Assessing main death pathways in T lymphocytes from HIV infected individuals
Assessing main death pathways in T lymphocytes from HIV infected individuals
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DOI：
10.1002/cyto.a.22299
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发表时间：
2013-07-01
期刊：
影响因子：
3.7
通讯作者：
Blanco, Julia
中科院分区：
文献类型：
--
作者：
Massanella, Marta;Curriu, Marta;Blanco, Julia
淋巴细胞死亡增加是人类免疫缺陷病毒(HIV)感染的一个标志。尽管病毒学因素与这一现象有关，但在病毒复制停止的治疗个体中，仍观察到细胞死亡率增加。为了了解剩余变化的细胞死亡的本质，我们开发了一种简单而快速的方法来评估从HIV感染者分离的淋巴细胞中的主要细胞死亡途径。结合三个因素：(I)抗体染色以识别CD3+CD4+和CD3+CD8+细胞，(Ii)使用DiOC6(3)或JC-1探针和活性染料评估线粒体和质膜功能，以及(Iii)caspase抑制，允许量化caspase依赖和依赖于caspase的CD4和CD8T细胞死亡。根据线粒体膜电位耗散对Z-VAD-fmk或Q-VD-Oph处理的敏感性，后一种机制可分为内源性和外源性两种途径。我们的数据显示，在接受治疗的感染者中，这两种半胱氨酸酶抑制剂的结果相似，而Q-VD-Oph在病毒症患者中显示出更强的抑制作用，产生较低水平的内在细胞凋亡。比较DiOC6(3)和JC-1探针在CD4T细胞中得到了类似的结果，从而对这些细胞的死亡机制有了明确的定义。然而，在CD8T细胞中，JC-1表现出异质性染色，并检测到明显较低的细胞死亡水平，而内源性凋亡的贡献较高。总之，我们提供了一种简单的方法来评估HIV感染者的CD4T细胞死亡机制。CD8T细胞线粒体异质性的原因和后果需要进一步研究。(C)2012年国际细胞测量促进会
Increased lymphocyte death is a hallmark of human immunodeficiency virus (HIV) infection. Although virological factors have been linked to this phenomenon, increased cell death rates are still observed in treated individuals in which viral replication is halted. To understand the nature of this remaining altered cell death, we have developed a simple and fast assay to assess major cell death pathways in lymphocytes isolated from HIV-infected individuals. The combination of three factors: (i) antibody staining to identify CD3+CD4+ and CD3+CD8+ cells, (ii) assessment of mitochondrial and plasma membrane function using DiOC6(3) or JC-1 probes and vital dyes, and (iii) caspase inhibition, allowed for the quantification of caspase-independent and -dependent cell death in CD4 and CD8 T cells. The latter mechanism was divided in intrinsic and extrinsic apoptotic pathways according to the sensitivity of the dissipation of mitochondrial membrane potential to Z-VAD-fmk or Q-VD-oPH treatment. Our data show similar results for both caspase inhibitors in treated infected individuals, whereas Q-VD-oPH showed a more potent inhibition in viremic individuals, yielding lower levels of intrinsic apoptosis. Comparison of DiOC6(3) and JC-1 probes yielded similar results in CD4 T cells, allowing for a clear definition of death mechanism in these cells. However, in CD8 T-cells, JC-1 showed heterogeneous staining and detected significantly lower levels of cell death with a higher contribution of intrinsic apoptosis. In conclusion, we provide a simple method to assess CD4 T-cell death mechanisms in HIV-infected individuals. The reasons and consequences of mitochondrial heterogeneity in CD8 T-cells require further evaluation. (c) 2012 International Society for Advancement of Cytometry