As an airborne pathogen, M.tb, besides exposure to airway epithelial cells, exposes to and infects alveolar macrophages where M.tb establishes an ideal niche for replication (Singh et al., 2012). Bacterial proliferation induces the production of a pro-inflammatory response, and subsequently recruitment of additional macrophages and immune cells, which results in the formation of a granuloma. With developing host adaptive immunity, the granuloma can limit the growth and expansion of bacteria. Nevertheless, within the granuloma, certain cells may undergo necrosis and create a necrotic core where M.tb can persist extracellularly (Ramakrishnan, 2012). On the other hand, during the infection, the pathogen also may reside in the phagolysosome which is formed by the fusion of the lysosome and the phagocytic vacuoles (Pethe et al., 2004). Shortly after infection, the mycobacterial components including cell wall components, lipoproteins, or glycolipids exit the phagosome through endocytic compartments and are subsequently released within host-derived EVs (Palacios et al., 2021). These EVs mostly share similar size and surface markers with exosomes (Prados-Rosales et al., 2011; Smith et al., 2015) and their components and properties have been studied in various experimental models including infected cells, animal models as well as in serum or plasma of tuberculosis patients (Giri et al., 2010; Kruh-Garcia et al., 2014; Singh et al., 2015; Mehaffy et al., 2017). Even though different studies have found different kinds and amounts of components inside these EVs, there is a consensus for the enrichment of the TLR2 ligands in the infected host-derived EVs, like those in bacterial-derived EVs. Many of these components have been characterized as highly immunogenic mycobacterial antigens including antigen 85 (Ag85) complex, early secreted antigenic target of 6 kDa (ESAT-6), and MPT63 (Rath et al., 2013).

Although there are some overlaps between bacterial components inside the EVs from the infected host or bacteria, interestingly, most of the mycobacterial proteins found in host-derived EVs are not present in bacterial-derived EVs (Kruh-Garcia et al., 2014; Mehaffy et al., 2022). So, it is worth knowing how bacterial components are targeted to host EVs in the infected cells. To address this question, Smith et al. looked at the role of the ubiquitylation process in this context. Interestingly, when mycobacterial proteins were introduced separately to both RAW264.7 mouse macrophages and Human Embryonic Kidney (HEK) 293 cells, they underwent endocytosis, ubiquitination, and subsequent release via host EVs. While suppression of the ubiquitylation using its corresponding inhibitors led to the loss of mycobacterial components in host-derived EVs. Furthermore, ubiquitylated proteins were found to bind to the endosomal sorting complexes required for transport (ESCRT-I) for their dissemination into MVBs (Singh et al., 2023). These findings suggested that ubiquitination is a necessary process for trafficking bacterial components from the phagocytic/endocytic network to host EVs (Smith et al., 2015). Interestingly M.tb can manipulate these processes by inducing the expression of the host ESCRT-dependent and independent genes in the infected cells and increasing the production of EVs with different protein and lipid composition in different stages of disease (Singh et al., 2023).

Characterization and separation of host and M.tb-derived vesicles based on their content and function can be informative in this regard. Athman et al. tried to separate two populations of bacterial EVs and host-derived EVs within macrophage culture supernatants using a sucrose density gradient based on their specific surface markers (Athman et al., 2015). However, their conclusion was challenging, and based on the data provided it was not possible to confirm that human and M.tb EVs were completely separated in their experiments. So further experiments using progressive techniques including advanced flow cytometry methods may help for efficient separating of different populations of EVs.

The importance of EVs released by Mycobacterium-infected cells in immune response regulation during intracellular bacterial infections was first reported by Sanchita Bhatnagar and colleagues (Bhatnagar and Schorey, 2007). They demonstrated that M. avium-infected macrophages released EVs containing glycopeptidolipids (GPLs) and could transfer these molecules from infected to uninfected macrophages and trigger proinflammatory responses by these macrophages (Bhatnagar and Schorey, 2007). Subsequently, they identified some of the antigenic mycobacterial components, including LAM and the 19-kDa lipoprotein, within the EVs purified from the bronchoalveolar lavage fluid (BALF) of BCG-infected mice (Bhatnagar et al., 2007). These EVs had proinflammatory properties and when applied intranasally into mice, they stimulated TNF and IL-12 production and enhanced the migration of neutrophils and macrophages to the lung of the animal (Bhatnagar et al., 2007). Interestingly, the proinflammatory properties of these EVs were dependent on the time of isolation. EVs isolated within 48- and 72 hours post-infection showed stimulatory activity, while at earlier or later time points, the isolated EVs failed to trigger the inflammatory responses. In addition, the stimulatory activity correlated with the levels of exosomal LAM and 19-kDa lipoprotein which suggested that the trafficking of mycobacterial components to multivesicular bodies (MVB) changed over time (Bhatnagar et al., 2007).

These studies for the first time highlighted the important role of EVs as the carrier of Pathogen-Associated Molecular Patterns (PAMPs) in the mechanism of immune responses against intracellular pathogens.

Furthermore, when macrophages from TLRs or MyD88-knockout mice were incubated with these EVs, there was no induction of the proinflammatory response which demonstrated that the mechanism underlying the proinflammatory activity of these EVs is dependent on TLRs and MyD88-related pathways and also highlighted that these EVs contained TLR ligands (Bhatnagar and Schorey, 2007; Bhatnagar et al., 2007) ( Figure 1A ). Interestingly, proteomic analysis showed that only EVs from the virulent strains contained TLR2 ligands (Prados-Rosales et al., 2011).

The determinant role of EVs’ TLRs in the modulation of immune response in the recipient cells was further confirmed by other studies. Singh, P.P., et al. showed these EVs can modulate IFN-γ related pathways in the bystander cells via modulation of TLR2 and Myd88-related pathways (Singh et al., 2011). Macrophages infected with M.tb gain resistance to IFN-γ stimulation (Ting et al., 1999). However, this effect is not limited to infected macrophages and can gain access to uninfected macrophages via EVs (Singh et al., 2011).

IFN-γ regulates host anti-TB responses by different mechanisms including the production of reactive nitrogen and oxygen species, upregulating antigen-presenting molecules including MHC molecules as well as induction of autophagy in the infected macrophages (Ghanavi et al., 2021). Interestingly, this study revealed that the EVs released by M.tb H37Rv-infected macrophages inhibited these mechanisms in the uninfected macrophages by suppressing their corresponding responsible genes at the transcriptional level and acting via TLR2 and Myd88-related pathways (Singh et al., 2011)( Figure 1B ).

Accordingly, Liu et al. showed that EVs secreted by M.tb-infected mesenchymal stem cells (MSCs) also triggered TLR2-Myd88 related pathways in the naïve macrophages which consequently enhanced the level of proinflammatory cytokines such as TNF, RANTES(CCL5), and iNOS (Liu et al., 2021).

EVs secreted by the M.tb infected neutrophils applied the same mechanism to activate naïve macrophages. These EVs triggered an inflammatory response in macrophages via TLR2/6 ligands, leading to increased expression of CD80 and PD-L1 on immature monocyte-derived dendritic cells (DCs). Additionally, they enhanced the permeability of epithelial monolayers and induced immune cell migration to the injection site in mice. Furthermore, these exosomes promoted macrophage autophagy and superoxide anion production, ultimately reducing intracellular mycobacterial survival (González-Cano et al., 2010; Walters et al., 2013; Alvarez-Jiménez et al., 2018).

Studies by Singh et al. showed that exosomes released from H37rv (M.tb)-infected macrophages after stimulation of TLRs, triggered MAPK-dependent signaling mechanisms through MK-2 and NF-κβ activation. This process then effectively induced the differentiation of naïve monocytes to the functionally active macrophages (Singh et al., 2023)( Figure 1C ). MyD88-dependent activation of MAPK via TLR2 signaling in addition to upregulation of the inflammatory cytokines led to actin cytoskeleton reorganization and the assembly/disassembly of focal adhesions (FAs), cell attachment, and ultimately generation of mature functional macrophages (Singh et al., 2023). The effects of inflammatory exosomes on the polarization of macrophages were also reported by other studies. The exosomes secreted by Brucella melitensis-infected macrophages increased the polarization of macrophages towards M1 subtype. These exosomes contained Brucella antigen components and stimulated secretion of M1 cytokines (TNF and IFN-γ) through the NF-κB signaling pathway, inhibited the secretion of M2 cytokines (IL-10), and subsequently reduced the intracellular survival of Brucella (Wang et al., 2023).

EVs derived from the M.tb- infected macrophages, disrupted the integrity of the monolayer of epithelial cells which subsequently increased the migration of inflammatory cells to the infection site in lung (Distler et al., 2006)( Figure 1D ). Furthermore, it has been shown that these EVs can activate endothelial cells (Li et al., 2018). Endothelial cells have an important role in regulating immune responses through the regulation of leukocyte adhesion, cell migration as well as secretion of some inflammatory cytokines such as TNF (Shao et al., 2020; Mostmans et al., 2021). Treatment of endothelial cells with M.tb-infected macrophage-derived EVs or serum of M.tb-infected mice increased cell monolayer permeability. This treatment changed the expression of the genes involved in cell adhesion and inflammatory-related pathways in the endothelial cells and subsequently increased macrophage migration through the endothelial cell monolayers (Li et al., 2018)( Figure 1E ). The process may be mediated by NF-κB since incubation with M.Tb-EVs, induced NF-κB nuclear localization in the endothelial cells (Li et al., 2018).

EVs produced by M.Tb-infected macrophages contain active mRNA and miRNA which were found to be specific to these infected cells including transcripts involved in the immune responses. Such exosomal RNAs were biologically active and could be expressed in recipient macrophages ( Figure 1F ). These active RNA molecules had mostly host origin but also included mycobacterial transcripts (Singh et al., 2015).

It was shown that the host EVs containing mycobacterial RNA stimulated the cytoplasmic RNA sensor RIG-I in the recipient cells and promoted the maturation of phagosomes and bacterial clearance (Cheng and Schorey, 2018; Cheng and Schorey, 2019). It was shown that M.tb releases its RNA into the host-derived EVs using the mycobacterial SecA2 and ESX-1 secretion systems which subsequently activates host Mitochondrial antiviral-signaling protein (MAVS)-dependent RNA sensing machinery which consequently induces the production of IFN-β in the recipient macrophages (Cheng and Schorey, 2018; Cheng and Schorey, 2019). TANK-binding kinase 1(TBK1) and Interferon regulatory factor 3 (IRF3) are two important molecules downstream of the RNA sensing machinery that are phosphorylated and transported into the nucleus upon stimulation by external RNA(e.g. viral RNA) to initiate transcription of type I IFNs (Li et al., 2018). ( Figure 1G ).

The EVs from the infected macrophages induced phosphorylation of TBK1 at Ser172 and subsequently IRF3 nuclear translocation (Pilli et al., 2012). This process also may play a role in the activation of autophagy in host cells as the production of type I interferon in the recipient macrophages, promotes the LC3-associated phagocytosis pathway and increases phagosome maturation and M.tb clearance (Martinez et al., 2015). Different mechanisms of function of EVs during M.tb infection are summarized in Figure 1 .

During infection with M.tb, DCs transport bacteria to local lymph nodes. However, infected macrophages poorly present antigens to CD4+ T cells to activate them. Therefore, there should be some mechanisms taken by bacteria for manipulation of the antigen presentation process in the infected host cells (Elieh Ali Komi et al., 2018).

Walters et al. observed that microparticles (MPs) released from BCG and M.tb-infected macrophages activated M. tb-specific CD4(+) T cells. These MPs contained mycobacterial proteins and stimulated Ag-specific CD4+ T cell proliferation in vitro and in vivo in a DC-dependent manner (Walters et al., 2013).

Vázquez-Flores et al. reported the presence of mycobacterial proteins containing epitopes for CD4+ T-cell activation with high concentration in the EVs released by the M.tb-infected human neutrophil (Vázquez-Flores et al., 2023).

However, Athman et al. observed that direct exposure to M.tb-released EVs inhibits T cell activation which was demonstrated with downregulation of IL-2 production and T cell proliferation (Athman et al., 2017). M.tb derived EVs also were shown to induce partial anergy demonstrated by increased expression of gene related to anergy in lymphocytes (GRAIL) during primary stimulation of naïve T cells which subsequently transiently inhibited effector T cells (Athman et al., 2017).

Interestingly, in a study by Srivastava et al., it was demonstrated that M.tb antigens are exported from infected DCs which will be uptaken by kinesin 2-dependent vesicular transport. This process reduces the quantity of the antigens available for presentation by MHC class II-related and subsequently, limits CD4 T cell activation (Srivastava et al., 2016). locking the release of “free” antigens by silencing kinesin-2 in the M.tb-infected cells decreased antigen export and increased the availability of antigen to the MHC class II antigen presentation processing, resulting in more effective CD4 T cell activation (Srivastava et al., 2016).

In addition, shuttling the mycobacterial antigens via EVs mediates the cross-presentation of mycobacterial antigens in distance. These EVs also contain MHC-I, MHC-II, and costimulatory molecules and it has been shown that they could active BCG-sensitized CD4+ and CD8+ T cells in vitro and naïve CD4+ and D8+ T cells in vivo (Giri and Schorey, 2008).

Moreover, when immature and mature DCs were exposed to these EVs, they induced the production of IFN-γ by autologous CD4+ T cells specific to M.tb antigens. The aforementioned results suggest that these EVs may act as antigen carriers and transfer mycobacterial proteins to the antigen-presenting cells (Vázquez-Flores et al., 2023).

In combination with these studies, the experiments by Smith et al. vividly confirmed the significant role of EVs as an extracellular source of antigen for T-cells during infection with M.tb (Smith et al., 2017). They utilized Rab27a-deficient mice as a model to investigate the role of EVs during TB infection. They observed that exosomes released during M.tb infection in mice significantly contributed to T-cell responses. Rab27a, a member of the Rab GTPases family (Elieh Ali Komi et al., 2020), is known to mediate exosome biogenesis and secretion (Ostrowski et al., 2010). The Rab27a-deficient mice exhibited reduced exosome concentrations in serum after TB infection, followed by an increase in bacterial burden. These changes were linked to the decreased mycobacterial antigen transport via exosomes, reduced activation of the lung and splenic T cell, and a decreased capacity to elicit a pro-inflammatory response. Additionally, the number of T cells producing IFN-γ upon antigen stimulation was also reduced (Smith et al., 2017).

During homeostasis, cells metabolize glucose mostly through the citric acid cycle (Krebs cycle) and oxidative phosphorylation (Lachmandas et al., 2016). However, during infection with M.tb, the bacteria perturb the host metabolism by inducing a metabolic switch toward aerobic glycolysis in CD14+ monocytes or CD3+ T cells isolated from human peripheral blood mononuclear cells (PBMCs). The metabolic shift was dependent on TLR2 and was facilitated by the activation of the AKT-mTOR (mammalian target of rapamycin) pathway (Lachmandas et al., 2016; Alipoor and Mirsaeidi, 2021). Regarding the presence and importance of EVs surface TLR ligands, it can be expected that these EVs could induce reprogramming of metabolic pathways in the recipient cells.

Our team previously reported that the exosomal miRNAs released by BCG-infected macrophages were enriched in a cluster of miRNAs that regulated host metabolism pathways (Alipoor et al., 2017). The cluster of dysregulated miRNAs upon infection with BCG included miR-1224, miR-1293, miR-425, miR−484, and miR−96 all were predicted to target metabolism and energy production-related pathways including the central carbon metabolism (CCM) pathways, genes involved in ketone body and amino acid synthesis, glycosaminoglycan biosynthesis and heparan sulfate/keratin sulfate metabolism pathways in recipient cells (Alipoor et al., 2017). Interestingly, this cluster of exosomal miRNAs also was enriched in the exosomes from the serum of TB patients (Alipoor et al., 2019a).

Modulation of metabolic exosomal miRNAs upon infection with mycobacteria suggests that mycobacteria may reprogram the expression of metabolic host miRNA network and metabolic-specific genes to recruit host metabolic pathways in favor of survival.

It has been shown that in TB patients, M.tb impacts the protein and lipid composition of circulating EVs, which vary according to the disease stage (Biadglegne et al., 2022). Therefore, M.tb may induce the enrichment of exosomes in a particular size and probably with a specific composition for modulating metabolic pathways for maintaining its survival (Singh et al., 2023).

EVs from both the pathogen and the host could influence the outcome of the infection. It seems that M.tb takes advantage of these active bio-vesicles as the main strategy to control the host’s physiological condition and immune responses for its survival.

One strategy taken by M.tb is increasing the production of EVs by host cells (Walters et al., 2013; Alvarez-Jiménez et al., 2018; Singh et al., 2023) through manipulation of the host ESCRT-dependent and independent genes (Singh et al., 2023). Considering the role of TLRs in EV-mediated mechanisms, the enhanced secretion of EVs by infected cells may have a contrary effect. Exosomal TLR-2 ligands primarily trigger the activation of CD4+ T cells and the formation of granulomas, however, this is not sufficient to clear the bacterial infection because of the consequences of long-term TLR-2 stimulation mainly immunosuppression. This effect is exerted by promoting the secretion of immunosuppressive cytokines and finally suppression of MHC-II antigen presentation (Tapping and Tobias, 2003; Gehring et al., 2004; Pecora et al., 2006; Prados-Rosales et al., 2011).

On the other hand, M.tb inhibits T-cell receptor signaling by delivering lipoglycan to T-cells within mycobacterial EVs. Athman et al. observed that upon incubation of T cells with M.tb infected Macrophages, T cells were associated with mycobacterial LAM. This was also observed in the T-cells isolated from the lungs of tuberculosis-infected mice (mice were infected with M.tb via aerosol infection). In a series of experiments, they showed that the trafficking of bacterial LAM to T cells is via the bacterial EVs that are produced inside the phagosomes (Athman et al., 2017). They concluded that M.tb EVs escape from infected macrophages and enter the extracellular environment. These bacterial EVs can transport M.tb molecules to uninfected immune cells including T cells, emphasizing the significance of EV production during M.tb infection (Athman et al., 2015).

In addition, the concentration of mycobacterium EVs during infection depends on the presence of extracellular mycobacteria and varies in the different stages of infection in tuberculosis patients (Schorey et al., 2021). In the later stages of infection more extracellular rather than intracellular bacteria are expected to be found in the center of the granuloma which suggests that during these later stages of infection, Mycobacterium-derived EVs may have the higher concentration and so may exert relatively more biological effect (Schorey et al., 2021).

It seems that bacteria strategically regulate the production, content, and release of these EVs to maintain the balance between immunity and immune evasion, which is a feature of latent M.tb infection and essential for the maintenance of bacterial intracellular survival ( Figure 2A ).

Noncoding RNAs are the most abundant and important regulatory molecules that are transferred via the EVs. These molecules have a paramount role in the immune phenotype of inflammatory niches (Alipoor et al., 2018). In silico analysis has shown that the dysregulated miRNAs in bovine tuberculosis have the potential to trigger the oncogenic pathways including mitogen-activated protein kinase, PI3K-Akt signaling, and fibroblast growth factors as the key factors (Ndzi et al., 2019a).

In another study, pathway analysis of the exosomal miRNAs released by the infected macrophages with BCG has shown that the target genes of these exosomal miRNAs are most associated with important cell cycle signaling pathways related to EGF/EGFR, WNT, Rap1, Ras, receptor tyrosine kinases ErbB, activated FGFRs, and VEGFA-VEGFR2 (Alipoor et al., 2017; Alipoor et al., 2019b). Disturbing these important pathways in cell proliferation and metabolism may make the recipient cells more susceptible to entering a cancerous stage. These clusters of M.tb-induced dysregulated miRNAs also were involved in amino acid, pyrimidine, and purine nucleotide biosynthesis which are increased in M.tb-infected lung tissue and are also an important factor in the progression of cancers (Alipoor et al., 2017). So, EVs released during M.tb infection have the potential to change the physiological condition in the recipient cells by targeting important pathways in cell cycle, metabolism, and proliferation that increase their susceptibility to tumor initiation and progression.

TB exosomal miRNAs may also induce epigenetic changes inside the inflammatory niche which may create a suitable environment for tumor initiation.

miR-29a is one of the most common exosomal miRNAs in the serum of TB patients (Ndzi et al., 2019b). This miRNA belongs to the family of epi-miRNAs and regulates the methylation of DNA by targeting DNA methyltransferases (DNMTs) including DNMT1 or DNMT3 (Amodio et al., 2015). These events subsequently change the expression profile of the epigenetics enzymes including Enhancer of Zeste homolog 2 (EZH2).

EZH2, as the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), modifies gene expression by trimethylating Lys-27 on histone 3 (H3K27me3). This modification leads to the silencing of its target genes (Duan et al., 2020).

Activation of EZH2 increases the proliferation rate of lung cancer cells (Entezari et al., 2023), and promotes metastasis and EMT resulting in lung cancer progression. Elevated EZH2 levels and an increased count of genes silenced via H3K27me3 were linked to the advanced stage of lung cancer (Agarwal et al., 2016).

miR-21 is a known oncomir that plays a critical role in lung cancer, and it is reported that it is upregulated within the serum EVs from TB patients (Wang et al., 2011; Latorre et al., 2015; Duffy et al., 2018).

miR-21 promotes lung cancer via the regulation of different mechanisms involved in epigenetics modulation (Melnik et al., 2020), metastasis, genetic instability of tumor cells, angiogenesis (Ni et al., 2019), and EGFR-regulated anti-apoptotic factor (Seike et al., 2009). The mir-21 promoter is robustly regulated by STAT3, which is activated by IL-6 (Löffler et al., 2007). IL-6 is considered a major driver of disease severity in TB and a key player in the tumorigenesis mechanisms in Lung cancer. The IL-6/STAT3 pathway via miR-21 is an epigenetic switch linking inflammation to cancer (Iliopoulos et al., 2010) and may contribute to the initial stage of the tumor (Löffler et al., 2007; Bi and Chng, 2014; Liu et al., 2016; Saltarella et al., 2021). Increased cellular levels of miR-21 either by endogenous upregulation or uptake of miR-21-rich exosomes can trigger these tumorigenic mechanisms (Xu et al., 2020).

miR-31, miR-125b, miR146a, and miR-155 are also the most reported circulating miRNAs upregulated in the exosome obtained from TB patients (Daniel et al., 2022). These miRNAs are considered oncomiR since their target gene plays a role in the promotion of tumors ( Table 1 ).

Therefore, during a chronic TB infection, exosomal miRNAs derived from the infected cells may change the epigenetic and transcription profile inside the inflammatory niche and facilitate the development of the TME.

During chronic TB, EVs may also change the phenotype of the inflammatory microenvironment by modulation of macrophage polarization. The increased production of EVs during TB infection and subsequently the continuous triggering of the TLRs by these EVs may start anti-immunity responses including an increase in the secretion of IL-10, IL-4, and TGF-β (Hirsch et al., 1996; Saraav et al., 2014). This event promotes polarization to TAM-like (M2-like) phenotype (Hu et al., 2016; Zhang et al., 2016) ( Figure 2B ).

TAMs play a significant role in tumor progression by producing substantial amounts of IL-10, TGF-β, and VEGF. These molecules facilitate immunosuppression and angiogenesis (Xu et al., 2020). Additionally, central transcription regulators, including STAT1, STAT3, and STAT6, which activate the M2 phenotype (Gong et al., 2017; Vergadi et al., 2017) may be modulated in the recipient cells by up-taking EVs during TB infection.

Triggering TLRs by EVs released by the infected macrophages may also increase the release of IL-6 (Flynn et al., 2019). IL-6 significantly increases invasion in lung cancer cells by regulating EMT (Liu et al., 2020). Additionally, IL-6 may stimulate the expression of IL-10, and together, macrophage polarization and TAM production (Dehai et al., 2014; Wang X. et al., 2017; Xu et al., 2020) ( Figure 2B ).

It also has been shown that infection of human monocyte cells with M.tb, increases the invasion and induces EMT in vitro (Gupta et al., 2016). Additionally, TB EVs shuttle a significant amount of vimentin and HSPs (Diaz et al., 2016) which respectively regulate key events during EMT and impact malignant progression in lung cancer (Usman et al., 2021).

EVs derived from the Mtb-infected cells harbor an array of immuno-stimulatory and immuno-inhibitory factors that can contribute to cellular reprogramming in recipient cells. These EVs play a pivotal role in regulating intracellular communication within the inflammatory niche during chronic tuberculosis. Additionally, they can modulate their surroundings, creating an optimal microenvironment for tumor initiation and progression (Schorey and Bhatnagar, 2008).