3.1. Exosomes in Intercellular Communication: Effects on Immune Response
Exosomes are small vesicles measuring approximately 30 to 150 nm in size secreted by different cell types, which have recently received great attention for their possible role as biomarkers in various pathological conditions [16
]. Despite having been previously cited as mere cell debris, recent studies have shown an active role of exosomes in intercellular communication by transporting proteins, RNA, and microRNAs that can significantly alter the function of target cells [17
]. The exosomes are now known to correspond to intraluminal vesicles of endosomal multivesicular bodies (MVBs), formed after endosome invagination and released into the extracellular space by fusion of the MVBs with the plasma membrane. Due to their cellular origin, these particles contain endosomal pathway-specific marker proteins such as tetraspanins (CD63, CD9, and CD81) and heat shock proteins (HSP70) [19
The exosomes were first visualized in medium collected from reticulocyte cultures [20
]; since then, several cell types have been identified as exosome sources, such as hematopoietic cells, epithelial cells, neurons, and adipocytes among others [21
]. Initially, exosomes were suggested to play the role of removing molecules unnecessary for cellular metabolism that were only partially degraded by the lysosomal system [22
]. However, as investigations progress, their functions appear to be considerably more complex: platelets secrete coagulation-regulating exosomes [23
], extracellular vesicles of cardiac progenitors are capable of inhibiting cardiomyocyte apoptosis following myocardial infarction [24
], and astrocyte-derived exosomes decrease neuronal damage caused by hypoxia through in vivo autophagy regulation [25
It is clear from these observations that since exosomes carry particular profiles of proteins, RNA, and microRNAS but recap the internal content of their source cells, these nanoparticles appear to undergo a process of “selective packaging” as a way of refining and enhancing intercellular communication at distant sites and thus regulating important biological functions [26
]. Exosomes are internalized by target cells through direct membrane fusion or endocytosis [27
] and act mainly by regulating the expression of specific proteins. These effects can be accomplished through the direct transport of mRNAs to be translated or the delivery of microRNAs that lead to transcriptional repression and consequent genetic silencing [28
Importantly, exosomes as molecular messengers have the potential to modulate several pathological scenarios, such as the maintenance of tumor microenvironments. This is accomplished through different biological processes, mainly those involved in immune responses and that include, for instance, signal transduction and antigen presentation [30
]. In fact, there is growing evidence about the modulation of immune cells functions by exosomes, which can be particularly observed in the case of tumor-derived exosomes. Tumor cells can secrete exosomes capable of attenuating the responses of lymphocytes, macrophages, NK cells, and DCs, as well as promoting the expansion of myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells involved in states of immunosuppression [29
However, some studies, in contrast, have pointed to a possible role of exosomes in antitumor immunovigilance as carriers of tumor antigens to be loaded into dendritic cells. Wolfers et al. showed that exosomes derived from solid tumors such as colon and breast cancer, when delivered to dendritic cells, lead to the activation of T-cell-mediated immune responses culminating in tumor rejection. Interestingly, there was cross-protection among the various tumors evaluated, pointing to the possibility that these exosomes carry common tumor antigens, which would be easily presented by MHC-I molecules in dendritic cells [34
]. A similar approach was tested using exosomes derived from heat-stressed carcinoembryonic antigen (CEA) positive tumor cells. These particles induced DC maturation that culminated in cytotoxic lymphocytic responses and reduced tumor burden [35
In our laboratory, we initially speculated that exosomes purified from serum of patients with hematologic malignancies could also be useful as an antigenic pulse in the development of new forms of immunotherapy. This seemed promising considering previous results with solid cancers and the fact that exosomes are released containing particular contents of proteins, RNAs, and microRNAs that recap the internal content of the maternal cells, and could thus contain specific antigenic material for the priming of dendritic cells. To evaluate this hypothesis, exosomes from serum of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) were purified and used as an antigenic source for DCs in co-cultures with lymphocytes and leukemic K562 cells. Surprisingly, our results demonstrated that incubation of DCs with patients’ exosomes decreased the lysis of target cells, probably corresponding to an immune tumor evasion mechanism in vivo [36
In fact, because they are responsible for T lymphocyte activation, DCs also play an important role in the sensitive balance between immune response and tolerance. Previous studies have shown that mature DCs can limit effector T-cell responses and promote immune tolerance in response to different signaling molecules such as IL-27 and IL-10 [37
]. In the case of cancer patients, circulating exosomes could possibly be generated in the tumor microenvironment, also containing immunosuppressive molecules, constituting an effective mechanism for the paracrine induction of tolerance and therefore tumor escape.
Our group also demonstrated that these DCs stimulated with exosomes from AML patients, despite not altering lymphocyte proliferation rates, led to a marked decrease in INF-γ production by these effector cells, and INF-γ levels were inversely related to CD86 expression in DCs. Therefore, we can speculate that in AML, the exosome-induced suppression of cytotoxicity may, at least in part, be the result of dysregulation in co-stimulatory molecules in DCs, such as CD86, leading to decreased activation of lymphocytes with impaired INF-γ production [36
Interestingly, a study with a murine model of AML had previously demonstrated the effectiveness of using exosomes as a pulse of DCs [39
]. One possible explanation for this discrepancy in relation to human patients may be that the tumor in mice was not autologous, but tumor cells had been injected into these animals. This approach may not necessarily mimic the systemic immunosuppressive environment involved in AML in humans, which may explain why promising preclinical results have not been reproduced in human trials.
Furthermore, our results are in line with the latest studies published on this topic, consolidating the idea that exosomes participate in the induction of immunotolerance in AML. Hong et al. evaluated AML patients treated with NK cell infusion and could observe the effect of patients’ exosomes present in pretreatment samples. These authors noted that there was a decrease in the cytotoxicity of these NK cells when they were incubated along with the serum containing exosomes that was extracted from patients prior to treatment [40
]. Moreover, specifically in relation to dendritic cells, a recent study with prostate cancer patients demonstrated that after incubation of DCs with patients’ exosomes, there was also a significant decrease in the release of inflammatory cytokines and less activation of INF-producing CD8+ lymphocytes [41
]. Recent studies have also demonstrated the immunosuppressive potential of extracellular vesicles in gliomas [42
], and melanoma-derived exosomes have been shown to inhibit the differentiation of monocytic precursors in dendritic cells, leading to increased TGF-β production and suppression of lymphocyte proliferation [43
3.2. Possible Roles Uncovered for Micrornas and Epigenetics?
Epigenetics describes cellular modifications other than DNA sequence variations that can be heritable and modified by environmental stimuli leading to changes in gene expression. These changes arise due to processes such as DNA methylation, histone modifications, chromatin remodeling proteins, miRNAs, and non-coding RNAs, and have the potential to modify the tumor microenvironment which in turns can influence cancer initiation, proliferation, and metastasis [44
DNA methylation is one of the best characterized epigenetic modifications. This process is dynamically regulated through the action of DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes. DNMTs add methyl groups to certain regions of the genome that contain clusters of CpG sequences (CpG islands); most of them are located upstream of promoters, thus resulting in the silencing of certain genes [47
]. In a transplant model, Zhu et al. demonstrated that microvesicles derived from K562 leukemia cells contained BCR-ABL1 mRNA, which can be transferred to mononuclear cells from the normal donor, resulting in the expression of a malignant phenotype [48
]. Genomic instability was the main mechanism observed in these cells, and this was achieved by upregulation of methyltransferases and global DNA hypermethylation. Furthermore, the treatment of microvesicles with RNase led to a decrease in DNMT3a and DNMT3b, proving that leukemia-derived microvesicles influenced the methylation pattern of target cells via transmission of microvesicular RNA. Thus, the microvesicular cargo derived from neoplastic cells may deliver enzymes involved in methylation and/or demethylation to recipient cells, inducing changes in the expression of tumor-related genes, which in turn leads to accelerated tumor proliferation and metastasis.
Chromatin structure is also crucial for gene expression regulation and may be modulated by histone modifications [49
]. However, the role of extracellular vesicles in posttranslational histones modifications remains controversial. Sharma et al. used bioinformatic analysis and observed an impressive overlap between genes relevant to transgenerational epigenetic inheritance and the cargo of exosomes released by different cell types, including cancer cells. Among such genes were those related to histone acetylation, deacetylation, ubiquitination, and other histone modifications, indicating that exosomal mRNAs and proteins may directly or indirectly participate in the response to the environmental exposure and epigenetic modification [50
]. Importantly, we may speculate that these changes in mononuclear cells may have some specific effects on dendritic cells, which should undergo investigation.
MicroRNAs (miRNAs) are small noncoding RNAs (17–25 nucleotides long) that also control gene expression by promoting degradation or repressing translation of target mRNAs. miRNAS are seen as master regulators, efficiently tuning fundamental cellular processes such as proliferation, apoptosis, and development [51
]. The biosynthesis of miRNAs is a multistep process that starts in the nucleus following transcription and continues to the cytoplasm, where the mature miRNA molecule exerts its main functions [52
Owing to their relatively small size, miRNAS are the most abundant RNAs in exosomes [53
], and novel functions for exosomal miRNAs are being revealed in the effectuation of immune responses. For instance, miR-21 and miR-29a can act as ligands to toll-like receptors (TLR) in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately leads to tumor growth and metastasis [54
]. Exosomes isolated from peripheral blood of systemic lupus erythematosus (SLE) patients have recently been demonstrated to have the ability to stimulate the secretion of INF-α by plasmocitoid dendritic cells (pDC), and the authors could show that this effect was related to the microRNAs isolated from these exosomes. Interestingly, synthetic microRNAs containing an IFN induction motif could also activate cytokine secretion and induce pDC maturation, revealing the possibility of manipulating exosomal microRNAs as a potential therapeutic target to be explored also in neoplastic diseases [55
In summary, extracellular vesicles released by tumoral cells into the microenvironment may influence the phenotype of receptor cells, including their epigenetic status, through delivery of mRNAs and miRNAs. Considering the lack of information on how these specific mechanisms may impact dendritic cells and thus favor tumor proliferation, this remains an exciting field for future scientific exploration.