RESUMEN
Mechanisms that regulate metabolites and downstream energy generation are key determinants of T cell cytokine production, but the processes underlying the Th17 profile that predicts the metabolic status of people with obesity are untested. Th17 function requires fatty acid uptake, and our new data show that blockade of CPT1A inhibits Th17-associated cytokine production by cells from people with type 2 diabetes (T2D). A low CACT:CPT1A ratio in immune cells from T2D subjects indicates altered mitochondrial function and coincides with the preference of these cells to generate ATP through glycolysis rather than fatty acid oxidation. However, glycolysis was not critical for Th17 cytokines. Instead, ß oxidation blockade or CACT knockdown in T cells from lean subjects to mimic characteristics of T2D causes cells to utilize 16C-fatty acylcarnitine to support Th17 cytokines. These data show long-chain acylcarnitine combines with compromised ß oxidation to promote disease-predictive inflammation in human T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos/metabolismo , Activación de Linfocitos/inmunología , Células Th17/inmunología , Adulto , Anciano , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Células Cultivadas , Estudios Transversales , Citocinas/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Glucólisis/genética , Humanos , Inflamación/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Obesidad/metabolismo , Oxidación-Reducción , Transfección , Adulto JovenRESUMEN
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.
