First, when bacterial EVs come into contact with or are phagocytosed by macrophages, the pathogen-associated molecular patterns (PAMPs) they carry interact with the pattern recognition receptors (PRRs) of macrophages (Asano et al., 2021). This interaction induces macrophages to produce various inflammatory factors (Ellis et al., 2010; Asano et al., 2021; Xu et al., 2022). For example, lipoproteins in S. aureus EVs can stimulate macrophages to release IL-6 and TNF-α (Kopparapu et al., 2021). LPS and flagellin in Pseudomonas aeruginosa EVs can stimulate macrophages to release MIP-2, TNF-α, and IL-6 (Ellis et al., 2010). Extracellular RNA in Aggregatibacter actinomycetemcomitans EVs can promote TNF-α production in macrophages (Han et al., 2019). These examples show that bacterial EVs can significantly promote macrophage-mediated inflammatory responses. However, in addition to their pro-inflammatory effects, EVs from some bacteria, such as Escherichia coli, Francisella tularensis, and Clostridium butyricum, can stimulate macrophages to release the anti-inflammatory cytokine IL-10 (Hu et al., 2020; Pavkova et al., 2021; Guangzhang et al., 2023). This indicates that EVs from certain bacteria have complex dual effects on macrophage-mediated inflammatory responses, capable of inducing the production of both pro-inflammatory and anti-inflammatory cytokines by macrophages. Moreover, besides directly inducing the production of various cytokines, studies have found that EVs from E. coli can induce macrophages to release pro-inflammatory EVs after interacting with them. This implies that EVs derived from E. coli have the ability to transmit inflammatory signals to macrophage EVs (Imamiya et al., 2023). Interestingly, although Porphyromonas gingivalis EVs can stimulate macrophages to release various inflammatory cytokines, the Arg- and Lys-gingipains enzymes in these EVs can degrade macrophage inflammatory cytokines, thereby achieving fine-tuned regulation of the immune response (Barth and Genco, 2016; Castillo et al., 2022).

Additionally, it is important to note that not all bacterial EVs exhibit pro-inflammatory effects. Current research indicates that EVs produced by some commensal bacteria, such as Lactobacillus plantarum, C. butyricum, and Bacteroides thetaiotaomicron, can induce macrophages to polarize towards the M2 type, leading to the release of the anti-inflammatory cytokine IL-10, thereby inhibiting inflammatory responses (Kim et al., 2020; Muller et al., 2021; Liang et al., 2022). Moreover, studies have found that certain mycobacteria, such as Mycobacterium avium and Mycobacterium kansasii, also produce EVs with anti-inflammatory properties that can induce macrophages to produce IL-10 (Kamran-Sarkandi et al., 2018; Tavassol et al., 2020). Therefore, these studies demonstrate that EVs from different bacterial sources have varying effects on macrophage-mediated inflammatory responses.

Additionally, as immune cells, macrophages possess strong antibacterial capabilities. When pathogens are recognized by macrophages, they form phagosomes on their cell membranes to engulf the pathogens, which then fuse with lysosomes to form phagolysosomes, thereby eliminating the pathogens (Hirayama et al., 2017). However, it is worth noting that bacterial EVs have varying effects on the antibacterial activity of macrophages. For example, Blancá et al (Blanca et al., 2022). investigated the effects of Bordetella pertussis-derived EVs on the antimicrobial activity of macrophages. The study found that B. pertussis EVs contain various toxins, including adenylate cyclase (CyaA). It was shown that pre-incubating macrophages with EVs significantly weakened their phagocytic and bactericidal activities against B. pertussis. This is because the CyaA in B. pertussis EVs inhibits the antibacterial capacity of macrophages by reducing the level of complement receptor 3 on their surface. Furthermore, Jung et al. (2016) studied how EVs from Legionella pneumophila affect the immune response of macrophages and promote bacterial replication within macrophages. Their research revealed that macrophages pre-treated with L. pneumophila EVs responded differently to subsequent Legionella infection. Initially, the EVs enhanced the immune defense response of macrophages and reduced bacterial replication. However, as the infection progressed, this effect gradually shifted, making macrophages more tolerant of bacterial replication. These findings suggest that L. pneumophila EVs have a complex dual role in macrophages, initially activating immune defenses and then creating an environment conducive to bacterial survival and replication. Interestingly, studies have shown that while EVs from Streptococcus pneumoniae can enhance macrophage phagocytosis of S. pneumoniae, this makes macrophages a “reservoir” for the bacteria, negatively impacting their antibacterial activity (Yerneni et al., 2021). On the other hand, bacterial EVs can also promote the antibacterial activity of macrophages. For example, EVs from P. gingivalis and E. coli can induce macrophages to produce reactive oxygen species (ROS) and nitric oxide (NO) upon interaction (Imayoshi et al., 2011; Guangzhang et al., 2023). These substances are important antibacterial weapons of macrophages, capable of exerting toxic effects directly on pathogens (Nathan and Hibbs, 1991; Canton et al., 2021; Herb and Schramm, 2021). Additionally, EVs from E. coli Nissle can increase the activity of acid phosphatase in macrophages (Hu et al., 2020). This enzyme is located in lysosomes and is closely related to the digestive functions of macrophages (Xu et al., 2012).

Additionally, macrophages possess important antigen-presenting functions. After pathogens are digested by macrophages, they are degraded into multiple small antigen fragments. These antigen fragments bind to major histocompatibility complex (MHC) molecules in macrophages, thereby triggering specific immune responses (Guerriero, 2019). However, it should be noted that some studies have indicated that EVs secreted by certain bacteria have the ability to affect the antigen-presenting function of macrophages. For example, Armstrong et al. (2020) investigated the impact of EVs secreted by P. aeruginosa on the expression of MHC-related molecules in human lung macrophages. The study found that P. aeruginosa EVs significantly inhibited the expression of 11 different MHC-related molecules in lung macrophages, including several subunits of MHC class II molecules (HLA-DRA, HLA-DPA1, etc.). Additionally, three CpG sites within the CFB gene showed significant hypomethylation under the influence of EVs, which was closely associated with the downregulation of MHC gene expression. These results suggest that P. aeruginosa EVs may regulate the antigen-presenting function of macrophages by altering the epigenetic state of their genes. Moreover, another study found that EVs from E. coli can promote the expression of the co-stimulatory molecule CD86 on the surface of macrophages (Guangzhang et al., 2023). This molecule plays a crucial role in the activation of T cells induced by macrophages (Smith et al., 2016; Nordlohne et al., 2021). However, current research on the impact of bacterial EVs on the antigen-presenting function of macrophages is still limited, and it has not been clearly identified which components in EVs interfere with this function. This area warrants further exploration in future research.

Programmed cell death is an orderly and controlled process of cell death, which includes apoptosis, pyroptosis, and autophagy (Newton et al., 2024). First, pyroptosis is an inflammation-related form of cell death characterized by the release of cellular contents and the subsequent induction of an inflammatory response (Rao et al., 2022). Current studies indicate that bacterial EVs can deliver a series of bacterial molecules to macrophages to induce pyroptosis. For instance, E. coli EVs can transport LPS into the cytoplasm, directly activating caspase-11 to induce pyroptosis in macrophages (Vanaja et al., 2016; Finethy et al., 2017). Similarly, lipoproteins and pore-forming toxins in S. aureus EVs can activate caspase-1 through the NLRP3 inflammasome, thereby inducing pyroptosis in macrophages (Wang et al., 2020). Additionally, bacterial EVs containing flagellin can induce pyroptosis in macrophages by activating the NLRC4 inflammasome rather than the NLRP3 inflammasome (Bitto et al., 2018). Unlike the NLRP3 inflammasome, the NLRC4 inflammasome specifically recognizes bacterial flagellin and flagellin-like molecules, leading to a more specific and rapid activation (Gram et al., 2021; Chebly et al., 2022). For example, Salmonella EVs containing flagellin can quickly activate the NLRC4 inflammasome in a shorter time compared to flagellin-deficient E. coli and Salmonella EVs (Yang et al., 2020). Interestingly, P. gingivalis itself cannot activate inflammasome formation in macrophages, but its EVs can, as observed in a study by Fleetwood et al. (2017). This might be due to the enrichment of gingipain in P. gingivalis EVs, which helps P. gingivalis survive within cells (Sheets et al., 2008; Mantri et al., 2015). After being phagocytosed by macrophages, P. gingivalis downregulates gingipain levels (Xia et al., 2007). Moreover, the activation of inflammasomes in macrophages by bacterial EVs requires the involvement of guanylate-binding proteins (GBPs) (Finethy et al., 2017; Santos et al., 2018). GBPs are a class of large GTPases that play a crucial role in inflammasome activation (Shenoy et al., 2012).

Additionally, bacterial EVs can also induce apoptosis in macrophages. Apoptosis is a non-inflammatory form of cell death that can occur via intrinsic and extrinsic pathways (Kiraz et al., 2016). Initial studies using Neisseria gonorrhoeae EVs to treat bone marrow-derived macrophages found that the toxin protein PorB in N. gonorrhoeae EVs was sufficient to cause mitochondrial dysfunction in macrophages, leading to the release of cytochrome c and inducing apoptosis (Deo et al., 2018). However, recent research has discovered that EVs from N. gonorrhoeae, uropathogenic E. coli, and P. aeruginosa cause mitochondrial dysfunction in macrophages, which inhibits intracellular protein synthesis without inducing rapid apoptosis (Deo et al., 2020). Nonetheless, prolonged inhibition of protein synthesis can regulate members of the BCL-2 family, thereby activating mitochondrial apoptosis (Speir et al., 2016). Consequently, over time, EVs indirectly activate the pro-apoptotic member BAK, leading to apoptosis (Czabotar et al., 2014).

Moreover, autophagy, as a form of programmed cell death, is also an important antibacterial mechanism in macrophages (Wu and Lu, 2019). However, to date, only one study by David et al. has reported on the effects of bacterial EVs on macrophage autophagy. This study found that EVs produced by E. coli expressing HlyF can inhibit autophagic flux by preventing the fusion of autophagosomes with lysosomes, thereby hindering the formation of acidic autolysosomes and the clearance of autophagosomes. This suggests that EVs from E. coli expressing HlyF can enhance bacterial virulence by inhibiting autophagy in macrophages (David et al., 2022).

It is worth noting that, in addition to the aforementioned impacts, current research indicates that bacterial EVs can also affect the antiviral immune response of macrophages. For instance, Bierwagen et al. (2023) explored the interaction between bacterial EVs and human macrophages and how this interaction influences viral replication, particularly concerning influenza virus. This study focused on EVs from different Gram-negative bacteria (such as Klebsiella, E. coli, and Salmonella) and found that these EVs can activate the antiviral immune response in macrophages via the TLR4-TRIF signaling axis. The research demonstrated that these bacterial EVs can induce macrophages to produce type I interferons and other antiviral molecules like Mx1, effectively inhibiting the replication of influenza virus A. Additionally, Bhar et al. (2022) investigated how commensal bacterial EVs influence murine norovirus (MNV) infection by modulating the antiviral immune response of macrophages. The study found that commensal bacterial EVs can bind to MNV and promote the co-inoculation of the virus and vesicles into host cells. This co-inoculation method significantly reduced viral replication in host cells compared to infection with the virus alone. Furthermore, these EVs increased the production of pro-inflammatory cytokines such as IL-6 and TNFα in macrophages during MNV infection, suggesting that EVs control viral infection by enhancing the immune response of host cells.

It is noteworthy that engineered bacterial EVs have also been reported to affect the antitumor immune effects of macrophages. Some tumor cells overexpress the CD47 molecule, which can inhibit the phagocytic function of macrophages, thus avoiding clearance (Kaur et al., 2016). Therefore, effectively activating tumor-associated macrophages is crucial in tumor therapy. In this regard, Feng et al. (2022) developed engineered bacterial OMVs designed to activate the phagocytic function of macrophages in the tumor microenvironment. These engineered vesicles were fused with CD47 nanobodies (CD47nb) on their surface and encapsulated with a layer of selenium-bonded polyethylene glycol (PEG/Se), enabling controlled release in the tumor site triggered by radiation. The results showed that OMV-CD47nb significantly enhanced the phagocytic ability of macrophages against tumor cells, primarily by inducing M1 polarization and blocking the “don’t eat me” signal on tumor cells. Additionally, Guo et al. (2021) developed a drug delivery system based on bacterial OMVs for sequential targeting of tumor cells and macrophages. This system utilized OMVs from Gram-negative bacteria loaded with paclitaxel (PTX) and siRNA targeting Redd1 in macrophages, achieving sequential drug release through a pH-sensitive linkage. The system first released PTX in the acidic environment of the tumor site, followed by siRNA delivery to M2 macrophages, enhancing glycolysis and promoting their polarization to the antitumor M1 type. The results showed that this OMV system demonstrated significant antitumor activity both in vitro and in vivo, effectively regulating macrophage metabolism and inhibiting tumor cell migration and invasion. These studies indicate that engineered bacterial EVs can achieve antitumor effects by influencing macrophages, providing new strategies for future cancer therapies.