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.
Highlights
Immune cells are main sources of the inflammation that supports obesity-associated insulin resistance and type 2 diabetes (T2D) [1, 2]
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 [7,8,9,10,11,12,13]
The field has not tested the possibility that shifts in the nutrient milieu that immerses immune cells in obesity/ T2D, alone or in combination with cell-intrinsic changes in fuel utilization, mechanistically explain the compromised immune function in such subjects leading to impaired wound healing and pathogen clearance
Summary
Immune cells are main sources of the inflammation that supports obesity-associated insulin resistance and type 2 diabetes (T2D) [1, 2]. 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 [7,8,9,10,11,12,13]. 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]. The field has not tested the possibility that shifts in the nutrient milieu that immerses immune cells in obesity/ T2D, alone or in combination with cell-intrinsic changes in fuel utilization, mechanistically explain the compromised immune function in such subjects leading to impaired wound healing and pathogen clearance
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