(11), 529-536. doi:10.1093/intimm/dxy032
Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2), 281-297. doi:10.1016/s0092-8674(04)00045-5
Bickel, M. (1993). The role of interleukin-8 in inflammation and mechanisms of regulation. J Periodontol, 64(5 Suppl), 456-460.
Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S., . . . Zychlinsky, A. (2004). Neutrophil extracellular traps kill bacteria. Science, 303(5663), 1532-1535. doi:10.1126/science.1092385
Chan, R. W., Tam, L. S., Li, E. K., Lai, F. M., Chow, K. M., Lai, K. B., . . . Szeto, C. C. (2003). Inflammatory cytokine gene expression in the urinary sediment of patients with lupus nephritis. Arthritis Rheum, 48(5), 1326-1331. doi:10.1002/art.11062
Chen, S. Y., Shiau, A. L., Li, Y. T., Lin, Y. S., Lee, C. H., Wu, C. L., & Wang, C. R. (2012). Suppression of collagen-induced arthritis by intra-articular lentiviral vector-mediated delivery of Toll-like receptor 7 short hairpin RNA gene. Gene Ther, 19(7), 752-760. doi:10.1038/gt.2011.173
Chen, Y. C., Chou, Y. C., Hsieh, Y. T., Kuo, P. Y., Yang, M. L., Chong, H. E., . . . Wang, C. R. (2021). Targeting Intra-Pulmonary P53-Dependent Long Non-Coding RNA Expression as a Therapeutic Intervention for Systemic Lupus Erythematosus-Associated Diffuse Alveolar Hemorrhage. Int J Mol Sci, 22(13). doi:10.3390/ijms22136948
Crow, M. K. (2014). Type I interferon in the pathogenesis of lupus. J Immunol, 192(12), 5459-5468. doi:10.4049/jimmunol.1002795
Fan, Y., Ji, Y., Wang, X., Hu, J., Zhang, Q., Xu, J., . . . Wang, A. (2020). Relationship of miRNA-146a to systemic lupus erythematosus: A PRISMA-compliant meta-analysis. Medicine (Baltimore), 99(40), e22444. doi:10.1097/md.0000000000022444
Fousert, E., Toes, R., & Desai, J. (2020). Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses. Cells, 9(4). doi:10.3390/cells9040915
Hakkim, A., Fuchs, T. A., Martinez, N. E., Hess, S., Prinz, H., Zychlinsky, A., & Waldmann, H. (2011). Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nat Chem Biol, 7(2), 75-77. doi:10.1038/nchembio.496
Harris, H. E., Andersson, U., & Pisetsky, D. S. (2012). HMGB1: a multifunctional alarmin driving autoimmune and inflammatory disease. Nat Rev Rheumatol, 8(4), 195-202. doi:10.1038/nrrheum.2011.222
Hawez, A., Al-Haidari, A., Madhi, R., Rahman, M., & Thorlacius, H. (2019). MiR-155 Regulates PAD4-Dependent Formation of Neutrophil Extracellular Traps. Front Immunol, 10, 2462. doi:10.3389/fimmu.2019.02462
Herman, S., Kny, A., Schorn, C., Pfatschbacher, J., Niederreiter, B., Herrmann, M., . . . Hoffmann, M. H. (2012). Cell death and cytokine production induced by autoimmunogenic hydrocarbon oils. Autoimmunity, 45(8), 602-611. doi:10.3109/08916934.2012.719948
Hochberg, M. C. (1997). Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum, 40(9), 1725. doi:10.1002/art.1780400928
Honarpisheh, M., Köhler, P., von Rauchhaupt, E., & Lech, M. (2018). The Involvement of MicroRNAs in Modulation of Innate and Adaptive Immunity in Systemic Lupus Erythematosus and Lupus Nephritis. J Immunol Res, 2018, 4126106. doi:10.1155/2018/4126106
Huarte, M., Guttman, M., Feldser, D., Garber, M., Koziol, M. J., Kenzelmann-Broz, D., . . . Rinn, J. L. (2010). A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell, 142(3), 409-419. doi:10.1016/j.cell.2010.06.040
Jarrot, P. A., Tellier, E., Plantureux, L., Crescence, L., Robert, S., Chareyre, C., . . . Kaplanski, G. (2019). Neutrophil extracellular traps are associated with the pathogenesis of diffuse alveolar hemorrhage in murine lupus. J Autoimmun, 100, 120-130. doi:10.1016/j.jaut.2019.03.009
Lara, A. R., & Schwarz, M. I. (2010). Diffuse alveolar hemorrhage. Chest, 137(5), 1164-1171. doi:10.1378/chest.08-2084
Liao, T. L., Chen, Y. M., Tang, K. T., Chen, P. K., Liu, H. J., & Chen, D. Y. (2021). MicroRNA-223 inhibits neutrophil extracellular traps formation through regulating calcium influx and small extracellular vesicles transmission. Sci Rep, 11(1), 15676. doi:10.1038/s41598-021-95028-0
Liu, M. F., & Wang, C. R. (2014). Increased Th17 cells in flow cytometer-sorted CD45RO-positive memory CD4 T cells from patients with systemic lupus erythematosus. Lupus Sci Med, 1(1), e000062. doi:10.1136/lupus-2014-000062
Mohan, C., & Putterman, C. (2015). Genetics and pathogenesis of systemic lupus erythematosus and lupus nephritis. Nat Rev Nephrol, 11(6), 329-341. doi:10.1038/nrneph.2015.33
Mortazavi-Jahromi, S. S., Aslani, M., & Mirshafiey, A. (2020). A comprehensive review on miR-146a molecular mechanisms in a wide spectrum of immune and non-immune inflammatory diseases. Immunol Lett, 227, 8-27. doi:10.1016/j.imlet.2020.07.008
Nahid, M. A., Benso, L. M., Shin, J. D., Mehmet, H., Hicks, A., & Ramadas, R. A. (2016). TLR4, TLR7/8 agonist-induced miR-146a promotes macrophage tolerance to MyD88-dependent TLR agonists. J Leukoc Biol, 100(2), 339-349. doi:10.1189/jlb.2A0515-197R
Peng, J. S., Chen, S. Y., Wu, C. L., Chong, H. E., Ding, Y. C., Shiau, A. L., & Wang, C. R. (2016). Amelioration of Experimental Autoimmune Arthritis Through Targeting of Synovial Fibroblasts by Intraarticular Delivery of MicroRNAs 140-3p and 140-5p. Arthritis Rheumatol, 68(2), 370-381. doi:10.1002/art.39446
Pieterse, E., Rother, N., Yanginlar, C., Hilbrands, L. B., & van der Vlag, J. (2016). Neutrophils Discriminate between Lipopolysaccharides of Different Bacterial Sources and Selectively Release Neutrophil Extracellular Traps. Front Immunol, 7, 484. doi:10.3389/fimmu.2016.00484
Reeves, W. H., Lee, P. Y., Weinstein, J. S., Satoh, M., & Lu, L. (2009). Induction of autoimmunity by pristane and other naturally occurring hydrocarbons. Trends Immunol, 30(9), 455-464. doi:10.1016/j.it.2009.06.003
Smith, S., Wu, P. W., Seo, J. J., Fernando, T., Jin, M., Contreras, J., . . . Jefferies, C. A. (2018). IL-16/miR-125a axis controls neutrophil recruitment in pristane-induced lung inflammation. JCI Insight, 3(15). doi:10.1172/jci.insight.120798
Swamydas, M., Luo, Y., Dorf, M. E., & Lionakis, M. S. (2015). Isolation of Mouse Neutrophils. Curr Protoc Immunol, 110, 3.20.21-23.20.15. doi:10.1002/0471142735.im0320s110
Taheri, M., Eghtedarian, R., Dinger, M. E., & Ghafouri-Fard, S. (2020). Exploring the Role of Non-Coding RNAs in the Pathophysiology of Systemic Lupus Erythematosus. Biomolecules, 10(6). doi:10.3390/biom10060937
Wang, C. R., Liu, M. F., Weng, C. T., Lin, W. C., Li, W. T., & Tsai, H. W. (2018). Systemic lupus erythematosus-associated diffuse alveolar haemorrhage: a single-centre experience in Han Chinese patients. Scand J Rheumatol, 47(5), 392-399. doi:10.1080/03009742.2017.1420817
Weng, C. T., Lee, N. Y., Liu, M. F., Weng, M. Y., Wu, A. B., Chang, T. W., . . . Wang, C. R. (2010). A retrospective study of catastrophic invasive fungal infections in patients with systemic lupus erythematosus from southern Taiwan. lupus, 19(10), 1204-1209. doi:10.1177/0961203310368969
Y. Deng, B. H. H. B. P. T. (2007). Systemic Lupus Erythematosus (Vol. 2): Elsevier.
Yu, F., Haas, M., Glassock, R., & Zhao, M. H. (2017). Redefining lupus nephritis: clinical implications of pathophysiologic subtypes. Nat Rev Nephrol, 13(8), 483-495. doi:10.1038/nrneph.2017.85

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