Image_4_Metabolic Profiling of Human Eosinophils.tif

<p>Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO₂ 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol–myristate–acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.</p

Image_3_Metabolic Profiling of Human Eosinophils.tif

<p>Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO₂ 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol–myristate–acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.</p

Image_2_Metabolic Profiling of Human Eosinophils.tif

<p>Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO₂ 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol–myristate–acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.</p

Image_5_Metabolic Profiling of Human Eosinophils.tif

<p>Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO₂ 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol–myristate–acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.</p

Mitochondrial function is impaired in m.547A>T fibroblasts and cybrids.

<p>Oxygen consumption rate (OCR) was measured to assess mitochondrial function in fibroblasts (A, C) and cybrids (B, D), normalised for cell number. Representative comparison of fibroblasts (A) and cybrids (B) from control individuals (red) with fibroblasts or cybrids from patients with the m.547A>T substitution (blue) showing a substantial change in respiratory profile. There was a significant decrease in baseline oxygen consumption, and in maximal oxygen consumption following addition of FCCP in both fibroblasts (C) and cybrids (D). Asterisks *p < 0.05, **p < 0.005, and ***p < 0.001 for control versus patient groups, represented as the mean ± SD of separate experiments performed in triplicate with four patient and four control cell lines.</p

mt.616T>C cybrids have a defect in respiration.

<p>(A) Two pedigrees showing potential maternal inheritance of renal disease. Individuals with kidney disease are represented by filled shapes as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006620#pgen.1006620.g001" target="_blank">Fig 1</a>. Family 8 from ref 13 forms part of pedigree II, and is indicated by the dotted box. Pedigree III is Family 6 from ref 13. Individuals from whom DNA was sequenced in the current study are marked with asterisks. All affected individuals who were sequenced were found to have homoplasmic levels of the m.616T>C substitution. The mitochondrial haplotype is T1a1. (B) Measurement of oxygen consumption in patient-derived and control cybrids showing a reduction in basal (before addition of oligomycin) and maximal respiration (after addition of FCCP) in patient-derived cybrids. (C) Expression levels of tRNAs quantified by Northern blot of three control and patient-derived cybrids show reduced mitochondrial tRNAPhe levels relative to the light strand encoded tRNAGln in mt.616T>C cells.(p = 0.006) The tRNA levels for valine and leucine were unaffected. (D) Conservation of mt tRNA Phe within vertebrates. The anticodon (GAA) is highlighted in bold, the nucleotides forming the last pair of the anticodon stem are underlined. Sequences were aligned with clustal omega and manually adjusted. The stem (s) and loop (l) secondary structure of the tRNA Phe of humans is indicated at the bottom.</p

Pedigree with maternally inherited renal disease and m.547A>T substitution.

<p>(A) Pedigree of family showing individuals affected with chronic kidney disease (CKD, red symbols), and end-stage renal disease (ESRD, black symbols). One individual (yellow symbol) died many years previously age 18 with a diagnosis of Batten’s disease based on electronmicroscopy of a skin biopsy showing characteristic curvilinear bodies. We consider this to be unrelated to the renal disease (B) Renal biopsy showing evidence of focal tubular atrophy by light microscopy (arrowed). (C) Renal biopsy showing mitochondria in a renal tubular epithelial cell which appear structurally normal on electron microscopy. (D) Sanger sequencing of mtDNA from a control individual. (E) Sequence of an affected individual showing the homoplasmic m.547A>T substitution.</p

m.547A>T fibroblasts display a marked reduction in complex I, III and IV protein expression.

<p>Four individual pairs of fibroblasts from different patients and controls were SILAC labelled and the mitochondrial fraction was analysed by LC-MS/MS. The ratio of protein levels in patient versus control cells is displayed on a log2 scale. Respiratory chain proteins which were quantified in at least two pairs are shown. All identified mitochondrial-encoded proteins are annotated (ND1-5: NADH dehydrogenase subunit 1–5, CYT-B: cytochrome b, COII: cytochrome c oxidase II, ATP6: ATP synthase 6. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006620#pgen.1006620.s004" target="_blank">S4 Fig</a> for complete annotation). The size of each dot indicates the significance (adjusted p value) of the difference in abundance of the protein in the patient and control samples. Location below the line of equivalence (0.0) indicates lower abundance in the patient samples compared to controls.</p

Reduced expression of mitochondrial heavy strand transcripts in m.547A>T patient-derived cells.

<p>(A, C) RNA from patient- and control-derived cells (n = 4 each) was analysed for expression of a panel of mitochondrial tRNAs by quantitative Northern blot. The graphs show mean expression of heavy strand tRNAs relative to light strand tRNA Gln(Q). Values are expressed relative to the mean for the control cell lines in each case. Error bars indicate the standard deviation. Heavy strand tRNAPhe and tRNALeu expression is reduced relative to light strand tRNA Gln in m.547A>T fibroblasts (A, p<0.001) and cybrids (C, p<0.001 for tRNA Phe(F), Val (V) and Leu (L)). Experiments were repeated three times with equivalent results. Labelling for 5S rRNA was used to confirm equal loading. Heavy chain transcripts RNR1 (B) and CO1 (D) were measured by quantitative RT-PCR and were reduced in m.547A>T cybrids relative to the light strand transcript ND6. Each point represents the mean of several independent cybrid cell lines derived from a single donor.</p