To date, only one cathelicidin gene (CAMP) has been described in mice and human, inducing a protein named CRAMP and LL-37, respectively (75). LL-37 exhibits a broad spectrum of antimicrobial activity, several immunomodulatory effects, anticancer activities, and also chemotactic and pro-angiogenic properties (35, 40). It has been detected in several types of cells, tissues, and fluids, and along with other AMPs, such as β-defensins, plays a central role in mucosal defense (76). An increased or decreased expression of LL-37 has been identified in several diseases, observing elevated levels in inflammatory pathologies like systemic lupus erythematosus and pulmonary diseases (77–79). In contrast, low production of LL-37 is associated with asthma and skin disorders (80–82).

The expression of LL-37 is driven by several stimuli such as inflammatory mediators and microbial structures, and varies depending on cell type (83) (Table 2). Vitamin D3 appears to be the major regulator of LL-37 expression in humans as the CAMP gene encoding LL-37 contains three VDREs (Vitamin D response element) on its promoter. Upregulating the expression of natural LL-37 or developing synthetic mimics might represent a potent new therapeutic option that needs to be carefully evaluated.

The defensin family of AMPs can be classified as α-defensins, β-defensins, or θ-defensins depending on their cellular origin, gene structure, and connectivity of the cysteine residues in their sequence (75). Defensins play important roles in innate and adaptive immunity against microbial and viral infections and also participate in wound repair, cytokines and chemokine expression, production of histamine, and enhancement of antibody responses (58).

β-Defensins, hBD-1, hBD-2, and hBD-3 are the mainly functional peptides in humans (35) expressed by many epithelial cells, granulocytes, and MSCs (24). Except for hBD-1, their expression is inducible (75), hBD-2 and hBD-3 are induced by pro-inflammatory stimuli or microorganisms (91, 146). hBD-1 and 2 are microbicidal predominantly against Gram-negative bacteria, while hBD-3 is a broad-spectrum antimicrobial peptide. Given their significant antimicrobial and antiviral activity, modulating endogenous defensin production through the use of specific regulatory stimuli makes defensins promising candidates for therapy (Table 2). Direct administration to sites such as the skin or cervico-vaginal mucosa for treatment of wounds or infection is of interest, particularly in conditions where antimicrobial peptide generation is reduced (76).

Hepcidin is a natural human host defense peptide, originally found in urine and plasma (147, 148) which is produced mainly by hepatocytes but also by other cells including MSCs and myeloid leukocytes (71).

Hepcidin is known to act as an iron regulatory hormone, as well as it exerts a broad spectrum of antimicrobial activity against fungal species and clinical relevant bacteria such as Escherichia coli, S. epidermidis, S. aureus, and group B streptococci; however, the role as an antimicrobial peptide remains to be further unraveled for most type of infections (51, 147). Both peptides forms of hepcidin (hep-20 and hep-25) exhibit antimicrobial properties (147); however, hep-20 is not involved in the regulation of iron use (149).

The expression of hepatocyte hepcidin is regulated by iron status through the BMP-SMAD pathway and by inflammation, where the binding of inflammatory cytokines like IL-6 triggers the JAK–signal transducer and activator of transcription 3 (STAT3) signaling pathway (150, 151). An hepcidin-mediated hypoferremia could functions as a host defense mechanism that evolved to restrict iron availability and slow the pathogen growth (70, 71). The enhancement of bactericidal activity at low pH renders these peptides interesting for the development of drugs specifically designed, for example, for bacterial and/or fungal infections occurring in body areas with acidic pH (51, 152). Thus, modulation of the hepcidin pathway (Table 2) has become an interesting target for new therapeutic strategies (140).

Lipocalins are a family of small soluble proteins, which are often secreted (88). Lcns functions as transporters binding small organic molecules that have been associated with many biological processes: the immune response, cell growth, proliferation and metabolism, synthesis of prostaglandins, and iron transportation (153). Lcn2 appears to have an important role in innate antimicrobial defense mechanism (154) and in contrast with other AMPs, it exerts an indirect function against pathogens.

Originally, Lcn2 was identified as a component of neutrophil granules; however, it was later shown to be also expressed in various cells such as macrophages, adipocytes, MSCs, and epithelial cells in response to inflammatory conditions (26, 115, 153, 155) (Table 2). The antimicrobial role is given by its ability to sequesters bacterial iron chelators, called siderophores, that consequently prevent the iron transfer to bacteria and arrested their growth (bacteriostatic effect) (34, 72). This function was demonstrated using mice genetically lacking Lcn2 that showed a marked increase in mortality in an E. coli-induced sepsis model (155, 156). Other studies revealed that Lcn2 also binds other types of siderophores, such as bacillibactin (B. anthracis) (157) and carboxy-mycobactin (mycobacteria) (158). Lcn2 is one of the most commonly studied novel biomarkers (159) and has emerged as a promising biomarker for different pulmonary diseases (97), kidney injury (160–163), liver failure (164), and tumorigenesis (144). Thus, the clinical relevance of Lcns is rapidly increasing even though the exact mechanisms behind their functions remain to be elucidated.

Most of the data about the antimicrobial properties of MSCs have been obtained from in vitro studies with bacteria, although little data exist about the effect of MSCs on viral, fungal, and parasite pathogens. For both unstimulated and stimulated MSCs, a direct antimicrobial effect has been described (Table 3).

The antimicrobial efficacy of MSCs mediated by AMPs has been described for different sources of stromal cells, although different MoA and antibacterial range have been reported for them. Probably, these variations in the antimicrobial spectrum of MSCs might be a specific response of MSCs to produce the most effective AMPs against a specific type of pathogen challenge. A summary of the types of AMPs detected and not detected in the different sources of MSCs are shown in Table 4. Likewise, the data available to date suggest notable species-specific difference between murine and human MSCs with regard to the MoA of the antimicrobial effector function of MSCs (16).

BMSCs are the most studied source regarding the intrinsic antimicrobial ability of MSCs. In humans, the antimicrobial effect of BMSCs is mediated by LL-37 (19, 23) and hepcidin (20).

These AMPs have been detected in both unstimulated and stimulated-BMSC cultures. Respect to LL-37, BMSCs as well as their conditioned medium has demonstrated the property to inhibit the bacterial growth of E. coli, Pseudomonas aeruginosa, S. aureus, and S. pneumonia (19, 23). In the study performed by Krasnodembskaya et al. (19), the authors showed that BMSCs are able to inhibit bacterial growth directly, also by means of conditioned culture medium, but only when BMSCs were previously challenged with bacteria. They also showed that BMSCs produce and secrete inducible quantities of LL-37, responsible for the inhibition of bacterial growth of E. coli and P. aeruginosa in vitro. In contrast, hBD-2-3, Lcn2, and surfactant protein D (SP-D) were negative in BMSCs or expressed very low protein levels, which were insufficient to elicit an antibacterial effect. Likewise, these cells exhibit direct antimicrobial activity against S. aureus, through a contact-independent mechanism and mediated in part by the release of LL-37. Recently, Sutton et al. (23). demonstrated that BMSCs constitutively secrete LL-37, which exert a potent antimicrobial effect in vitro against P. aeruginosa, S. aureus, and S. pneumonia, controlling the rate of bacterial growth and transition into colony forming units. These data suggest that BMSCs supernatants can be useful as an adjunct treatment to conventional ABs. Interestingly, the levels of LL-37 were enhanced after the preconditioning with inflammatory stimulus, as IFNγ, IL-1β, or IL-12. However, in line with previous report (19, 20), the levels of hBD-2-3 were not detected in unstimulated nor stimulated BMSCs (23). Furthermore, the levels of LL-37 were detected in BMSCs only when the cell growth was performed under FBS (fetal bovine serum) condition, since the serum-free or platelet lysate conditions reduce the antimicrobial efficacy of BMSCs and their production of LL-37 (23). Conversely with the aforementioned data, in which expression of the antimicrobial peptide LL-37 was detected constitutively in BMSCs or following stimulation with E. coli or inflammatory stimulus, data from our research group did not show any basal or induced expression of LL-37 under both bacterial mixture and LPS stimulations in BMSCs. However, we detected in bacteria-stimulated BMSCs the expression of hepcidin, which was involved in the antimicrobial effect of MSCs both direct and in conditioned medium (20).

The antimicrobial properties of MSCs is not only limited to AMPs activity. In fact, upon stimulation with inflammatory cytokines, BMSCs through a substantial increase in IDO expression, exhibit a cell autonomous, broad-spectrum antimicrobial effector function directed against clinically relevant bacteria (S. aureus; S. epidermidis; E. faecium; Group B streptococci), protozoal parasites (Toxoplasma gondii), and viruses (CMV and HSV-1) (16). Also, it has been reported that IL-17-positive murine(mu)BMSCs exert a potent antifungal activity with respect to IL-17-negative or bulk muBMSCs. The growth inhibition of Candida albicans is mediated by IL-17 in a dose-dependent manner, while anti-IL-17 antibodies partially reduce anti-C. albicans effect of muBMSCs (32).

Potent antimicrobial effects of muBMSCs have also been described on E. coli and C. albicans but with a different MoA with respect to their human counterpart. As reviewed by Balan et al. (165), no data indicate secretion of LL-37 by muBMSCs, but the preconditioning with E. coli significantly increases the production of the antimicrobial protein Lcn2 (26). Furthermore, in contrast to BMSCs of human origin, muBMSCs fail to express IDO even after stimulation with inflammatory cytokines such as IFNγ, tumor necrosis alpha, and IL-1β, and they consequently do not inhibit bacterial growth (16). However, it has been reported that unstimulated muBMSCs directly exhibit phagocytic activity for E. coli or S. aureus in a cell dose-dependent manner (25), although further studies are required to confirm these claims.

More recently, menstrual fluid-derived MSCs (MenSCs) have emerged as an attractive alternative for cell therapy since they are isolated in a non-invasive manner with the possibility of periodical collections from the same donor, ensuring high amounts of cells at low culture passages and from the same genetic background (2, 6). Our group has recently shown that MenSCs have an important antimicrobial effect mediated by hepcidin, both directly and through the peptide present in their conditioned medium. Polybacterial stimulation significantly increased the expression of hepcidin in MenSCs, whereas other antimicrobial peptides such as LL-37 and hBD-1-2-3 remained below the limit of detection (20). Interestingly, the downregulation of hepcidin through hypoxic condition resulted in the loss of the antimicrobial property of MenSCs, indicating a hepcidin-dependent mechanism (20). Therefore, culture condition need to be carefully considered when expanding MenSCs as any modification in the oxygen levels can affect their biological activities and consequently, their antimicrobial potential. In that perspective, the effect of hypoxia on MenSCs functions could be considered as a negative regulator of their antimicrobial activity (20) but is contrasted with a positive effect on their angiogenic property (2). Nevertheless, the extend of the effect of this type of conditioning is largely dependent on the source of MSCs, as an example, it has been observed that the immunosuppressive capacity of AT-MSCs remains unchanged under hypoxic conditions (166).

Sung et al. (24) described that the growth of E. coli in vitro was significantly inhibited by UC-MSCs or their conditioned medium after bacterial exposure. In microarray analysis performed to detect gene expression changes in MSCs responsible for their antibacterial action, a significant upregulation of toll-like receptor (TLR)-2 and TLR-4, and hBD-2 were identified in UC-MSCs after E. coli exposure, suggesting that the antimicrobial effects of MSCs might be mediated by the secretion of hBD-2 via TLR signaling pathway. However, the increased hBD-2 level and the in vitro antibacterial effects of MSCs were abolished by specific antagonist or by silencing of the TLR-4, but not TLR-2. The effect was restored by hBD-2 supplementation, indicating that hBD-2 secreted by MSCs only via TLR-4 mediates the antibacterial effects. In this study, the gene expression profiles of other antimicrobial proteins, such as LL-37 (19, 23) or Lcn2 (26), were not significantly upregulated.

AT-MSCs or their conditioned medium have also shown an antimicrobial effect. Sutton et al. (23) report that the conditioned medium of AT-MSCs decreased the P. aeruginosa growth rate to levels comparable with the AB geneticin, a broad-spectrum aminoglycoside AB. Importantly, the combination of AT-MSCs with geneticin showed a potent antimicrobial effect against P. aeruginosa, revealing that this type of stromal cells have an AB-enhancing effect.

The in vitro data demonstrating the antimicrobial effect of MSCs have also been supported by in vivo studies. MSCs from different sources or origins have the capability to reduce the burden of pathogens in different preclinical models, independently of the route of administration, doses, or time of injections, having been shown in blood, spleen, peritoneum, lung, and bronchoalveolar lavage (BAL) fluid (17–23, 25, 26, 30). In Table 5, we summarize the current in vivo data regarding antimicrobial effector function of MSCs mediated by AMPs.

In an immunocompetent model of pneumonia by E. coli, the treatment with 1 × 10⁶ BMSCs after 4 h of E. coli instillation induced a sharp reduction in total bacterial counts in lung homogenates (LH) and BAL fluid compared with vehicle (19). Total BAL cell counts and absolute neutrophil counts were also lower in the BMSCs-treated group, suggesting that bacterial clearance in the BAL of BMSC-treated mice did not primarily depend on the recruitment of immune cells. In fact, the administration of BMSCs together with neutralizing anti-LL-37 antibody resulted in a 10-fold increase in bacterial number both in LH and BAL, revealing that LL-37 effect is needed for the antimicrobial activity of BMSCs in vivo (19). These results are in line with Sung’s work (24), where in the same model, the treatment with 1 × 10⁵ UC-MSCs administered intratracheally after 3 h of E. coli instillation provoke a downregulation of the inflammatory response and enhanced bacterial clearance, increased hBD-2 secretion in BAL fluid and the resultant protection against E. coli-induced pneumonia. Since hBD-2 secreted by MSCs are via the TLR-4 pathway, the antibacterial effects of UC-MSCs were abolished with TLR-4 siRNA transfection of UC-MSCs. Likewise, the detection of hBD-2 in BAL fluid, suggests that hBD-2 secreted by transplanted UC-MSCs plays a pivotal role in mediating the in vivo antibacterial effects of UC-MSCs (24). Similarly, another study with similar preclinical model setting described that the concentration of the antimicrobial molecule Lcn2 in BAL fluid is increased after the transplantation of muBMSCs; and the blocking of Lcn2 by a monoclonal antibody results in the elimination of the antibacterial effect of MSCs treatment in vivo (26).

In immunocompetent mice with a polymicrobial sepsis induced by cecal ligation and puncture (CLP), the administration of MSCs, either of human or murine origin, induced a reduction of the burden of pathogens and also a significant improvement in the survival rate (17, 20, 22, 25, 30). Our group has recently shown that the administration of 7.5 × 10⁵ MenSCs 3 h after CLP-induced sepsis is able to reduce the animal mortality regulating different aspects of the septic process, such as the organ dysfunction, modulation of the inflammatory response without severe immunosuppression, and promotion of bacterial clearance in blood, in a similar degree to the present standard of treatment with AB therapy (20). Notably, the synergism between MenSCs + AB resulted in the highest improvement of survival (20). Although the in vivo role of hepcidin was not evaluated, the in vitro data suggest a potential antibacterial effect of hepcidin in the resolution of the bacteremia. In line with these data, Gonzalez-Rey et al. (17) described that AT-MSCs-treated septic mice have lower peritoneal bacterial counts than untreated mice, suggesting that AT-MSCs could promote bactericidal activities by themselves or on other cell types. Concordantly, Mei et al. (25) reported that 2.5 × 10⁵ muBMSCs administered 6 h after CLP-induced sepsis results in a higher bacterial clearance in part due to enhanced phagocytotic activity of the host immune cells.

Cystic fibrosis is a genetic disease in which the battle between pulmonary infection and inflammation becomes the major cause of morbidity and mortality. In a mouse model of cystic fibrosis infected with P. aeruginosa and S. aureus, the administration of 1 × 10⁶ BMSCs or its conditioned medium results in a reduction of the CFU of these two pathogens in BAL fluid. Although the role of the peptide LL-37 was not evaluated in vivo, the in vitro data showing the constitutive or stimulated expression of LL-37 suggest a potential role for this peptide in the reduction of bacterial growth in vivo (23).

Recent studies have ignited significant interest on EVs released by MSCs accounting, at least in part, on their paracrine effect resulting in a horizontal transfer of the nucleic acids and proteins between the injured cells and MSCs. This cell-to-cell interaction is doubt to be bi-directional, resulting in reprogramming of their secretome to respond to specific need of the tissue by transferring to the injured cells, factors restraining injury, and leading to tissue regeneration. MSCs-derived EVs have been implicated in the tissue restoring effects of MSCs including, anti-apoptotic (172), wound healing (173), and anti-tumoral activities (168).

In the last few years, the beneficial role of MSCs-derived EVs has been described in several preclinical models of inflammatory/infectious diseases. Zhu et al. (174) reported that MVs released by BMSCs were therapeutically effective following E. coli endotoxin-induced acute lung injury (ALI) in mice, in part through the transfer of keratinocyte growth factor (KGF) mRNA from the MVs to the injured alveolar epithelium and lung endothelium. In line, Monsel et al. showed that the administration of MVs secreted by BMSCs improved survival, and decreased the influx of inflammatory cells and bacteria in a bacterial pneumonia mouse model. The antimicrobial effect of BMSCs-derived MVs was in part through enhancement of monocyte phagocytosis of bacteria, which could be further increased by pre-stimulation of BMSCs with a TLR-3 agonist before the release of MVs (175). However, it remains to be explored whether MVs released from MSCs conserve the cell antimicrobial effect through their AMPs content. Future experimental designs are required to explicitly address their antimicrobial relevance in the previously evaluated infectious disease models. Furthermore, the exposure of MSCs to conditions that are known to upregulate the expression of AMPs such as cytokines (IFNγ, TNFα, IL-22, etc.), growth factors, and UV exposure should be considered (Figure 1). Additionally, the effect of the conditioning on increasing the MVs cargo content with AMPs will also need to be evaluated. Nevertheless, in other applications, it has been demonstrated that MVs can transport drugs to target sites and maintain a higher drug concentration than conventional dosage forms. Tang et al. engineered drug-packaging MVs by incubating cells with chemotherapeutic agents. These hybrid MVs were used to effectively kill tumor cells in murine tumor models without typical side effects (176). More recently, another group assessed the feasibility of an exosome-based drug by loading a chemotherapeutic agent through sonication to treat multidrug-resistant cancer. Interestingly they observed a 50-fold increase in the cytotoxicity in drug-resistant cancer cells (177). This is a relevant outcome as it limits the side effect related to the conventional administration of chemotherapies and advance a novel and sustained drug release method.

Similar drug loading strategy could be evaluated based on AMPs packaging in EVs. The rationale for EVs loaded with AMPs is the possibility to enhance their therapeutic potential through increasing their concentrations at the site of infection and/or by reducing the toxicity of ABs.