Background
Ageing has been defined as the time-dependent functional decline in living organisms, which is primarily caused by the time-dependent accumulation of cellular and molecular damage [
1]. Dysfunctional tissue homeostasis as a result of an age-dependent reduction of stem cell function is considered a central physiological characteristic of ageing [
2]. Hematopoietic stem cells (HSCs) undergo either self-renewal or differentiation into multilineage committed progenitor cells, such as lymphoid or myeloid lineages of the immune system including T cells, B cells, neutrophils, natural killer (NK) cells, and antigen presenting cells (APCs) including monocytes, macrophages and dendritic cells (DCs) [
3]. Reduction of HSC function with age leads to age-related deficiencies of both adaptive and innate immune systems, a process called “immunosenescence” [
2,
4]. Immunosenescence leads to subclinical accumulation of pro-inflammatory factors and inflammageing, which is defined as a chronic, sterile, low-grade inflammation [
5]. Immunosenescence and inflammageing collectively contribute to most of the diseases of the elderly, with increasing risk of infections, cancer, autoimmune disorders, and chronic inflammatory diseases [
5].
Recently, extracellular vesicles (EVs) have emerged as key indicators and effectors of immune function [
6]. EVs are released by almost all mammalian cells, and carry surface markers and effectors (eg. cytoplasmic proteins, DNA, mRNA, miRNA, small non-coding RNAs, mitochondria, and cytokines) from their parent cells [
7‐
9]. Although EVs are delimited by a lipid bilayer [
7,
8], they are distinguished from their parent cells by an inability to replicate [
10]. Characterizing EVs in plasma during ageing may help to understand the lifespan and healthspan of their parent cells and presumably the organism. Plasma concentrations of EVs decline with age, while mechanisms for this are still largely unknown. Increased internalization of EVs from older individuals by B cells is one of the mechanisms contributing to this decline [
11]. EVs have been posited to be mediators of cell-to-cell communication and paracrine effectors through transfer of proteins, lipids and nucleic acids in their cargo [
7,
8] to other cells. EVs also carry both damaged and functional mitochondria in their cargo [
12]. Ageing is generally accompanied by a decrease in respiratory capacity of mitochondria [
13]. Defective mitochondrial function can contribute to ageing in mammals through reactive oxygen species generation [
1]. Impaired mitochondrial function is commonly observed in many types of ageing-associated neurodegenerative diseases [
14]. We hypothesized that specific surface markers and mitochondrial content of circulating EVs reflect the state of immune function of their parent cells that can be used to monitor age-related changes of immune function at an organismal level. Therefore, our goal in this study was to develop a methodology to extensively characterize the size, surface markers and cargo of EVs from stem cells and immune cells across the lifespan of healthy individuals to better understand the process of immunosenescence with ageing.
Discussion
In this study, we identified three major subsets of plasma EVs derived from HCs distinguished on the basis of their size, including LEV, MEV and SEV. The three EV populations share some surface markers but can be differentiated based on the combination of size, surface markers and cargo. Consistent with findings from another group [
49], we identified traditional EV markers CD81, CD9, CD29 and CD63 on all sizes of plasma EVs from HCs. This contrasts with previous studies in which CD9, CD63 and CD81 were considered specific markers for small EVs (20–100 nm) termed exosomes [
7,
8,
10,
49]. Among these traditional EV markers, CD81 was highly expressed by LEV, while the percentage of CD9
+, CD29
+ and CD63
+ EVs were low in plasma of HCs. Of note, using the same detection antibodies, we have observed higher percentages of CD81
+,CD9
+, CD29
+ and CD63
+ EVs in human synovial fluid compared to matched plasma in another cohort (higher by 4.3-fold, 3.7-fold, 3.6-fold and, 10-fold, respectively;
n = 16 pairs). Therefore, the observed low expression in plasma appears not to be related to technical limitations but rather is dependent upon and varies by bio-fluid type and individual tissue and cell phenotypes.
CD34+ EVs associated with HSCs and progenitor cells were abundant in plasma from HCs and present in all sizes of EVs. In addition, plasma LEVs highly expressed HLA-ABC, CD81 and CD14, and carried pro-inflammatory cytokines IL-1β, IL-6 and IL-17A. In contrast, SEVs mainly carried IFN-γ and anti-inflammatory cytokine IL-10. The distinct surface marker expression pattern and cytokine cargo, particularly comparing LEV versus MEV and SEV, suggested that plasma EV subsets may carry different bio-messages and originate by different biogenesis pathways.
We noted several specific EVs of immune cells that declined with ageing, including multiple MEV and SEV subsets carrying surface markers of B cells, T cells, NK cells, and APCs. Many immune cells decline with age, including B cells, T cells, neutrophils, NK cells, and APCs [
2,
4]. The age-related decline of plasma EVs of these immune cells during normal human ageing may be a sensitive indicator for the age-associated defects in their parent cells during immunosenescence and inflammageing [
2,
4]. The selective decline in plasma EVs of immune cells might also result from increased demands with ageing for the parent cells or uptake of EVs at a tissue level, thereby reducing the measurable circulating EV population [
11].
Since all hematopoietic cells are derived from HSCs, immunosenescence, the age-dependent decline in immune cells, could be attributed to the dysfunctional activity of HSCs in the aged. However, although hematopoiesis declines with age [
2,
4], CD34
+ plasma EVs, potential indicators of HSCs, did not decrease with age in healthy controls. In addition, a high number of CD34
+ plasma EVs carried functional respiring mitochondria, which were also not affected by ageing. By contrast, we observed age-associated declines in respiring mitochondria in the cargo of EVs associated with certain types of stem cells (such as CD29
+ adipose-derived stem cells, CD29
+HLA-G
+ mesenchymal stem cells and CD31
+ HSCs) and immune cells. The decline with age in the respiring mitochondrial cargo of multiple EV subpopulations occurred in EVs whose total number declined with age as well as in EVs that did not decline with age.
EVs have been classified into three main groups: shedded vesicles that originate directly from the plasma membrane, apoptotic body-vesicles that originate from disintegrating cells during controlled cell-death, and exosome-vesicles that originate from multivesicular organelles/multivesicular bodies inside cells [
54]. Since the majority of EVs isolated in our study contained functional mitochondria, our data suggest that the vast majority of circulating EVs in plasma are exosome vesicles and/or apoptotic-body vesicles since it is known that subsets of apoptotic bodies can contain mitochondria [
55].
Mitochondria contribute to cellular ageing through the modulation of the metabolic profile of the cell [
13]. When a depletion of the mitochondrial genome or accumulation of misfolded proteins within the mitochondria induce a stress response pathway, mitophagy, the autophagy of mitochondria, appears to mitigate deleterious consequences of mitochondria DNA mutation accumulation and promote longevity [
13]. Our observation of decreased production of EVs with respiring mitochondria with age may indicate age-associated defects of mitophagy with age; this would lead to accumulation of mitochondria DNA mutations and misfolded proteins inside cells, which may in turn cause cellular ageing. Mitochondrial dysfunction-mediated-hyperproduction of reactive oxygen species [
56], together with immunosenescence and inflammageing can lead to apoptosis of immune cells [
57]; as a result, plasma EVs from these immune cells may decrease. This observation further supports the hypothesis that the parent cells of these EVs may experience age-dependent mitochondrial dysfunction-induced cell death during immunosenescence [
2,
14]. Mitochondrial respiration generates adenosine triphosphate (ATP), which is essential for regulation of cell death by apoptosis [
58]. Mitochondria are swollen and disrupted in cells with necrosis [
59]. Although mitochondria have nearly normal appearance in apoptotic bodies and cells with apoptosis, mitochondria release cytochrome c to the cytosol where it participates in the formation of the caspase-activating complex––apoptosome [
59]. Disruption of the electrochemical gradient across the mitochondrial transmembrane can promote caspase activation and execution of apoptosis [
60]. Thus, our observation of a marked decreased production of EVs in specific immune cells with age may represent a cause and/or effect of ageing.
We developed a methodology to characterize plasma EVs of a wide range of sizes using high resolution flow cytometry. High resolution flow cytometry is one of the recommended methods for measuring EVs from large to small sizes [
11]. Because a clear, meticulous definition of EVs has not yet been established [
61], it is currently difficult to define EV subtypes, such as a type related to ageing with a particular biogenesis pathway [
10]. Terms such as “exosomes”, “microvesicles” and “apoptotic bodies” are frequently used in the literature to describe EV subtypes, although there are currently no uniform definitions of these EV subtypes based on size, density, shape, cargo or function [
7,
8]. With increased understanding of EVs, it is now generally agreed that there are no specific markers of EV subtypes [
10]. Therefore, the International Society for Extracellular Vesicles has recommended use of operational terms for EV subtypes that refer to: physical characteristics of EVs, such as size or density, with ranges defined; or biochemical composition, such as CD81
+EVs; or descriptions of conditions or cell of origin such as podocyte EVs or hypoxic EVs [
10]. For this reason, we characterized plasma EVs by a combination of their size, surface markers and cargo. Calibration beads can be useful tools for forward scatter-based sizing of nano-sized vesicles by flow cytometry. However, results from forward scatter-based sizing of nano-sized biological vesicles and similarly sized calibration beads can conflict, depending on multiple factors including the composition of the studied vesicles and beads, and the forward scatter collection angles of the flow cytometer [
16]. Therefore, our reported EV sizes represent approximations, although the relative sizes (small to large EVs) are valid.
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