Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly defined immune cell subtypes, we quantify the natural variation in mitochondrial DNA copy number (mtDNAcn), citrate synthase, and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naïve and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning four decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte subpopulations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

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Published in eLife, v. 10, e70899.

© 2021, Rausser et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Funding Information

Work of the authors is supported by the Wharton Fund and NIH grants MH119336, GM119793, MH122706, AG066828, AG056635, AG026307, and UL1TR001873. These studies used the resources of the Irving Cancer Center Core Facility funded in part through center grant P30CA013696.

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All data generated and analyzed during this study, including mitochondrial biochemistry, mtDNA content, and blood chemistry, cell counts from CBC and flow cytometry, and de-identified participant information are included in the supporting data files. Source data files have been provided for Figures 1–9, and for figure supplements (Figure 1—figure supplement 3, Figure 2—figure supplement 1, Figure 4—figure supplement 1, Figure 4—figure supplement 2, Figure 6—figure supplement 1, Figure 6—figure supplement 2, Figure 6—figure supplement 3, and Supplementary file 3). Requests for resources or other information should be directed to and will be fulfilled by the corresponding author.

elife-70899-supp1-v2.xlsx (11 kB)
Supplementary file 1: Leukocyte subtypes included in the study. Immune cell subtypes included in this study, including a brief summary of their functions and cell surface markers used for immunolabeling and FACS. FACS, fluorescence-activated cell sorting.

elife-70899-supp2-v2.xlsx (14 kB)
Supplementary file 2: CBC- and FACS-based cell proportions for all study participants and time points. Participants are ordered by age. CBC measurements were performed using a Sysmex XN-9000 instrument, and FACS-based cell proportions were determined using a BD Influx cell sorter (see Appendix 1 for details). CBC, complete blood count; FACS, fluorescence-activated cell sorting.

elife-70899-supp3-v2.xlsx (9 kB)
Supplementary file 3: Technical variation for each mitochondrial assay and calculated MHI by cell type. Coefficients of variation (CVs) across 2–5 biological replicates (different 5 M cell pellet isolated from the same blood draw) for each cell subtype and PBMCs (see Appendix 1 for details). MHI, mitochondrial health index; PBMC, peripheral blood mononuclear cell.

elife-70899-supp4-v2.xlsx (10 kB)
Supplementary file 4: Recipes for antibody cocktails used to detect cell surface markers for FACS-based cell proportions and sorting (see Appendix 1 for details). FACS, fluorescence-activated cell sorting.

elife-70899-transrepform1-v2.docx (247 kB)
Transparent reporting form

elife-70899-supp5-v2.xlsx (55 kB)
Source data 1: Technical variation for each mitochondrial assay and calculated MHI by cell type.

elife-70899-app2-fig1-data1-v2.xlsx (30 kB)
Appendix 2—figure 1—source data 1: Mitochondrial health index and coherence of mitochondrial features across cell subtypes.