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颗粒佐剂诱导的NLRP3炎性小体激活和细胞毒性。

NLRP3 inflammasome activation and cytotoxicity induced by particulate adjuvants.

作者信息

Yang Marie, Hearnden Claire H A, Oleszycka Ewa, Lavelle Ed C

机构信息

Adjuvant Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College, Dublin 2, Ireland.

出版信息

Methods Mol Biol. 2013;1040:41-63. doi: 10.1007/978-1-62703-523-1_5.

Abstract

The ability of particulate materials to provoke inflammatory immune responses has been well documented. In the case of endogenous and environmental particulates, these effects can often lead to pathological disorders. In contrast, particulate adjuvants incorporated into vaccines promote immune responses, which in turn provide efficient protection against infectious diseases. In recent years, studies have revealed that the NLRP3 inflammasome plays a key role in particulate-driven inflammation and its associated cytotoxicity. Hence, this chapter covers protocols useful to (1) assess NLRP3 inflammasome activation triggered by particulate adjuvants or materials in mouse bone marrow-derived dendritic cell (BMDCs) differentiated cultures, and (2) measure particle-induced cytotoxicity. More specifically, protocols are described for the preparation and differentiation of BMDCs, their priming and stimulation using particulate NLRP3 agonists such as monosodium urate monohydrate (MSU) and the vaccine adjuvant alum. We then detail protocols to assess particulate-driven cytotoxicity via flow cytometry using annexin V-propidium iodide (PI) and novel dye LIVE/DEAD(®) aqua stain. General considerations are provided that warn against the use of endotoxin-contaminated particles and emphasize the use of experimental controls. Suggestions are also outlined for further assessment of the immunomodulatory effects of particulate materials in vivo using the mouse peritonitis model.

摘要

颗粒物质引发炎症免疫反应的能力已有充分记载。就内源性和环境颗粒而言,这些影响常常会导致病理紊乱。相比之下,疫苗中所含的颗粒佐剂可促进免疫反应,进而提供针对传染病的有效保护。近年来,研究表明NLRP3炎性小体在颗粒驱动的炎症及其相关细胞毒性中起关键作用。因此,本章涵盖了有助于(1)评估颗粒佐剂或物质在小鼠骨髓来源的树突状细胞(BMDC)分化培养物中触发的NLRP3炎性小体激活,以及(2)测量颗粒诱导的细胞毒性的方案。更具体地说,描述了BMDC的制备和分化、使用颗粒NLRP3激动剂(如尿酸单钠一水合物(MSU)和疫苗佐剂明矾)对其进行启动和刺激的方案。然后,我们详细介绍了通过使用膜联蛋白V-碘化丙啶(PI)和新型染料LIVE/DEAD® Aqua stain进行流式细胞术评估颗粒驱动的细胞毒性的方案。提供了一些一般性注意事项,提醒注意使用受内毒素污染的颗粒,并强调使用实验对照。还概述了使用小鼠腹膜炎模型在体内进一步评估颗粒物质免疫调节作用的建议。

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