- Email: [email protected]
Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species
Microbes and Infection 16 (2014) 80e85 www.elsevier.com/locate/micinf
Short communication
Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species Attila Ga´cser a,*, Zolta´n Tiszlavicz b, Tibor Ne´meth a, Gyo¨rgy Sepre´nyi c, Yvette Ma´ndi b b
a Department of Microbiology, University of Szeged, Kozep Fasor 52., H-6726 Szeged, Hungary Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged, Hungary c Department of Medical Biology, University of Szeged, Szeged, Hungary
Received 27 June 2013; accepted 23 September 2013 Available online 2 October 2013
Abstract In this study we investigated the effects of Candida albicans, Candida krusei, Candida tropicalis and Candida parapsilosis on human betadefensin 2 (HBD-2) production in Caco-2 intestinal cell line, and the production of alpha-defensins (human neutrophil peptides, HNP 1e3) in peripheral blood. Opportunistic pathogen yeasts can modulate the host immune function by inducing defensins, the natural antimicrobial peptides. Here we show that Candida spp. stimulated HBD-2 expression in and release from Caco-2 cells, with C. albicans inducing the highest levels of HBD-2. Similarly, HNP 1e3 secretion was significantly increased in whole blood after exposure to Candida yeast cells, with C. albicans producing the greatest effect. Our investigations underscore the important role of beta and alpha defensins produced by intestinal epithelial cells locally and neutrophils systemically in the antifungal defense against Candida. Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Human defensins; Candida; HBD-2; Fungal infection
1. Introduction Fungal infections represent an increasing health problem globally. In developed countries, high rates of invasive fungal infections are associated with the increasing number of immunocompromized individuals. Candida spp. are a normal component of the human intestinal flora, and are involved in intestinal homeostasis [1]. However, in the setting of damage or immunosuppression, endogenous candidasis may arise from the commensal flora of the colonized gastrointestinal tract [2]. For instance, defective innate immune defense mechanisms can lead to intestinal inflammation due to fungal translocation through the gut wall, and systemic infection can develop [3]. There is exceptional diversity as well as significant variation in the fungal species within the intestines of different individuals [4]. Intestinal mucosal epithelial cells play a crucial role in the
* Corresponding author. E-mail address: [email protected] (A. Ga´cser).
local antimicrobial defense. These cells represent not only a physiologic barrier for pathogens, but also function as a part of the innate immune system as they, for example, produce cytokines and antimicrobial peptides. Human defensins, short cysteine-rich cationic proteins, are key components of the innate immune system [5,6]. The inducible human beta-defensins are antimicrobial peptides with a broad spectrum of antibacterial and antifungal activity, including against Candida [7e9]. Human beta-defensin 2 (HBD-2) is produced by epithelial cells primarily, but monocytes, macrophages and dendritic cells are also capable of HBD-2 production. The peptide is highly inducible due to various stimuli, such as inflammatory cytokines and bacterial, viral, fungal or protozoal infection. HBD-2 has a broad spectrum antimicrobial activity that is cidal for Candida [10]. In humans, six alpha defensins have been identified. Four of them, human neutrophil proteins (HNP) 1e4, are mainly produced by neutrophil granulocytes and are localized in azurophilic granules of neutrophil granulocytes [7]. HNP 1e3 share identical amino acid sequences except for the first
1286-4579/$ - see front matter Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2013.09.003
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
N-terminal residue. HNP 1e3 are involved in the destruction of microbes following phagocytosis, degranulation or the formation of neutrophil extracellular traps (NETs) [11,12]. HNPs have been shown to have an in vitro antimicrobial activity against Gram-negative and Gram-positive bacteria, enveloped viruses and certain fungi, including Candida albicans [11]. The aim of our study was to examine the effects of various Candida spp. on the production of HBD-2 by Caco-2 intestinal epithelial-like cells. Additionally, as Candida translocation through the gut wall can result in systemic infections, we investigated whether the different Candida spp. could influence the release of HNP 1e3 from peripheral blood neutrophils. We determined that Candida spp. induced the production and release of defensins. 2. Materials and methods 2.1. Fungal strains and culture conditions In our experiments, C. albicans SC5314, Candida krusei CBS 573, Candida parapsilosis GA1 [13] and Candida tropicalis CBS 94 isolates were used. The yeast strains were inoculated in 2 ml YPD medium (0.5% yeast extract, 1% bactopeptone, 1% glucose) supplemented with 1% PenicillineStreptomycin (Sigma), and were incubated overnight at 30 C in an orbital shaker (200 rpm). 2.2. Cell line and in vitro infection The human intestinal epithelial cell line Caco-2 was maintained in Eagle culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS, GIBCO), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells (1 106 cells/well) were seeded into 6-well tissue culture plates (Costar) and maintained at 37 C in 5% CO2. Confluent cells were then cultivated without (negative control) or with different Candida spp. at a multiplicity of infection (MOI) of 100 for 4 h or 24 h. As a positive control, Caco-2 cells were induced with Escherichia coli strain Nissle 1917, a potent inducer of HBD-2 [14]. 2.3. LDH assay To determine whether Caco-2 cells were damaged during prolonged cultivation with or without Candida spp., 200 ml samples of the supernatants were assayed for lactate dehydrogenase (LDH) activity using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega). 2.4. RNA isolation and PCR amplification Total RNA was isolated with TRI Reagent (Molecular Research Center, Inc.). RNA concentration was determined by the A260 value of the sample. Complementary DNA (cDNA) was generated from 1 mg total RNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) in a
81
final volume of 20 ml in a thermal cycler (Bio-Rad MJ Mini Thermal Cycler). After reverse transcription, real time amplification was carried out using LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche) in a fluorescence thermocycler (LightCycler). Initial denaturation at 95 C for 10 min was followed by 45 cycles of 95 C for 15 s, the primer-specific annealing temperature for 5 s, and elongation at 72 C for 15 s. For hBD-2 (sense, 50 -ATC AGC CAT GAG GGT CTT GT-30 ; antisense, 50 -GAG ACC ACA GGT GCC AAT TT-30 ), the annealing temperature was set to 62 C. For the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase; sense, 50 -AAG GTC GGA GTC AAC GGA TTT-30 ; antisense, 50 -TGG AAG ATG GTG ATG GGATTT-30 ) primers were used. Melting curves were generated after each run, to confirm amplification of specific transcripts. The specificity of the amplification products was verified by subjecting the amplification products to electrophoresis on a 2% agarose gel. The fragments obtained were visualized by ethidium bromide staining. Relative expression is given as a ratio between target gene and the housekeeping GAPDH gene. 2.5. Western blot assays Cells (1 106) were homogenized in ice-cold lysis buffer containing RIPA buffer and protease inhibitor cocktail (Sigma), and the mixture was then centrifuged at 10,000 g for 10 min to remove cell debris. Protein concentrations of cell lysates were determined using the Bio-Rad protein assay (BioRad, Hercules, CA, USA). Supernatants were mixed with Laemmli’s sample buffer and boiled for 3 min. To detect beta defensin, aliquots of the supernatants containing 25 mg of total protein were resolved by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad). Preblocked blots were reacted for 4 h with goat IgG anti-human beta-defensin 2 antibody (R&D Systems, Minneapolis, MN, USA) in PBS containing 0.05% (v/v) Tween-20, 1% (w/v) dried non-fat milk (Difco Laboratories, Detroit, MI, USA) and 1% (w/v) BSA (fraction V; Sigma). Blots were then incubated for 2 h with a peroxidase-conjugated anti-goat IgG antibody (SigmaeAldrich). Filters were washed five times in PBSTween for 5 min after each step and were developed using a chemiluminescence detection system (Amersham, Buckinghamshire, UK). 2.6. Immunofluorescence and semiquantitative assessment of fluorescence intensities Expressions of HBD-2 peptide in Caco-2 cells infected with Candida spp. were investigated from Cytospin preparations by immunofluorescence analysis. Cells were fixed in 1% acetone for 15 min at room temperature. Cells were stained with goat anti-HBD-2 antibody (R&D Systems) for 1 h. The secondary antibody was anti-goat IgG NorthernLights 493 (NL493) fluorochrome-labeled antibody (R&D Systems), which was applied for 45 min. After each incubation step, the cells were washed three times with PBS containing 0.2% BSA. Fluorescence signals were analyzed via confocal microscopy.
82
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
Fig. 1. Panel A: HBD-2 mRNA relative expression in Caco-2 cells after infection with Candida spp. Caco-2 cells were infected with Candida albicans, C. krusei, C. parapsilosis and C. tropicalis, at a multiplicity of infection (MOI) of 100:1 for 4 h. Uninfected Caco-2 cells or cells challenged with E. coli Nissle 1917 served as negative and positive controls, respectively. The HBD-2 mRNA levels were normalized to GAPDH, using quantitative real-time PCR. Results are expressed as mean SD of the data from three separate experiments. Significance: p ¼ 0.0085 according one way ANOVA *p < 0.05 vs. control, **p < 0.01 vs. control. Panel B: HBD2 peptide concentration in culture supernatants from Caco-2 cells infected with Candida spp. Caco-2 cells were infected with Candida albicans, C. krusei, C. parapsilosis and C. tropicalis at a multiplicity of infection (MOI) of 100:1 for 24 h. Uninfected Caco-2 cells or cells challenged with E. coli Nissle 1917 served as negative and positive controls, respectively. The secreted HBD-2 peptide levels in the supernatants were measured by ELISA. Results are expressed as mean SD of the data from three individual experiments. Significance: p ¼ 0.002 according one way ANOVA test *p < 0.05 vs. control **p < 0.01 vs. control. Panel C: Immunoblot analysis of HBD-2 peptide expression in Candida spp.-infected intestinal epithelial cells. Caco-2 cell cultures were infected with Candida spp. at a MOI of 100:1 for 24 h. Total cellular protein (25 mg) from each culture was loaded on a 15% SDS-PAGE gel for immunoblotting. After transfer, the membranes were incubated with antibody to HBD-2, and the labeling was detected with ECL. Lane 1 represents non-infected Caco-2 cells whereas the other lanes are Caco-2 cells incubated with different Candida spp. for 24 h. These results are representative of three experiments performed independently. Relative density to negative control cells were set at 1.
Eight serial images of each immunostained sample were captured by Olympus FV1000 confocal laser scanning microscope with standard parameter settings. The immunofluorescence of control and Candida infected cells was quantitatively analyzed by ImageQuant software (Molecular Dynamics) as follows: 6-6 equally sized circular areas covering the cells were randomly selected on each image. The backgrounds of the selected areas were eliminated by threshold set up and the fluorescence intensities/pixel values of the randomly selected cells were quantified. 2.7. HBD-2 ELISA For the measurement of the secreted HBD-2 protein in the tissue culture medium, a sensitive HBD-2 ELISA (Alpha Diagnostic International, San Antonio, TX, USA) was used. The detection limit of this ELISA kit was 0.8 pg/HBD-2 protein/ml. 2.8. Whole blood incubation method EDTA-anticoagulated freshly drawn peripheral blood samples (1 ml) from healthy blood donors (whose granulocyte
count varied from 3.8 106/ml to 4.7 106/ml) were incubated in the presence of Candida spp. for 4 h at 37 C. Blood donation was approved by the Institutional Review Board of the University of Szeged. Negative control samples were blood samples without fungal cells. Staphylococcus aureus (heat killed), a potent HNP-1 inducer, served as positive control [15]. 2.9. HNP 1e3 ELISA Whole blood samples were centrifuged at 300 rpm, and the supernatants were collected. Samples were immediately tested for HNP1e3 content using an HNP 1e3 ELISA kit (Hycult Biotechnology, The Netherlands). 2.10. Statistical analysis All values are expressed as mean SD. For the quantification of HBD-2 expression using RT-PCR and ELISA, results from Candida spp.-infected samples were compared with noninfected controls. The data were subjected to one way ANOVA test with Dunnett and Bonferroni post test according the data set. For immunofluorescence intensity analysis, intensity data
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
83
Candida spp. compared to uninfected samples after 4 h of the infection (Fig. 1A). Among the Candida spp., C. tropicalis, C. albicans and C. krusei were the most potent HBD-2 mRNA inducers (4.1 0.4, 3.5 0.3, 3.2 0.2 fold increase, respectively), although non of them reached the level induced by the positive control, E. coli Nissle 1917 strain (8.4 0.3 fold increase). C. parapsilosis induced the least HBD-2 mRNA expression in this system (2.3 0.2 fold increase). ANOVA test was used to evaluate whether the increase in HBD-2 expression was statistically significant. There was a significant difference between the values: p ¼ 0.0085; but according the Dunnett post test the difference was statistically significant ( p < 0.05) only between control vs. C. albicans, and between control vs. C. tropicalis. The differences between species were not statistically significant according the Bonferroni post test. 3.2. Candida spp. induce HBD-2 secretion by Caco-2 cells
Fig. 2. Panel A: Immunofluorescence staining of HBD-2 in Caco-2 cells without infection (control). Panel B: Immunfluorescence labeling of HBD-2 in Caco-2 cells infected with C. albicans. Caco-2 cells were infected with C. albicans for 24 h. Cells were fixed in 1% acetone for 15 min at room temperature and stained with goat anti-HBD-2 antibody (R&D Systems) for 1 h. The secondary antibody was anti-goat IgG NorthernLights 493 (NL493) fluorochrome-labeled antibody. Panel C: Fluorescence intensity of HBD-2 peptide in Caco-2 cells infected with Candida spp. Expression of HBD-2 in Candida spp.-infected and non-infected Caco-2 cells was investigated by immunofluorescence analysis. The immunofluorescence of control and Candida-infected cells was quantitatively analyzed by ImageQuant software. Control: non-infected Caco-2 cells. Results are expressed as mean SD of the data from three individual experiments Statistical analysis. ANOVA one way test, p ¼ 0.0008, and Dunnett post test. *p < 0.05 vs. control, **p < 0.01 vs. control.
from Candida spp.-infected cells were compared with those of the non-infected control. For all statistical evaluations, P < 0.05 was considered statistically significant. Data analyses were performed by Graph PadPrism 5 (GraphPad Software Inc., San Diego, CA, USA) statistical program. 3. Results 3.1. Expression of HBD-2 mRNA in Caco-2 cells infected with Candida spp RT-PCR was performed to test whether HBD-2 gene expression is induced in Caco-2 cells upon infection with Candida spp. The relative expression of inducible HBD-2 mRNA was significantly increased in the cells infected with
Experiments were performed to determine whether HBD-2 is secreted by the intestinal epithelial cells following Candida spp. infection. HBD-2 ELISAs showed that the concentration of HBD-2 protein was significantly elevated in the supernatant 24 h after infection (Fig. 1B). C. albicans induced the highest release of HBD-2 relative to the other Candida species, with C. krusei, C. parapsilosis and C. tropicalis producing similar results. HBD-2 levels were low in the uninfected control supernatant. These results imply that human epithelial cells release HBD-2 upon stimulation with Candida spp., especially C. albicans. Again, ANOVA test was used to assessment of whether the increase in HBD-2 protein levels were statistically significant. There was a significant difference between the values: p ¼ 0.0025; but according the Dunnett post test the difference was statistically significant only between control vs. C. albicans ( p < 0.05), and between control vs. C. tropicalis ( p < 0.01). The differences between species were not statistically significant according the Bonferroni post test. 3.3. HBD-2 peptide expression in Caco-2 cells infected with Candida spp The expression of HBD-2 peptide in Candida spp.-infected epithelial cells was determined by Western blot analysis. Using the HBD-2 binding antibody, we identified a single band of about 4 kDa, from epithelial cell lysates (Fig. 1C). The signal intensity of the band was increased in lysates of cells challenged with Candida yeast cells, with C. albicans inducing the largest increase. The level of HBD-2 was only weakly detectable after the processing of the control cell lysate without Candida infection. 3.4. Immunofluorescent staining of Caco-2 cells for HBD-2 Immunofluorescent staining images of Candida spp. infected Caco-2 cells revealed that HBD-2 was diffusely distributed within the cytoplasm of the epithelial cells, as
84
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
expected for a peptide within granules (Fig. 2B). Fluorescence was minimal in the uninfected controls (Fig. 2A). The calculated fluorescence intensity data (Fig. 2C) indicate that the HBD-2 peptide was highly inducible upon infection with Candida species. Considerable fluorescence intensity was detected following infection with C. albicans, and also with C. krusei, and C. parapsilosis ( p < 0.01 vs. control according the ANOVA test with Dunnett post test, respectively) and with C. tropicalis ( p < 0.05). 3.5. HNP 1e3 peptide secretion in whole blood incubated with Candida spp Neutrophils are the primary effector cells in the host response against invasive Candida. Since HNP 1e3 are the principle defensins produced by neutrophils, we examined the levels of HNP 1e3 in whole blood exposed to Candida yeast cells. HNP 1e3 secretion was significantly higher in the whole blood samples incubated with Candida compared to the control samples. C. albicans produced the greatest release of HNP 1e3. Moreover, significantly higher concentration of HNP 1e3 was induced by C. albicans and C. tropicalis than by S. aureus, which served as a positive control (600 50 ng/ml, 300 25 ng/ml vs. 150 25 ng/ml). p < 0.01 as a Bonferroni post test of ANOVA statistical test). The other Candida spp. exerted also higher induction of HNP 1e3, with a statistical significance of 0.05 vs. values of S. aureus Fig. 3. 4. Discussion There is increasing evidence that opportunistic pathogenic yeasts stimulate the intestinal immune system to the benefit of the host. However, some Candida spp. can also cause severe
infections in immunocompromised individuals or when natural barriers are damaged [3]. In addition to their barrier function, intestinal epithelial cells participate in the host’s innate immune response to microorganisms and secrete cytokines as well as antimicrobial peptides in response to pathogenic and nonpathogenic bacterial and/or fungal cells [16]. In this study, we examined the HBD-2 production by Caco-2 intestinal epithelial-like cells upon infection with several different opportunistic pathogen Candida species. Our results show that all four Candida species used in our study (C. albicans, C. krusei, C. parapsilosis and C. tropicalis) are able to induce HBD-2 mRNA and protein production in human intestinal epithelial cells, suggesting that locally released HBD-2 may have a role in regulating the amount of commensal yeasts in the gut. In our models, C. albicans was the most potent inducer of HBD-2 secretion. Interestingly, this species-dependent induction of HBD-2 could only be seen at the protein and not at the mRNA level. This is most probably the consequence of differences in posttranslational regulation of defensin synthesis, as these molecules are known to be sythesized as inactive precursors in human cells [17]. However, the high induction of HBD-2 by C. albicans may be associated with the fact that C. albicans is one the most prominent Candida species in the human intestinal tract [4,18]. Additionally, our results demonstrate that alpha defensin secretion can also be induced by Candida spp. In our model system, whole blood was used as source of peripheral granulocytes [19]. In our experimental setup, Candida spp. were more potent stimulators of HNP 1e3 secretion than S. aureus, which highlights the role of yeasts in the induction of alpha defensin in the blood. Although all Candida species were able to induce the production of alpha defensins, C. albicans induced the highest defensin levels. Although there are several studies investigating the antifungal activity of defensins [20], little is known about the induction of these antimicrobial peptides by different Candida species in the intestines. Notably, it may be interesting to examine the defensin-inducing capacity of different strains of these species in the future, as strong strain-to-strain differences have been noted among Candida species regarding susceptibility to defensins [20]. A deeper understanding of the mechanisms by which fungi stimulate immune responses in the gut may lead even to better therapies for inflammatory bowel disease (IBD) in the future [21]. Acknowledgments
Fig. 3. HNP1e3 peptide levels in whole blood challenged with Candida spp. Peripheral whole blood was incubated with Candida spp. for 4 h, at a multiplicity of infection (MOI) of 100:1 for 4 h. Whole blood without fungal challenge and cells exposed to S. aureus served as negative and positive controls, respectively. Secreted HNP 1e3 levels in supernatant were measured by ELISA. Results are expressed as mean SD of the data from three different experiments. Statistical test was performed by one way ANOVA with Bonferroni post test *p < 0.05 vs. control. **p < 0.01 vs. control þp < 0.05 vs. S. aureus þþp < 0.01 vs. S. aureus þp < 0.05 vs. S. aureus.
The authors thank Mrs Gyo¨rgyi Mu¨ller for expert technical ´ MOP-4.2.2.B1/ assistance. This work was supported by TA ´ KONV2010/12 and by TAMOP 4.2.2.A-11-1-KONV-20120035. AG was supported in part by OTKA NF 84006, NN100374 (ERA-Net PathoGenomics Program) and an EMBO Installation Grant. References [1] L. Yan, C. Yang, J. Tang, Disruption of the intestinal mucosal barrier in Candida albicans infections, Microbiol. Res. 168 (2013) 389e395.
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85 [2] J.W. van der Meer, F.L. van de Veerdonk, L.A. Joosten, B.J. Kullberg, M.G. Netea, Severe Candida spp. infections: new insights into natural immunity, Int. J. Antimicrob. Agents 36 (Suppl. 2) (2010) S58eS62. [3] N.A. Gow, B. Hube, Importance of the Candida albicans cell wall during commensalism and infection, Curr. Opin. Microbiol. 15 (2012) 406e412. [4] S.J. Ott, T. Kuhbacher, M. Musfeldt, P. Rosenstiel, S. Hellmig, A. Rehman, O. Drews, W. Weichert, K.N. Timmis, S. Schreiber, Fungi and inflammatory bowel diseases: alterations of composition and diversity, Scand. J. Gastroenterol. 43 (2008) 831e841. [5] M.E. Selsted, A.J. Ouellette, Mammalian defensins in the antimicrobial immune response, Nat. Immunol. 6 (2005) 551e557. [6] D. Yang, O. Chertov, J.J. Oppenheim, Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37), J. Leukocyte Biol. 69 (2001) 691e697. [7] T. Ganz, Defensins: antimicrobial peptides of innate immunity, Nat. Rev. Immunol. 3 (2003) 710e720. [8] E. Guani-Guerra, T. Santos-Mendoza, S.O. Lugo-Reyes, L.M. Teran, Antimicrobial peptides: general overview and clinical implications in human health and disease, Clin. Immunol. 135 (2010) 1e11. [9] D. Yang, A. Biragyn, D.M. Hoover, J. Lubkowski, J.J. Oppenheim, Multiple roles of antimicrobial defensins, cathelicidins, and eosinophilderived neurotoxin in host defense, Annu. Rev. Immunol. 22 (2004) 181e215. [10] A.M. Aerts, I.E. Francois, B.P. Cammue, K. Thevissen, The mode of antifungal action of plant, insect and human defensins, Cell Mol. Life Sci. 65 (2008) 2069e2079. [11] R.I. Lehrer, T. Ganz, D. Szklarek, M.E. Selsted, Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations, J. Clin. Invest. 81 (1988) 1829e1835. [12] V. Papayannopoulos, A. Zychlinsky, NETs: a new strategy for using old weapons, Trends Immunol. 30 (2009) 513e521.
85
[13] A. Gacser, D. Trofa, W. Schafer, J.D. Nosanchuk, Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence, J. Clin. Invest. 117 (2007) 3049e3058. [14] J. Wehkamp, J. Harder, K. Wehkamp, B. Wehkamp-von Meissner, M. Schlee, C. Enders, U. Sonnenborn, S. Nuding, S. Bengmark, K. Fellermann, J.M. Schroder, E.F. Stange, NF-kappaB- and AP-1mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium, Infect Immun. 72 (2004) 5750e5758. [15] T. Ganz, M.E. Selsted, D. Szklarek, S.S. Harwig, K. Daher, D.F. Bainton, R.I. Lehrer, Defensins. Natural peptide antibiotics of human neutrophils, J. Clin. Invest. 76 (1985) 1427e1435. [16] S. Saegusa, M. Totsuka, S. Kaminogawa, T. Hosoi, Cytokine responses of intestinal epithelial-like Caco-2 cells to non-pathogenic and opportunistic pathogenic yeasts in the presence of butyric acid, Biosci. Biotechnol. Biochem. 71 (2007) 2428e2434. [17] C.L. Wilson, A.P. Schmidt, E. Pirila, E.V. Valore, N. Ferri, T. Sorsa, T. Ganz, W.C. Parks, Differential processing of {alpha}- and {beta}defensin precursors by matrix metalloproteinase-7 (MMP-7), J. Biol. Chem. 284 (2009) 8301e8311. [18] M. Knoke, Gastrointestinal microecology of humans and Candida, Mycoses 42 (Suppl. 1) (1999) 30e34. [19] A.K. Kocsis, I. Ocsovszky, L. Tiszlavicz, Z. Tiszlavicz, Y. Mandi, Helicobacter pylori induces the release of alpha-defensin by human granulocytes, Inflammation Res. 58 (2009) 241e247. [20] S. Joly, C. Maze, P.B. McCray Jr., J.M. Guthmiller, Human betadefensins 2 and 3 demonstrate strain-selective activity against oral microorganisms, J. Clin. Microbiol. 42 (2004) 1024e1029. [21] I.D. Iliev, V.A. Funari, K.D. Taylor, Q. Nguyen, C.N. Reyes, S.P. Strom, J. Brown, C.A. Becker, P.R. Fleshner, M. Dubinsky, J.I. Rotter, H.L. Wang, D.P. McGovern, G.D. Brown, D.M. Underhill, Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis, Science 336 (2012) 1314e1317.
Short communication
Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species Attila Ga´cser a,*, Zolta´n Tiszlavicz b, Tibor Ne´meth a, Gyo¨rgy Sepre´nyi c, Yvette Ma´ndi b b
a Department of Microbiology, University of Szeged, Kozep Fasor 52., H-6726 Szeged, Hungary Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged, Hungary c Department of Medical Biology, University of Szeged, Szeged, Hungary
Received 27 June 2013; accepted 23 September 2013 Available online 2 October 2013
Abstract In this study we investigated the effects of Candida albicans, Candida krusei, Candida tropicalis and Candida parapsilosis on human betadefensin 2 (HBD-2) production in Caco-2 intestinal cell line, and the production of alpha-defensins (human neutrophil peptides, HNP 1e3) in peripheral blood. Opportunistic pathogen yeasts can modulate the host immune function by inducing defensins, the natural antimicrobial peptides. Here we show that Candida spp. stimulated HBD-2 expression in and release from Caco-2 cells, with C. albicans inducing the highest levels of HBD-2. Similarly, HNP 1e3 secretion was significantly increased in whole blood after exposure to Candida yeast cells, with C. albicans producing the greatest effect. Our investigations underscore the important role of beta and alpha defensins produced by intestinal epithelial cells locally and neutrophils systemically in the antifungal defense against Candida. Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Human defensins; Candida; HBD-2; Fungal infection
1. Introduction Fungal infections represent an increasing health problem globally. In developed countries, high rates of invasive fungal infections are associated with the increasing number of immunocompromized individuals. Candida spp. are a normal component of the human intestinal flora, and are involved in intestinal homeostasis [1]. However, in the setting of damage or immunosuppression, endogenous candidasis may arise from the commensal flora of the colonized gastrointestinal tract [2]. For instance, defective innate immune defense mechanisms can lead to intestinal inflammation due to fungal translocation through the gut wall, and systemic infection can develop [3]. There is exceptional diversity as well as significant variation in the fungal species within the intestines of different individuals [4]. Intestinal mucosal epithelial cells play a crucial role in the
* Corresponding author. E-mail address: [email protected] (A. Ga´cser).
local antimicrobial defense. These cells represent not only a physiologic barrier for pathogens, but also function as a part of the innate immune system as they, for example, produce cytokines and antimicrobial peptides. Human defensins, short cysteine-rich cationic proteins, are key components of the innate immune system [5,6]. The inducible human beta-defensins are antimicrobial peptides with a broad spectrum of antibacterial and antifungal activity, including against Candida [7e9]. Human beta-defensin 2 (HBD-2) is produced by epithelial cells primarily, but monocytes, macrophages and dendritic cells are also capable of HBD-2 production. The peptide is highly inducible due to various stimuli, such as inflammatory cytokines and bacterial, viral, fungal or protozoal infection. HBD-2 has a broad spectrum antimicrobial activity that is cidal for Candida [10]. In humans, six alpha defensins have been identified. Four of them, human neutrophil proteins (HNP) 1e4, are mainly produced by neutrophil granulocytes and are localized in azurophilic granules of neutrophil granulocytes [7]. HNP 1e3 share identical amino acid sequences except for the first
1286-4579/$ - see front matter Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2013.09.003
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
N-terminal residue. HNP 1e3 are involved in the destruction of microbes following phagocytosis, degranulation or the formation of neutrophil extracellular traps (NETs) [11,12]. HNPs have been shown to have an in vitro antimicrobial activity against Gram-negative and Gram-positive bacteria, enveloped viruses and certain fungi, including Candida albicans [11]. The aim of our study was to examine the effects of various Candida spp. on the production of HBD-2 by Caco-2 intestinal epithelial-like cells. Additionally, as Candida translocation through the gut wall can result in systemic infections, we investigated whether the different Candida spp. could influence the release of HNP 1e3 from peripheral blood neutrophils. We determined that Candida spp. induced the production and release of defensins. 2. Materials and methods 2.1. Fungal strains and culture conditions In our experiments, C. albicans SC5314, Candida krusei CBS 573, Candida parapsilosis GA1 [13] and Candida tropicalis CBS 94 isolates were used. The yeast strains were inoculated in 2 ml YPD medium (0.5% yeast extract, 1% bactopeptone, 1% glucose) supplemented with 1% PenicillineStreptomycin (Sigma), and were incubated overnight at 30 C in an orbital shaker (200 rpm). 2.2. Cell line and in vitro infection The human intestinal epithelial cell line Caco-2 was maintained in Eagle culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS, GIBCO), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells (1 106 cells/well) were seeded into 6-well tissue culture plates (Costar) and maintained at 37 C in 5% CO2. Confluent cells were then cultivated without (negative control) or with different Candida spp. at a multiplicity of infection (MOI) of 100 for 4 h or 24 h. As a positive control, Caco-2 cells were induced with Escherichia coli strain Nissle 1917, a potent inducer of HBD-2 [14]. 2.3. LDH assay To determine whether Caco-2 cells were damaged during prolonged cultivation with or without Candida spp., 200 ml samples of the supernatants were assayed for lactate dehydrogenase (LDH) activity using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega). 2.4. RNA isolation and PCR amplification Total RNA was isolated with TRI Reagent (Molecular Research Center, Inc.). RNA concentration was determined by the A260 value of the sample. Complementary DNA (cDNA) was generated from 1 mg total RNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) in a
81
final volume of 20 ml in a thermal cycler (Bio-Rad MJ Mini Thermal Cycler). After reverse transcription, real time amplification was carried out using LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche) in a fluorescence thermocycler (LightCycler). Initial denaturation at 95 C for 10 min was followed by 45 cycles of 95 C for 15 s, the primer-specific annealing temperature for 5 s, and elongation at 72 C for 15 s. For hBD-2 (sense, 50 -ATC AGC CAT GAG GGT CTT GT-30 ; antisense, 50 -GAG ACC ACA GGT GCC AAT TT-30 ), the annealing temperature was set to 62 C. For the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase; sense, 50 -AAG GTC GGA GTC AAC GGA TTT-30 ; antisense, 50 -TGG AAG ATG GTG ATG GGATTT-30 ) primers were used. Melting curves were generated after each run, to confirm amplification of specific transcripts. The specificity of the amplification products was verified by subjecting the amplification products to electrophoresis on a 2% agarose gel. The fragments obtained were visualized by ethidium bromide staining. Relative expression is given as a ratio between target gene and the housekeeping GAPDH gene. 2.5. Western blot assays Cells (1 106) were homogenized in ice-cold lysis buffer containing RIPA buffer and protease inhibitor cocktail (Sigma), and the mixture was then centrifuged at 10,000 g for 10 min to remove cell debris. Protein concentrations of cell lysates were determined using the Bio-Rad protein assay (BioRad, Hercules, CA, USA). Supernatants were mixed with Laemmli’s sample buffer and boiled for 3 min. To detect beta defensin, aliquots of the supernatants containing 25 mg of total protein were resolved by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad). Preblocked blots were reacted for 4 h with goat IgG anti-human beta-defensin 2 antibody (R&D Systems, Minneapolis, MN, USA) in PBS containing 0.05% (v/v) Tween-20, 1% (w/v) dried non-fat milk (Difco Laboratories, Detroit, MI, USA) and 1% (w/v) BSA (fraction V; Sigma). Blots were then incubated for 2 h with a peroxidase-conjugated anti-goat IgG antibody (SigmaeAldrich). Filters were washed five times in PBSTween for 5 min after each step and were developed using a chemiluminescence detection system (Amersham, Buckinghamshire, UK). 2.6. Immunofluorescence and semiquantitative assessment of fluorescence intensities Expressions of HBD-2 peptide in Caco-2 cells infected with Candida spp. were investigated from Cytospin preparations by immunofluorescence analysis. Cells were fixed in 1% acetone for 15 min at room temperature. Cells were stained with goat anti-HBD-2 antibody (R&D Systems) for 1 h. The secondary antibody was anti-goat IgG NorthernLights 493 (NL493) fluorochrome-labeled antibody (R&D Systems), which was applied for 45 min. After each incubation step, the cells were washed three times with PBS containing 0.2% BSA. Fluorescence signals were analyzed via confocal microscopy.
82
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
Fig. 1. Panel A: HBD-2 mRNA relative expression in Caco-2 cells after infection with Candida spp. Caco-2 cells were infected with Candida albicans, C. krusei, C. parapsilosis and C. tropicalis, at a multiplicity of infection (MOI) of 100:1 for 4 h. Uninfected Caco-2 cells or cells challenged with E. coli Nissle 1917 served as negative and positive controls, respectively. The HBD-2 mRNA levels were normalized to GAPDH, using quantitative real-time PCR. Results are expressed as mean SD of the data from three separate experiments. Significance: p ¼ 0.0085 according one way ANOVA *p < 0.05 vs. control, **p < 0.01 vs. control. Panel B: HBD2 peptide concentration in culture supernatants from Caco-2 cells infected with Candida spp. Caco-2 cells were infected with Candida albicans, C. krusei, C. parapsilosis and C. tropicalis at a multiplicity of infection (MOI) of 100:1 for 24 h. Uninfected Caco-2 cells or cells challenged with E. coli Nissle 1917 served as negative and positive controls, respectively. The secreted HBD-2 peptide levels in the supernatants were measured by ELISA. Results are expressed as mean SD of the data from three individual experiments. Significance: p ¼ 0.002 according one way ANOVA test *p < 0.05 vs. control **p < 0.01 vs. control. Panel C: Immunoblot analysis of HBD-2 peptide expression in Candida spp.-infected intestinal epithelial cells. Caco-2 cell cultures were infected with Candida spp. at a MOI of 100:1 for 24 h. Total cellular protein (25 mg) from each culture was loaded on a 15% SDS-PAGE gel for immunoblotting. After transfer, the membranes were incubated with antibody to HBD-2, and the labeling was detected with ECL. Lane 1 represents non-infected Caco-2 cells whereas the other lanes are Caco-2 cells incubated with different Candida spp. for 24 h. These results are representative of three experiments performed independently. Relative density to negative control cells were set at 1.
Eight serial images of each immunostained sample were captured by Olympus FV1000 confocal laser scanning microscope with standard parameter settings. The immunofluorescence of control and Candida infected cells was quantitatively analyzed by ImageQuant software (Molecular Dynamics) as follows: 6-6 equally sized circular areas covering the cells were randomly selected on each image. The backgrounds of the selected areas were eliminated by threshold set up and the fluorescence intensities/pixel values of the randomly selected cells were quantified. 2.7. HBD-2 ELISA For the measurement of the secreted HBD-2 protein in the tissue culture medium, a sensitive HBD-2 ELISA (Alpha Diagnostic International, San Antonio, TX, USA) was used. The detection limit of this ELISA kit was 0.8 pg/HBD-2 protein/ml. 2.8. Whole blood incubation method EDTA-anticoagulated freshly drawn peripheral blood samples (1 ml) from healthy blood donors (whose granulocyte
count varied from 3.8 106/ml to 4.7 106/ml) were incubated in the presence of Candida spp. for 4 h at 37 C. Blood donation was approved by the Institutional Review Board of the University of Szeged. Negative control samples were blood samples without fungal cells. Staphylococcus aureus (heat killed), a potent HNP-1 inducer, served as positive control [15]. 2.9. HNP 1e3 ELISA Whole blood samples were centrifuged at 300 rpm, and the supernatants were collected. Samples were immediately tested for HNP1e3 content using an HNP 1e3 ELISA kit (Hycult Biotechnology, The Netherlands). 2.10. Statistical analysis All values are expressed as mean SD. For the quantification of HBD-2 expression using RT-PCR and ELISA, results from Candida spp.-infected samples were compared with noninfected controls. The data were subjected to one way ANOVA test with Dunnett and Bonferroni post test according the data set. For immunofluorescence intensity analysis, intensity data
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
83
Candida spp. compared to uninfected samples after 4 h of the infection (Fig. 1A). Among the Candida spp., C. tropicalis, C. albicans and C. krusei were the most potent HBD-2 mRNA inducers (4.1 0.4, 3.5 0.3, 3.2 0.2 fold increase, respectively), although non of them reached the level induced by the positive control, E. coli Nissle 1917 strain (8.4 0.3 fold increase). C. parapsilosis induced the least HBD-2 mRNA expression in this system (2.3 0.2 fold increase). ANOVA test was used to evaluate whether the increase in HBD-2 expression was statistically significant. There was a significant difference between the values: p ¼ 0.0085; but according the Dunnett post test the difference was statistically significant ( p < 0.05) only between control vs. C. albicans, and between control vs. C. tropicalis. The differences between species were not statistically significant according the Bonferroni post test. 3.2. Candida spp. induce HBD-2 secretion by Caco-2 cells
Fig. 2. Panel A: Immunofluorescence staining of HBD-2 in Caco-2 cells without infection (control). Panel B: Immunfluorescence labeling of HBD-2 in Caco-2 cells infected with C. albicans. Caco-2 cells were infected with C. albicans for 24 h. Cells were fixed in 1% acetone for 15 min at room temperature and stained with goat anti-HBD-2 antibody (R&D Systems) for 1 h. The secondary antibody was anti-goat IgG NorthernLights 493 (NL493) fluorochrome-labeled antibody. Panel C: Fluorescence intensity of HBD-2 peptide in Caco-2 cells infected with Candida spp. Expression of HBD-2 in Candida spp.-infected and non-infected Caco-2 cells was investigated by immunofluorescence analysis. The immunofluorescence of control and Candida-infected cells was quantitatively analyzed by ImageQuant software. Control: non-infected Caco-2 cells. Results are expressed as mean SD of the data from three individual experiments Statistical analysis. ANOVA one way test, p ¼ 0.0008, and Dunnett post test. *p < 0.05 vs. control, **p < 0.01 vs. control.
from Candida spp.-infected cells were compared with those of the non-infected control. For all statistical evaluations, P < 0.05 was considered statistically significant. Data analyses were performed by Graph PadPrism 5 (GraphPad Software Inc., San Diego, CA, USA) statistical program. 3. Results 3.1. Expression of HBD-2 mRNA in Caco-2 cells infected with Candida spp RT-PCR was performed to test whether HBD-2 gene expression is induced in Caco-2 cells upon infection with Candida spp. The relative expression of inducible HBD-2 mRNA was significantly increased in the cells infected with
Experiments were performed to determine whether HBD-2 is secreted by the intestinal epithelial cells following Candida spp. infection. HBD-2 ELISAs showed that the concentration of HBD-2 protein was significantly elevated in the supernatant 24 h after infection (Fig. 1B). C. albicans induced the highest release of HBD-2 relative to the other Candida species, with C. krusei, C. parapsilosis and C. tropicalis producing similar results. HBD-2 levels were low in the uninfected control supernatant. These results imply that human epithelial cells release HBD-2 upon stimulation with Candida spp., especially C. albicans. Again, ANOVA test was used to assessment of whether the increase in HBD-2 protein levels were statistically significant. There was a significant difference between the values: p ¼ 0.0025; but according the Dunnett post test the difference was statistically significant only between control vs. C. albicans ( p < 0.05), and between control vs. C. tropicalis ( p < 0.01). The differences between species were not statistically significant according the Bonferroni post test. 3.3. HBD-2 peptide expression in Caco-2 cells infected with Candida spp The expression of HBD-2 peptide in Candida spp.-infected epithelial cells was determined by Western blot analysis. Using the HBD-2 binding antibody, we identified a single band of about 4 kDa, from epithelial cell lysates (Fig. 1C). The signal intensity of the band was increased in lysates of cells challenged with Candida yeast cells, with C. albicans inducing the largest increase. The level of HBD-2 was only weakly detectable after the processing of the control cell lysate without Candida infection. 3.4. Immunofluorescent staining of Caco-2 cells for HBD-2 Immunofluorescent staining images of Candida spp. infected Caco-2 cells revealed that HBD-2 was diffusely distributed within the cytoplasm of the epithelial cells, as
84
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85
expected for a peptide within granules (Fig. 2B). Fluorescence was minimal in the uninfected controls (Fig. 2A). The calculated fluorescence intensity data (Fig. 2C) indicate that the HBD-2 peptide was highly inducible upon infection with Candida species. Considerable fluorescence intensity was detected following infection with C. albicans, and also with C. krusei, and C. parapsilosis ( p < 0.01 vs. control according the ANOVA test with Dunnett post test, respectively) and with C. tropicalis ( p < 0.05). 3.5. HNP 1e3 peptide secretion in whole blood incubated with Candida spp Neutrophils are the primary effector cells in the host response against invasive Candida. Since HNP 1e3 are the principle defensins produced by neutrophils, we examined the levels of HNP 1e3 in whole blood exposed to Candida yeast cells. HNP 1e3 secretion was significantly higher in the whole blood samples incubated with Candida compared to the control samples. C. albicans produced the greatest release of HNP 1e3. Moreover, significantly higher concentration of HNP 1e3 was induced by C. albicans and C. tropicalis than by S. aureus, which served as a positive control (600 50 ng/ml, 300 25 ng/ml vs. 150 25 ng/ml). p < 0.01 as a Bonferroni post test of ANOVA statistical test). The other Candida spp. exerted also higher induction of HNP 1e3, with a statistical significance of 0.05 vs. values of S. aureus Fig. 3. 4. Discussion There is increasing evidence that opportunistic pathogenic yeasts stimulate the intestinal immune system to the benefit of the host. However, some Candida spp. can also cause severe
infections in immunocompromised individuals or when natural barriers are damaged [3]. In addition to their barrier function, intestinal epithelial cells participate in the host’s innate immune response to microorganisms and secrete cytokines as well as antimicrobial peptides in response to pathogenic and nonpathogenic bacterial and/or fungal cells [16]. In this study, we examined the HBD-2 production by Caco-2 intestinal epithelial-like cells upon infection with several different opportunistic pathogen Candida species. Our results show that all four Candida species used in our study (C. albicans, C. krusei, C. parapsilosis and C. tropicalis) are able to induce HBD-2 mRNA and protein production in human intestinal epithelial cells, suggesting that locally released HBD-2 may have a role in regulating the amount of commensal yeasts in the gut. In our models, C. albicans was the most potent inducer of HBD-2 secretion. Interestingly, this species-dependent induction of HBD-2 could only be seen at the protein and not at the mRNA level. This is most probably the consequence of differences in posttranslational regulation of defensin synthesis, as these molecules are known to be sythesized as inactive precursors in human cells [17]. However, the high induction of HBD-2 by C. albicans may be associated with the fact that C. albicans is one the most prominent Candida species in the human intestinal tract [4,18]. Additionally, our results demonstrate that alpha defensin secretion can also be induced by Candida spp. In our model system, whole blood was used as source of peripheral granulocytes [19]. In our experimental setup, Candida spp. were more potent stimulators of HNP 1e3 secretion than S. aureus, which highlights the role of yeasts in the induction of alpha defensin in the blood. Although all Candida species were able to induce the production of alpha defensins, C. albicans induced the highest defensin levels. Although there are several studies investigating the antifungal activity of defensins [20], little is known about the induction of these antimicrobial peptides by different Candida species in the intestines. Notably, it may be interesting to examine the defensin-inducing capacity of different strains of these species in the future, as strong strain-to-strain differences have been noted among Candida species regarding susceptibility to defensins [20]. A deeper understanding of the mechanisms by which fungi stimulate immune responses in the gut may lead even to better therapies for inflammatory bowel disease (IBD) in the future [21]. Acknowledgments
Fig. 3. HNP1e3 peptide levels in whole blood challenged with Candida spp. Peripheral whole blood was incubated with Candida spp. for 4 h, at a multiplicity of infection (MOI) of 100:1 for 4 h. Whole blood without fungal challenge and cells exposed to S. aureus served as negative and positive controls, respectively. Secreted HNP 1e3 levels in supernatant were measured by ELISA. Results are expressed as mean SD of the data from three different experiments. Statistical test was performed by one way ANOVA with Bonferroni post test *p < 0.05 vs. control. **p < 0.01 vs. control þp < 0.05 vs. S. aureus þþp < 0.01 vs. S. aureus þp < 0.05 vs. S. aureus.
The authors thank Mrs Gyo¨rgyi Mu¨ller for expert technical ´ MOP-4.2.2.B1/ assistance. This work was supported by TA ´ KONV2010/12 and by TAMOP 4.2.2.A-11-1-KONV-20120035. AG was supported in part by OTKA NF 84006, NN100374 (ERA-Net PathoGenomics Program) and an EMBO Installation Grant. References [1] L. Yan, C. Yang, J. Tang, Disruption of the intestinal mucosal barrier in Candida albicans infections, Microbiol. Res. 168 (2013) 389e395.
A. Ga´cser et al. / Microbes and Infection 16 (2014) 80e85 [2] J.W. van der Meer, F.L. van de Veerdonk, L.A. Joosten, B.J. Kullberg, M.G. Netea, Severe Candida spp. infections: new insights into natural immunity, Int. J. Antimicrob. Agents 36 (Suppl. 2) (2010) S58eS62. [3] N.A. Gow, B. Hube, Importance of the Candida albicans cell wall during commensalism and infection, Curr. Opin. Microbiol. 15 (2012) 406e412. [4] S.J. Ott, T. Kuhbacher, M. Musfeldt, P. Rosenstiel, S. Hellmig, A. Rehman, O. Drews, W. Weichert, K.N. Timmis, S. Schreiber, Fungi and inflammatory bowel diseases: alterations of composition and diversity, Scand. J. Gastroenterol. 43 (2008) 831e841. [5] M.E. Selsted, A.J. Ouellette, Mammalian defensins in the antimicrobial immune response, Nat. Immunol. 6 (2005) 551e557. [6] D. Yang, O. Chertov, J.J. Oppenheim, Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37), J. Leukocyte Biol. 69 (2001) 691e697. [7] T. Ganz, Defensins: antimicrobial peptides of innate immunity, Nat. Rev. Immunol. 3 (2003) 710e720. [8] E. Guani-Guerra, T. Santos-Mendoza, S.O. Lugo-Reyes, L.M. Teran, Antimicrobial peptides: general overview and clinical implications in human health and disease, Clin. Immunol. 135 (2010) 1e11. [9] D. Yang, A. Biragyn, D.M. Hoover, J. Lubkowski, J.J. Oppenheim, Multiple roles of antimicrobial defensins, cathelicidins, and eosinophilderived neurotoxin in host defense, Annu. Rev. Immunol. 22 (2004) 181e215. [10] A.M. Aerts, I.E. Francois, B.P. Cammue, K. Thevissen, The mode of antifungal action of plant, insect and human defensins, Cell Mol. Life Sci. 65 (2008) 2069e2079. [11] R.I. Lehrer, T. Ganz, D. Szklarek, M.E. Selsted, Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations, J. Clin. Invest. 81 (1988) 1829e1835. [12] V. Papayannopoulos, A. Zychlinsky, NETs: a new strategy for using old weapons, Trends Immunol. 30 (2009) 513e521.
85
[13] A. Gacser, D. Trofa, W. Schafer, J.D. Nosanchuk, Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence, J. Clin. Invest. 117 (2007) 3049e3058. [14] J. Wehkamp, J. Harder, K. Wehkamp, B. Wehkamp-von Meissner, M. Schlee, C. Enders, U. Sonnenborn, S. Nuding, S. Bengmark, K. Fellermann, J.M. Schroder, E.F. Stange, NF-kappaB- and AP-1mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium, Infect Immun. 72 (2004) 5750e5758. [15] T. Ganz, M.E. Selsted, D. Szklarek, S.S. Harwig, K. Daher, D.F. Bainton, R.I. Lehrer, Defensins. Natural peptide antibiotics of human neutrophils, J. Clin. Invest. 76 (1985) 1427e1435. [16] S. Saegusa, M. Totsuka, S. Kaminogawa, T. Hosoi, Cytokine responses of intestinal epithelial-like Caco-2 cells to non-pathogenic and opportunistic pathogenic yeasts in the presence of butyric acid, Biosci. Biotechnol. Biochem. 71 (2007) 2428e2434. [17] C.L. Wilson, A.P. Schmidt, E. Pirila, E.V. Valore, N. Ferri, T. Sorsa, T. Ganz, W.C. Parks, Differential processing of {alpha}- and {beta}defensin precursors by matrix metalloproteinase-7 (MMP-7), J. Biol. Chem. 284 (2009) 8301e8311. [18] M. Knoke, Gastrointestinal microecology of humans and Candida, Mycoses 42 (Suppl. 1) (1999) 30e34. [19] A.K. Kocsis, I. Ocsovszky, L. Tiszlavicz, Z. Tiszlavicz, Y. Mandi, Helicobacter pylori induces the release of alpha-defensin by human granulocytes, Inflammation Res. 58 (2009) 241e247. [20] S. Joly, C. Maze, P.B. McCray Jr., J.M. Guthmiller, Human betadefensins 2 and 3 demonstrate strain-selective activity against oral microorganisms, J. Clin. Microbiol. 42 (2004) 1024e1029. [21] I.D. Iliev, V.A. Funari, K.D. Taylor, Q. Nguyen, C.N. Reyes, S.P. Strom, J. Brown, C.A. Becker, P.R. Fleshner, M. Dubinsky, J.I. Rotter, H.L. Wang, D.P. McGovern, G.D. Brown, D.M. Underhill, Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis, Science 336 (2012) 1314e1317.