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Human Lactoferrin-Derived Peptide's Antifungal Activities against Disseminated Candida albicans Infection

  1. Antonella Lupetti1,5,a,
  2. Carlo P. J. M. Brouwer4,a,
  3. Sylvia J. P. Bogaards1,4,
  4. Mick M. Welling2,
  5. Emile de Heer3,
  6. Mario Campa5,
  7. Jaap T. van Dissel1,
  8. Robert H. E. Friesen4,b and
  9. Dr. Peter H. Nibbering1
  1. 1 Department of Infectious Diseases, Leiden University Medical Center, Leiden
  2. 2 Department of Radiology, Division of Nuclear Medicine, Leiden University Medical Center, Leiden
  3. 3 Department of Pathology, Leiden University Medical Center, Leiden
  4. 4 AM-Pharma, Netherlands
  5. 5 Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università di Pisa, Pisa, Italy
  1. Reprints or correspondence: Dr. Peter H. Nibbering, Dept. of Infectious Diseases, C5-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands (p.h.nibbering{at}lumc.nl)

  1. Presented in part: 33rd National Meeting of the Italian Society of Microbiology, Naples, Italy, 16–19 October 2005; 7th International Conference on Lactoferrin: Structure, Function and Applications, Honolulu, Hawaii, 16–19 October 2005; 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 16–19 December 2005; 29th European Peptide Symposium, Gdansk, Poland, 3–8 September 2006.

  • ↵Present affiliation: Morphosys AG, Martinsried/Planegg, Germany

  1. ↵A.L. and C.P.J.M.B. contributed equally to this study

 
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Abstract

Background.Because the human lactoferrin-derived peptide, hLF(1–11), exerts potent in vitro candidacidal activity, we investigated whether it displays antifungal activity against disseminated Candida albicans infections.

Methods.Neutropenic mice were intravenously infected with C. albicans and, 24 h later, were injected with hLF(1–11); 18 h later, the number of viable yeasts in the kidneys was determined microbiologically, the size and number of infectious foci were determined histologically, and serum cytokine levels were determined by immunoassays.

Results.hLF(1–11) was effective (maximum reduction, 1.5 logs) against disseminated C. albicans infections, and its antifungal activity leveled off at a concentration of 0.4 ng of hLF(1–11)/kg of body weight. The antifungal activity of hLF(1–11) was increased in mice injected with interleukin (IL)–10 neutralizing antibodies, which suggests that IL-10 reduces the antifungal activity of hLF(1–11). In agreement with this result was the finding that injection of high doses of hLF(1–11) into infected mice was accompanied by increased levels of IL-10 in serum. Microscopic analysis revealed that infectious foci in kidneys of hLF(1–11)-treated mice contained mainly blastoconidia, whereas filamentous forms were abundant in untreated mice. The peptide inhibited the in vitro morphological transition of C. albicans, in a dose-dependent manner

Conclusions.hLF(1–11) is effective against disseminated C. albicans infections; and its effects on C. albicans viability and virulence and on host cells may explain this antifungal activity.

The frequency of Candida infections has increased during the past 2 decades, as a consequence of the rising number of immunocompromised hosts. An international program of surveillance of disseminated infections caused by Candida reported that 53% were due to Candida albicans [1], with an overall crude mortality rate of 38% [2]. Moreover, in recipients of bone-marrow transplants and in patients undergoing intensive chemotherapy, the crude mortality rate of disseminated Candida infections is >80% [3].

In healthy individuals, C. albicans is a commensal of skin and mucosal surfaces. This commensal status is ensured by an equilibrium between the yeast, the microflora, and the innate and acquired immune systems. The most relevant components of the innate immune system are phagocytes, NK cells, mast cells, and proteins such as complement factors, cytokines, and antimicrobial proteins/peptides (e.g., human lactoferrin [hLF]) [4–8]. hLF, a 77-kDa iron-binding glycoprotein synthesized by mucosal-gland epithelial cells and neutrophils, is present in body secretions such as milk and tears and in specific granules of neutrophils. This protein plays a role in the host defense by (1) releasing (during pepsinolysis) lactoferricin (residues 1–47) and various smaller N-terminal peptides with potent antimicrobial activities [5, 7, 8] and (2) modulating immune responses [9, 10]—for example, by inhibiting complement activation [11], affecting the production of reactive oxygen intermediates [12] and cytokines by phagocytes [5, 13, 14] and T lymphocytes [15], and increasing the phagocytosis and intracellular killing of Staphylococcus aureus by bovine phagocytes [16]. Furthermore, hLF triggers the production of antimicrobial peptides by mucosal epithelial cells [17], and it is able to prevent biofilm formation [18]. Therefore, analysis of the mechanisms underlying the antimicrobial activities of (peptides derived from) hLF in experimental animals and humans should consider all biological properties of this peptide.

Elsewhere we have reported that a synthetically prepared, naturally occurring, cationic N-terminal peptide, referred to as "hLF(1–11)," displays fungicidal activity against various Candida species, including fluconazole-resistant C. albicans strains in vitro [19, 20]. The mechanism of action of hLF(1–11) is as follows: via mitochondrial calcium uptake [21], it stimulates an increase in the mitochondrial membrane's potential and permeability [19], resulting in the synthesis and secretion of adenosine triphosphate and the production of reactive oxygen species [22], thereby leading to C. albicans cell death. In addition, hLF and lactoferricin inhibit filamentous growth of some azole-resistant strains of C. albicans [23]. This is important because the phenotypic transition of C. albicans from the blastoconidial to the filamentous form (together with both expression of adhesion molecules in the cell wall and protease production) is involved in tissue invasion by C. albicans [24–27]. The present study was undertaken to (1) determine whether hLF(1–11) displays antifungal activities in mice with disseminated fluconazole-resistant C. albicans infection and (2) gain more insight into the mechanisms underlying the antifungal activities of this peptide.

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Materials and Methods

Lactoferrin-derived peptides.The synthetic peptide corresponding to residues 1–11 of hLF (amino acids, GRRRRSVQWCA; molecular mass, 1374 daltons), hLF(1–11), was purchased from Peptisyntha; the synthetic peptide comprising alanines at position 2, 3, 6, and 10 of hLF(1–11), hereinafter referred to as a "control peptide" (Peptisyntha), was included as a negative control. The purity of these 2 peptides exceeded 97%, as determined by reverse-phase high-performance liquid chromatography. Stocks of the peptides at a concentration of 1 mg/mL of 0.01% acetic acid (pH 3.7) were stored at -20°C and were dried in a Speed-Vac (Savant Instruments) immediately before being used in in vitro experiments. In addition, synthetic peptides were freshly dissolved at a concentration of 1 mg/mL of saline and were diluted to desired concentrations immediately before being used in in vivo experiments.

Source of C. albicans strain.Fluconazole-resistant C. albicans strain Y01–19 was purchased from Pfizer. The yeast was identified by Candiselect (Sanofi Pasteur) and was confirmed by the pattern of sugar utilization (API, ID 32C; bioMerieux). Fluconazole resistance was evaluated, as MIC >256 μg/mL, by the Etest (AB Biodisk). Yeasts were cultured overnight at 37°C and then was subcultured, for 2.5 h at 37°C, on a rotary wheel in Sabouraud broth (Oxoid). Virulent strains of the yeast were obtained after 2 passages in Swiss mice. In brief, ∼ 1 × 105 cfu of yeasts in 0.1 mL of saline were injected into a tail vein, and the mice were killed 24 h later. The spleen was aseptically removed and homogenized, and serial dilutions of the homogenate were plated onto Sabouraud agar. A single colony-forming unit was transferred into 25 mL of Sabouraud broth and was incubated for 24 h at 37°C, and aliquots of this suspension, containing ∼ 5 × 108 virulent cfu of yeasts/mL of broth, supplemented with 30% (vol/vol) glycerol, were stored at -80°C.

Animals.Specific pathogen-free, female Swiss mice, 8–10 weeks old and weighing 20–25 g (Charles River), were used in the study. The mice were housed in local animal facilities for ⩾1 week before the start of the experiments. Food and water were given ad libitum. The animal studies were approved by the Leiden Experimental Animal Committee (approval DEC 02909) and were performed in compliance with Dutch laws related to the conduct of animal experiments.

Treatment of disseminated fluconazole-resistant C. albicans infections in neutropenic mice.The mice were rendered neutropenic by intraperitoneal injection, at days -3 and 0 of infection, of 100 mg of cyclophosphamide (Sigma-Aldrich Chemie BV)/kg of body weight. After the second injection of cyclophosphamide, blood-cell counts were randomly performed to confirm neutropenia, and then the mice were anesthetized with a single intraperitoneal injection of 0.1 mL of water containing 11 μg of fluanisone and 330 μg of fentanyl citrate (Hypnorm; Janssen Pharmaceutics). Next, ∼ 1 × 105 cfu of fluconazole-resistant C. albicans in 0.1 mL of saline were aseptically injected into a tail vein; 24 h later, the mice were intravenously injected with various doses (range, 0.04 ng–4 mg/kg of body weight) of hLF(1–11). Control mice were injected with saline. The control peptide and fluconazole (Pfizer) were included as negative controls. Fluconazole dissolved in 0.1 mL of saline was subcutaneously injected into mice at a concentration of 5 mg/kg of body weight. Amphotericin B (intraperitoneal injection of 0.5 μg/kg of body weight; Bristol-Meyers Squibb Group) was included as a positive control. After 18 h of treatment, the mice were killed; their kidneys were removed, weighed, and homogenized; and the number of viable yeasts was determined by plating serial dilutions of each sample on Sabouraud agar. The detection limit was 667 cfu. Results, expressed as the number of colony-forming units per gram of kidney, are from 2–4 independent experiments. Values for each mouse, as well as the median for its group, are reported. In some experiments, one kidney was used for microbiological analysis of the antifungal activities of the peptide whereas the other was processed for histological analysis (see below).

Measurement of cytokines.Levels of interleukin (IL)—10 and tumor necrosis factor (TNF)—α in serum were measured by ELISA (Sanquin), according to manufacturer's instructions. In addition, we took advantage of the Cytometric Bead Assay (Becton-Dickinson), to simultaneously measure multiple cytokines (IL-4, IL-5, IL-6, IL-10, monocyte chemotactic protein [MCP]-1, interferon [IFN]-γ, and IL-12p70) in small samples of serum. This assay was performed according to the manufacturer's instructions and used FACSCalibur (Becton-Dickinson) for quantitation of the levels in serum. The lower limit of detection of these immunological assays was ∼5 ng/mL. Results for each mouse, as well as the median (of 2–4 independent experiments) for its group, are reported.

Treatment with IL-10 neutralizing monoclonal antibody (mAb), in mice with disseminated fluconazole-resistant C. albicans infection.The mice were intravenously injected with 250 μg of IL-10 neutralizing mAb (clone JES052A5, rat IgG1; R&D Systems) 2 h before and 4 h after infection. As a control, the isotype-matched mAb anti-keyhole limpet hemocyanin (clone 43414, rat IgG1; R&D Systems) was injected into infected mice. Results for each mouse, as well as the median for its group (4–7 mice/group, in a single experiment), are reported.

Histology.Tissue samples taken at autopsy were fixed in 70% alcohol for 24 h and then in 4% formaldehyde and were processed and embedded in paraffin wax. Next, 5-μm—thick sections were stained with Grocott's modification of Gomori's methanamine-silver stain [28] and were counterstained with 0.2% light green (Fluka Sigma-Aldrich), for microscopic examination both of the number and size of infectious foci and of the morphology of C. albicans at sites of infection.

Assay for filamentous C. albicans. The assay used for determination of the transition of C. albicans from the blastoconidial to the filamentous form has been described elsewhere [29]. In brief, C. albicans cells were harvested in stationary phase by centrifugation at 1500 g for 10 min and then were washed twice in distilled water and were diluted to a concentration of 2 × 106 cfu/mL of Hanks' balanced salt solution (HBSS; GIBCO BRL, Paisley). The formation of filamentous C. albicans was induced in blastoconidia by 10% (vol/vol) heat (56°C for 30 min)–inactivated human plasma prepared from citrate blood. After being incubated for 45 min at 37°C, C. albicans cells were washed twice in HBSS and were reincubated at various concentrations of hLF(1–11) (range, 0–300 μg/mL), for 1 h at 37°C, on a rotary wheel. The test tubes then were transferred onto melting ice, and the percentage of filamentous C. albicans was scored by light microscopy. Results are means ± SEs of 5 independent experiments.

Statistical analyses.The antifungal activities of the various treatments—different doses of hLF(1–11), amphotericin B, fluconazole, control peptide, and saline—in mice with disseminated C. albicans infection, as well as those of different doses of hLF(1–11) in mice either injected or not injected with IL-10 neutralizing antibodies, were compared by Bonferroni's post-hoc multiple-comparison test. The levels of cytokine in sera of mice treated with the different doses of the hLF(1–11) peptide were also compared by this test. Analysis of variance was used to determine the effect that hLF(1–11) has on the morphological transition of C. albicans in vitro. Correlations between the number of viable C. albicans and the levels of TNF-α and IL-6 in serum were calculated by Spearman's rank test. In all tests, the level of significance was set at P < .05.

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Results

hLF(1–11)'s antifungal activity against disseminated fluconazole-resistant C. albicans infection in neutropenic mice.The results revealed that hLF(1–11) was effective (P < .05) against disseminated C. albicans infections, with a maximum antifungal effect of an ∼1.5-log reduction (figure 1). It is noteworthy that the antifungal activity of this peptide leveled off at concentrations as low as 0.4 ng of peptide/kg of body weight. As expected, the control peptide and fluconazole had no antifungal effect. In addition, 0.4 μg of hLF(1–11)/kg of body weight and an optimal dose of amphotericin B were equally effective (figure 1). The antifungal activity of hLF(1–11) could already be seen 4 h after treatment; for example, the median level of C. albicans in mice treated with 40 μg of peptide/kg of body weight was 1.9 × 105 (range, 1.7–3.1 × 105) cfu/g of kidney and that in control mice was 8.6 × 106 (range, 2.9 × 106–1.4 × 107) cfu/g of kidney (n = 3 mice; P < .05).

Figure 1.
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Figure 1.

Human lactoferrin-derived peptide hLF(1–11)'s antifungal activity against disseminated infection with fluconazole-resistant Candida albicans in neutropenic mice. The mice were rendered neutropenic by intraperitoneal injection, at days -3 and 0 of infection, of 100 mg cyclophosphamide/kg of body weight. Next, the mice were intravenously infected with 1 × 105 cfu of fluconazole-resistant C. albicans in 0.1 mL of saline and, 24 h later, were intravenously injected with saline containing various amounts (range, 0.04 ng–4 mg/kg of body weight) of hLF(1–11). Control mice were injected with saline. Amphotericin B (intraperitoneal injection of 0.5 μg/kg of body weight) was used as the positive control, and fluconazole dissolved in saline (subcutaneous injection of 5 mg/kg of body weight) and the control peptide (intravenous injection of 40 μg/mL) were used as the negative controls. After 18 h of treatment, the mice were killed; their kidneys were removed, weighed, and homogenized; and the number of viable yeasts was determined microbiologically. Values for each mouse, as well as the median for its group, are shown. The results are from 3–4 independent experiments, except in the case of mice treated with 0.0004 μg of hLF(1–11)/kg of body weight and amphotericin B (2 experiments). P values are for the differences between the values for mice treated with the indicated dose of either the peptide or the antifungal drug and those for mice that were injected with saline.

All mice treated with doses inadequate to reduce antifungal activity displayed clinical symptoms, such as fever and ruffed fur, and 27% of these mice died on day 2 of infection. None of the mice treated with an effective dose of hLF(1–11) peptide were severely ill during the period of analysis.

Effect of hLF(1–11) on levels of cytokine in sera of neutropenic mice with disseminated fluconazole-resistant C. albicans infection.Because we considered the possibility that the leveling off of the antifungal activity of hLF(1–11) peptide can be attributed to the levels of IL-10 in serum, we measured the levels of IL-10 in serum samples collected 18 h after hLF(1–11) was injected into mice with disseminated C. albicans infection. The levels of IL-10 in serum samples from mice that were injected with 4 mg of hLF(1–11)/kg of body weight were significantly (P < .001) greater than those in serum samples from mice injected with ⩽40 μg of peptide/kg of body weight (figure 2). In addition, the levels of TNF-α in serum samples from infected mice treated with 4 mg of hLF(1–11)/kg of body weight did not differ from those in serum samples from untreated infected mice, whereas they were decreased (P < .03) in infected mice injected with ⩽40 μg of peptide/kg of body weight (figure 3). No detectable levels of IL-10 and TNF-α were seen in uninfected neutropenic mice, and injection of the peptide into them did not induce detectable serum levels of these cytokines (data not shown).

Figure 2.
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Figure 2.

Effect that human lactoferrin-derived peptide (hLF(1–11)) has on levels of interleukin (IL)–10 in sera of neutropenic mice with disseminated fluconazole-resistant Candida albicans infection. The mice were intravenously injected with 0.1 mL of saline containing various doses (range, 0.4 ng–4 mg/kg of body weight) of hLF(1–11); 18 h later, the mice were killed, and blood samples were collected via cardiac puncture. Next, the levels of IL-10 in the sera were measured by ELISA. Values for each mouse, as well as the median for its group, are shown. The results are from 2–4 independent experiments. P values are for the differences between the values for mice that were treated with the indicated dose of peptide and those for mice that were injected with saline.

Figure 3.
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Figure 3.

Effect that human lactoferrin-derived peptide (hLF(1–11)) has on levels of tumor necrosis factor (TNF)—α in sera of neutropenic mice with disseminated fluconazole-resistant Candida albicans infection. The mice were intravenously injected with saline containing various doses (range, 0.4 ng–4 mg/kg of body weight) of hLF(1–11); 18 h later, the mice were killed, and blood samples were collected via cardiac puncture. Next, the levels of TNF-α in the sera were measured by ELISA. Values for each mouse, as well as the median for its group, are shown. The results are from 2–3 independent experiments. P values are for the differences between the values for mice that were treated with the indicated dose of peptide and those for mice that were injected with saline.

To gain more insight into the cytokine profile of hLF(1–11)-treated mice, we measured the effects that this peptide had on the levels of type 1 (IFN-γ, IL-12p70), type 2 (IL-4, IL-5), and other cytokines (e.g., MCP-1 and IL-6) in the sera of infected mice; we found detectable levels of IFN-γ, IL-12p70, MCP-1, and IL-6 (table 1) but not of IL-4 and IL-5. The levels of IL-12 and MCP-1 in the sera of infected mice were not affected by this peptide, and the levels of IFN-γ in the sera of mice injected with 0.0004–0.4 μg of peptide/kg of body weight were significantly greater than those in untreated mice, whereas the levels of IL-6 were significantly (P < .05) decreased by these doses of hLF(1–11). Interestingly, significant correlations were found between the number of viable C. albicans and the levels of both TNF-α (P < .01) and IL-6 (P < .01) that were induced by treatment with 0.0004–40 μg of hLF(1–11)/kg of body weight.

Effect of IL-10 neutralizing mAb on hLF(1–11)'s antifungal activity against disseminated C. albicans infection.To discover whether the level of IL-10 in the mice reduces the antifungal activity of hLF(1–11) peptide, we determined the antifungal activities of 2 different doses—4 μg and 4 mg of hLF(1–11) peptide/kg of body weight—in mice injected with and in mice not injected with IL-10 neutralizing mAb. Both doses of peptide completely eliminated C. albicans from the kidneys of mice injected with IL-10 neutralizing mAb, whereas 4 μg and 4 mg of peptide/kg of body weight resulted, respectively, in 4-log and 3-log reductions in the number of colony-forming units per gram of kidney in mice not injected with this mAb (figure 4). Interestingly, the number of C. albicans in mice injected with IL-10 neutralizing mAb was 1.5 log lower (P < .01) than that in mice not injected with this antibody. It is noteworthy that the antifungal activity of the hLF(1–11) peptide in infected mice did not differ from that in infected mice injected with the isotype-matched control mAb (data not shown).

Figure 4.
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Figure 4.

Effect that interleukin (IL)—10 neutralizing monoclonal antibody (mAb) has on human lactoferrin-derived peptide hLF(1–11)'s ) antifungal activity against disseminated infection with fluconazole-resistant Candida albicans in neutropenic mice. The mice were rendered neutropenic by intraperitoneal injection, at days -3 and 0 of infection, of 100 mg of cyclophosphamide/kg of body weight. Next, the mice were intravenously infected with 1 × 105 cfu of fluconazole-resistant C. albicans in 0.1 mL of saline and, 24 h later, were intravenously injected with 0.1 mL of saline containing various amounts of hLF(1–11). Some groups of mice were intravenously injected, 2 h before and 4 h after infection, with 250 μg of either IL-10 neutralizing mAb or an isotype-matched control mAb; 18 h after treatment, the mice were killed; their kidneys were removed, weighed, and homogenized; and the number of viable yeasts was determined microbiologically. Values for each mouse, as well as the median for its group (7 mice/group, in a single experiment), are shown.

Effect of hLF(1–11) on the size and number of infectious foci and on the morphology of C. albicans in kidney tissue sections of mice with disseminated fluconazole-resistant C. albicans infection.Light-microscopic examination of Grocott-stained kidney-tissue sections of mice with disseminated fluconazole-resistant C. albicans infection revealed that the size and number of infectious foci decreased with increasing doses of hLF(1–11), up to a dose of 40 μg/kg of body weight; for example, the number and size of infectious foci in mice injected with 40 μg of peptide/kg of body weight (figure 5A and 5B) were considerably lower than those in untreated mice (figure 5C and 5D). Doses >40 μg of peptide/kg of body weight were not more effective (results not shown). In addition, the morphology of C. albicans in these tissue sections was studied. The results revealed that C. albicans cells, which were mainly filamentous in infectious foci in untreated mice (figure 5D), were growing as blastoconidia in mice effectively treated with hLF(1–11) (figure 5B).

Figure 5.
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Figure 5.

Infectious foci in kidneys of neutropenic mice with disseminated Candida albicans infection. The mice were intravenously injected with 40 μg of either human lactoferrin-derived peptide (hLF(1–11))/kg of body weight (A and B) or saline (C and D); 18 h later, the mice were killed, and their kidneys were removed, fixed, and embedded in paraffin wax. Next, 5-μm sections of kidney were cut and stained with Grocott's modification of Gomori's methamine-silver stain and were counterstained with light green. Arrows indicate the presence of C. albicans. Magnifications, 50× (A and C) and 400× (B and D).

Effect of hLF(1–11) on the in vitro transition, induced by human plasma, in C. albicans morphology.In order to evaluate whether hLF(1–11) inhibits the transition in C. albicans morphology, blastoconidia were incubated with 10% (vol/vol) heat-inactivated human plasma in vitro and then were washed and reincubated in buffer in the presence of increasing concentrations of the peptide. Microscopic analysis revealed that the percentage of C. albicans germ-tube formation in vitro decreased (P < .05) with increasing concentrations of hLF(1–11) (figure 6).

Figure 6.
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Figure 6.

Effect that human lactoferrin-derived peptide (hLF(1–11)) has on the plasma-induced transition in Candida albicans morphology in vitro. In brief, transition to the filamentous form was induced in 2 × 106 blastoconidia/mL of Hanks' balanced salt solution by incubation, for 45 min at 37°C, with 10% heat-inactivated human plasma. Next, the cells were washed and were reincubated, for 1 h at 37°C, with various concentrations (range, 0–300 μg/mL) of hLF(1–11) and were scored by light microscopy. Results, expressed as the percentage of filamentous C. albicans, are means + SEs of 5 independent experiments; an asterisk (*) indicates a result significantly different (P < .05) from that for Candida not treated with this peptide.

Table 1.
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Table 1.

Effect that human lactoferrin–derived peptide (hLF(1–11)) has on levels of cytokine in sera of mice with disseminated Candida albicans infection.

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Discussion

The main conclusion to be drawn from the results of the present study is that the hLF(1–11) peptide is effective against disseminated (fluconazole-resistant) C. albicans infections in cyclophosphamide-treated mice. This conclusion is based on the following observations. First, compared with the number of viable yeasts in the kidneys of untreated mice, those in the kidneys of mice treated with a wide range of hLF(1–11) doses was significantly reduced, and the results are in agreement with our earlier reports showing that in vitro hLF(1–11) kills Candida species in a dose-dependent manner [19, 20]. Second, microscopic analysis of kidney-tissue sections showed that infectious foci in hLF(1–11)-treated mice contained considerably fewer C. albicans than did those in untreated mice, and both the size and number of infectious foci in the kidneys of untreated mice also were greater than those in hLF(1–11)-treated mice. Moreover, it appeared that C. albicans was growing mainly as blastoconidia in hLF(1–11)-treated mice, whereas filamentous forms prevailed in untreated mice; consistent with this observation is that the percentage of C. albicans germ-tube formation in vitro decreased with increasing concentrations of hLF(1–11). Because, both in animal studies [18] and in cell cultures [30, 31], the filamentous form is associated with the virulence of C. albicans, it is conceivable that inhibition of the morphological transition of C. albicans effected by this peptide contributes to the amelioration of clinical signs and symptoms of C. albicans infection in mice.

It should be noted that the dose of hLF(1–11) peptide necessary to provide antifungal effects (killing and inhibition of the morphological transition of C. albicans) in mice was much lower than that in in vitro experiments [19, 20]. A possible explanation for this observation is that the antifungal effects of hLF(1–11) in animals with experimental infections involve synergistic/additive effects between this peptide and host-derived factors, including other antimicrobial molecules, as has been reported for hLF and lysozyme and secretory leukoprotease inhibitor [32], reactive oxygen intermediates [33], and other local factors, such as pH and Ca2+ and Zn2+ concentrations. Another possible explanation is that hLF(1–11) stimulates the antifungal activities of host cells, as has been reported for the effects that lactoferrin has on the antimicrobial activities of macrophages and neutrophils [14, 34, 35] and the production of antimicrobial peptides by mucosal epithelial cells [17].

One of the most remarkable findings of the present study is that the antifungal activity of hLF(1–11) leveled off after a dose as low as 0.4 ng/kg of body weight. This finding is in agreement with our observation that mice treated with low doses of hLF(1–11) did not have significant clinical signs and symptoms of the infection. The most likely explanation for the leveling off of the antifungal effects of hLF(1–11) is that the latter induces multiple processes that contribute differently to its antifungal activity against disseminated C. albicans infection in mice and that activation of some of these processes was seen only when high doses of this peptide were used. Consistent with this explanation is our finding that, compared with the levels of IL-10 in the sera of untreated mice, those in the sera of mice treated with high doses—that is, ⩾4 mg/kg of body weight—of hLF(1–11) were elevated whereas those in the sera of mice injected with ⩽40 μg/kg of body weight were not. Because IL-10 suppresses immunity against Candida infections [36–39], it could be that the leveling off of its antifungal effects in mice is due partly to the inhibitory effects that IL-10 has on host defense against C. albicans infections. Indeed, we found that, compared with the antifungal effects in mice that were injected with either the isotype-matched control mAb or no antibody, those in mice injected with IL-10 neutralizing mAb were increased, indicating that, in this latter group of mice, IL-10 counteracts hLF(1–11)'s antifungal effects in a dose-dependent manner. Further investigation into the effects that hLF(1–11) has on the levels of cytokines in the sera of mice with disseminated C. albicans infection have shown that, compared with the levels of TNF-α and IL-6 in the sera of untreated mice, those in the sera of mice treated with effective doses of hLF(1–11) were significantly decreased whereas those in mice treated with the highest tested dose (i.e., 4 mg/kg of body weight) of this peptide were not. These data are in agreement both with the observation that bovine lactoferrin reduces the production of TNF-α and IL-6 by circulating blood cells in mice [13] and with the finding that bovine lactoferricin suppresses the lipopolysaccharide-stimulated production of IL-6 and TNF-α by human monocytic cells [40]. Our observation that the levels of IFN-γ in the sera of mice treated with 0.4 ng–0.4 μg of hLF(1–11)/kg of body weight were significantly greater than those in the sera of untreated mice may indicate that this peptide acts on T lymphocytes, as has elsewhere been reported for intact human lactoferrin [15]. Obviously, further investigations are necessary to establish whether cytokine production at sites of infection is reflected by the levels of cytokines in serum.

In conclusion, the antifungal activities of hLF(1–11) may involve both its actions on C. albicans viability and virulence and its effects on host cells that are crucial in the defense against disseminated C. albicans infections, as has elsewhere been reported for amphotericin B [41].

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Acknowledgment

We thank Mrs. Suzan Teeuwen (Department of Infectious Diseases, Leiden University Medical Center) for technical assistance.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: SenterNovem (contract ISO 44096 to R.H.E.F. and P.H.N.); Italian "Ministerio dell'Istruzione, dell'Universia e della Ricerca" (contract 2003062784 to A.L.).

  • Received August 16, 2006.
  • Accepted April 16, 2007.
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References

  1. 1.↵
    1. Pfaller MA,
    2. Jones RN,
    3. Doern GV,
    4. Sader HS,
    5. Hollis RJ,
    6. Messer SA
    . International surveillance of bloodstream infections due to Candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the United States, Canada, and South America for the SENTRY Program. The SENTRY Participant Group. J Clin Microbiol 1998;36:1886-9.
    Abstract/FREE Full Text
  2. 2.↵
    1. St-Germain G,
    2. Laverdiere M,
    3. Pelletier R,
    4. et al
    . Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada. J Clin Microbiol 2001;39:949-53.
    Abstract/FREE Full Text
  3. 3.↵
    1. Eggimann P,
    2. Garbino J,
    3. Pittet D
    . Management of Candida species infections in critically ill patients. Lancet Infect Dis 2003;3:772-85.
    CrossRefMedlineWeb of Science
  4. 4.↵
    1. van der Strate BW,
    2. Harmsen MC,
    3. et al.
    ; The TH. Plasma lactoferrin levels are decreased in end-stage AIDS patients. Viral Immunol 1999;12:197-203.
    MedlineWeb of Science
  5. 5.↵
    1. Wakabayashi H,
    2. Takakura N,
    3. Teraguchi S,
    4. Tamura Y
    . Lactoferrin feeding augments peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Microbiol Immunol 2003;47:37-43.
    MedlineWeb of Science
  6. 6.
    1. Soukka T,
    2. Tenuovuno J,
    3. Lenander-Lumikari M
    . Fungicidal effect of human lactoferrin against Candida albicans. FEMS Microbiol Lett 1992;69:223-8.
    Medline
  7. 7.↵
    1. Andersen JH,
    2. Jenssen H,
    3. Gutteberg TJ
    . Lactoferrin and lactoferricin inhibit herpes simplex 1 and 2 infection and exhibit synergy when combined with acyclovir. Antiviral Res 2003;58:209-15.
    CrossRefMedlineWeb of Science
  8. 8.↵
    1. Nibbering PH,
    2. Ravensbergen E,
    3. Welling MM,
    4. et al
    . Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria. Infect Immun 2001;69:1469-76.
    Abstract/FREE Full Text
  9. 9.↵
    1. Guillen C,
    2. McInnes IB,
    3. Vaughan DM,
    4. et al
    . Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice. J Immunol 2002;168:3950-7.
    Abstract/FREE Full Text
  10. 10.↵
    1. Haversen LA,
    2. Baltzer L,
    3. Dolphin G,
    4. Hanson LA,
    5. Mattsby-Baltzer I
    . Anti-inflammatory activities of human lactoferrin in acute dextran sulphate-induced colitis in mice. Scand J Immunol 2003;57:2-10.
    CrossRefMedlineWeb of Science
  11. 11.↵
    1. Samuelsen O,
    2. Haukland HH,
    3. Ulvatne H,
    4. Vorland LH
    . Anti-complement effects of lactoferrin-derived peptides. FEMS Immunol Med Microbiol 2004;41:141-8.
    CrossRefMedlineWeb of Science
  12. 12.↵
    1. Mocsai A,
    2. Ligeti E,
    3. Lowell CA,
    4. Berton G
    . Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck. J Immunol 1999;162:1120-6.
    Abstract/FREE Full Text
  13. 13.↵
    1. Zimecki M,
    2. Spiegel K,
    3. Wlaszczyk A,
    4. Kübler A,
    5. Kruzel ML
    . Lactoferrin increases the output of neutrophil precursors and attenuates the spontaneous production of TNF-alpha and IL-6 by peripheral blood cells. Arch Immunol Ther Exp (Warsz) 1999;47:113-8.
    Medline
  14. 14.↵
    1. Tanida T,
    2. Rao F,
    3. Hamada T,
    4. Ueta E,
    5. Osaki T
    . Lactoferrin peptide increases the survival of Candida albicans-inoculated mice by upregulating neutrophil and macrophage functions, especially in combination with amphotericin B and granulocyte-macrophage colony-stimulating factor. Infect Immun 2001;69:3883-90.
    Abstract/FREE Full Text
  15. 15.↵
    1. Legrand D,
    2. van Berkel PH,
    3. Salmon V,
    4. et al
    . The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells. Biochem J 1997;327:841-6.
  16. 16.↵
    1. Kai K,
    2. Komine K,
    3. Komine Y,
    4. et al
    . Lactoferrin stimulates a Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland. Microbiol Immunol 2002;46:187-94.
    MedlineWeb of Science
  17. 17.↵
    1. Velliyagounder K,
    2. Kaplan JB,
    3. Furgang D,
    4. et al
    . One of two human lactoferrin variants exhibits increased antibacterial and transcriptional activation activities and is associated with localized juvenile periodontitis. Infect Immun 2003;71:6141-7.
    Abstract/FREE Full Text
  18. 18.↵
    1. Singh PK,
    2. Parsek MR,
    3. Greenberg EP,
    4. Welsh MJ
    . A component of innate immunity prevents bacterial biofilm development. Nature 2002;417:552-5.
    CrossRefMedline
  19. 19.↵
    1. Lupetti A,
    2. Paulusma-Annema A,
    3. Welling MM,
    4. Senesi S,
    5. van Dissel JT,
    6. Nibbering PH
    . Candidacidal activities of human lactoferrin peptides derived from the N terminus. Antimicrob Agents Chemother 2000;44:3257-63.
    Abstract/FREE Full Text
  20. 20.↵
    1. Lupetti A,
    2. Paulusma-Annema A,
    3. Welling MM,
    4. et al
    . Synergistic activity of the N-terminal peptide of human lactoferrin and fluconazole against Candida species. Antimicrob Agents Chemother 2003;47:262-7.
    Abstract/FREE Full Text
  21. 21.↵
    1. Lupetti A,
    2. Brouwer CP,
    3. Dogterom-Ballering H,
    4. et al
    . Release of calcium from intracellular stores and subsequent uptake by mitochondria are essential for the candidacidal activity of a N-terminal peptide of human lactoferrin. J Antimicrob Chemother 2004;54:603-8.
    Abstract/FREE Full Text
  22. 22.↵
    1. Lupetti A,
    2. Paulusma-Annema A,
    3. Senesi S,
    4. Campa M,
    5. van Dissel JT,
    6. Nibbering PH
    . Internal thiols and reactive oxygen species in candidacidal activity exerted by an N-terminal peptide of human lactoferrin. Antimicrob Agents Chemother 2002;46:1634-9.
    Abstract/FREE Full Text
  23. 23.↵
    1. Wakabayashi H,
    2. Abe S,
    3. Teraguchi S,
    4. Hayasawa H,
    5. Yamaguchi H
    . Inhibition of hyphal growth of azole-resistant strains of Candida albicans by triazole antifungal agents in the presence of lactoferrin-related compounds. Antimicrob Agents Chemother 1998;42:1587-91.
    Abstract/FREE Full Text
  24. 24.↵
    1. Kretschmar M,
    2. Hube B,
    3. Bertsch T,
    4. et al
    . Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis. Infect Immun 1999;67:6637-42.
    Abstract/FREE Full Text
  25. 25.
    1. Fratti RA,
    2. Belanger PH,
    3. Sanati H,
    4. Ghannoum MA
    . The effect of a new triazole, voriconazole (UK-109,496), on the interactions of Candida albicans and Candida krusei with endothelial cells. J Chemother 1998;10:7-16.
    MedlineWeb of Science
  26. 26.
    1. Lopez-Ribot JL,
    2. Vespa MN,
    3. Chaffin WL
    . Adherence of Candida albicans germ tubes to murine tissues in an ex vivo assay. Can J Microbiol 1994;40:77-81.
    MedlineWeb of Science
  27. 27.↵
    1. Odds FC
    . Pathogenesis of Candida infections. J Am Acad Dermatol 1994;31(3 Pt 2):S2-5.
    MedlineWeb of Science
  28. 28.↵
    1. Stevens A
    1. Bancroft JD
    . Theory and practice of histological techniques. 2nd ed. Stevens A, editor. Edinburgh: Churchill Livingstone; 1982.
  29. 29.↵
    1. Cutuli M,
    2. Cristiani S,
    3. Lipton JM,
    4. Catania A
    . Antimicrobial effects of alpha-MSH peptides. J Leukoc Biol 2000;67:233-9.
    Abstract
  30. 30.↵
    1. Lo H-J,
    2. Kohler JR,
    3. DiDomenico B,
    4. Loebenberg D,
    5. Cacciapuoti A,
    6. Fink GR
    . Nonfilamentous Candida albicans mutants are avirulent. Cell 1997;90:939-49.
    CrossRefMedlineWeb of Science
  31. 31.↵
    1. Rocha CR,
    2. Schroppel K,
    3. Harcus D,
    4. et al
    . Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 2001;12:3631-43.
    Abstract/FREE Full Text
  32. 32.↵
    1. Singh PK,
    2. Tack BF,
    3. McCray PB Jr.,
    4. Welsh MJ
    . Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Am J Physiol Lung Cell Mol Physiol 2000;279:L799-805.
    Abstract/FREE Full Text
  33. 33.↵
    1. Porro GA,
    2. Lee JH,
    3. de Azavedo J,
    4. et al
    . Direct and indirect bacterial killing functions of neutrophil defensins in lung explants. Am J Physiol Lung Cell Mol Physiol 2001;281:L1240-7.
    Abstract/FREE Full Text
  34. 34.↵
    1. Miyauchi H,
    2. Hashimoto S,
    3. Nakajima M,
    4. Shinoda I,
    5. Fukuwatari Y,
    6. Hayasawa H
    . Bovine lactoferrin stimulates the phagocytic activity of human neutrophils: identification of its active domain. Cell Immunol 1998;187:34-7.
    CrossRefMedlineWeb of Science
  35. 35.↵
    1. Manev V,
    2. Maneva A,
    3. Sirakov L
    . Effect of lactoferrin on the phagocytic activity of polymorphonuclear leukocytes isolated from blood of patients with autoimmune diseases and Staphylococcus aureus allergy. Adv Exp Med Biol 1998;443:321-30.
    MedlineWeb of Science
  36. 36.↵
    1. Vazquez-Torres A,
    2. Jones-Carson J,
    3. Wagner RD,
    4. Warner T,
    5. Balish E
    . Early resistance of interleukin-10 knockout mice to acute systemic candidiasis. Infect Immun 1999;67:670-4.
    Abstract/FREE Full Text
  37. 37.
    1. Tonnetti L,
    2. Spaccapelo R,
    3. Cenci E,
    4. et al
    . Interleukin-4 and -10 exacerbate candidiasis in mice. Eur J Immunol 1995;25:1559-65.
    MedlineWeb of Science
  38. 38.
    1. Netea MG,
    2. Sutmuller R,
    3. Hermann C,
    4. et al
    . Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol 2004;172:3712-8.
    Abstract/FREE Full Text
  39. 39.↵
    1. Romani L,
    2. Puccetti P,
    3. Mencacci A,
    4. et al
    . Neutralization of IL-10 up-regulates nitric oxide production and protects susceptible mice from challenge with Candida albicans. J Immunol 1994;152:3514-21.
    Abstract
  40. 40.↵
    1. Haversen L,
    2. Ohlsson BG,
    3. Hahn-Zoric M,
    4. Hanson LA,
    5. Mattsby-Baltzer I
    . Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 2002;220:83-95.
    CrossRefMedlineWeb of Science
  41. 41.↵
    1. Vonk AG,
    2. Netea MG,
    3. Denecker NEJ,
    4. Verschueren CMM,
    5. van der Meer JWM,
    6. Kullberg BJ
    . Modulation of the pro- and anti-inflammatory cytokine balance by amphotericin B. J Antimicrob Chemother 1998;42:469-74.
    Abstract/FREE Full Text
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