Research Article: Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis

Date Published: February 8, 2017

Publisher: Public Library of Science

Author(s): Dequina Nicholas, Elizabeth A. Proctor, Forum M. Raval, Blanche C. Ip, Chloe Habib, Eleni Ritou, Tom N. Grammatopoulos, Devin Steenkamp, Hans Dooms, Caroline M. Apovian, Douglas A. Lauffenburger, Barbara S. Nikolajczyk, Nades Palaniyar.

http://doi.org/10.1371/journal.pone.0170975

Abstract

Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.

Partial Text

Immune cells are main sources of the inflammation that supports obesity-associated insulin resistance and type 2 diabetes (T2D) [1, 2]. Lymphocytes such as T cells and B cells contribute to obesity-associated inflammation in unhealthy adipose tissue [3–6], but the paucity of lymphocytes, and especially B cells, in human adipose tissue remains a challenge that limits functional and mechanistic studies on these cells. Several lines of evidence indicate that blood lymphocytes are a reasonable surrogate to guide studies aimed at understanding the roles T cells and B cells play in obesity-associated complications like insulin resistance and T2D [7–13]. These studies include our recently published T cell cytokine signature, which distinguishes samples from T2D and body mass index-matched non-T2D subjects, and was developed from analysis of peripheral blood mononuclear cells [14].

We have developed a reproducible method for XF96 analysis of human PBMCs and CD4+ T cells. Perhaps most importantly, we have published a free downloadable XF analysis tool, SHORE, which has automated quality control and can handle data from an unrestricted number of samples exclusive of operator bias. SHORE is a critical advance for analysis of outcomes from the large number of samples required to make strong conclusions given the known variability of human samples, and the importance of analyzing outcomes from multiple treatments in parallel. We also generated preliminary evidence that our systematic approaches will facilitate analysis of both PBMCs, which are relatively plentiful, and lymphocytes, which can be available in relatively low numbers. The ability to ascertain detailed outcomes from a relatively limited number of cells is an important advance given that the Seahorse platform has only been somewhat miniaturized since the original design for cell line usage. Our work builds on earlier detailed descriptions of Seahorse protocols and the commercially available Wave and Mito Stress Test Generator programs [31, 42] that although initially useful, needed to be modified to more fully exploit the technology.

 

Source:

http://doi.org/10.1371/journal.pone.0170975

 

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