Existence of Circulating Mitochondria in Human and Animal Peripheral Blood Mitochondria are usually located in the cytoplasm of cells where they generate adenosine triphosphate (ATP) to empower cellular functions.

Xiang Song, Wei Hu, Haibo Yu, Honglan Wang, Yelu Zhao, Robert Korngold and Yong Zhao *
Center for Discovery and Innovation,

Hackensack Meridian Health, Nutley, 07110 NJ, USA; Xiang.Song@HMH-CDI.org (X.S.); whu2@stevens.edu (W.H.); Haibo.Yu@HMH-CDI.org (H.Y.); yeluzh@gmail.com (Y.Z.); Robert.Korngold@HMH-CDI.org (R.K.) * Correspondence: Yong.Zhao@HMH-CDI.org; Tel.: +201 880 3460
Received: 18 February 2020; Accepted: 17 March 2020; Published: 19 March 2020

Abstract

Mitochondria are usually located in the cytoplasm of cells where they generate adenosine triphosphate (ATP) to empower cellular functions. However, we found circulating mitochondria in human and animal blood. Electron microscopy confirmed the presence of mitochondria in adult human blood plasma. Flow cytometry analyses demonstrated that circulating mitochondria from the plasma of human cord blood and adult peripheral blood displayed the immune tolerance-associated membrane molecules such as CD270 and PD-L1 (programmed cell death-ligand 1).

Similar data were obtained from fetal bovine serum (FBS) and horse serum of different vendors.

Mitochondria remained detectable even after 56 °C heat inactivation. A real-time PCR array revealed purified mitochondria from animal sera expressed several genes that contribute to human T- and B-cell activation.

Transwell experiments confirmed the migration capability of mitochondria through their expression of the chemokine receptor CXCR4 in responses to its ligand stromal-derived factor-1α (SDF-1α).

Functional analysis established that human plasma mitochondria stimulated the proliferation of anti-CD3/CD28 bead-activated PBMC, up-regulated the percentage of activated CD4+ T and CD8+ T cells, and reduced the production of inflammatory cytokines.

These findings suggested that the existence of circulating mitochondria in blood may function as a novel mediator for cell-cell communications and maintenance of homeostasis.

Plasma-related products should be cautiously utilized in cell cultures due to the mitochondrial contamination.

1. Introduction

For decades, mitochondria have been considered as “a cellular power plant” to generate ATP through oxidative phosphorylation and capable of energizing cellular activities.

Dysfunction of mitochondria can result in cell apoptosis/death or various cellular stresses including elevated reactive oxygen species (ROS) production, which can contribute to metabolic syndrome, diabetes, autoimmune diseases, and cancer.

To date, increasing evidence has revealed the diverse functions of mitochondria in maintaining cell hemostasis through dynamic mitochondrial fusion/fission, metabolic control, interactions with other organelles and the modulation of the nuclear genome [1,2].

Recently, the horizontal transfer of mitochondria have been reported between adjacent cells through the formation of tunneling nanotubes (TNT), cell fusion, GAP junctions, and microvesicles [3].

Under pathological conditions, free or microvesicle-associated mitochondria could be released by the activated monocytes [4], diseased organ cells [5], and oxidative-stressed mesenchymal stem cells [6]. However, it is unknown whether mitochondria are physiologically presented in the blood.

We previously identified multipotent stem cells from human cord blood (designated cord blood-derived multipotent stem cells, CB-SC) [7] that phenotypically exhibit embryonic transcription factors (e.g., OCT3/4 and SOX2) that distinguish them from mesenchymal stem cell (MSC) and hematopoietic stem cells (HSC) [8].

Based on the comprehensive immune modulation features of CB-SC [9,10], we developed a “stem cell educator (SCE) therapy” that uses allogeneic CB-SC to “educate” a patient’s immune cells, affecting the progression of autoimmunity in subjects with type 1 diabetes (T1D) [9] and other autoimmune diseases such as alopecia areata [11].

SCE therapy provided lasting improvement of pancreatic islet β-cell function and C-peptide (a by-product of insulin production) levels [9].

While exploring clinical mechanisms underlying the long-lasting therapeutic effects of SCE therapy [12] in ex vivo studies, we found that mitochondria released from platelets can migrate to pancreatic islets and be taken up by human islet β cells, leading to the improvement of islet β-cell function and C-peptide production [12], supporting the clinical outcomes of the improved health status in patients with type 1 [9,13] and type 2 diabetes [14].

Platelets along with erythrocytes (red blood cells, RBC) are the largest components of blood and whereas platelets have functional mitochondria, mature RBC do not have them [15]. Therefore, we hypothesized that there were platelet-released mitochondria circulating in the blood. Here, we demonstrate the presence of mitochondria in human and animal blood by flow cytometry and electronic microscopy.

2. Results

2.1. Characterization of Cord Blood (CB)- and Adult Peripheral Blood (PB) Plasma-Derived Mitochondria Initially, plasma mitochondria were purified from fresh plasma of cord blood and adult peripheral blood by using the optimized protocol with a serial centrifugation and filtration (Figure 1A). Using human platelets’ mitochondrial staining as positive control, flow cytometry established that MitoTracker Deep Red-labeled CB- and PB-derived mitochondria expressed the mitochondrion-specific marker HSP60 (Figure 1B). The percentages of MitoTracker Deep Red+HSP60+ mitochondria were 80.11% ± 10.16% for CB-derived mitochondria (N = 4) and 74.73% ± 5.06% for PB-derived mitochondria (N = 4), relative to the positive control from purified platelet-derived mitochondria (81.85% ± 15.72%, N = 4).

Additional phenotypic characterization confirmed that these purified, CB- and PB-plasma-derived mitochondria displayed immune tolerance-associated membrane molecules such as CD270 (HVEM, Herpesvirus entry mediator), with 85.76% ± 13.22% and 79.37% ± 8.18% of MitoTracker Deep Red+CD270+ mitochondria for CB- (N = 8) and PB-derived mitochondria (N = 9) respectively, and CD274 (programmed cell death-ligand 1, PD-L1), with 81.32% ± 7.39% and 64.33% ± 10.45% of MitoTracker Deep Red+CD274+ mitochondria for CB- (N = 8) and PB-derived mitochondria (N = 9) respectively (Figure 1C), as previously characterized on platelet-derived mitochondria (Figure 1C, top panels) [12]. Electron microscopy confirmed the presence of mitochondria in adult blood plasma and showed that the double membranes and mitochondrial cristae were intact with oval (Figure 1D) or elongated morphology (Figure 1E). These data proved the existence of mitochondria circulating human peripheral blood. . . . .