体内结肠细胞 Photolabeling 对新生小鼠造血细胞迁移潜能的评价
Summary
这里描述的协议使用了 photolabeling 方法在新生老鼠特别辨认免疫细胞移居从结肠到肠外的站点。这一策略将有助于研究早期生命中宿主-微生物的相互作用。
Abstract
肠道细菌群落是早期建立的, 对免疫细胞的发育和功能有影响。新生儿微生物群易受多种外部影响, 包括抗生素的使用和饮食, 这会影响对自身免疫和炎症性疾病的敏感性。炎症性肠病 (IBD) 等疾病的特点是大量的免疫细胞涌入肠道。然而, 由微生物群所调节的免疫细胞可能会另外从肠道中移出, 以影响肠道的免疫应答。因此, 有必要识别和鉴定可能携带微生物信息从肠道到远端部位的细胞。在这里, 我们描述了一种方法来标记细胞在结肠的新生小鼠在体内, 使他们的身份, 在肠外的地方, 迁移后鉴定。
Introduction
哺乳动物胃肠道有数以百计的细菌存在于与宿主1的共生关系中。在当地环境中存在的免疫细胞加强了与这些微生物的和平共处, 并对病菌入侵建立了保护性屏障。因此, 免疫细胞和微生物群之间的双向相互作用是建立一个共生社区, 教育宿主免疫系统并设定免疫反应性的阈值的关键。微生物组成的变化, 或 dysbiosis, 可能扰乱免疫动态平衡和扰乱调节电路, 抑制肠道炎症导致免疫介导的疾病, 如1型糖尿病和 IBD2,3.
出生后的时间是一个独特的发展窗口, 肠道微生物群落开始建立的同时免疫系统成熟4。产后微生物群是不稳定的, 与转移在社区构成自然地和频繁地发生5。与微生物群相互作用的免疫细胞位于肠道的两个不同的解剖位置–固有层和肠道上皮6。许多类型的免疫细胞存在于肠道, 包括淋巴细胞 (如 T 细胞, B 细胞, 和先天淋巴细胞) 以及髓细胞 (包括树突状细胞, 单核, 和巨细胞)。这些细胞, 也称为造血细胞, 执行多种功能, 保持肠道屏障和维持动态平衡。
除了在肠道部位的调节功能外, 粘膜的免疫细胞还可能携带微生物信息到肠外的部位, 以调节全身免疫7、8、9。这是一个日益增长的研究兴趣的领域, 并强调需要的方法, 以确定免疫细胞迁移出肠道组织, 以探讨其功能。这里报告的协议使用了一个商业上可用的老鼠模型, 其中 photoconvertible 荧光蛋白被利用来标记细胞。范切除小鼠无所不在表达绿色荧光 Dendra2 蛋白, 是不可逆转地切换到红色荧光激活时紫外线 (UV) 光10。利用光纤套管将405纳米光传送到新生小鼠结肠中, 我们证明了 photoconverted 造血细胞 (起源于或通过结肠过渡) 可以在脾脏中找到。
Protocol
所有动物程序都是在马萨诸塞州总医院的机构动物护理和使用委员会 (IACUC) 批准和遵守的情况下进行的。 注意: 此协议涉及使用3b 类激光 (LG3)。LG3 激光安全护目镜在操作激光时必须始终使用。必须遵循适当的培训和安全准则, 以避免受伤的危险。 1. 激光的设计与装配 通过连接到 led 的4针 M8 连接器电缆, 将发光二极管 (LED) (405 nm、19.3 兆瓦、1…
Representative Results
用光纤电缆将405纳米光传送到2天的老范切除小鼠的冒号中。在以前的实验中, 三十年代的暴露被确定为最大 photoconversion 的结肠细胞具有最小的细胞毒性 (图 1a)。因此, 按照《议定书》的规定, 对结肠的不同部分进行连续三十年代的曝光。激光照射后, 老鼠立即被安乐死, 细胞的 photoconversion 被确定。采用荧光共轭 anti-CD45 抗体对全?…
Discussion
细胞的识别和鉴定与结肠菌群的影响是重要的, 应该有助于理解如何将信息从粘膜微环境传递到身体的其余部分。研究肠道相关细胞迁移的一种方法, 需要将肠道相关细胞分离出来, 然后将移植到受体小鼠体内, 以确定它们的组织自导模式和功能12,13。此方法是有限的, 因为仅可以跟踪传输的特定单元格类型。此外, 由于从组织中分离细胞和纯化过程而产生…
Divulgations
The authors have nothing to disclose.
Acknowledgements
尼亚耆那得到了 NIH/NIAID 职业转型奖1K22AI116661-01 的支持。
Materials
| Laser | |||
| Light Emitting Diode (LED) | THORLABS | M405FP1 | CAUTION: this is a Class 3b laser. Safety goggles must be worn when using the laser. It emits a 405 nm wavelength with a current of 1400 mA. It is fiber-coupled. It accepts SMA connector. https://www.thorlabs.com/thorproduct.cfm?partnumber=M405FP1 |
| LED driver | THORLABS | LEDD1B | Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616 |
| Optogenetics patch cable | THORLABS | M87L01 | 1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&pn=M87L01#11454 |
| Fiber optic cannula | Doric lenses | MFC_480/500-0.5_5mm_ZF1.25_C45 | 5 mm long cannula with an outer diameter of 500 µm and an inner diameter of 480 µm. The NA value is 0.5. The ferrule is zirconia, 1.25 mm OD. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6036 |
| Power supply | THORLABS | KPS101 | Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101 |
| LG3 laser safety goggles | THORLABS | LG3 | Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&pn=LG3#2523 |
| Red light | Electron Microscopy Sciences | 74327-10 | 15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences |
| Intestinal cell isolation | |||
| Isoflurane | Patterson Veterinary | 07-893-1389 | CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood. https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3 |
| 1X HBSS | Gibco | 14025076 | Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&searchTerm=14025076&searchType=RAPID&matchedCatNo=14025076 |
| Calf Serum | Hyclone AZM | 197696 | |
| EDTA | Invitrogen | 15575020 | 0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020 |
| DTT | Sigma | 10197777001 | CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en®ion=US |
| HEPES | Gibco | 15630080 | 1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080 |
| Petri dish | Corning | 353004 | https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&searchTerm=08772F&searchType=RAPID&matchedCatNo=08772F |
| 70 micron cell strainer | Falcon | 352350 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712 |
| Micro magnetic stir bar | Fisherbrand | 1451364 | Rinse in 70% ethanol after each use. Rinse several times in distilled water prior to each use. The bar is 8 mm long with an octagonal shape. https://www.fishersci.com/shop/products/fisherbrand-octagonal-magnetic-stir-bars-12/1451364#?keyword=1451364 |
| Magnetic stir plate | Corning Laboratory Stirrers | 440826 | https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&persist=true&ip=no&gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE |
| Collagenase | Roche | 5401020001 | https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en®ion=US&gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE |
| DNase I | Sigma | 10104159001 | https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en®ion=US |
| 1X PBS | Gibco | 20012-027 | https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027 |
| Pipet aid | Thermo Scientific | 14387165 | https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165 |
| 10 mL serological pipet | Falcon | 357530 | https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659 |
| 25 mL serological pipet | Falcon | 357515 | https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659 |
| 15 mL conical centrifuge tube | Thermo Scientific | 339651 | https://www.thermofisher.com/order/catalog/product/339650 |
| 50 mL conical centrifuge tube | Thermo Scientific | 339653 | https://www.thermofisher.com/order/catalog/product/339650 |
| Single cell suspension | |||
| Eppendorf tubes | Seal-Rite | 1615-5500 | Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx |
| Tissue homogenizer | Kimble | K7495400000 | Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000 |
| Homogenizer tips | Kimble | 7495210590 | Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590 |
| ACK lysing buffer | Gibco | A10492-01 | https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01 |
| 40 micron cell strainer | Falcon | 08-771-1 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711 |
| Antibodies | |||
| BV786 anti-mouse CD45 | BD | 564225 | Clone 3O-F11 https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-mouse-antibodies/cell-surface-antigens/bv786-rat-anti-mouse-cd45-30-f11/p/564225 |
| Live/Dead | Invitrogen | L34962 | https://www.thermofisher.com/order/catalog/product/L34962 |
| Other | |||
| Razor blades | VWR | 55411-050 | Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades |
References
- Maynard, C. L., Elson, C. O., Hatton, R. D., Weaver, C. T. Reciprocal interactions of the intestinal microbiota and immune system. Nature. 489, 231-241 (2012).
- Paun, A., Yau, C., Danska, J. S. The Influence of the Microbiome on Type 1 Diabetes. Journal of Immunology. 198, 590-595 (2017).
- Mathis, D., Benoist, C. Microbiota and autoimmune disease: the hosted self. Cell Host Microbe. 10, 297-301 (2011).
- Kollmann, T. R., Kampmann, B., Mazmanian, S. K., Marchant, A., Levy, O. Protecting the Newborn and Young Infant from Infectious Diseases: Lessons from Immune Ontogeny. Immunity. 46, 350-363 (2017).
- Jain, N., Walker, W. A. Diet and host-microbial crosstalk in postnatal intestinal immune homeostasis. Nature Reviews in Gastroenterology and Hepatology. 12, 14-25 (2015).
- Mowat, A. M., Agace, W. W. Regional specialization within the intestinal immune system. Nature Reviews in Immunology. 14, 667-685 (2014).
- Macpherson, A. J., Uhr, T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science. 303, 1662-1665 (2004).
- Mowat, A. M. Anatomical basis of tolerance and immunity to intestinal antigens. Nature Reviews in Immunology. 3, 331-341 (2003).
- Diehl, G. E., et al. Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells. Nature. 494 (3), 116-120 (2013).
- Pham, A. H., McCaffery, J. M., Chan, D. C. Mouse lines with photo-activatable mitochondria to study mitochondrial dynamics. Genesis. 50, 833-843 (2012).
- Conway, K. L., et al. ATG5 regulates plasma cell differentiation. Autophagy. 9, 528-537 (2013).
- Buzoni-Gatel, D., Lepage, A. C., Dimier-Poisson, I. H., Bout, D. T., Kasper, L. H. Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii. Journal of Immunology. 158, 5883-5889 (1997).
- Guo, X., Muite, K., Wroblewska, J., Fu, Y. X. Purification and Adoptive Transfer of Group 3 Gut Innate Lymphoid Cells. Methods in Molecular Biology. 1422, 189-196 (2016).
- Morton, A. M., et al. Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut. Proceedings of the National Academy of Sciences of the United States of America. 111, 6696-6701 (2014).
Citer Cet Article
Porter, C., Ennamorati, M., Jain, N. In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice. J. Vis. Exp. (138), e57929, doi:10.3791/57929 (2018).