1. Introduction
Currently, it is well-known that extracellular vesicles (EVs) play a role in diverse cellular communication processes. Initially, when they were discovered, no function could be identified. As shown in the timeline (
Figure 1), the first observation of multivesicular bodies (MVBs), and thus of (intraluminal) exosomes which are the smallest class among EVs, occurred in the 1950s. MVBs were first recognized in algae [
1] and mammalian cells [
2]. At the same time, outer membrane vesicles (OMVs) were found in bacteria [
3,
4]. Nearly ten years after the detection in algae, in 1965, MVBs were found in higher plants [
5]. Again, after almost a decade, in 1973, EVs were reported in fungi [
6]. At that time, probably none of the researchers recognized the significance of the discovered structures. About two decades later, by investigating reticulocytes, exosomes were thought to facilitate cells getting rid of garbage. It was assumed that they simply “defenestrate” remnants, instead of degradation [
7,
8,
9,
10]. This assumption of waste disposal probably resulted from the juvenile state of research on EVs at that time. The year 1996 marked a turning point in the thinking of EVs, when Raposo et al. assumed EVs influenced antigen presentation in vivo [
11]. Since then, EVs were no longer thought to function only as “trash cans”.
The ubiquity of EVs in all empires of life was confirmed in 2000, when they were accounted for in archaea [
12]. In recent years, much knowledge on EVs has been gathered. Loaded with bioactive proteins, lipids, and nucleic acids, EVs enable long distance communication between cells. Information can either be transmitted by EV uptake (membrane fusion, endocytosis) into the recipient cell or via receptor interaction on the cell surface [
13,
14]. Because EVs shuttle nucleic acids, they facilitate post-transcriptional regulation of the recipient cell metabolism. It was revealed, in 2007, that EVs carry messenger RNA (mRNA) and small noncoding RNA (sRNA), enabling cells to exchange genetic information, and therefore the thinking of intercellular communication was renewed [
15,
16,
17,
18]. The key regulators are sRNAs, a class of single stranded RNA, comprising around 22 nucleotides. This sRNA binds complimentarily to mRNA, resulting in post-transcriptional gene silencing [
19,
20,
21]. EVs protect this fragile cargo from degradation by RNases and appear to be responsible for targeting recipient cells [
20,
22,
23,
24,
25,
26,
27,
28]. Compared to cell-cell communication, utilized by low-molecular messenger substances (e.g., hormones), EVs further provide vehicles transmitting cargo in high concentrations, unaffected by diffusion or dilution [
29].
EVs were observed throughout all empires of life, underlining their high evolutionary importance, however, to date, the major question about EVs remains unanswered, i.e., "What is their elementary function?” Although we cannot solve this conundrum, in this review, we have compiled information on EVs from different origins and vesicular cross talk between individuals, species, and even kingdoms.
4. Inter-Individual, Interspecies, and Inter-Kingdom Regulation
After the observation that EV-mediated information transfer is not limited to one organism, species or kingdoms, the one central question of EV research became, “Why can EVs overcome kingdom boundaries?” Investigations on EV-mediated regulation processes, from mother–infant to host–pathogen interaction, might elucidate this query (
Table 2).
In addition to mitogenic lipids and signaling proteins, sRNAs are considered to be crucial regulatory elements in EV-mediated (inter-kingdom) communication [
99]. They are able to manipulate various biological processes, such as cell growth, differentiation, development, metabolism, and apoptosis [
19,
20]. Stability and absorption of sRNA are obviously critical aspects of bioavailability for recipient organisms or cells. In contrast to traditional persuasions on the stability of extracellular RNA, a few studies have shown surprisingly high pH-, temperature-, and RNase-resistances for sRNA in mammalian body fluids [
26,
190,
191,
192,
193,
194], as well as for plant sRNAs [
21,
80,
96,
195,
196]. The vesicular envelope of EVs is thought to be decisive for the enhanced sRNA stability. This assumption is strongly underlined by the fact that severe losses of sRNA are detectable after pasteurization and homogenization or after ultrasonic exosome depletion of bovine milk [
28,
175,
197]. Furthermore, the envelope also provides a vehicle for cellular uptake of the cargo, not only in the intestine [
28,
175,
176,
192,
198,
199,
200].
Since EVs have been found in the milk of distinct mammals, such as pork, cow, or human, increasing numbers of inter-individual and interspecies regulation processes are being assumed highly probable [
28,
192,
201,
202,
203,
204]. Moreover, increased serum levels of bovine milk specific sRNA were detected in humans after consumption of cow´s milk [
175]. Until today, we are lacking reliable studies on physiological or pathological effects of ingested EVs on humans, while a broad range of such effects is conceivable. This assumption is supported by investigations that have shown that a breastfed infant profits from ingested milk-derived sRNAs by elevated T-cell levels and enhanced differentiation of B cells [
20,
28,
192,
201].
Although there has been previous evidence for inter-kingdom regulation mediated by sRNAs [
205,
206,
207,
208], the study by Zhang et al., 2012 was somehow paradigm shifting. Their finding, that the dietary uptake of a particular plant-derived micro RNA can measurably affect the metabolism of a mammal [
189], quickly ignited increased interest in this field.
Probably, fungal cells send EVs in order to downregulate host immune response. Observations in both human–fungus and plant–fungus interactions suggest fungal virulence to be strongly enhanced by inter-kingdom RNA interference, enabled by sRNA containing EVs [
124,
172,
179,
181,
209]. Conversely, plants send sRNA to silence fungal virulence genes, which has recently also been related to EVs [
27,
91,
97,
106,
210,
211].
In the area of difficult-to-treat infections, OMVs play a major role in drug resistance because they transfer resistance genes (DNA) between bacteria, even of different origin [
148]. Many OMVs from pathogenic bacteria were found to have surface proteins, which can readily interact with mammalian host cells. These interaction mechanisms make OMVs a pivotal element of trans-kingdom and host-cell communication by letting them interact in a highly specific manner [
212]. OMVs have been shown to carry PAMPs, including lipopolysaccharides, and can transfer other virulence associated factors [
213]. These factors can trigger strong immune responses in host cells, while OMVs act as immunomodulators, for example, by leading to expression of receptors on macrophages to specifically recognize the pathogen [
214]. As OMVs can help pathogenic bacteria to persist attack by the mammalian immune system, they strongly contribute to the cause of infectious disease [
174,
182]. Prokaryotic pathogens such as
Bacillus anthracis Cohn [
183],
Helicobacter pylori (
Marshall) Goodwin [
184],
Neisseria gonorrhoeae (Zopf) Trevisan [
185],
Pseudomonas aeruginosa (Schroeter) Migula [
186], and
Streptococcus pneumoniae (Klein) Chester [
187], as well as eukarytotic pathogens such as
Leishmania spp.
Ross [
215],
Plasmodium spp.
Marchiafava et Celli [
216], and
Trichomonas vaginalis Donné [
217] similarly send EVs to increase their contagiousness [
188,
218,
219,
220]. This phenomenon is not limited to unicellular organisms, since helminths also modulate host immunity, as
Heligosomoides polygyrus Dujardin [
26] and
Dicrocoelium dendriticum Rudolphi [
177].
Overall, EVs appear to be potent agents in regulation processes, crossing not only the borders of species but rather of kingdoms or even empires. Therefore, they enhance an arms race in host–pathogen interaction [
106,
180]. But do exosomes also facilitate intercellular communication beyond the animal kingdom? Especially host–pathogen interactions imply the possibility of host-host and pathogen-pathogen signaling, intended to improve the chance of survival on each side (
Figure 4). A better understanding of host-pathogen interactions can elucidate unknown mechanisms, and therefore future targets, improving therapies of infectious diseases.
5. Conclusions
Because shedding of EVs has been found to be ubiquitous throughout all empires of life, it appears to be evolutionarily advantageous. The abundance of homologous proteins in distinct kingdoms clearly indicates that the release of membranous vesicles is evolutionary highly conserved. Independently from their origin, EVs can be loaded with a wide range of drugs, including chemotherapeutic compounds, DNA expression vectors, sRNA, and proteins such as antibodies, and have been shown in vivo to deliver their cargo and to protect the therapeutic agent from degradation [
71,
102,
221,
222]. Because the application of EVs can either increase or decrease the in vitro viability of cells, the bioactive cargo seems to be responsible for the triggered effects. Lacking cytotoxic effects, edible plant-derived EV lipids are interesting for the development of nanovectors regarding drug delivery. But since our knowledge of the comparability of EVs from different kingdoms is limited, comprehensive EV research regarding multiple organisms offers a better understanding of the entire field.
The EV shell is generally assumed to be crucial for the stability of sRNA or rather the complete cargo. In animals, EVs are widely thought to facilitate intercellular communication, but we can only speculate about the “genuine intention” of EV release. There is evidence that EV-mediated inter-kingdom regulation is more than a random event. It seems to be more likely that cells release EVs in order to control (remotely) or influence their environment. Possibly, plants are using EVs as a defense strategy against invading fungi, while fungi for their part enhance their own virulence. Therefore, the composition of membrane lipids and proteins seems to be crucial for addressing the intended target cell, tissue, or organism. EVs consist of a complex and mutually well-coordinated mixture of biomolecules. They can be assumed to be Janus-faced natural products and it is on us to use this instrument in a responsible manner. Currently, we are at the beginning of a developing field and a comprehensive view on the issue could help overseeing complex linkage. Around 20 years ago, a couple of researchers realized EVs to be more than tiny garbage bags. They recognized their broad capability and kept going deeper into the unknown. As a result, we find ourselves today with very detailed knowledge on human exosomes. Unfortunately, this knowledge cannot be transferred one-for-one from animal EVs to other kingdoms, but those other fields can profit from well-established methods. This will ease the way towards unpredictable findings. Thus, now, we need the same pioneering spirit and courageousness to create a more general point of view, in order to fully exploit the potential of the cross-linking vehicles we try to decrypt.