在 Vivo 增强的古特-霍姆调节 T 细胞诱导
Summary
在这里，我们提出了一个方案，在体内扩增的肠道-细胞调节T细胞诱导。在此协议中，树突状细胞被设计成局部产生高浓度活性维生素D（1，25-二羟基维生素D或1，25[OH]2 D）和活性维生素A（视黄酸或RA）的活性。
Abstract
炎症性肠病 （IBD） 是胃肠道 （GUT） 中的炎症性慢性疾病。在美国，大约有140万IBD患者。人们普遍认为，对肠道细菌的免疫反应失调会引发疾病并破坏粘膜上皮屏障。我们最近表明，肠道调节T（Treg）细胞是IBD的一种有前途的治疗方法。因此，本文提出了一种在体内扩增肠道Treg细胞诱导的协议。在此协议中，树突状细胞被设计成产生局部高浓度的两种分子，活性维生素D（1，25-二羟基维生素D或1，25[OH]2D）和活性维生素A（视黄酸或RA）。我们根据先前的发现选择1，25（OH）2 D和RA，表明1，25（OH）2 D可以诱导调节分子的表达（例如，叉头盒P3和白细胞介素-10），RA可以刺激T细胞中肠道宿主受体的表达。为了产生这种工程树突状细胞，我们使用慢病毒载体来转导树突状细胞来过度表达两个基因。一个基因是细胞色素P450家族27亚家族B成员1，编码25-羟基维生素D1+羟基酶，这在生理上催化1，25（OH）2 D的合成。另一个基因是甲醛脱氢酶1家族成员A2，编码视网膜醛脱氢酶2，这在生理上催化RA的合成。该协议可用于今后对体内肠道细胞的考察。
Introduction
炎症性肠病 （IBD） 是胃肠道 （GUT） 中的炎症性慢性疾病。在美国，大约有140万IBD患者。人们普遍认为，对肠道细菌的免疫反应失调会引发疾病，并破坏粘膜上皮屏障1，2。因此，目前美国食品和药物管理局（FDA）批准的药物可以抑制炎症中介的作用，或阻止免疫细胞进入肠道。然而，被攻击的炎症介质和免疫细胞也是免疫防御所必需的。因此，炎症中介抑制剂损害全身免疫防御和免疫细胞阻滞剂削弱肠道免疫防御，两者都可能导致严重后果3，4。此外，免疫细胞阻滞剂还可以阻止调节性T（Treg）细胞进入肠道，从而恶化IBD患者已经受损的肠道免疫耐受性。此外，由于Treg细胞在血液中的积累5，阻断Treg细胞进入肠道也可能导致全身免疫抑制。最后，抑制剂和阻滞剂在瞬时作用下发挥作用，因此需要频繁的管理。频繁使用这些抑制剂和阻滞剂可能会进一步加剧不良的副作用。
最近，我们提出了一个新的策略，可以潜在地减轻甚至消除与当前药物IBD治疗6相关的副作用。这种策略增加了周围淋巴组织6的肠道细胞的诱导。这种策略的原理是，肠道细胞专门归于肠道，因此不会损害全身免疫防御。此外，由于Treg细胞可能形成记忆7，8，肠道细胞可能提供一个稳定的控制慢性肠道炎症在IBD患者，因此，治疗不应该经常进行。此外，由于这种策略增强了体内肠道培养Treg细胞的诱导，因此在高度促进炎的环境中，它与体外产生的Treg细胞的收养转移有关，它不存在体内不稳定的问题。在这方面，体外产生的Treg细胞是治疗自身免疫性疾病11、12、13和移植排斥14、15的拟议策略之一。最后，在此策略中，树突状细胞 （DC） 被设计成产生局部高浓度的两种分子：活性维生素 D（1，25-二羟基维生素 D 或 1，25[OH]2D）和活性维生素 A（视黄酸或 RA）。我们选择了1，25（OH）2 D和RA，因为1，25（OH）2 D可以诱导调节分子的表达（例如，叉头盒P3[foxp3]和白细胞介素-10[IL-10]）16，17，并且RA可以刺激T细胞18的肠道宿主受体的表达。因为1，25（OH）2D和RA也可以容忍DC28，29，我们有理由认为，工程DC将稳定地维持在体内的耐原状态，从而规避与体外产生的耐性DC（TolDC）19、20、21相关的体内不稳定问题。在这方面，TolDCs也是建议在体内增强Treg细胞功能19，20，21的策略之一。为了支持我们的推理，我们已经表明，工程的DC，在体内分娩时，可以增强周围淋巴组织6的肠道胃肠道Treg细胞的诱导。
我们提议的战略的另一个优点是 1，25（OH）2D 还具有其他功能，可能有益于 IBD 患者。这些其他功能包括1，25（OH）2 D的能力，刺激抗菌素22的分泌和抑制致癌23。感染和癌症经常与IBD24，25相关。
为了生成能够产生本地高浓度的1，25（OH）2 D和RA denovo的DC，我们使用慢病毒载体来设计DC，以过度表达两个基因。一个基因是细胞色素P450家族27亚家族B成员1（CYP27B1），编码25-羟基维生素D1+羟基酶（1+-羟基酶），这在生理上催化1，25（OH）2 D的合成。另一个基因是甲醛脱氢酶1家族成员A2（ALDH1a2），编码视网膜醛脱氢酶2（RALDH2），这在生理上催化RA6的合成。
由于在体内扩增肠道Treg细胞诱导在IBD的治疗中可能很重要，在以下协议中，我们将详细说明1+-羟基酶-RALDH2过度表达DC（DC-CYP-ALDH细胞）的生成程序，可用于将来对体内肠道细胞的考察。
Protocol
所有体内动物研究协议都经过洛马琳达大学机构动物护理和使用委员会（IACUC）以及美国陆军医学研究与物资司令部动物护理和使用审查办公室（ACURO）的审查和批准。国防部 1. 制备同时表达1-羟基酶和RALDH2（拉比-CYP-ALDH病毒）的Lenti病毒 第0天：在清晨，在CM-10-D细胞培养基中制备5 x 105细胞/mL的293T细胞。 种子 20 mL/板在 150 毫米 x 25 毫米培养皿。在37°C和…
Representative Results
DC-CYP-ALDH细胞表达的1-羟基酶的量显著增加。为了确定BMDC产生的DC-CYP-ALDH细胞是否表示1-羟基酶的含量显著增加，BMDC与慢透-CYP-ALDH病毒一起转导，以产生骨髓衍生的DC-CYP-ALDH细胞（BMDC-CYP-ALDH细胞）。随后，FACS对BMDC-CYP-ALDH细胞的1-羟基酶的表达进行了检查。我们的数据表明，BMDC-CYP-ALDH细胞与亲子细胞相比，显示了1°-羟基酶的增强表达（图1A）。我们还…
Discussion
在本文中，我们将描述DC-CYP-ALDH细胞的使用，用于增强周围淋巴组织内肠道细胞的诱导。我们的数据表明，DC-CYP-ALDH细胞可以在存在相应基质（即分别为25[OH]D和视黄醇）的情况下，在体外合成1，25（OH）2 D和RA的局部高浓度。由于在有30、31缺症的患者中，分别可以通过维生素D和A补充轻松获得足够的血液浓度，因此，当存在25（OH）D和视黄醇的正?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了负责卫生事务的助理国防部长办公室的支持，该计划通过”同行评审医学研究计划”获得第1号奖。W81XWH-15-1-0240 （XT）。意见、解释、结论和建议是作者的意见、解释、结论和建议，不一定得到国防部的认可。这项工作还部分得到了洛马琳达大学医学系的研究创新赠款（681207-2967[XT和GG]、681205-2967[XT]和325491[DJB]的支持。
Materials
| 10 mL syringes | ThermoFisher Scientific | Cat# 03-377-23 | |
| 100 mm x 20 mm culture dishes | Sigma-Aldrich | Cat# CLS430167 | |
| 12-well culture plates | ThermoFisher Scientific | Cat# 07-200-82 | |
| 150 mm x 25 mm culture dishes | Sigma-Aldrich | Cat# CLS430559 | |
| 25-hydroxycholecalciferol (25[OH]D) | Sigma-Aldrich | Cat# H4014 | |
| 293T cells | ATCC | CRL-3216 | |
| 2-mercaptoethanol | ThermoFisher Scientific | Cat#: 21985023 | |
| 6-well culture plates | ThermoFisher Scientific | Cat# 07-200-83 | |
| ALDEFLUOR kit | Stemcell Technologies | Cat# 01700 | |
| Anti-CYP27B1 | Abcam | Cat# ab95047 | |
| BD FACSAria II | BD Biosciences | N/A | |
| CaCl2 | Sigma-Aldrich | Cat# C1016 | |
| CM-10-D cell culture medium | DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine. | ||
| CM-10-R cell culture medium | RPMI 1640 medium (no glutamine) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine. | ||
| CM-4-D cell culture medium | DMEM medium containing 4% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine. | ||
| Corning bottle-top vacuum filters, 0.22 mM, 500 mL | Sigma-Aldrich | Cat# CLS430513 | |
| Corning bottle-top vacuum filters, 0.45 mM, 500 mL | Sigma-Aldrich | Cat# CLS430514 | |
| Dissecting scissor | ThermoFisher Scientific | Cat# 08-940 | |
| DMEM medium | ThermoFisher Scientific | Cat# 11960044 | |
| Fetal bovine serum | ThermoFisher Scientific | Cat# 16000044 | |
| Forceps | ThermoFisher Scientific | Cat# 22-327379 | |
| Gibco ACK lysing buffer | ThermoFisher Scientific | Cat# A1049201 | |
| Glycerol | Sigma-Aldrich | Cat# G5516 | |
| Goat anti-rabbit IgG | Abcam | Cat# ab205718 | |
| HEPES | Millipore | Cat# 391340 | |
| Lenti-CYP-ALDH | Custom-made | 1.6-kb mouse CYP27B1 and ALDH1a2 cDNAs were amplified by PCR using a plasmid containing the CYP27B1 cDNA and a plasmid containing the ALDH1a2 cDNA respectively (GeneCopoeia). The amplified CYP27B1 cDNA fragment with a 5' KOZAK ribosome entry sequence was cloned into the pRRL-SIN.cPPt.PGKGFP.WPRE lentiviral vector (Addgene). The resulting construct was designated as lenti-CYP-GFP. The amplified ALDH1a2 cDNA fragment was cloned into the lenti-CYP-GFP to replace the GFP and was designated as lenti-CYP-ALDH. This bicistronic plasmid expresses CYP27B1 controlled by SFFV promoter and ALDH1a2 controlled by PGK promoter. | |
| L-glutamine | ThermoFisher Scientific | Cat#25030081 | |
| Lipopolysaccharide | Sigma-Aldrich | Cat# L3755 | |
| Murine GM-CSF | Peprotech | Cat# 315-03 | |
| Murine IL-4 | Peprotech | Cat# 214-14 | |
| Na2HPO4 | Sigma-Aldrich | Cat# NIST2186II | |
| NaCl | Sigma-Aldrich | Cat# S9888 | |
| Needles | ThermoFisher Scientific | Cat# 14-841-02 | |
| Nonessential Amino Acids | ThermoFisher Scientific | Cat#: 11140076 | |
| pCMVR8.74 | Addgene | Plasmid# 22036 | |
| Penicillin/Streptomycin | ThermoFisher Scientific | Cat#15140148 | |
| Phoshate Balanced Solution (PBS) | ThermoFisher Scientific | Cat#: 20012027 | |
| PMD2G | Addgene | Plasmid# 12259 | |
| Polypropylene tube, 15 mL | ThermoFisher Scientific | Cat# AM12500 | |
| Polypropylene tube, 50 mL | ThermoFisher Scientific | Cat# AM12502 | |
| Protamine sulfate | Sigma-Aldrich | Cat# P3369 | |
| Rabbit polycloncal IgG isotype control | Abcam | Cat# ab171870 | |
| Radioimmunoassay for 1,25(OH)2D measurement | Heartland Assays | ||
| RPMI 1640 medium, no glutamine | ThermoFisher Scientific | Cat# 21870076 | |
| Sodium pyruvat | ThermoFisher Scientific | Cat#: 11360070 | |
| Sorvall Legend XTR Centrifuge | ThermoFisher Scientific | Cat# 75004521 | |
| Sterile Cell strainers, 40 mm | ThermoFisher Scientific | Cat# 07-201-430 | |
| Sterile storage bottles, 500 mL | ThermoFisher Scientific | Cat# CLS431432 |
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Cite This Article
Bi, H., Wasnik, S., Baylink, D. J., Liu, C., Tang, X. In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction. J. Vis. Exp. (155), e60585, doi:10.3791/60585 (2020).