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The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.
Antimicrobial proteins/peptides have attracted attention as candidates for the development of new agents to treat infections with drug-resistant pathogens, as their modes of action differ from those of current anti-infectives (2, 7, 19, 20). In this connection, we reported that a peptide comprising the first 11 N-terminal residues of human lactoferrin (hLF1-11) is active in (neutropenic) mice with methicillin-resistant Staphylococcus aureus (MRSA) (12), multidrug-resistant Acinetobacter baumannii (4), and invasive fluconazole-resistant Candida albicans infections (9). The peptide also displays antimicrobial activity against these pathogens in vitro (10-12), but only at subphysiological salt concentrations. The antimicrobial properties of hLF1-11 in vivo may therefore be explained by a modulatory effect of the peptide on the hosts' immune cells. hLF1-11, which has a half-life in mice of approximately 9 min (13), is effective 24 h after intravenous (i.v.) injection, and considering that the actions of macrophages are crucial in the defense against these pathogens, we hypothesized that hLF1-11 modulates monocyte-macrophage differentiation.
The mononuclear phagocyte system includes blood monocytes that have differentiated from committed myeloid progenitor cells. Monocytes circulate in the blood and, upon entering tissues, undergo further differentiation to become macrophages. The phenotype and functional activities of these macrophages are controlled by local factors, such as cytokines, chemokines, and growth factors (1, 6, 8, 16). Most macrophages in the tissues are in an anti-inflammatory state. However, upon encountering pathogens, tissue damage, or other danger signals, granulocyte-macrophage colony-stimulating factor (GM-CSF) is rapidly produced. This directs the differentiation of monocytes toward macrophages with a proinflammatory phenotype that produce large amounts of the proinflammatory cytokines interleukin 12p40 (IL-12p40) and tumor necrosis factor alpha (TNF-α) and small amounts of the anti-inflammatory cytokine IL-10 (6, 16).
Monocytes and macrophages display distinct biological functions, enabling them to undertake crucial actions in the innate immune response when pathogen-associated molecular patterns (PAMPs) are detected through pathogen recognition receptors, such as toll-like receptors (TLRs) and C-type lectin receptors (5, 17). These interactions lead to activation of the macrophage, which is associated with the production of cytokines, chemokines, and growth factors, attracting more immune cells to the site of infection, thereby regulating the immune response. In addition, macrophages clear an infection by binding, phagocytosis, and intracellular killing of pathogens.
To further unravel the mechanisms of action of the hLF1-11 peptide, we investigated whether hLF1-11 modulates the differentiation of monocytes into macrophages and, if so, what are the functional characteristics of these macrophages. We found that hLF1-11 directs GM-CSF-driven monocyte differentiation toward a macrophage subset that produces both pro- and anti-inflammatory cytokines, demonstrates enhanced recognition of pathogenic structures, and shows increased clearance of bacteria and fungi.
The human lactoferrin-derived peptide (hLF1-11; GRRRRSVQWCA) was obtained from Peptisyntha (Torrance, CA), and the control peptide (GAARRAVQWAA) lacking in vitro and in vivo antimicrobial activity (9) was obtained from Isogen (De Meern, The Netherlands). The purity of both peptides was >97% as determined by high-performance liquid chromatography (HPLC). Stocks of the peptides were made in phosphate-buffered saline (PBS) (Department of Pharmacy, LUMC, Leiden, The Netherlands) and stored at −20°C. Endotoxin concentrations were below detection level.
The following stimuli were used: lipopolysaccharide (LPS) from Escherichia coli (Sigma-Aldrich, Zwijndrecht, The Netherlands), ultrapure lipoteichoic acid (LTA) from Staphylococcus aureus (Invivogen, San Diego, CA), and heat-killed (30 min at 100°C) Candida albicans (strain Y01-19; Pfizer Inc., Groton, CT).
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors using Ficoll-Hypaque density gradient centrifugation (ρ = 1.077 g/ml). Monocytes were further purified by CD14-positive selection using magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol (>95% pure; viability, >96%). Macrophages were prepared by culturing monocytes with recombinant human GM-CSF (rhGM-CSF) according to the protocol of Verreck et al. (16). In short, monocytes were incubated for 7 days in RPMI 1640 (Gibco Invitrogen, Breda, The Netherlands) containing 10% heat-inactivated fetal bovine serum (FBSi) (Greiner Bio-one), 2 mM penicillin, 2 mM streptomycin (both PAA GmbH, Pasching, Germany), 2 mM l-glutamine (Gibco Invitrogen), and 5 ng/ml rhGM-CSF (Biosource, Camarillo, CA), referred to below as culture medium.
To investigate the effects of hLF1-11 on GM-CSF-driven monocyte-macrophage differentiation, monocytes were incubated in culture medium with hLF1-11, control peptide, or saline. On day 6, the cells were stimulated with LPS (up to 100 ng/ml), LTA (up to 1 μg/ml), or heat-killed C. albicans (up to 1 × 107 CFU/ml) for 20 h; thereafter, the supernatants were collected for assessment of the production of cytokines, i.e., IL-10, IL-12p40, and TNF-α levels, using commercially available enzyme-linked immunosorbent assay (ELISA) cytosets (Biosource) and MCP-1 using a singleplex assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturers' instructions. The surface molecule expression and antimicrobial activities of macrophages were determined on day 7 without stimulation on day 6. Where indicated, monocytes were exposed to hLF1-11 for 10 or 60 min, washed, reincubated in culture medium, and stimulated with LPS or not on day 6. hLF1-11 did not affect the viability of GM-CSF-incubated monocytes after 24 h, as determined by annexin V/propidium iodide (PI) staining.
Macrophages were collected and resuspended in cold PBS with 0.2% bovine serum albumin (BSA), washed twice, and incubated with the selected fluorescently labeled monoclonal antibodies for 30 min on ice in the dark. The following monoclonal antibodies were used: phycoerythrin (PE)-conjugated antibodies against CD11b and CD163 and fluorescein isothiocyanate (FITC)-labeled antibodies against CD14 and CD206 were obtained from BD Biosciences (Heidelberg, Germany). PE-conjugated antibody against Dectin-1 was obtained from R&D Systems. FITC-conjugated antibody against CD282, and CD284 were from HyCult (Uden, The Netherlands). Cell surface molecule expression was assessed by flow cytometry using a FACSCalibur and BD CellQuest software (BD Biosciences).
Phagocytosis of C. albicans by macrophages was assessed by fluorescence-activated cell sorter (FACS) analysis, as described for the phagocytosis of apoptotic cells by macrophages (18), with modifications. In short, overnight-cultured C. albicans cells were washed twice in PBS and then labeled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) for 30 min at 37°C in the dark, centrifuged, and resuspended in RPMI 1640 supplemented with 20% human serum (HuS). The macrophages were collected, washed twice in PBS-0.2% BSA, and labeled with PE-conjugated antibody against CD11b for 30 min (on ice in the dark). Next, the macrophages were mixed with C. albicans in a 1:1 ratio and incubated for various intervals at 37°C under slow rotation in the dark. The percentage of macrophages associated with C. albicans was assessed by determining the percentage of double-positive (CD11b+ CFSE+) macrophages by two-color flow cytometry. Furthermore, experiments were performed at 4°C to quantify the binding of C. albicans to the macrophages. In addition, the percentage decrease in the number of free C. albicans cells over 60 min, as determined by FACS, was taken as a measure of the capacity of the macrophages to clear C. albicans.
For phagocytosis of S. aureus, pHrodo-labeled S. aureus was obtained from Invitrogen and resuspended according to the manufacturer's protocol. pHrodo is a dye that is nonfluorescent at neutral pH and bright red in acidic environments (e.g., phagolysosomes). In short, equal volumes of macrophages (1 × 105) with 5-times-diluted pHrodo-labeled S. aureus stock were incubated for several intervals at 37°C or as a control at 4°C. Thereafter, the pHrodo fluorescence of the macrophages was assessed by FACS analysis. The percentage of pHrodo-positive macrophages and the median of the pHrodo fluorescence intensity of this population were obtained. In addition, equal numbers of S. aureus LUH2141 (LUMC, The Netherlands) and macrophages were incubated at 37°C under rotation. At several intervals, macrophages were lysed using ice-cold water with BSA (0.01%) and vortexed for 30 s. Thereafter, the lysates were serially diluted and plated onto agar plates. The next day, the viable bacteria were counted, and the percentage of killed bacteria was determined.
The Friedman nonparametric test, followed by Dunn's multiple-comparison post test and, where indicated, by the Wilcoxon signed-rank test, was used to determine the differences between the various groups. Data are expressed as median and range. Two-sided P values are reported, and the level of significance was set at P < 0.05.
To find out if hLF1-11 modulates GM-CSF-driven macrophage differentiation, we first assessed the cytokine production of macrophages differentiated in the absence or presence of hLF1-11 (referred to below as hLF1-11 macrophages) in response to LPS or heat-killed C. albicans using a multiplex cytokine assay (data not shown). Since the production of a variety of cytokines and chemokines by hLF1-11 macrophages was enhanced, we selected for convenience three cytokines to read out pro- and anti-inflammatory cytokine production. Our results showed that hLF1-11 macrophages produced significantly (P < 0.05) larger amounts of IL-10 (Fig. (Fig.1A),1A), but not IL-12p40 (Fig. (Fig.1B)1B) and TNF-α (Fig. (Fig.1C),1C), in response to LPS than control macrophages and control peptide-macrophages did. When stimulated with LTA, the production of both IL-10 (P < 0.05) and IL-12p40 (P < 0.001), but not TNF-α, was significantly upregulated by hLF1-11 macrophages compared to control macrophages (Fig. 1D to F). In addition, hLF1-11 macrophages produced larger amounts of IL-10 (P < 0.01) and TNF-α (P < 0.001) in response to heat-killed C. albicans than control macrophages did, while IL-12p40 (P < 0.05) was upregulated only at 10 μg/ml hLF1-11 (Fig. 1G to I).
To gain insight into the responsiveness of hLF1-11 macrophages to LPS, we measured the production of IL-10 in response to increasing concentrations of LPS. Our results revealed that 0.1 ng/ml of LPS was sufficient to significantly (P < 0.01) enhance IL-10 production by hLF1-11 macrophages, but not by control macrophages (Fig. (Fig.2A).2A). Interestingly, expression of CD14 and TLR-4, which are involved in the recognition of LPS, was significantly (P < 0.05) upregulated by hLF1-11 macrophages compared to control (peptide)-macrophages (Fig. 3A and B).
Next, we investigated the responsiveness of hLF1-11 and control macrophages to LTA. Since macrophages hardly produce cytokines in response to lower concentrations of LTA, we chose the chemokine monocyte chemotactic protein 1 (MCP-1), based on the multiplex results, as a readout. The results revealed that upon stimulation with 10 ng/ml LTA, hLF1-11 macrophages, but not control macrophages, produced significantly (P < 0.05) more MCP-1 than upon stimulation with 1 ng/ml (Fig. (Fig.2B).2B). hLF1-11 macrophages and control macrophages did not differ in expression of TLR-2 (Fig. (Fig.3C3C).
Additionally, we investigated the responsiveness of the macrophages to heat-killed C. albicans using TNF-α production as a readout. The results showed that hLF1-11 macrophages, but not control macrophages, produced significantly (P < 0.05) more TNF-α when stimulated with ~1 × 106 heat-killed C. albicans cells (Fig. (Fig.2C).2C). Interestingly, receptors involved in C. albicans recognition, such as the C-type lectin receptor Dectin-1 and the complement receptor CD11b, but not the macrophage mannose receptor CD206, were significantly (P < 0.05) upregulated on hLF1-11 macrophages (Fig. 3D and F).
Together, these data indicate that hLF1-11 promotes the development of macrophages with increased responsiveness to different microbial stimuli.
Since hLF1-11 macrophages showed increased responsiveness to several pathogenic structures, we next determined whether the antimicrobial activities of hLF1-11 macrophages were also enhanced compared to control macrophages. Our results revealed that after 15 and 30 min, significantly (P < 0.05) more hLF1-11 macrophages than control macrophages had taken up C. albicans. At 60 min, no difference was observed in the percentages of hLF1-11 and control macrophages associated with C. albicans. Experiments performed at 4°C revealed that less than 5% of the macrophages bound C. albicans at all intervals. Moreover, at 60 min, significantly (P < 0.05) fewer free C. albicans cells were present in samples containing hLF1-11 macrophages than in those containing control macrophages, indicating that hLF1-11 macrophages had cleared more C. albicans within 1 h than control macrophages (Table (Table11).
In addition, we determined whether hLF1-11 macrophages displayed enhanced antimicrobial activities against S. aureus compared to control macrophages. The results revealed that at 60 and 120 min of incubation, the percentage of S. aureus-phagocytosing hLF1-11 macrophages was significantly (P < 0.05) higher than that of control macrophages (Table (Table1).1). However, fluorescence levels within hLF1-11 macrophages and control macrophages did not differ, indicating that the phagocytosis per macrophage was not enhanced (Table (Table1).1). When these experiments were performed at 4°C, no bacterial fluorescence could be detected in macrophages. Notably, after 60 min of incubation, hLF1-11 macrophages killed S. aureus significantly better than control macrophages; however, after 120 min, the cells were equally effective (Table (Table11).
Since our in vivo results suggested the possibility of a priming effect by hLF1-11 on mononuclear phagocytes (13), we investigated whether a short exposure of monocytes to the hLF1-11 peptide during differentiation could induce effects similar to those of the continuous presence of this peptide during culture. Our results showed that the presence of hLF1-11 for 60 min, but not 10 min, at the start of differentiation was sufficient to induce a significant (P < 0.05) increase in LPS-induced IL-10 production similar to that of cells that were exposed to hLF1-11 during the whole period of differentiation (Fig. (Fig.4A).4A). In agreement with this, the presence of hLF1-11 for 60 min, but not 10 min, during differentiation was sufficient to significantly (P < 0.05) enhance the phagocytosis of C. albicans by the resulting macrophages (Fig. (Fig.4B).4B). These data indicate that a short exposure of monocytes to hLF1-11 is sufficient to direct GM-CSF-driven differentiation of monocytes toward macrophages with an altered phenotype.
The main conclusion to be drawn from the present results is that hLF1-11 directs GM-CSF-driven monocyte differentiation toward macrophages demonstrating enhanced effector functions. This conclusion is based on the following observations.
First, hLF1-11 was able to modulate the production of a variety of cytokines and chemokines by macrophages in response to three different microbial stimuli. Interestingly, IL-10 production was significantly enhanced by hLF1-11 macrophages in response to all these stimuli; proinflammatory cytokine production was either enhanced or unchanged. This indicates a general enhancement of the inflammatory response by hLF1-11 rather than a shift toward an anti-inflammatory phenotype. No definitive conclusion can be drawn as to whether the additional IL-10 production is beneficial for the control of infection (3). In this connection, Scott et al. (14) suggested that increased IL-10 levels (induced by the IDR-1 peptide) could help control inflammation, while immune responses were enhanced. Second, hLF1-11 macrophages can detect lower concentrations of the microbial stimuli than control macrophages, suggesting that these cells respond to an infection more adequately than control cells. Third, the results of the phagocytosis and killing experiments showed that hLF1-11 directs GM-CSF-driven differentiation toward a subset of macrophages that are highly effective against C. albicans and S. aureus.
The fourth main finding of this study is that the effects of hLF1-11 on monocyte-macrophage differentiation were already achieved by 60 min of exposure of the monocytes to the peptide at the start of a 7-day culture system. These actions may contribute to the anti-infective effects of the peptide against infections in mice, as reported earlier (9). However, further investigations are required to discover if this mechanism could play a role in vivo. The optimal concentration of hLF1-11 in this study (100 μg/ml) is within the therapeutic range of hLF1-11 used in animals. A dose of 5 mg had a favorable side-effect profile (15) and though this is lower than what we used, we do have good reasons to suppose that the present dose does not lead to undue adverse effects. Moreover, the present concentration of hLF1-11 is similar to that of other cationic antimicrobial peptides in in vitro experiments (e.g., IDR-1 ).
Unfortunately as our attempts to unravel the signal transduction in monocytes after incubation with hLF1-11 have not been successful, we cannot offer a mechanistic explanation for the ability of hLF1-11 to affect human monocyte-macrophage differentiation.
Interestingly, the functional properties of hLF1-11 macrophages show striking similarities to a set of responses of macrophages resulting from stimulation with foreign or nonself molecular patterns, characterized as the adaptive component of innate immunity as described by Bowdish et al. (1). This set includes upregulation of certain receptors on macrophages, resulting in a general enhancement of host immunity, such as the ability to clear bacteria and to produce cytokines. Our results indicate that the hLF1-11 peptide induces such properties during GM-CSF-driven differentiation of monocytes into macrophages, creating a subset with increased affinity for (a broader class of) pathogens, thereby possibly strengthening the innate immune response of the host to a subsequent infectious challenge. Furthermore, the present results are important for the further development of this peptide as a therapeutic agent for treatment of infections in patients with compromised immune systems.
This work was financially supported by SenterNovem grant ISO44096.
Published ahead of print on 23 November 2009.