Metabolic substrate utilization in stress-induced immune cells

Abstract

Immune cell activation leads to the acquisition of new functions, such as proliferation, chemotaxis, and cytokine production. These functional changes require continuous metabolic adaption in order to sustain ATP homeostasis for sufficient host defense. The bioenergetic demands are usually met by the interconnected metabolic pathways glycolysis, TCA cycle, and oxidative phosphorylation. Apart from glucose, other sources, such as fatty acids and glutamine, are able to fuel the TCA cycle.Rising evidence has shown that cellular metabolism has a direct effect on the regulation of immune cell functions. Thus, quiescent immune cells maintain a basal metabolic state, which shifts to an accelerated metabolic level upon immune cell activation in order to promote key effector functions.This review article summarizes distinct metabolic signatures of key immune cell subsets from quiescence to activation and demonstrates a methodical concept of how to assess cellular metabolic pathways. It further discusses why metabolic functions are of rising interest for translational research and how they can be affected by the underlying pathophysiological condition and/or therapeutic interventions.

Keywords: Catecholamines; Glycolysis; Immunometabolism; Oxidative phosphorylation; Pentose phosphate pathway; Reactive oxygen species; Tricarboxylic acid cycle.

Fig. 1

ATP-producing metabolic pathways in distinct immune cell subsets. Glucose oxidation to pyruvate via glycolysis is a fast reaction generating 2 mol of ATP per mol glucose. This aerobic glycolysis is complemented by the PPP that can produce further metabolic precursor molecules and is involved in ROS production. Pyruvate can be converted to lactate or can be further oxidized to acetyl-CoA entering the mitochondrial TCA cycle (yellow box). The TCA cycle (red box) generates reducing equivalents NADH and FADH₂ which are utilized in the mitochondrial respiratory chain to build up the proton gradient across the mitochondrial inner membrane by complexes I–IV of the respiratory chain. As a by-product, ROS and RNS are produced. Oxidative phosphorylation produces larger amounts of ATP (36–38 mol/mol glucose) by complex V. Immune cells are also able to utilize substrates such as glutamine, which enters these pathways via the TCA metabolite α-ketoglutarate, and fatty acids, which are oxidized to acetyl-CoA via β-oxidation. Granulocytes and M1 macrophages have a highly glycolytic metabolism (yellow box) even when oxygen is available. Their TCA cycle and respiratory chain activity is kept at low level. T_n, T_m, and T_reg cells as well as monocytes and M2 macrophages primarily perform OXPHOS and are also able to metabolize fatty acids and glutamine in order to fuel the TCA cycle (red box). T_eff cells have a highly active metabolism including all of the pathways described (green box). These pathways do not only culminate in ATP production but also provide other biosynthetic pathways with metabolic precursors thus supporting different functional necessities of the immune cell populations. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; CoA, coenzyme A; FADH₂/FAD, flavin adenine dinucleotide in its reduced/oxidized form; IMS, intermembrane space; MM, mitochondrial membrane; NADH/NAD⁺, nicotinamide adenine dinucleotide in its reduced/oxidized form; OXPHOS, oxidative phosphorylation; PPP, pentose phosphate pathway; ROS, reactive oxygen species; RNS, reactive nitrogen species; TCA, tricarboxylic acid cycle; T_eff, effector T cell; T_n, naïve T cell; T_m, memory T cell; T_reg, regulatory T cell

Fig. 2

Metabolic pathways of distinct immune cell subsets in quiescence and upon activation. Under quiescent conditions, neutrophils rely on aerobic glycolysis, whereas T_n cells, monocytes, and B cells preferentially rely on OXPHOS for ATP production. Immune cell activation reshapes the metabolic demands of immune cells performing their various effector functions, differentiation to T_eff, T_reg, or T_m cells, and migration across the endothelium into the tissue, where substrate and oxygen availability can be limited. Abbreviations: ATP, adenosine triphosphate; OXPHOS, oxidative phosphorylation; T_eff, effector T cell; T_n, naïve T cell; T_m, memory T cell; T_reg, regulatory T cell

Fig. 3

Assessing cellular metabolism with stable isotope-labeled substrates. a ¹³C labeling patterns of TCA cycle metabolites resulting from utilization of 1,2-¹³C₂-glucose (upper left), ¹³C₆-glucose (upper right), and ¹³C₅-glutamine (lower left). Depending on the substrate used, further conclusions can be drawn on the involvement of the PPP or the cycling within the TCA cycle. b ¹³CO₂ production in PBMCs from a porcine model of acute subdural hematoma at MP before (MP1), 12 h (MP2), and 24 h (MP3) after hematoma induction. 5 × 10⁶ PBMCs/mL were incubated in chemically identical RPMI media containing either 1,2-¹³C₂-glucose, ¹³C₆-glucose, or ¹³C₅-glutamine. Supernatant was transferred to airtight vials and acidified to release CO₂ into the gas phase where ¹³CO₂ enrichment was determined by gas chromatography-mass spectrometry. Data are presented as median and interquartile range; ***p < 0.0001 according to a 2-way ANOVA and a Sidak’s multiple comparisons test, n = 5–6. Abbreviations: CO₂, carbon dioxide; MP, measurement timepoint; PBMCs, peripheral blood mononuclear cells; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle

Fig. 4

Assessment of immune cell metabolism. Panel a shows the mitochondrial oxygen consumption in a porcine model of acute subdural hematoma combined with hemorrhagic shock assessed in the uncoupled state (electron transport system capacity) before (MP1), as well as 12 (MP2) and 24 h (MP3) after trauma induction in PBMCs (gray bars) and granulocytes (white bars). 10 × 10⁶ cells/1 mL RPMI medium were added to the measurement chamber of the Oxygraph O2K (Oroboros Instruments, Innsbruck, Austria) which measures oxygen concentration with a Clark electrode. After addition of the ATP synthase inhibitor oligomycine (0.5 μL of a 0.5 μM stock solution), the uncoupling agent FCCP (0.5 μL of a 0.5 μM stock solution) was added stepwise to reach the level of maximum oxygen consumption (electron transfer system capacity). b O₂⁻ production quantified by electron spin resonance spectroscopy in PBMCs (gray bars) and granulocytes (white bars) isolated from pigs before trauma induction. Therefore, 2.5 × 10⁶ cells (in 1 mL RPMI) were mixed with the superoxide-targeted spin probe CMH. A serial measurement over 30 min enables calculation of superoxide radical production rate. An equally treated sample of RPMI with CMH served as blank value that was subtracted from the cell suspensions’ production rate. c Before trauma induction, H₂O₂ production of 1 × 10⁶ PBMCs (gray bars) and granulocytes (white bars) in 2 mL RPMI medium was electrochemically quantified with a Pt-black modified microelectrode. The data presented in b and c are adapted from [72]. d outlines the concept for our methodical approach to analyze the energy metabolism in granulocytes and PBMCs. Usually, granulocytes preferentially utilize glucose to produce ATP and have a low mitochondrial oxygen consumption but a high ROS production. PBMCs on the other hand prefer glutamine over glucose utilization, have a higher mitochondrial oxygen consumption and low ROS production. It is important to note that the metabolic pathways shift depending on the state of activation and that immune cells use different substrates (fatty acids, amino acids) in order to safeguard ATP homeostasis. Data are presented as a median with interquartile range and b, c mean with SD, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ^###p < 0.001 vs PBMC; ^####p < 0.0001 vs PBMC; according to a a 1-way ANOVA and Tukey’s multiple comparisons test, n = 6–14, and b Wilcoxon signed-rank test, n = 7–9, c n = 3 per group. Abbreviations: ATP, adenosine triphosphate; CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (spin probe); FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (uncoupling agent); MP, measurement timepoint; PBMCs, peripheral blood mononuclear cells; Pt-black, platinum black; ROS, reactive oxygen species; SD, standard deviation