Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly-defined immune cell subtypes, we quantify the natural variation in citrate synthase, mitochondrial DNA copy number (mtDNAcn), 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 4 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 sub-populations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

Background:Hair greying is a hallmark of aging generally believed to be irreversible and linked to psychological stress.Methods:Here, we develop an approach to profile hair pigmentation patterns (HPPs) along individual human hair shafts, producing quantifiable physical timescales of rapid greying transitions.Results:Using this method, we show white/grey hairs that naturally regain pigmentation across sex, ethnicities, ages, and body regions, thereby quantitatively defining the reversibility of greying in humans. Molecularly, grey hairs upregulate proteins related to energy metabolism, mitochondria, and antioxidant defenses. Combining HPP profiling and proteomics on single hairs, we also report hair greying and reversal that can occur in parallel with psychological stressors. To generalize these observations, we develop a computational simulation, which suggests a threshold-based mechanism for the temporary reversibility of greying.Conclusions:Overall, this new method to quantitatively map recent life history in HPPs provides an opportunity to longitudinally examine the influence of recent life exposures on human biology.Funding:This work was supported by the Wharton Fund and NIH grants GM119793, MH119336, and AG066828 (MP).

How mitochondria functionally differ between immune cell subtypes, between the sexes, across ages, and whether they dynamically change over time has not been characterized. Here we deploy a high-throughput mitochondrial phenotyping platform to define cell-type specific mitochondrial features in circulating immune cell subtypes. In women and men spanning four decades of life, we find that mitochondrial content, mitochondrial DNA copy number (mtDNAcn), and respiratory chain enzymatic activities vary by up to 3.5-fold between neutrophils, monocytes, B and T lymphocyte subtypes. Within individuals, mitochondrial content and mtDNAcn, and to a lesser degree respiratory chain function, are strongly correlated with each other across immune cell types, suggesting their systemic harmonization. Moreover, repeated weekly measurements of mitochondrial features in the same individual reveal remarkable variation in mitochondrial content and respiratory chain function over time. This suggests that immune cell mitochondria exhibit dynamic state properties, which we find to be partially correlated with circulating biomarkers. We also define multivariate mitochondrial phenotypes - mitotypes - that distinguish lymphoid from myeloid cell types, naïve-to-memory lymphocyte states, and moderately differ between women and men. Finally, we compare mitochondrial features of purified cell subtypes to peripheral blood mononuclear cells (PBMCs) and determine the influence of contaminating platelets and of variable cell type composition. Our results invite caution in using cell type mixtures to infer person-level mitochondrial behavior. Together, these findings identify dynamic cell-type specific variation in mitochondrial biology among circulating leukocytes and provide foundational knowledge to develop interpretable blood-based assays of mitochondrial health.

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Mitochondria release their genome as cell-free mitochondrial DNA (cf-mtDNA) in multiple biofluids including in the blood, where it predicts mortality and is a marker of mental and physical stress. Here we report cf-mtDNA in human saliva, an accessible biofluid used to study dynamic neuroendocrine changes. To map the natural dynamics of salivary cf-mtDNA over time, we examine cf-mtDNA and steroid hormones in a small cohort of healthy adults, and perform an intensive repeated-measures analysis of two healthy men studied at 4 daily timepoints over 53-60 consecutive days (n=412-420 observations). Salivary cf-mtDNA exhibits a robust awakening response reaching up to two orders of magnitude 30-45 minutes after awakening, varies from day-to-day, and moderately correlates with the cortisol awakening response. Moreover, we find no evidence that salivary cf-mtDNA has pro-inflammatory effects. The dynamic behavior of salivary cf-mtDNA opens the door to non-invasive studies examining the relevance of mtDNA signaling on human health.