短发夹RNA介导的基因敲低在造血干和祖细胞系体外发育的树突状细胞发育研究
Summary
在这里，我们提供了一种方案，用于筛选参与树突状细胞（DC）发展的潜在转录因子，使用shRNA的慢病毒转导来获得稳定的敲低细胞系，用于 体外 DC分化。
Abstract
树突状细胞（DC）是连接先天性和适应性免疫应答的重要抗原呈递细胞。DC是异质的，可分为常规DC（cDC）和浆细胞样DC（pDC）。cDC专门向幼稚T细胞呈递抗原并激活它们。另一方面，pDC可以在病毒感染期间产生大量的I型干扰素（IFN-I）。DC的规范发生在骨髓中DC祖细胞（BM）的早期阶段，由转录因子（TF）网络定义。例如，cDC高度快速ID2，而pDC高度快速E2-2。由于越来越多的DC子集被识别出来，人们越来越有兴趣了解控制DC发展的特定TF。在这里，我们建立了一种方法，通过将携带短发夹RNA（shRNA）的慢病毒递送到永生化的造血干细胞和祖细胞（iHSPCs）系 中，在体外 筛选对DC分化至关重要的TF。在选择和 体外 分化后，通过流式细胞术分析稳定敲低细胞系的cDC和pDC电位。这种方法提供了一个平台，可以在 体外识别可能控制祖细胞DC命运的基因。
Introduction
DC是先天免疫和适应性免疫的关键调节因子1。发展中国家主要分为两个功能不同的群体，即pDC和cDC。此外，cDC包括两个子集，即I型和II型cDC或cDC1和cDC2s2。pDC在小鼠中表达BST2，Siglec-H和CD11c的中间水平3，4，是在炎症和病毒感染期间可以分泌大量IFN-I的细胞5。由于其强大的IFN-I产生能力，它们也被怀疑在自身免疫性疾病的进展中起关键作用，包括系统性红斑狼疮（SLE）6。cDC1s由小鼠中XCR1，CD8a，CLEC9A和CD103的表面表达定义7，专门用于通过抗原交叉递送激活和极化细胞毒性CD8 + T细胞（CTL），从而启动I型免疫以响应细胞内病原体和癌症8，9。另一方面，在人类和小鼠中表达CD11b和CD172α（也称为Sirpα）的cDC2s可以激活CD4 + T细胞并促进针对过敏原和寄生虫的II型免疫反应10，以及调节细胞外细菌和微生物群识别后的III型免疫11，12。
DC的多样化由BM中造血干和祖细胞（HSPCs）的一组TF决定.E2-2（由 Tcf4编码）是pDCs分化和功能的主调节因子13，14。相反，DNA结合2（ID2）的抑制剂通过阻断E蛋白活性来驱动cDC规范并抑制pDC的发展15。此外，cDC1s的开发需要IRF8和BATF3，而cDC2的区分高度依赖于IRF416。最近的工作探索了pDC17 和cDC的异质性及其转录调节18。由于直流网络的复杂性，需要建立一个平台来识别控制DC开发和功能的其他TF。
在这里，我们使用一种iHSPC，该iHSPC是通过在BM细胞（也称为Hoxb8-FL细胞）中表达Hoxb8的雌激素调节核易位而产生的19。在β雌二醇和Flt3配体（FL）存在下，iHSPCs可以增殖并保持在未分化阶段，而当β雌二醇退出时，它们在FL存在下开始分化成不同的DC类型19。基于这一特征，我们可以在祖细胞阶段敲低感兴趣的基因，然后检查对pDC和cDC 体外 分化的影响。因此，这种方法是发现调节DC发育和功能的基因的有力工具。
Protocol
Representative Results
Discussion
基于慢病毒的shRNA载体通常用于通过病毒转导到细胞中的基因沉默，并允许稳定地整合到宿主基因组中。然而，需要考虑不同细胞类型的各种转导效率，并且已经采取了许多方法来克服这个问题。
聚苯乙烯是一种聚阳离子聚合物，可以中和细胞膜上的电荷，从而增强病毒粒子在转导过程中与细胞的结合20。虽然它是增加转导率的有效方法，但当添加过量时，…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们非常感谢陈志玲博士的技术支持。我们感谢国家RNAi核心设施（台湾中央研究院）提供shRNA慢病毒（http://rnai.genmed.sinica.edu.tw）。这项工作得到了台湾科技部（MOST 108-2320-B-002-037-MY3和MOST 109-2320-B-002-054-MY3）的支持。
Materials
| Antibodies | |||
| APC/Cy7 anti-mouse CD11c Antibody | Biolegend | 117324 | (Clone: N418) |
| FITC anti-mouse/human CD11b Antibody | Biolegend | 101206 | (Clone: M1/70) |
| PE anti-mouse/human B220 Antibody | Biolegend | 103208 | (Clone: RA3-6B2) |
| Cell culture | |||
| 1.5 mL Micro tube | ExtraGene | TUBE-170-C | |
| 12-well tissue culture-treated plate | Falcon | 353043 | |
| Fetal bovine serum (FBS) | Corning | 35-010-CV | |
| RPMI 1640 medium | gibco | 11875-085 | |
| Reagent | |||
| β-estradiol | Sigma-Aldrich | E2758-250MG | |
| β-mercaptoethanol (β-ME) | Sigma-Aldrich | M6250 | |
| FACS buffer | home-made | Formula: 1xPBS+0.5 %FBS+0.1%NaN3 | |
| Flt3 ligand (FL) | home-made | ||
| Polybrene | Sigma-Aldrich | TR-1003-G | |
| Puromycin | Invivogen | ant-pr-1 | |
| TRIsure | BIOLINE | BIO-38032 | |
| shRNA (Taregt sequence/clone ID) | Company | ||
| shId2 (GCTTATGTCGAATGATAGCAA/TRCN0000054390) | The RNAi Consortium (TRC) | ||
| shLacZ (CGCGATCGTAATCACCCGAGT/TRCN0000072224) | The RNAi Consortium (TRC) | ||
| shTcf4 (GCTGAGTGATTTACTGGATTT/TRCN0000012094) | The RNAi Consortium (TRC) | ||
Referências
- Steinman, R. M., Witmer, M. D. Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proceedings of the National Academy of Sciences. 75 (10), 5132-5136 (1978).
- Guilliams, M., et al. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nature Reviews Immunology. 14 (8), 571-578 (2014).
- Blasius, A. L., Cella, M., Maldonado, J., Takai, T., Colonna, M. Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12. Blood. 107 (6), 2474-2476 (2006).
- Swiecki, M., Colonna, M. Unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance. Immunology Reviews. 234 (1), 142-162 (2010).
- Liu, Y. -. J. IPC: Professional Type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annual Review of Immunology. 23 (1), 275-306 (2005).
- Panda, S. K., Kolbeck, R., Sanjuan, M. A. Plasmacytoid dendritic cells in autoimmunity. Current Opinion in Immunology. 44, 20-25 (2017).
- Durai, V., Murphy, K. M. Functions of murine dendritic cells. Immunity. 45 (4), 719-736 (2016).
- Mashayekhi, M., et al. CD8α(+) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites. Immunity. 35 (2), 249-259 (2011).
- Anderson, D. A., Dutertre, C. -. A., Ginhoux, F., Murphy, K. M. Genetic models of human and mouse dendritic cell development and function. Nature Reviews Immunology. 21 (2), 101-115 (2021).
- Plantinga, M., et al. Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen. Immunity. 38 (2), 322-335 (2013).
- Persson, E. K., et al. IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity. 38 (5), 958-969 (2013).
- Kumamoto, Y., et al. CD301b+ dermal dendritic cells drive T helper 2 cell-mediated immunity. Immunity. 39 (4), 733-743 (2013).
- Cisse, B., et al. Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development. Cell. 135 (1), 37-48 (2008).
- Grajkowska, L. T., et al. Isoform-specific expression and feedback regulation of E protein TCF4 control dendritic cell lineage specification. Immunity. 46 (1), 65-77 (2017).
- Jackson, J. T., et al. Id2 expression delineates differential checkpoints in the genetic program of CD8α+ and CD103+ dendritic cell lineages. The EMBO Journal. 30 (13), 2690-2704 (2011).
- Suzuki, S., et al. Critical roles of interferon regulatory factor 4 in CD11bhighCD8alpha- dendritic cell development. Proceedings of the National Academy of Science U. S. A. 101 (24), 8981-8986 (2004).
- Rodrigues, P. F., et al. Distinct progenitor lineages contribute to the heterogeneity of plasmacytoid dendritic cells. Nature Immunology. 19 (7), 711-722 (2018).
- Brown, C. C., et al. Transcriptional basis of mouse and human dendritic cell heterogeneity. Cell. 179 (4), 846-863 (2019).
- Redecke, V., et al. Hematopoietic progenitor cell lines with myeloid and lymphoid potential. Nature Methods. 10 (8), 795-803 (2013).
- Abe, A., Miyanohara, A., Friedmann, T. Polybrene increases the efficiency of gene transfer by lipofection. Gene Therapy. 5 (5), 708-711 (1998).
- Sorgi, F. L., Bhattacharya, S., Huang, L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Therapy. 4 (9), 961-968 (1997).
- Kirkling, M. E., et al. Notch signaling facilitates in vitro generation of cross-presenting classical dendritic cells. Cell Reports. 23 (12), 3658-3672 (2018).
- Pearce, E. J., Everts, B. Dendritic cell metabolism. Nature Reviews. Immunology. 15 (1), 18-29 (2015).
- Wculek, S. K., Khouili, S. C., Priego, E., Heras-Murillo, I., Sancho, D. Metabolic Control of Dendritic Cell Functions: Digesting Information. Frontiers in Immunology. 10 (775), (2019).
- Saxton, R. A., Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell. 168 (6), 960-976 (2017).
