Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: J Appl Microbiol. 2014 Nov 25;118(1):212–221. doi: 10.1111/jam.12666

Oral lactoferrin protects against experimental candidiasis in mice

Kabilan Velliyagounder 1,*, Wijdan Alsaedi 1, Waad Alabdulmohsen 1, Kenneth Markowitz 1, Daniel H Fine 1
PMCID: PMC4469205  NIHMSID: NIHMS699200  PMID: 25319508

Abstract

Aims

To determine the role of lactoferrin in protecting the oral cavities of mice against Candida albicans infection in lactoferrin knockout (LFKO−/−) mice were compared to wild-type (WT) mice. We also determine the protective role of human lactoferrin in the LFKO−/− mice.

Methods and Results

Antibiotic treated immunosuppressed mice were inoculated with C. albicans (or sham infection) by oral swab and evaluated for the severity of infection after 7 days of infection. To determine the protective role of hLF, we added 0.3% solution of hLF to the drinking water given to some of the mice. CFU count, scoring of lesions and microscopic observations were carried out to determine the severity of infection. LFKO−/−I mice showed a 2 log (P=0.001) higher CFUs of C. albicans in the oral cavity compared to the WTI mice. LFKO−/−I mice given hLF had a 3 log (P=0.001) reduction in CFUs in the oral cavity compared to untreated LFKO−/−I mice. The severity of infection, observed by light microscopy revealed that the tongue of the LFKO−/−I mice showed more white patches compared to WTI and LFKO−/−I+hLF mice. Scanning electron microscopic observation revealed that more filiform papillae were destroyed in LFKO−/−I mice when compared to WTI or LFKO−/−I +hLF mice.

Conclusions

Human lactoferrin is important in protecting mice from oral C. albicans infection. Administered hLF may be used to prevent C. albicans infection.

Significance and Impact of the Study

Human lactoferrin, a multifunctional iron-binding glycoprotein can be used as a therapeutic active ingredient in oral health care products against C. albicans.

Keywords: human lactoferrin, Candida albicans, oral candidiasis, lactoferrin knockout

Introduction

Candida albicans the most prevalent fungal biofilm forming pathogen, is responsible for several types of oral and systemic infections, including oropharyngeal candidiasis (OPC) or thrush, denture stomatitis (DS) and bloodborne candidemia infections (Drobacheff et al. 1996; Cannon and Chaffin 1999; Ruhnke 2006). In the United States, candidemia is the fourth most common bloodstream infection seen in hospitalized patients (Edmond et al. 1999). Sixty to ninety percent of HIV-infected patients are susceptible to fungal infections, specifically oropharyngeal candidiasis (Moore and Chaisson 1996). C. albicans bloodstream infections are polymicrobial and often associate with a variety of pathogenic bacterial species, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis (Morales and Hogan 2010; Peleg et al. 2010; Kovac et al. 2013). The mortality rate of systemic Candida infection was reported to be 71–97% (Fraser et al. 1992). Also, it can cause infections in diabetic patients and those who have a long history of antibiotic and immunosuppressive drug usage. C. albicans’ main targets are immunocompromised patients, neonates and the elderly, especially denture wearers (Akpan and Morgan 2002). It has been reported that 65% of denture wearers develop Candida-associated denture stomatitis (Daniluk et al. 2006). C. albicans pathogenicity mainly relies on the immune status of the host and the virulence factors of the fungus. There are several virulence factors that are responsible for the pathogenicity of C. albicans including; host cell adhesion, hydrolytic enzymes production, dimorphic phenotype and the ability to form a biofilm on biotic and abiotic surfaces (Cutler et al. 1991).

There are several reports showing resistant strains of C. albicans due to the recurrent episodes of oropharyngeal candidiasis and the prolonged use of azole antifungal treatment (White and Goetz 1994; Ruhnke et al. 1994; Drobacheff et al. 1996; Hamza et al. 2008). Currently different antimicrobials and antifungal or a combination of both are being used to treat oral and systemic infections caused by C. albicans. However, being an adherent organism with a tendency to invade tissues, it is extremely difficult to eradicate C. albicans from the oral cavity and other organs. Also the emergence of resistant strains makes it difficult to treat C. albicans resulting in a high percentage of recurrence. Frequent use of antifungal drugs not only increases fungal resistance but also increases the susceptibility to unpleasant side effects. Therefore, new strategies are required for the development of antifungal agents and combined treatments against C. albicans infection.

Lactoferrin is an important component of the innate immune system found in high levels in human milk, especially high concentration in the colostrum. It is also freely available in the subgingival environment as a result of neutrophil degranulation (Tomita et al. 2009). HLF exhibits bacteriostatic and bactericidal activities against a wide range of Gram-negative and Gram-positive bacteria (Gonzalez-Chavez et al. 2009). The anti-Candida activity of hLF was first reported by Kirkpatrick et al. (1971) and after that there have been several reports showing that hLF has anti-candidal activity demonstrated in vitro and in vivo (Abe et al, 2000; Samaranayake et al. 2001; Takakura et al. 2003; Lupetti et al. 2003; Takakura et al. 2004; Lupetti et al. 2007; Andres et al. 2008). It has also been reported that hLF prevented Candida species from producing pseudeohyphea for 3 days (Al-Sheikh 2009). The mechanism of hLF anticandidal activity against C. albicans may be related to cell surface alterations, leakage of proteins and formation of surface blebs (Nikawa et al. 1993; Nikawa et al. 1995).

A study showed that prophylactic treatment of talactoferrin alfa (TLF) had a significant effect on neonatal rats with C. albicans and S. epidermidis coinfection (Venkatesh et al. 2007) Another study demonstrated the synergic effect of LF and lysozyme against C. albicans (Samaranayake et al. 2001). Moreover, it has been reported that transgenic porcine LF milk-fed mice were protected against E. coli, S. aureus and C. albicans infections of their digestive tract (Yen et al. 2009). Masci (2000) had reported that including LF and lysozyme in a mouthwash was useful in treating refractory oral candidiasis in HIV patients (Masci 2000). Another clinical study also showed that LF in mucoadhesive tablets reduced oropharyngeal candidiasis (Kuipers et al. 2002).

There have been many studies demonstrating the protective effect of LF and LF derived peptides against in vivo infections in mouse models where the animals are capable of producing LF. Candida infection has not been studied in animal models where LF was lacking or where LF deficient mice were given exogenous hLF. We have demonstrated in our previous study that the lactoferrin knockout (LFKO−/−) mice had a higher risk and severity of infection with oral bacteria including higher alveolar bone loss with increased expression of proinflammatory cytokines as well as the chemokine expression during infection with Aggregatibacter actinomycetemcomitans (Velusamy et al. 2013). We also reported that IV administration of hLF rapidly cleared A. actinomycetemcomitans and S. mutans in blood and other organs in LFKO−/− mice compared to untreated group (Velusamy et al. 2014a; Velusamy et al. 2014b).

Based on our preliminary data and previous studies we hypothesize that LFKO−/− mice are more susceptible to C. albicans infections compared to wild-type mice. It is of our interest to establish a model to examine the use of hLF to control oral candidiasis in the absence of endogenous LF. Therefore, in the present study, we examined the experimental oral candidiasis and the eventual patterns of the anticandidal activities in response to hLF treatment in a LFKO−/− mouse model. This study will provide data necessary to help us understand the use of hLF as an anticandidal agent and its mechanisms of action in vivo in an experimentally induced oral candidiasis in mouse models.

Materials and Methods

C. albicans and growth condition

Candida albicans strain (ATCC 90028) obtained from Dr. Gill Diamond (University of Florida, Gainesville, FL) was stored at −80°C in YPD broth containing 0.5% yeast extract and 10% glycerol until the experiment was performed. The yeast was grown on YPD agar plates at 37°C for 24 h and harvested by microspatula and then suspended in RPMI 1640 medium (Sigma Chemical Co., St. Louis, Mo) containing 2.5% fetal calf serum. The yeast cell number of the suspension was adjusted to 2.5×107 cells/ml by using a hemocytometer. In this study the following chemicals and reagents were used including tetracycline (5mg/ml), prednisolone (0.83 mg/ml) (Sigma), human milk (a generous gift from Dr. Plaut, Tufts-New England Medical Center, Boston, MA) and hLF (Sigma).

In vitro effect of human milk and hLF on C. albicans

Based on the previous study, LF concentration in human milk (HM) was adjusted to 200 µg/ml as previously described (Plaut et al. 2000). C. albicans was incubated either with HM or purified LF from HM (200 µg/ml) for different time points (6, 12, 24, 48 and 72 h) at 37 °C. At each time point the yeast cells were then washed three times with phosphate buffered saline (PBS) (Morrill et al. 2003). The samples were then serially diluted and plated on YPD agar plates and viable colonies were enumerated after 2 days.

In vivo experimental oral candidiasis

Mice

Experimental groups comprised of 7–8 week-old male wild-type (C57BL/6J) and lactoferrin knockout (LFKO−/−) mice, a generous gift from Dr. Orla Conneely, (Baylor College of Medicine, Houston, TX, USA). All animal experimental protocols were approved by the campus Institutional Animal Care and Use Committee (IACUC) of Rutgers School of Dental medicine, Newark, NJ, USA.

In the murine model of oral candidiasis that was previously developed, oral candidiasis was observed to be non-lethal (Takakura et al. 2003; Wakabayashi et al. 2003). Based on the patterns of infections in these studies, we used 6 mice for each group and total of 30 mice were used to test our hypothesis. The following experimental groups were studied; 1) Wild-type control mice (WTC), 2) Wild-type mice infected with C. albicans (WTI), 3) LFKO−/− mice control (LFKO−/−C), 4) LFKO−/− mice infected with C. albicans (LFKO−/−I) and 5) LFKO−/− mice infected with C. albicans and treated with hLF (LFKO−/−I+hLF).

Experimental oral candidiasis

The animals were randomized, assigned to groups of 6 and given food and water ad libitum. C. albicans experimental oral infection was performed according to the published method (Takakura et al. 2003). Tetracycline hydrochloride in the drinking water at a concentration of 0.83 mg/ml was given to the mice beginning one day before the infection and continued throughout the study to reduce oral bacteria. The oral cavity was swabbed before infection and plated on YPD-tetracycline (5 mg/ml) agar plates to verify the absence of bacteria. Mice were immunosuppressed with two subcutaneous injections of prednisolone at a dose of 100 mg/kg of body weight one day prior to and 3 days after the infection with C. albicans. Mice were anesthetized by an intramuscular injection with 50 µl of 2-mg/ml chlorpromazine chlorides in the thigh. Small cotton bud was soaked in a C. albicans cell suspension (2.5×107 cells/ml) and the entire oral cavity of the anesthetized mice was swabbed to produce oral infection. The oral cavities of mice in the control group were swabbed with C. albicans-free media.

Human lactoferrin administration

To determine the protective role of hLF against C. albicans, hLF at a concentration of 0.3% solution in drinking water (equivalent to 0.5 g/kg/day) was consecutively administered from one day before the infection and throughout the study as previously described (Takakura et al. 2003).

Evaluation of severity of infection

Microbiological evaluation

The mice in each experimental group were sacrificed with CO2 7 days after C. albicans or sham infection. Microbiological evaluations of the progression of infection were carried out as follows. The whole oral cavity, including the buccal mucosa, the tongue, the soft palate, and other oral mucosal surfaces were swabbed with a cotton bud and placed in a tube containing 5 ml of sterile PBS. The samples were then serially diluted, plated on YPD agar plates with tetracycline incubated at 37°C for 2 days followed by enumeration of CFU of Candida cells (Takakura et al. 2003).

Microscopic Analysis

Light microscopy & scanning electron microscopy

The tongues from the experimental groups were removed, washed with PBS and then photographed using light microscopy (Olympus SZ61, Tokyo, Japan) to observe the severity of the infection. Also, scanning electron microscopy (SEM) analysis was performed to examine morphological changes of the dorsal surfaces of tongues after the Candida infection or sham infection. After washing the tongues with PBS, the tongues were fixed with 2% glutaraldehyde for 2 h, dehydrated with different concentrations of ethanol (25, 50, 75, 100%) with each for 25 min, and processed for SEM according to a previously published method using an accelerating voltage of 30 kV (Hitachi SEM S2500; Hitachi High Technologies America, Inc.) (Velliyagounder et al. 2012).

Macroscopic evaluation

Macroscopic evaluation of the infection was indicated by a lesion score from 0 to 4 on the basis of the extent and severity of whitish, curd-like patches on the tongue surface as follows; 0, normal; 1, white patches less than 20%; 2, white patches less than 90% but more than 21%; 3, white patches more than 91% and 4, thick white patches like pseudo membranes more than 91% of the tongue (Takakura et al. 2003).

Complete blood count (CBC)

Heparinized blood was obtained by retro-orbital phlebotomy under anesthesia, and the CBC was determined by using the automated H1 Technicon system. (Antech Diagnostics, New Hyde Park, NY).

Statistical analysis

The CFU of C. albicans isolated from the mouths of infected mice were analyzed by using Student’s t test, one-way analysis of variance (ANOVA) and post hoc Tukey’ HST test between the groups with the JMP software SAS 9.1. The data are reported as means±standard errors of the means (means±SEM). Values of P<0.05 were considered as statistically significant. The lesion scores were analyzed by using the Kruskal-Wallis post hoc Steel-Dwass tests for comparison between the groups (JUMP 9.1, SAS).

Results

Effect of HM and hLF against C.albicans growth

To determine the in vitro effect of LF in human milk (HM) and purified hLF (200 µg/ml) from HM against C. albicans, we incubated the fungal cells either with HM or hLF for different time periods. The results showed that HM did not have any effect on the growth of C. albicans up to 6 h of incubation. In contrast, after 12 h of incubation it started to inhibit fungal growth. The most significant reduction of C. albicans growth was observed after 12 h (P=0.001) compared to PBS treated cells. This inhibition trend was also seen after 48 h of incubation. Whereas hLF significantly reduced the growth of C. albicans even at 6 h and increasingly reduced the growth up to 72 h of incubation when compared to PBS and HM treated cells. These in vitro results showed that hLF was more effective against C. albicans growth when compared to HM (Fig. 1).

Fig. 1. In vitro time course efficacy of human milk and hLF on C. albicans growth.

Fig. 1

Open in a new tab

The fungal cells were incubated either with HM or hLF at 37 °C in different time point and plated on YPD agar plate. The viable fungal CFUs were enumerated after 2 days. C) Control, HM) human milk and hLF) human lactoferrin. The data shown are means±SEM. Asterisks indicate significant (P<0.05) difference between the groups.

In vivo effect of hLF on experimental oral candidiasis

In this study, we determined whether immunocompromised LFKO−/− mice are more susceptible to oral candidiasis compared to the WT mice and also investigated the therapeutic efficacy of oral administration of hLF in a LFKO−/− mouse model of oral candidiasis. No fungal cells were detected in the oral cavity of the mice in any experimental group before infection. We determined the C. albicans CFUs in the oral cavity after 7 days of infection or sham infection. As seen in figure 2, the C. albicans recovered from the oral cavity of the LFKO−/−I mice was significantly higher (2-log) compared to counts obtained from WTI mice (P=0.001). In the LFKO−/−I mice given hLF there was a significant (P=0.001) 3-log reduction in oral C. albicans CFUs compared to infected LFKO−/−I mice that were not given hLF (Fig. 2).

Fig. 2. In vivo therapeutic effect of hLF on oral candidiasis.

Fig. 2

Open in a new tab

The number of viable C. albicans CFUs was enumerated after 7 days of oral infection in WT, LFKO−/− and hLF treated LFKO−/− mice. The data shown are means±SEM for 6 mice. Asterisks indicate significant (P<0.05) difference between the groups.

Light microscopic evaluation of severity of infection

Grossly, the dorsums of the tongues of the experimental groups (WTC, WTI, LFKO−/−C, LFKO−/−I and LFKO−/−I+hLF mice) were examined using light microscope. It showed that the control groups (LFKO−/−C and WTC) had normal pink colored tongues, due to the presence of intact lingual papillae. In contrast, the dorsum of the tongue of the LFKO−/−I mice had irregular red lesions alternating with white lesions. The irregular red lesions represented a form of candidiasis known as erythematous (atrophic) candidiasis. In the erythematous form of candidiasis, there is loss of lingual papillae (depapillation), leaving a smooth area on the tongue. While other LFKO−/I mice presented with another form of candidiasis called pseudomembrane candidiasis. These lesions were white thrush-like lesions which when scraped off, revealed a red and smooth area. The white lesions are basically desquamated epithelium that has been invaded by the hyphae. These pathologic changes were observed to a lesser extent in infected LFKO−/− mice treated with hLF (Fig. 3).

Fig. 3. Morphological features of the murine tongue dorsum with oral candidiasis after 7 days of infection observed under the light microscope.

Fig. 3

Open in a new tab

A&B) More white patches covered the surface of WTI mice tongue compared to WTC, C&D) More than 90% of tongue was covered by white patches in LFKO−/−I mice group compared to LFKO−/−C mice tongue, E) Due to the treatment of hLF to LFKO−/−I mice, the white patches were significantly reduced on the surface of the tongue when compared to LFKO−/−I mice.

Scoring of tongue lesions

We determined the severity of oral candidiasis by scoring the white patches on the dorsal surface of the tongue after 7 days of actual or sham infection. The tongues of the uninfected WTC and LFKO−/−C were normal in appearance and received score of 0. Scores of the three infected groups are shown in figure 4. The dorsum of the tongue of LFKO−/− I mice had more white patches covering most of the tongue scoring 4, compared to the tongues of the WTI group that had fewer white patches covering only up to 20% giving it a score of 1. When we compared the scores of the tongue lesions of the LFKO−/−I and WTI groups with the Kruskal-Wallis post hoc Steel-Dwass tests, the score of the tongue lesions of the LFKO−/−I mice were more than 2 when compared to WTI (P=0.001). After the addition of hLF, there was a progressive lesion reduction with fewer white patches in LFKO−/−I+hLF (P=0.001) group supporting the fact that hLF is beneficial effect in treating Candida infection (Fig. 4).

Fig. 4. Scores of the tongue lesions.

Fig. 4

Open in a new tab

Severity of oral candidiasis was scored based on the white patches covering on the tongue surface as follows; 0, normal; 1, white patches less than 20%; 2, white patches less than 90% but more than 21%; 3, white patches more than 91% and 4, thick white patches like pseudo membranes more than 91% of the tongue. The data shown are means±SEM for 6 mice. Asterisks indicate significant (P<0.05) difference between the groups.

Scanning electron microscopy

When the mice tongues were observed under the SEM, the tongues of LFKO−/−C and WTC mice showed long, thin filiform papillae representing a normal-looking tongue. WTI mice showed tongues that had less intact filiform papillae with decreased density and the hyphae were present. The dorsal surfaces of the tongues of the LFKO−/−I mice showed destruction of the filiform papillae with fungal elements. In general, the lingual papillae, and the normal architecture of the tongue were disturbed. Microscopically, the dorsal surfaces of the tongues of the hLF treated (LFKO−/−I+hLF) mice showed more intact filiform papillae and less fungus occupation when compared to LFKO−/−I and WTI mice (Fig. 5).

Fig. 5. Scanning electron microscopic observation of mice tongue dorsum.

Fig. 5

Open in a new tab

After 7 days of infection, the tongues were dissected and processed for SEM as described in the materials and methods. A) Tongues of the mice of WTC and LFKO−/−C groups represent a normal architecture of a tongue, B) WTI mice tongue under SEM showing the presence of C. albicans and less intact filiform papilla. C). LFKO−/−I tongue under SEM showing destroyed filiform papilla. D) After administration of hLF to LFKO−/−I mice show less C. albicans occupation and more intact filiform papilla.

Peripheral blood count

Peripheral blood was examined after 7 days of infection or sham infection. It revealed that in both WTI (P=0.001) and LFKO−/−I (P=0.001) mice total lymphocyte counts were significantly decreased compared to sham-infected mice. There was also a significant increase in neutrophil counts in WTI, LFKO−/−I and LFKO−/−+hLF mice when compared to sham-infected mice. There was no difference seen in the cell counts of monocytes, eosinophils and basophils among any group of mice (Table 1).

Table 1. Peripheral leukocytes count during experimental oral candidiasis.

The blood samples were analyzed for the differential cell count 7 days after infection.

Treatment group Percentage of leukocytes in the blood
Lymphocyte Neutrophils Monocytes Eosinophils Basophiles
WTC 60.0±2.1 36.5±3.5 2.0±0.0 1.5±0.0 0.0±0.0
WTI 39.5±3.5* 61.0±2.8* 1.5±0.7 1.5±0.7 0.0±0.0
LFKO−/−C 53.0±0.8* 28.0±3.5* 1.5±0.7 3.5±0.7 1.0±0.0
LFKO−/−I 2.5±4.9* 74.5±4.9* 2.5±2.1 2.0±0.0 0.0±0.0
LFKO−/−I+hLF 50.0±17.7* 49.0±19.8* 1.8±0.7 1.5±1.4 0.0±0.0
Open in a new tab

The data shown are means ± SEM.

Asterisks indicate significant (P<0.05) difference between the groups.

Discussion

Animal studies have demonstrated that the presence of LF can protect animals from oral C. albicans infection. In this study, we used C. albicans strain (ATCC 90028) to induce oral candidiasis as there are studies showing that this fungus was the most predominant isolate associated with oral candidiasis (Akpan and Morgan 2002; Yan et al. 2011). C. albicans has a number of virulence factors including high cytotoxicity in gingival epithelial cells (Li and Dongari-Bagtzoglou 2007). We showed here that LFKO−/− mice are more susceptible to orally infected C. albicans when compared to WT mice and treatment with hLF reduced the severity of infection in LFKO−/−I mice. This study demonstrates that hLF has an important role in protecting the oral cavity from C. albicans infection and the destructive effects of this infection to oral soft tissue.

LF is an important component found in high concentrations in milk. It is well known for its antibacterial, antiviral and antifungal effect. Data taken from our in vitro study demonstrated that both HM and hLF kill C. albicans in a time dependant manner. However, when we compared the C. albicans killing ability of HM and hLF delivered at the same LF concentration, we observed that HM had no effect on the growth of the C. albicans up to 6 h. It’s effectiveness in inhibiting the growth of fungus was observed after 12 h of incubation, whereas hLF was active at 6 h. Several in vitro studies have also demonstrated a similar effect (Andersson et al. 2000; Morrill et al. 2003). Al-sheikh (2009) also showed a similar result that hLF decreased the growth of C. albicans after 1, 2 and 3 days of incubation (Al-Sheikh 2009). These studies showed that hLF was more effective in preventing the growth of fungus when compared to LF in HM. Our previous study also showed that HM has partially degraded Aae an outer membrane protein of periodontopathogen A. actinomycetemcomitans when compared to purified lactoferrin (Velliyagounder et al. 2010). Also it has been showed that the bactericidal activity of frozen HM was reduced as evidenced by decreased activity against bacteria due to long-term storage. The authors also suggested that the ability of HM fat globule membrane to adhere to suspended bacteria was gradually reduced due to long-term storage (Ogundele 2002). We believe that long-term storage may be the reason for reduced effect of HM against C. albicans when compared to LF. Further investigation is required to establish behind the ineffectiveness of LF in HM.

There are several reports demonstrating experimental oral candidiasis in an animal model (Takakura et al. 2003; Mima et al. 2010; Martins Jda et al. 2011; Hayama et al. 2012; Ninomiya et al. 2012; Costa et al. 2012). In this study, the severity of oral candidiasis caused by C. albicans was determined by both measuring the CFUs in the oral cavity and by scoring the white patches on the surface of the tongue and SEM. Previous studies done by Takakura et al. (Takakura et al. 2003; Takakura et al. 2004) used bovine LF, whereas in this study we used hLF. Both these lactoferrins have more than 77% nucleotide similarity and both of the proteins reduced the oral candidiasis effectively (Kirkpatrick et al. 1971). In vivo anticandidal effect of bLF was first seen in cyclophosphamide-immunosuppressed mice with systemic candidiasis (Abe et al. 2000). A study reported that intravenous injection of LF peptide (1–11) in cyclophosphamide-treated mice showed reduced fluconazole-resistant C. albicans infections and an increased IL-10 level in the serum. This effect was dose-dependent and was seen at concentration of 0.4 ng of LF/kg of body weight (Lupetti et al. 2007).

In this study, we demonstrated that LFKO−/−I mice are more susceptible to oral candidiasis when compared to WTI mice assuming that the absence of LF led to a decrease in the mice immunity. In WTI mice, LF plays an important role in combating the infection. Takakura et al. (2003) has reported that oral administration of LF and LF-derived peptides one day before the infection reduced the severity of oral candidiasis in mouse model (Takakura et al. 2003). We also administered hLF to LFKO−/−I mice one day before infection and administration was continued for 7 days. The treatment of hLF to LFKO−/−I+hLF mice resulted in a significant reduction in the severity of infection when compared to untreated LFKO−/−I mice. This protective role of LF contrasted with the rapid effect of antifungal agents such as fluconazole and amphotericin B, which demonstrated improvement on day 2 or 3 (Takakura et al. 2003). These results suggest that LF against oral candidiasis acts in a different way from those of the chemotherapeutic agents with different antifungal activities. It has been reported in previous in vitro studies that LF up-regulates the anti-inflammatory cytokines and down-regulates the proinflammatory cytokines (Sorimachi et al. 1997). It is well known that macrophages and neutrophils are stimulated by TNF-α and IFN- γ that directly kill C. albicans (Crouch et al. 1992; Tansho et al. 1994). TNF-α in oral mucosa has shown to be an important mediator of oropharyngeal candidiasis in mice (Farah et al. 2002). It has also been reported that LF enhanced nitric oxide production in C. albicans infected mice following significant increase of the level of IL-12 in the peritoneal fluid. Our hematological analysis revealed that neutrophil and lymphocyte counts were higher in the WTI and LFKO−/−I+hLF groups suggesting protective role of hLF. It has been reported that oral administration of hLF enhances murine peyer’s patches to secrete IgA and IgG (Debbabi et al. 1998). Recently it has been reported that fungal infections increased with gene defects in IL-17 pathway (Puel et al. 2011; Puel et al. 2012). Another study have shown that IL-17 deficient mice developed severe chronic mucocutaneous candidiasis (CMC) (Conti and Gaffen 2010). Mice that were depleted of IL-17R and TH17 showed severe oropharyngeal candidiasis (OPC) (Conti et al. 2009). Reports also indicated that IL-17 enhanced lipocalin 2, which has a role in mediating immunity against C. albicans (Ferreira et al. 2014). Microarray analysis revealed that IL-17 is down regulated in LFKO−/− mice when compared to WTC mice (unpublished data). We assume that deficient or defective IL-17 signaling may be another reason that LFKO−/− mice are more susceptible to oral candidiasis. The LFKO−/− mouse model is an excellent model for studying the effect of hLF. This could explain the essential role of endogenous LF in the system for host protection as well as immunomodulatory properties.

In conclusion, we have shown in this study that the LFKO−/− mice are more susceptible to C. albicans compared to wild-type mice. Future studies may focus on the cytokine patterns that occur in LFKO−/− mice with Candida infection. Other LF peptides may be studied to determine the most significant peptide in killing C. albicans in an animal model.

Acknowledgments

The corresponding author thanks Dr. Orla M. Conneely for providing us the LFKO−/− mice. This research was supported by the NIH NIDCR grant R21 DE019548 to V.K.

Footnotes

Conflict of Interest

None of the authors have a commercial or other association that might pose a conflict of interest.

References

  1. Abe S, Okutomi T, Tansho S, Ishibashi H, Wakabayashi H, Teraguchi S, Hayasawa H, Yamaguchi H. Augmentation by lactoferrin of host defense against Candida infection in mice. In: Shimazaki K, Tsuda H, Tomita M, Kuwata T, Perraudin J-P, editors. Lactoferrin: Structure, Function and Applications. Amsterdam: Elsevier; 2000. pp. 195–201. [Google Scholar]
  2. Akpan A, Morgan R. Oral candidiasis. Postgrad Med J. 2002;78:455–459. doi: 10.1136/pmj.78.922.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Al-Sheikh H. Effect of lactoferrin and iron on the growth of human pathogenic Candida species. Pak J Biol Sci. 2009;12:91–94. doi: 10.3923/pjbs.2009.91.94. [DOI] [PubMed] [Google Scholar]
  4. Andersson Y, Lindquist S, Lagerqvist C, Hernell O. Lactoferrin is responsible for the fungistatic effect of human milk. Early Hum Dev. 2000;59:95–105. doi: 10.1016/s0378-3782(00)00086-4. [DOI] [PubMed] [Google Scholar]
  5. Andres MT, Viejo-Diaz M, Fierro JF. Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K+-channel-mediated K+ efflux. Antimicrob Agents Chemother. 2008;52:4081–4088. doi: 10.1128/AAC.01597-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cannon RD, Chaffin WL. Oral colonization by Candida albicans. Crit Rev Oral Biol Med. 1999;10:359–383. doi: 10.1177/10454411990100030701. [DOI] [PubMed] [Google Scholar]
  7. Conti HR, Gaffen SL. Host responses to Candida albicans: Th17 cells and mucosal candidiasis. Microbes Infect. 2010;12:518–527. doi: 10.1016/j.micinf.2010.03.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conti HR, Shen F, Nayyar N, Stocum E, Sun JN, Lindemann MJ, Ho AW, Hai JH, Yu JJ, Jung JW, Filler SG, Masso-Welch P, Edgerton M, Gaffen SL. Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis. The Journal of experimental medicine. 2009;206:299–311. doi: 10.1084/jem.20081463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Costa AC, Campos Rasteiro VM, da Silva Hashimoto ES, Araujo CF, Pereira CA, Junqueira JC, Jorge AO. Effect of erythrosine- and LED-mediated photodynamic therapy on buccal candidiasis infection of immunosuppressed mice and Candida albicans adherence to buccal epithelial cells. Oral Surg Oral Med Oral Pathol Oral Radiol. 2012;114:67–74. doi: 10.1016/j.oooo.2012.02.002. [DOI] [PubMed] [Google Scholar]
  10. Crouch SP, Slater KJ, Fletcher J. Regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin. Blood. 1992;80:235–240. [PubMed] [Google Scholar]
  11. Cutler CW, Eke P, Arnold RR, Van Dyke TE. Defective neutrophil function in an insulin-dependent diabetes mellitus patients. A case report. J Periodontol. 1991;62:394–401. doi: 10.1902/jop.1991.62.6.394. [DOI] [PubMed] [Google Scholar]
  12. Daniluk T, Tokajuk G, Stokowska W, Fiedoruk K, Sciepuk M, Zaremba ML, Rozkiewicz D, Cylwik-Rokicka D, Kedra BA, Anielska I, Gorska M, Kedra BR. Occurrence rate of oral Candida albicans in denture wearer patients. Adv Med Sci. 2006;51(Suppl 1):77–80. [PubMed] [Google Scholar]
  13. Debbabi H, Dubarry M, Rautureau M, Tome D. Bovine lactoferrin induces both mucosal and systemic immune response in mice. J Dairy Res. 1998;65:283–293. doi: 10.1017/s0022029997002732. [DOI] [PubMed] [Google Scholar]
  14. Drobacheff C, Manteaux A, Millon L, Reboux G, Barale T, Laurent R. Oropharyngeal candidiasis resistant to fluconazole in patients infected by HIV. Annales de dermatologie et de venereologie. 1996;123:85–89. [PubMed] [Google Scholar]
  15. Edmond MB, Wallace SE, McClish DK, Pfaller MA, Jones RN, Wenzel RP. Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clin Infect Dis. 1999;29:239–244. doi: 10.1086/520192. [DOI] [PubMed] [Google Scholar]
  16. Farah CS, Gotjamanos T, Seymour GJ, Ashman RB. Cytokines in the oral mucosa of mice infected with Candida albicans. Oral Microbiol Immunol. 2002;17:375–378. doi: 10.1034/j.1399-302x.2002.170607.x. [DOI] [PubMed] [Google Scholar]
  17. Ferreira MC, Whibley N, Mamo AJ, Siebenlist U, Chan YR, Gaffen SL. Interleukin-17-induced protein lipocalin 2 is dispensable for immunity to oral candidiasis. Infect Immun. 2014;82:1030–1035. doi: 10.1128/IAI.01389-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fraser VJ, Jones M, Dunkel J, Storfer S, Medoff G, Dunagan WC. Candidemia in a tertiary care hospital: epidemiology, risk factors, and predictors of mortality. Clin Infect Dis. 1992;15:414–421. doi: 10.1093/clind/15.3.414. [DOI] [PubMed] [Google Scholar]
  19. Gonzalez-Chavez SA, Arevalo-Gallegos S, Rascon-Cruz Q. Lactoferrin: structure, function and applications. Int J Antimicrob Agents. 2009;33:301 e301–308 e301. doi: 10.1016/j.ijantimicag.2008.07.020. [DOI] [PubMed] [Google Scholar]
  20. Hamza OJ, Matee MI, Moshi MJ, Simon EN, Mugusi F, Mikx FH, Helderman WH, Rijs AJ, van der Ven AJ, Verweij PE. Species distribution and in vitro antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis. BMC Microbiol. 2008;8:135. doi: 10.1186/1471-2180-8-135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hayama K, Ishibashi H, Ishijima SA, Niimi K, Tansho S, Ono Y, Monk BC, Holmes AR, Harding DR, Cannon RD, Abe S. A D-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model. FEMS Microbiol Lett. 2012;328:130–137. doi: 10.1111/j.1574-6968.2011.02490.x. [DOI] [PubMed] [Google Scholar]
  22. Kirkpatrick CH, Green I, Rich RR, Schade AL. Inhibition of growth of Candida albicans by iron-unsaturated lactoferrin: relation to host-defense mechanisms in chronic mucocutaneous candidiasis. J Infect Dis. 1971;124:539–544. doi: 10.1093/infdis/124.6.539. [DOI] [PubMed] [Google Scholar]
  23. Kovac J, Kovac D, Slobodnikova L, Kotulova D. Enterococcus faecalis and Candida albicans in the dental root canal and periapical infections. Bratisl Lek Listy. 2013;114:716–720. [PubMed] [Google Scholar]
  24. Kuipers ME, Heegsma J, Bakker HI, Meijer DK, Swart PJ, Frijlink EW, Eissens AC, de Vries-Hospers HG, van den Berg JJ. Design and fungicidal activity of mucoadhesive lactoferrin tablets for the treatment of oropharyngeal candidosis. Drug Deliv. 2002;9:31–38. doi: 10.1080/107175402753413154. [DOI] [PubMed] [Google Scholar]
  25. Li L, Dongari-Bagtzoglou A. Oral epithelium-Candida glabrata interactions in vitro. Oral Microbiol Immunol. 2007;22:182–187. doi: 10.1111/j.1399-302X.2007.00342.x. [DOI] [PubMed] [Google Scholar]
  26. Lupetti A, Brouwer CP, Bogaards SJ, Welling MM, de Heer E, Campa M, van Dissel JT, Friesen RH, Nibbering PH. Human lactoferrin-derived peptide's antifungal activities against disseminated Candida albicans infection. J Infect Dis. 2007;196:1416–1424. doi: 10.1086/522427. [DOI] [PubMed] [Google Scholar]
  27. Lupetti A, Paulusma-Annema A, Welling MM, Dogterom-Ballering H, Brouwer CP, Senesi S, Van Dissel JT, Nibbering PH. Synergistic activity of the N-terminal peptide of human lactoferrin and fluconazole against Candida species. Antimicrob Agents Chemother. 2003;47:262–267. doi: 10.1128/AAC.47.1.262-267.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martins Jda S, Junqueira JC, Faria RL, Santiago NF, Rossoni RD, Colombo CE, Jorge AO. Antimicrobial photodynamic therapy in rat experimental candidiasis: evaluation of pathogenicity factors of Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;111:71–77. doi: 10.1016/j.tripleo.2010.08.012. [DOI] [PubMed] [Google Scholar]
  29. Masci JR. Complete response of severe, refractory oral candidiasis to mouthwash containing lactoferrin and lysozyme. Aids. 2000;14:2403–2404. doi: 10.1097/00002030-200010200-00023. [DOI] [PubMed] [Google Scholar]
  30. Mima EG, Pavarina AC, Dovigo LN, Vergani CE, Costa CA, Kurachi C, Bagnato VS. Susceptibility of Candida albicans to photodynamic therapy in a murine model of oral candidosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010;109:392–401. doi: 10.1016/j.tripleo.2009.10.006. [DOI] [PubMed] [Google Scholar]
  31. Moore RD, Chaisson RE. Natural history of opportunistic disease in an HIV-infected urban clinical cohort. Ann Intern Med. 1996;124:633–642. doi: 10.7326/0003-4819-124-7-199604010-00003. [DOI] [PubMed] [Google Scholar]
  32. Morales DK, Hogan DA. Candida albicans interactions with bacteria in the context of human health and disease. PLoS Pathog. 2010;6:e1000886. doi: 10.1371/journal.ppat.1000886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Morrill JF, Pappagianis D, Heinig MJ, Lonnerdal B, Dewey KG. Detecting Candida albicans in human milk. J Clin Microbiol. 2003;41:475–478. doi: 10.1128/JCM.41.1.475-478.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nikawa H, Samaranayake LP, Hamada T. Modulation of the anti-Candida activity of apo-lactoferrin by dietary sucrose and tunicamycin in vitro. Arch Oral Biol. 1995;40:581–584. doi: 10.1016/0003-9969(94)00195-h. [DOI] [PubMed] [Google Scholar]
  35. Nikawa H, Samaranayake LP, Tenovuo J, Pang KM, Hamada T. The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei. Arch Oral Biol. 1993;38:1057–1063. doi: 10.1016/0003-9969(93)90167-k. [DOI] [PubMed] [Google Scholar]
  36. Ninomiya K, Maruyama N, Inoue S, Ishibashi H, Takizawa T, Oshima H, Abe S. The essential oil of Melaleuca alternifolia (tea tree oil) and its main component, terpinen-4-ol protect mice from experimental oral candidiasis. Biol Pharm Bull. 2012;35:861–865. doi: 10.1248/bpb.35.861. [DOI] [PubMed] [Google Scholar]
  37. Ogundele MO. Effects of storage on the physicochemical and antibacterial properties of human milk. Br J Biomed Sci. 2002;59:205–211. doi: 10.1080/09674845.2002.11783661. [DOI] [PubMed] [Google Scholar]
  38. Peleg AY, Hogan DA, Mylonakis E. Medically important bacterial-fungal interactions. Nat Rev Microbiol. 2010;8:340–349. doi: 10.1038/nrmicro2313. [DOI] [PubMed] [Google Scholar]
  39. Plaut AG, Qiu J, St Geme JW., 3rd Human lactoferrin proteolytic activity: analysis of the cleaved region in the IgA protease of Haemophilus influenzae. Vaccine. 2000;19(Suppl 1):S148–S152. doi: 10.1016/s0264-410x(00)00296-6. [DOI] [PubMed] [Google Scholar]
  40. Puel A, Cypowyj S, Bustamante J, Wright JF, Liu L, Lim HK, Migaud M, Israel L, Chrabieh M, Audry M, Gumbleton M, Toulon A, Bodemer C, El-Baghdadi J, Whitters M, Paradis T, Brooks J, Collins M, Wolfman NM, Al-Muhsen S, Galicchio M, Abel L, Picard C, Casanova JL. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science. 2011;332:65–68. doi: 10.1126/science.1200439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Puel A, Cypowyj S, Marodi L, Abel L, Picard C, Casanova JL. Inborn errors of human IL-17 immunity underlie chronic mucocutaneous candidiasis. Curr Opin Allergy Clin Immunol. 2012;12:616–622. doi: 10.1097/ACI.0b013e328358cc0b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ruhnke M. Epidemiology of Candida albicans infections and role of non-Candida-albicans yeasts. Curr Drug Targets. 2006;7:495–504. doi: 10.2174/138945006776359421. [DOI] [PubMed] [Google Scholar]
  43. Ruhnke M, Eigler A, Tennagen I, Geiseler B, Engelmann E, Trautmann M. Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodeficiency virus infection. J Clin Microbiol. 1994;32:2092–2098. doi: 10.1128/jcm.32.9.2092-2098.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Samaranayake YH, Samaranayake LP, Pow EH, Beena VT, Yeung KW. Antifungal effects of lysozyme and lactoferrin against genetically similar, sequential Candida albicans isolates from a human immunodeficiency virus-infected southern Chinese cohort. J Clin Microbiol. 2001;39:3296–3302. doi: 10.1128/JCM.39.9.3296-3302.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sorimachi K, Akimoto K, Hattori Y, Ieiri T, Niwa A. Activation of macrophages by lactoferrin: secretion of TNF-alpha, IL-8 and NO. Biochem Mol Biol Int. 1997;43:79–87. doi: 10.1080/15216549700203841. [DOI] [PubMed] [Google Scholar]
  46. Takakura N, Wakabayashi H, Ishibashi H, Teraguchi S, Tamura Y, Yamaguchi H, Abe S. Oral lactoferrin treatment of experimental oral candidiasis in mice. Antimicrob Agents Chemother. 2003;47:2619–2623. doi: 10.1128/AAC.47.8.2619-2623.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Takakura N, Wakabayashi H, Ishibashi H, Yamauchi K, Teraguchi S, Tamura Y, Yamaguchi H, Abe S. Effect of orally administered bovine lactoferrin on the immune response in the oral candidiasis murine model. J Med Microbiol. 2004;53:495–500. doi: 10.1099/jmm.0.05505-0. [DOI] [PubMed] [Google Scholar]
  48. Tansho S, Abe S, Yamaguchi H. Inhibition of Candida albicans growth by murine peritoneal neutrophils and augmentation of the inhibitory activity by bacterial lipopolysaccharide and cytokines. Microbiol Immunol. 1994;38:379–383. doi: 10.1111/j.1348-0421.1994.tb01794.x. [DOI] [PubMed] [Google Scholar]
  49. Tomita K, Hosokawa K, Yano K, Takada A, Kubo T, Kikuchi M. Dermal vascularity of the auricle: implications for novel composite grafts. J Plast Reconstr Aesthet Surg. 2009;62:1609–1615. doi: 10.1016/j.bjps.2008.06.073. [DOI] [PubMed] [Google Scholar]
  50. Velliyagounder K, Ganeshnarayan K, Furgang D, Fine D. Lactoferrin Exhibits Proteolytic Activity Against Aae. Aggregatibacter actinomycetemcomitans; AADR conference; March 3–6; Washington DC. 2010. [Google Scholar]
  51. Velliyagounder K, Ganeshnarayan K, Velusamy SK, Fine DH. In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemc omitans. Antimicrob Agents Chemother. 2012;56:2397–2407. doi: 10.1128/AAC.00020-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Velusamy SK, Ganeshnarayan K, Markowitz K, Schreiner H, Furgang D, Fine DH, Velliyagounder K. Lactoferrin knockout mice demonstrates greater susceptibility to Aggregatibacter actinomycetemcomitans-induced periodontal disease. J Periodontol. 2013;84:1690–1701. doi: 10.1902/jop.2013.120587. [DOI] [PubMed] [Google Scholar]
  53. Velusamy SK, Poojary R, Ardeshna R, Alabdulmohsen W, Fine DH, Velliyagounder K. Protective Effects of Human Lactoferrin during Aggregatibacter actinomycetemcomitans-Induced Bacteremia in Lactoferrin-Deficient Mice. Antimicrob Agents Chemother. 2014a;58:397–404. doi: 10.1128/AAC.00020-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Velusamy SK, Fine DH, Velliyagounder K. Prophylactic effect of human lactoferrin against Streptococcus mutans bacteremia in lactoferrin knockout mice. Microbes Infect. 2014b doi: 10.1016/j.micinf.2014.07.009. http://dx.doi.org/10.1016/j.micinf.2014.07.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Venkatesh MP, Pham D, Kong L, Weisman LE. Prophylaxis with lactoferrin, a novel antimicrobial agent, in a neonatal rat model of coinfection. Adv Ther. 2007;24:941–954. doi: 10.1007/BF02877698. [DOI] [PubMed] [Google Scholar]
  56. Wakabayashi H, Takakura N, Teraguchi S, Tamura Y. Lactoferrin feeding augments peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Microbiol Immunol. 2003;47:37–43. doi: 10.1111/j.1348-0421.2003.tb02783.x. [DOI] [PubMed] [Google Scholar]
  57. White A, Goetz MB. Azole-resistant Candida albicans: report of two cases of resistance to fluconazole and review. Clin Infect Dis. 1994;19:687–692. doi: 10.1093/clinids/19.4.687. [DOI] [PubMed] [Google Scholar]
  58. Yan Z, Young AL, Hua H, Xu Y. Multiple oral Candida infections in patients with Sjogren's syndrome -- prevalence and clinical and drug susceptibility profiles. J Rheumatol. 2011;38:2428–2431. doi: 10.3899/jrheum.100819. [DOI] [PubMed] [Google Scholar]
  59. Yen CC, Lin CY, Chong KY, Tsai TC, Shen CJ, Lin MF, Su CY, Chen HL, Chen CM. Lactoferrin as a natural regimen for selective decontamination of the digestive tract: recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonates from pathogenic challenge in the gastrointestinal tract. J Infect Dis. 2009;199:590–598. doi: 10.1086/596212. [DOI] [PubMed] [Google Scholar]

RESOURCES