Exosomes: intriguing mediators of intercellular communication in the organism’s response to noxious agents

Exosomes are highly active during the pathological processes in an organism. They are predominantly released by immune cells, which thereby regulate the course of various pathogen infections. Research has revealed and confirmed an extremely important regulatory mechanism of exosome action, which is based on a bidirectional process: promoting infection and promoting antiinfection. In both cases, exosomes serve as a bridge for intercellular communication or the connection between pathogens and host cells by transferring various signalling molecules, thus facilitating the transmission of information (1).

Exosomes can promote infection through three mechanisms: (1) by mediating further infection through the transfer of pathogenassociated molecules, (2) by participating in immune evasion by the pathogen, and (3) by inhibiting immune responses through the apoptosis of immune cells. On the other hand, exosomes can stimulate anti-infection through two mechanisms: (1) by inhibiting pathogen proliferation and (2) through direct infection, inducing an immune response related to the function of monocytemacrophages, natural killer (NK) cells, T cells, and B cells (1, 5).

There is concrete evidence, based on studies involving bacteria, that exosomes mediate further infection in the organism by transferring pathogen-derived molecules (1, 20–22).

The potent pathogen Staphylococcus aureus causes pneumonia and, in some cases, sepsis, as well as infections of the bones and joints. It has been demonstrated that exosomes derived from neutrophils treated with S. aureus can induce an anti-inflammatory response in macrophages, leading to the production of interleukins such as IL-6 and IL-1β (21). Exosomes derived from Staphylococcus aureus-infected cells contain bacterial virulence factors, specifically the α-toxin molecule, and deliver it to distant cells within the organism (1, 23). Similarly, exosomes from Bacillus anthracis-infected cells transfer the virulence factor of lethal toxin to remote sites from the infection (24). A particularly intriguing mechanism is observed in the digestive system regarding the role of exosomes in Helicobacter pylori infection. Exosomes secreted from gastric epithelial cells expressing the Cytotoxin-associated gene A (CagA) contain the CagA virulence factor. These exosomes enter the circulation and “deliver” the CagA virulence factor to other organs and tissues (Table 1) (1, 25).

Furthermore, research focused on the lipid content of exosomes derived from macrophages before and after infection with Salmonella typhimurium revealed significant differences. The variations primarily involved glycerophospholipids, sphingolipids, and prenol lipids. These findings further support the signalling role of exosomes in pathogenic infections, contributing to the understanding of hostpathogen interactions (20).

Exosomes have been extensively studied in bacterial pneumonia. It has been established that in tuberculosis, exosomes isolated from cells infected with Mycobacterium tuberculosis promote the recruitment and activation of immune T cells. In this way, they contribute to the innate immune response (21, 26).

In pneumonia caused by Streptococcus pneumoniae, or pneumococcal pneumonia, the bacterium produces the pneumolysin toxin, which is the primary causative agent of the inflammatory processes. In this case, it was found that exosomes derived from neutrophils exposed to pneumolysin can cause direct activation of platelets. This subsequently results in a significant increase in the number of platelets expressing CD62P (P-selectin present in megakaryocytes) and a higher surface expression of CD62P compared to platelets incubated with exosomes derived from untreated neutrophils (21, 27).

Pseudomonas aeruginosa is a pathogen known to form antibioticresistant biofilms. Studies have shown that the microRNA (miRNA) type let-7b-5b, carried by exosomes derived from respiratory epithelial cells, can act as RNA interference to reduce the ability of P. aeruginosa to form biofilms, thereby increasing the susceptibility of this pathogen to the beta-lactam antibiotic aztreonam (21, 28).

Another causative agent of pneumonia, Legionella pneumophila, has been studied in the context of exosome function. Exosomes collected from the supernatant of Tamm-Horsfall Protein-1 (THP- 1) cells infected with L. pneumophila were used to infect healthy cells. The results showed an enhanced response in cells with increased expression of interleukin IL-8, tumour necrosis factor-alpha (TNF-α), interleukin IL-1β, and monocyte chemoattractant protein-1 (MCP-1), compared to uninfected cells (29).

The connection between viral infections and the formation of exosomes in the body was proposed as a theory around 20 years ago. Given that early definitions of exosomes referred to them as “cellular waste”, some studies even suggested that viruses and exosomes were essentially the same, with viruses being fully exosomes in every sense of the word (30). During the period of heightened scientific interest in the HIV virus, some studies suggested that retroviruses had “hijacked” and utilized the intercellular communication system for their spread, and even for biogenesis and replication (21). It is undeniable that this communication system referred to exosomes, and in this regard, research began to take the correct direction (30).

Subsequent findings (1, 31) revealed that exosomes isolated from cells infected with human immunodeficiency virus-1 (HIV-1) and human T-cell leukaemia virus-1 (HTLV-1) contain proteins of both viral and cellular origin, which inhibit the function of target cells, as well as dsRNA/ssRNA, and can increase the expression of certain genes, thereby promoting the spread of infection.

Target cells can be altered by exosomes at the protein or nucleic acid level, and the result is pathological consequences within cells and tissues (Table 1) (5, 32).

Recent studies have shown that exosomes serve as carriers of various viral components, including proteins, mRNA, microRNA (miRNA), and lipids. Exosomes transport these components to distant locations, delivering them to specific target cells, which then take up this bioinformation. For example, exosomes originating from the immune system, specifically T-cell lines infected with HTLV-1, carry and “deliver” the viral transactivator Tax, which can activate transcription in target cells (1, 33). Exosomes derived from cells infected with human herpesvirus 6 (HHV-6) contain fully mature virions and, through their transport role, facilitate more efficient viral spread (Table 1) (34, 35).

Exosomes can also transport prions. It is well known that prions are infectious particles or macromolecules that can cause transmissible spongiform encephalopathies (TSEs) in mammals. The yeast Saccharomyces cerevisiae, which can contain several types of prions, represents an ideal research model in that regard. The yeast prion prototype contains the translation termination factor Sup35. Findings of a study (36) demonstrated that cytosolic Sup35 NM prions were “packaged” into exosomes. Exosomes with such content can transfer the prion phenotype to neighbouring cells.

When we talk about the nucleic acid content carried by exosomes, it may originate from viral particles or be circular RNA (cRNA), which – based on research – has been strongly associated with tumour progression. In this context, exosomes are considered potential biomarkers for early diagnosis and disease monitoring, particularly in cancer (37–40). Epstein-Barr virus has been studied in the context of its transfer and correlation with exosomes. It was discovered that the latent membrane protein derived from cancer cells contaminated with Epstein-Barr virus can be exported in exosomes. Exosomes with such content inhibit the activity of natural killer cells and T-cell proliferation. Exosomes play a crucial role in the pathogenesis of human papillomavirus (HPV) tumours. In patients with this disease, different levels of miRNA content have been observed compared to the healthy population. It has been established that exosomes containing this miRNA content can regulate cell proliferation and apoptosis (35, 41).

Recently, research related to the correlation between exosomes and COVID-19 was conducted in several patient groups (fully recovered, asymptomatically recovered, moderately recovered, and severely/critically recovered) (42). It was primarily focused on lipid profiles in exosomes. Significant correlations were found regarding lipid content differences across the patient groups. Lipid abnormalities were also linked to glycerophospholipid metabolism and the biosynthesis of glycosylphosphatidylinositol. Furthermore, lipid analysis revealed that recovered patients from all groups were at a higher risk for developing diabetes and liver damage. Abnormalities in immune modulation were also observed in recovered patients, suggesting that such issues may persist after the illness. Research in this direction could further explain mechanisms that contribute to organ dysfunction during the disease and potentially aid in the development of new therapeutic approaches (42).

As evident from the literature review, viruses exploit exosome biogenesis systems to package their capsids, use exosomes for transport to target non-infected cells, and regulate virion production and viral particle formation (32). It could be argued that viruses also use exosomes for camouflage (mimicry) to evade or escape the host immune system’s attack. In this sense, exosomes play a critical role in cellular communication during viral infections, and thus in immune modulation.

Parasites have been present in the human population since its origin. Their existence has been documented in texts dating back 3,000 to 4,000 years. Approximately 400 species of parasites use humans as their hosts, with about 90 species being highly dangerous and associated with a significant mortality rate (43, 44).

A fundamental scientific question is how parasites evade the host’s defence mechanisms and successfully maintain their life cycle. One of the primary and potent tools they use is exosomes, which are released into the microenvironment they inhabit, thereby modulating it to their advantage (43, 45).

The interaction between the host and the parasite is bidirectional and is based on three types of communication: (1) exosomes originating from the parasite, (2) exosomes originating from host cells, and (3) exosomes originating from host cells stimulated by antigens derived from the parasite. The communication with the greatest impact on the parasite-host relationship, and consequently on the development of pathological conditions, is that mediated by exosomes originating from the parasite (43, 46).

Accumulating data from scientific research highlights the signalling and bioinformational roles of exosomes derived from parasites, which are aimed at maintaining parasitic physiology within a suitable microenvironment (46, 47).

Through their exosomes, parasites transmit bioinformational content to the host, thereby often modulating the immune response, which is their key target. It has been conclusively shown that the action of exosomes plays a critical, even decisive role in the development of diseases such as Chagas disease (caused by Trypanosoma cruzi), leishmaniasis, giardiasis, trichomoniasis, amebiasis, malaria, and neosporosis (48–50) (Table 1).

Given the extent of this topic, this literature review is limited to the most recent scientific studies. The ways in which parasites modulate the host’s immune response have been best demonstrated in a research study on the gastrointestinal nematode Heligmosomoides polygyrus (51). This parasite releases exosomes that are “eaten” by host macrophages. Upon uptake, these macrophages are modulated, resulting in the reduction of molecules associated with both type 1 and type 2 immune responses (IL-6 and TNF, as well as Ym1 and RELMa) and inhibition of ST2 receptor subunit expression for IL-33 (51).

On the other hand, the malaria parasite Plasmodium berghei releases exosomes that block the T-cell immune response. Exosomes derived from Leishmania can direct monocytes and dendritic cells toward anti-inflammatory phenotypes, thereby increasing Th2 cell polarization (52, 53).

The effects of exosomes from Leishmania on innate and adaptive immune responses have also been studied. Exosomes derived from Leishmania donovani modulated cytokine responses in human monocytes to IFN-γ, promoting IL-10 production while inhibiting TNF-α production. Additionally, these exosomes inhibited cytokine responses (IL-12p70, TNF-α, and IL-10) in dendritic cells originating from monocytes. A shift in Th cell polarization toward Th2 differentiation was also observed, which inevitably worsened the disease. Exosomes from Trichomonas vaginalis modulate the expression of cytokines IL-6 and IL-8 in ectocervical cells (50).

African sleeping sickness, caused by Trypanosoma brucei rhodesiense, has an aetiology based on immune evasion facilitated by exosomes. Specifically, trypanosomes in the bloodstream produce membrane nanotubes originating from the flagellar membrane. These nanotubes are incorporated into exosomes. In addition to their function in evading the innate immune response, these exosomes can fuse with host erythrocytes, leading to their rapid loss and anaemia. In other words, the exosomes cause modulation of erythrocyte function, rendering them dysfunctional (48).

Trematodes of the genus Schistosoma, the causative agents of schistosomiasis, also modulate the host immune response. This involves shifting the balance of Th cells toward Th2 production and macrophage polarization. The disruption of these strategic immune system targets has far-reaching consequences for health (47).

Such parasitic infections are chronic and often asymptomatic, with the communication between the parasite and host via exosomes, as well as the modulation of the host’s immune response, playing a crucial role in the infection’s course and the parasite’s life cycle. All of this points to the immunosuppressive action of parasitic exosomes.

Biotoxins, or biological toxins, are metabolites produced by various organisms that have toxic effects on the human body. Since these are macromolecules, predominantly of protein nature, they cannot replicate within the organism. Biotoxins can be classified based on their origin into animal toxins, marine toxins, phytotoxins, and microbial toxins (Table 1) (8). In general, biotoxins interfere with normal intercellular signalling, induce oxidative stress and apoptosis, and most commonly affect the kidneys, liver, nervous system, and reproductive system. In the process of intoxication by biotoxins, exosomes play an active role as signal carriers. On a molecular level, the expression of exosomes in intoxicated receptor cells varies depending on the type of cell and the type of biotoxin. There are three main roles of exosomes in biotoxin intoxication within the organism: receptor-mediated, vehicle of delivery, and regulation of immune response (Table 1). The receptor-mediated role of exosomes primarily involves the activation of receptors on the surface of target cells, thereby initiating intracellular signalling pathways. This effect may lead to the target cells producing more or fewer exosomes, depending on the nature of the biotoxin and the type of recipient cell. This role of exosomes is most commonly observed in intoxication by bacterial toxins, such as cholera toxin, diphtheria toxin, or mycotoxins such as T2 (8, 54, 55).

Exosomes can also serve as delivery vehicles, where they fuse with the plasma membrane of cells or are directly endocytosed, introducing proteins, lipids, and other active molecules into the cells. In this manner, they regulate cellular function and biological behaviour. This role of exosomes is observed in plant toxins, anthrax, and shiga toxin (8, 56).

The role of exosomes in the regulation of the immune response begins with antigen presentation, followed by immune regulation through the expression of activation molecules or complement factors. Toxin-induced interactions in vivo involve complex biological processes. The presence of toxins can stimulate cells to produce exosomes, which are enriched with signalling molecules related to inflammation, or to recruit surrounding signalling molecules such as cytokines and chemokines (e.g., MHC-I and MHC II). When neighbouring or distant cells uptake these exosomes, they can activate or enhance inflammatory responses, thereby promoting the body’s immune response to the toxin (8, 57). Generally, various immune cells (e.g., dendritic cells, lymphocytes) are exposed to the release of immunomodulatory exosomes. This role of exotoxins is also observed in animal toxins and Staphylococcus aureus alpha toxin (8, 58).

From the review of this literature, it can be concluded that exosomes play a dual role in immune system regulation; (1) as bioinformatic programs to induce innate or adaptive immunity and (2) for tactical evasion and resolution of inflammation (8, 46).