Psoriasin, a novel anti-Candida albicans adhesin

Psoriasin, a novel anti-Candida albicans adhesin Candida albicans belongs to the normal microbial flora on epithelial surfaces of humans. However, under certain, still not fully understood conditions, it can become pathogenic and cause a spectrum of diseases, from local infections to life-threatening septicemia. We investigated a panel of antimicrobial proteins and peptides (AMPs), potentially involved in mucosal immunity against this pathogen. Out of six studied AMPs, psoriasin was most up-regulated during a mucosal infection, an acute episode of recurrent Candida vulvovaginitis, although candidacidal activity has not been demonstrated. We here show that psoriasin binds to β-glucan, a basic component of the C. albicans cell wall, and thereby inhibits adhesion of the pathogen to surfaces and increases IL-8 production by mucosal epithelial cells. In conclusion, we show a novel mechanism of action of psoriasin. By inhibiting C. albicans adhesion and by enhancing cytokine production, psoriasin contributes to the immune response against C. albicans. Key messages & The antimicrobial peptide psoriasin is highly up-regulated during a local mucosal infection, Candida albicans vulvovaginitis. & Psoriasin binds to β-glucan in the Candida albicans cell wall and thereby inhibits adhesion of the pathogen. & Binding of psoriasin to Candida albicans induces an im- mune response by mucosal epithelial cells. . . . . Keywords Candida albicans Psoriasin β-Glucan Adhesion Immune response Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00109-018-1637-6) contains supplementary material, which is available to authorized users. Candida albicans is a commensal fungus, colonizing mucosal and cutaneous surfaces of healthy individuals. As an opportunis- * Annelie Brauner tic pathogen, C. albicans can cause a variety of diseases, ranging Annelie.Brauner@ki.se from trivial local to life-threatening systemic infections. Immunocompromised patients are mostly at risk for severe fun- Department of Microbiology, Tumor and Cell Biology, Division of gal infections [1]. Indwelling catheters constitute another risk Clinical Microbiology, Karolinska Institutet and Karolinska factor, due to the ability of the fungus to adhere and form biofilm University Hospital, Stockholm, Sweden on foreign surfaces [2]. Yet, factors of the host-pathogen interac- Department of Clinical Sciences, Division of Obstetrics and tions that lead to asymptomatic carriage, to local infection, or to Gynecology, Danderyd Hospital and Karolinska Institutet, invasive systemic infection are still not fully understood. Stockholm, Sweden The mucosal epithelium with its normal microbial flora con- Department of Clinical Science, Intervention and Technology, stitutes an important dynamic barrier for invading microorgan- Division of Pediatrics, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden isms. The epithelial cells produce mucus, anti-adhesins, antimi- crobial proteins and peptides (AMPs), chemokines, and cyto- Department of Dermatology, Venerology and Allergology, University Hospital Schleswig-Holstein, Kiel, Germany kines [3–7]. AMPs are mostly cationic, amphipatic, and typically 538 J Mol Med (2018) 96:537–545 with between 12 and 50 amino acid residues in length [8]. The contraceptives did not differ between patients and control par- antimicrobial protein psoriasin (S100A7), however, is larger, not ticipants (Supplementary Table 1). cationic (MW 11,457; pI 6.77) [9] and has highly potent E. coli-cidal activity [10]. Unlike most AMPs, it is not acting Isolation and identification of Candida albicans via membrane effects, but similar to calprotectin [11], in a zinc- dependent fashion [10]. In an approach to explain the resistance For identification of C. albicans, vaginal swabs were cultured of lesional psoriatic skin towards dermatophyte infection, we on CHROMagar Candida and typical colonies were further recently identified the cysteine-reduced form of psoriasin as a analyzed by latex surface antigen agglutination (Bichro- fungicidal AMP, which acts via intracellular zinc-depletion and Dubli) at the Department of Clinical Microbiology, induction of apoptosis-like fungal cell death [12]. Cysteine- Karolinska University Hospital, Stockholm, Sweden. reduced psoriasin, but not its oxidized form, is a powerful Absence of fungal and bacterial growth in the samples from broad-spectrum fungicide for many dermatophytes and control participants was confirmed according to the same pro- Aspergillus fumigatus. Interestingly, however, both the oxidized cedure. Isolates were kept frozen at − 80 °C. For experiments, and the cysteine-reduced forms of psoriasin lack activity towards if not stated otherwise, isolates were cultured on Sabouraud C. albicans [12]. agar for 1–2 days at 37 °C and then in yeast peptone dextrose In this study, we sought to investigate the interaction be- (YPD) medium overnight at 30 °C. This culture was diluted tween C. albicans and epithelial defense mechanisms, in par- 1:100 in fresh medium and grown for another 3 h at 30 °C to ticular AMPs, during a typical mucosal C. albicans infection, the logarithmic growth phase. Under these conditions, vulvovaginal candidiasis. Although lacking a direct antifungal C. albicans grew as yeast cells. effect, psoriasin was strongly up-regulated during the acute C. albicans infection. This AMP supported the mucosal im- Vaginal lavage munity by alternative mechanisms, namely by inhibition of C. albicans adhesion and by affecting the immune response. Vaginal lavage was obtained according to a modified method by Valore et al. [14]. Briefly, 5 ml of distilled water was ad- ministered intravaginally, and lavage samples were collected Materials and methods 5 min later. The fluid was centrifuged at 300×g for 10 min, and the cell pellet and the sterile-filtered supernatant were stored at Participants and samples − 80 °C. The clinical part of the study was approved by the Regional Biopsy samples Ethical Review Board in Stockholm. All participants were recruited at the gynecology outpatient clinic at Danderyd Two biopsies were taken from the vaginal wall of each patient Hospital, Stockholm, Sweden, and informed consent was ob- with 3-mm forceps at the 3 and 9 o’clock positions approxi- tained. Recurrent vulvovaginal candidiasis (RVVC) was de- mately 3–4 cm from the vaginal introitus. One biopsy was fined according to Fidel [13], and only cases of primary fixed in 4% paraformaldehyde at 4 °C overnight, transferred RVVC were included, i.e., no known predisposing underlying to 70% ethanol and processed for immunohistochemical anal- conditions including pregnancy, antibiotic treatment, diabetes, ysis; a second biopsy was placed in RNAlater RNA and immunosuppression. Infection with C. albicans was con- Stabilization Reagent (Qiagen) and subjected to RNA isola- firmed microbiologically; patients infected with other tion and gene expression analysis. Candida spp. or bacterial vaginosis were excluded from the study. None of the patients had received any antibacterial or Immunofluorescence staining of sections antifungal treatment at the time of the first sampling. At the first visit, all patients showed typical symptoms of an acute Biopsies collected for immunohistochemical analysis were vulvovaginal C. albicans infection, and oral antifungal treat- paraffin-embedded and cut at 4 μm. Sections were ment was initiated, consisting of 50 mg/day fluconazole for deparaffinized in xylene or Neo-Clear, rehydrated in an etha- 1 week and 150 mg/week for 5 weeks. Samples were collected nol gradient and washed in water and phosphate-buffered sa- as described below, before and 2–3 weeks after antifungal line (PBS). Heat-mediated antigen retrieval was performed in treatment was completed. A total of 16 women, aged 20– citrate buffer (10 mM, pH 6). Sections were blocked for 39 years (median 32 years), were included in the study. All 30 min with Image-iT FX Signal Enhancer (Life patients were culture-negative for C. albicans and other uro- Technologies) and for 60 min with 10% serum from the spe- genital infections at the time of the second visit. Control sam- cies that the secondary antibody was raised in. Overnight in- ples were collected once from 27 healthy female volunteers, cubation with primary antibodies was carried out at 4 °C. aged 19–40 years (median, 25 years). The usage of Primary antibodies used were rabbit anti-HBD1 (1:100, J Mol Med (2018) 96:537–545 539 Santa Cruz Biotechnology), goat anti-HBD2 (1:50, R&D Enzyme-linked immunosorbent assays (ELISA) Systems), rabbit anti-HBD3 (1:100, Santa Cruz Biotechnology), rabbit anti-psoriasin (1:800, Abcam), and Vaginal lavage samples were analyzed using ELISA kits for rabbit anti-RNase 7 (1:500, Novus Biologicals). Sections were human β-defensins (HBD) 1, 2, and 3 (Alpha Diagnostic then incubated with Alexa Fluor-conjugated secondary anti- International); the human cathelicidin LL-37 (Hycult bodies (Life Technologies) for 30–60 min at room temperature Biotech); human ribonuclease (RNase) 7 (Icosagen); and IL- and mounted in ProlongGold Antifade mounting medium 8 (R&D Systems) according to the manufacturer’s instruc- with DAPI (Life Technologies). Images were acquired on a tions. Samples were tested in appropriate dilutions to fall into Leica SP5 confocal microscope using a ×40 objective. the concentration range covered by the internal standards. For HBD3, samples with signals above the limit of the detection system (2000 pg/ml) were assigned a value of 3000 pg/ml to Total RNA extraction and RT real-time PCR allow statistical evaluation. Total RNA extraction from vaginal biopsies and cultured cells Chemicals and reagents was performed using the RNeasy Mini kit (Qiagen). Tissue pieces were lysed in RLT lysis buffer and homogenized with Psoriasin was purified from human skin [17], dissolved in mortar and pestle and then passed through a QIAshredder. 0.01% acetic acid to a concentration of 1 mg/ml, and stored Cells were lysed in lysis buffer; samples were further processed at − 20 °C. A vaginal fluid simulant (VFS) was used as medi- according to the manufacturer’s protocol. Reverse transcription um for all reactions to mimic the vaginal milieu. This previ- with up to 1 μg of RNA was carried out using the DyNAmo ously described formulation resembles the vaginal material cDNA Synthesis Kit (Finnzymes) or the High Capacity cDNA with respect to pH, osmolarity, and chemical composition Reverse Transcription Kit (Applied Biosystems) according to the [18]; organic acids are represented by lactic and acetic acid manufacturers’ instructions. Gene expression was analyzed with and proteins by albumin. All mono- and polysaccharides were TaqMan Gene Expression Assays for HBD1 (DEFB1, purchased from Sigma; working dilutions were prepared Hs00608345_m1), HBD2 (DEFB4A, Hs00175474_m1) and freshly in VFS. HBD3 (DEFB103A, Hs00218678_m1), psoriasin (S100A7, Hs00161488_m1), human cathelicidin LL-37/hCAP-18 Candida albicans adhesion assays (CAMP, Hs00189038_m1), IL-1β (IL1B, Hs01555410_m1), IL-6 (IL6, Hs00985639_m1), and IL-8 (IL8, Hs00174103_m1). The adhesive capacity of C. albicans isolates was determined Expression of RNase 7 was examined using a SYBR Green- in a microtiter plate assay, using VFS as medium or vaginal based assay (Qiagen) with previously described primers [15] lavage. C. albicans adhesion was measured according to a and conditions [16]. GAPDH and 18S rRNA were used as inter- previously described protocol with modifications [19]. nal controls to calculate relative gene expression. C. albicans yeast cells from the logarithmic growth phase were collected by centrifugation, washed twice in PBS, the pellet was suspended in VFS, and the density was adjusted Western blot detection of psoriasin spectrophotometrically. A 100-μl volume containing approx- imately 1 × 10 colony-forming units (CFU) was added to For detection of psoriasin in cell pellets, cells were lysed in an wells on 96-well sterile, non-treated, flat-bottomed microtiter equal volume of Triton X-100 (1% in PBS, with protease plates (Corning) and incubated for 30 min at 37 °C with low inhibitors). The lysate was cleared by centrifugation and the agitation (100 rpm). Thereafter, wells were washed three times protein concentration was determined by BCA assay (Thermo with PBS to remove non-adherent cells, and the number of Scientific). Equal amounts of protein were heated in twofold adherent cells was quantified by metabolic activity using an tricine sample buffer (Bio-Rad) at 95 °C for 5 min and sub- XTT assay. To evaluate the influence of psoriasin on jected to polyacrylamide-gel-electrophoresis using 10–20% C. albicans adhesion, psoriasin (0.001–1 μM or vector) was tris-tricine gels (Bio-Rad), and the separated samples were added to duplicate wells. In selected experiments, psoriasin transferred to PVDF membranes (Invitrogen). After transfer, (1 μM or vector) was pre-incubated with polysaccharides membranes were blocked with milk-TBSTand incubated with β-(1,3)-D-glucan from baker’syeast, D-mannan from baker’s a polyclonal rabbit anti-psoriasin (1:800 in milk-TBST) anti- yeast, and chitin from crab shells (1 mg/ml in VFS, all from body for 1 h at room temperature at 4 °C. Incubation with an Sigma) for 30 min at 4 °C prior to the addition of C. albicans. anti-rabbit HRP-conjugated secondary antibody was carried To evaluate the effect of vaginal lavage on adhesion, VFS was out at room temperature for 60 min. Signals were detected replaced by vaginal lavage fluid from patients and control using SuperSignal West Pico Chemiluminescent reagents participants. The lavage contributed to 40% of the final reac- (Thermo Scientific). tion volume. In some experiments, lavage samples were 540 J Mol Med (2018) 96:537–545 treated with polysaccharides in the same manner as described Statistical analysis for psoriasin. All statistical analyses were performed using GraphPad Prism, Version 6. Data obtained from clinical material were analyzed Psoriasin-Candida binding assays with non-parametric tests; results are presented as individual values with median. For matched comparisons, the Wilcoxon A pull-down assay was adapted from a previously described matched-pairs signed rank test or Friedman test was used; protocol [19]. To demonstrate binding between C. albicans 7 comparison between groups was performed by Mann- cells and psoriasin, approximately 1.5 × 10 cells were incu- Whitney U test or Kruskal-Wallis test as appropriate. bated with 10 μg psoriasin in VFS (final volume 250 μl) and Variations in the number of participants included were due incubated for 30 min at 4 °C with agitation; alternatively, to limited material, and biological outliers were not excluded. 250 μl of vaginal lavage was used. Cells were collected by Data from other in vitro experiments are presented with mean centrifugation and washed twice with VFS. Binding of and standard deviation from at least three independent exper- psoriasin to polysaccharides was tested in a similar manner; iments (n ≥ 3). Results were analyzed by unpaired t test, one- because of the solubility of mannan, mannan-agarose was way ANOVA with Dunnett’s multiple comparison tests, or used. β-(1,3)-D-Glucan from baker’s yeast, chitin from crab two-way ANOVAwith Bonferroni’s multiple comparison cor- shells (0.25 mg/ml), or mannan-agarose (30 μl) were mixed rection, as appropriate. Technical outliers were detected by with 10 μg psoriasin in VFS (final volume 750 μl) and incu- Grubb’s test and excluded. All tests were performed two- bated overnight at 4 °C with agitation; samples without any sided, and differences with P values of < 0.05 were considered polysaccharide or psoriasin were kept alongside for control statistically significant. purposes. Polysaccharides were then pelleted by centrifuga- tion and washed twice with VFS. The final pellets were heated in tricine sample buffer (Bio-Rad) at 95 °C for 5 min and Results Western blotting was performed as described above. Candida albicans induces production of psoriasin Cell culture during mucosal infection The vaginal epithelial cell line AO was kindly provided by To describe the expression of epithelial AMPs at baseline and David J. Klumpp, Northwestern University, Chicago, IL, and in response to C. albicans, mRNA and protein levels of six was used as model of the vaginal epithelium. Cells were cul- AMPs were analyzed in vaginal biopsies from healthy women tured in EpiLife Medium with 60 μM calcium and supple- (n =27, Fig. 1a), from patients during acute C. albicans vul- mented with Human Keratinocyte Growth Supplement vovaginitis and from the same patients after successful anti- (Gibco) in a humidified incubator at 37 °C with 5% CO . fungal treatment (n =16; Fig. 1b and Supplementary Fig. S1). Out of six analyzed AMPs, psoriasin (encoded by the S1007A Cell infection gene) and RNase 7 showed the highest mRNA and protein levels (Fig. 1a). RNase 7 expression was not influenced by C. albicans from logarithmic growth phase were collected by infection with C. albicans (Supplementary Fig. S1). Psoriasin, centrifugation, washed in PBS. Aliquots of 3 × 10 cells were on the other hand, was strongly up-regulated during the acute transferred in low-binding 1.5-ml tubes. Cells were collect- infection episode, both on the mRNA (Fig. 1b) and on the ed by centrifugation, and the pellet was suspended in 98 μl protein levels as shown by immunohistochemistry (Fig. 1c) VFS with 2 μl psoriasin (1 mg/ml) or vector. The mixture in vaginal biopsies and by Western blot in the cell pellet from was incubated 30 min on ice, with occasional mixing. Cells the vaginal lavage samples (Fig. 1d–e). Increased production were collected by centrifugation at 4 °C, the supernatant of psoriasin was primarily detected in the pellet of the vaginal was discarded, and the pellet suspended in 200 μl0.2% lavage containing desquamated mucosal cells and yeast, and paraformaldehyde in PBS. Cells were fixed for 10 min at the levels of psoriasin in the lavage supernatant were not sig- room temperature, washed once, and suspended in cell cul- nificantly affected by infection (Supplementary Fig. S2). ture medium to a final concentration of 10 CFU/ml. From this suspension, 100 μl was added to a well of confluent Psoriasin interacts with β-glucan in the Candida AO cells containing 900 μl of fresh cell culture medium albicans cell wall and incubated for 6 h at 37 °C with 5% CO in a humidi- fied incubator. The medium was removed, and cells were Since psoriasin produced during acute mucosal infection was collected for RNA extraction and gene expression analysis. associated with the cell pellet and not increased in the super- All conditions were analyzed in duplicate. natant (Fig. 1d and Supplementary Fig. S2), we analyzed a J Mol Med (2018) 96:537–545 541 Fig. 1 Psoriasin dominates the response to vulvovaginal infection with green; cell nuclei, blue; ×40 objective). d Western blot of lavage Candida albicans. Vaginal biopsies and lavage samples were collected samples. e Psoriasin-specific bands on Western blots were quantified from healthy women (controls, Co) (a–e) and patients with recurrent using ImageJ, and signal intensity is expressed in arbitrary units (AU); vulvovaginal candidiasis (patients, P) (b–e). a Expression of one representative blot with patients’ samples during infection (inf) and antimicrobial proteins and peptides in vaginal tissue was determined on after treatment (after), and samples from controls are shown. Individual the mRNA level by RT-PCR and is expressed in relation to 18S rRNA or values with median are shown (a, b, e); analyses were performed with GAPDH levels (open diamonds, left axis); secreted proteins and peptides Mann-Whitney test to compared data from controls and patients, and were detected by ELISA (filled diamonds, right axis). b S100 mRNA in Wilcoxon matched-pairs signed rank test was used to evaluate data controls (open circles) and patients during infection and after treatment from patients during acute infection and after treatment; **P < 0.01, (filled circles). c Immunohistochemistry of tissue sections (psoriasin, ***P < 0.001, ****P <0.0001 possible interaction between psoriasin and C. albicans.We under conditions mimicking the vaginal milieu [18]. performed a pull-down assay using whole C. albicans cells Adherence of C. albicans cells to polystyrene was reduced and the polysaccharides β-glucan, chitin and mannan, the by psoriasin in a concentration-dependent manner (Fig. 3a). most important components of the fungal cell wall. Both pu- β-Glucan had the ability to completely neutralize the anti- rified psoriasin and the psoriasin present in vaginal lavage adhesive properties of psoriasin, which was not the case for strongly bound to whole C. albicans cells and to β-glucan, chitin or mannan (Fig. 3b). while chitin and mannan displayed only weak binding activity To confirm the effect also in vaginal lavage, samples from to psoriasin (Fig. 2a). healthy volunteers and from patients with acute C. albicans Electron microscopic investigation demonstrated a signifi- vulvovaginitis were analyzed in a similar adhesion assay. The cant thinning of the cell wall in psoriasin-exposed C. albicans presence of anti-adhesive activity was confirmed in vaginal (Fig. 2b, c). Moreover, immunogold-labelling revealed dimin- lavage fluid and could partially be blocked by β-glucan ished content of β-glucan in the affected cells (Fig. 2b). (Fig. 3c). Psoriasin inhibits Candida albicans adhesion Epithelial immune response to Candida albicans is to surfaces enhanced by psoriasin To evaluate the effect of psoriasin-mediated changes in the To further analyze the consequences of binding of psoriasin to candidal cell wall, we investigated adhesion of C. albicans C. albicans, vaginal epithelial cells were infected with 542 J Mol Med (2018) 96:537–545 Fig. 2 Psoriasin binds to Candida β-glucan and reduces its content in the cell wall. a C. albicans cells or cell wall polysaccharides were incubated with purified psoriasin or vaginal lavage (VL), the pellet was subjected to SDS-PAGE, and membranes were probed for psoriasin. b Due to the solubility of mannan, a mannan-agarose conjugate was used. C. albicans was incubated with psoriasin or vector (control) for 30 min at 4 °C and investigated by transmission electron microscopy, scale bars 1 μm(upperpanel); β-glucan was marked with immunogold-labeled anti-β-glucan antibodies (lower panel, enlarged). c The cell wall thickness from non-treated (Co) and psoriasin-exposed cells (Psoriasin) in five to eight images per experiment was measured as exemplified in the close-up; mean results from three independent experiments are presented as mean and standard deviation and compared by unpaired t test; *P <0.05 psoriasin-treated and untreated fungal cells. Overall, psoriasin women suffering from sporadic or recurrent VVC are other- treatment of C. albicans had an impact on the induction of wise healthy without known underlying conditions [23]. pro-inflammatory cytokines, with the most pronounced effect Epithelial cells lining the body surfaces are the first to come on IL-8 (Fig. 4a). Accordingly, IL8 mRNA levels in patients into contact with invading pathogens and are equipped with during acute infection correlated positively with expression AMPs to fend off potential intruders [6]. We analyzed a panel levels of psoriasin mRNA (Fig. 4b). However, psoriasin alone of AMPs potentially involved in mucosal immunity against did not influence IL-8 expression in epithelial cells in vitro C. albicans. Overall, the pattern resembled a previously re- (Supplementary Fig. S3). ported expression profile [24]. To our surprise, the most abundantly expressed and up- regulated AMP during acute C. albicans infection was Discussion psoriasin. This S100 protein is a powerful antimicrobial for E. coli and to a less degree also some other bacteria [10]. In its We here describe the expression of the antimicrobial protein cysteine-reduced form, psoriasin is a potent fungicidal for fil- psoriasin during mucosal infection with C. albicans and a amentous fungi. However, unexpectedly and for unknown novel mechanism of action of this protein. Psoriasin contrib- reasons, it is not killing C. albicans [10, 12]. utes to the inhibition of C. albicans adhesion and to the in- In search for alternative actions of psoriasin, we performed creased immune response to C. albicans by mucosal epithelial a pull-down assay using whole C. albicans cells or the major cells. components of the fungal cell wall. Psoriasin strongly bound C. albicans is part of the normal flora on skin and mucosal to whole yeast cells and to β-glucan. Accordingly, C. albicans membranes of humans, but as an opportunistic pathogen can cells were also able to precipitate psoriasin in vaginal lavage cause both local and systemic infections. Vulvovaginal candi- samples. diasis (VVC) is a common local infection in women of repro- The mode of antibacterial action by psoriasin is still not ductive age [20], and some will experience recurrent VVC, completely understood. Its activity appears to depend on its with several episodes of acute infections per year [21, 22]. The zinc-binding motifs [10, 25, 26], which also mediate binding majority of mucosal as well as skin infections caused by to E. coli but not Staphylococcus aureus [25]. Membrane in- C. albicans can be linked to impaired immunity or the pres- tegrity, however, was only affected in a Gram-positive species and at low pH [27]. The fungicidal activity of cysteine- ence of foreign bodies like indwelling catheters. In contrast, J Mol Med (2018) 96:537–545 543 apoptosis-like cell death in filamentous fungi [12]. It is possi- ble that the absence of hydrophobin-like molecules in C. albicans contributes to psoriasin’s inability to kill C. albicans. To study potential consequences of psoriasin-mediated changes of the C. albicans cell wall, two major steps in the pathogenesis of Candida infection were investigated, adhesion and induction of a cytokine response [28]. First, our experi- ments with vaginal lavage from healthy women and from patients with recurrent VVC confirmed the presence of anti- adhesive activity in vivo. This activity could partly be linked to psoriasin as demonstrated by an in vitro assay. Both the anti-adhesive activity present in vivo and the activity demon- strated when using purified psoriasin could partially be blocked by β-glucan. Vaginal lavage is known to have several inhibitory factors, such as proteins from commensal lactobacilli [29]. In the current study, the blocking effect of β-glucan in the lavage samples was not complete, suggesting the presence of additional anti-adhesins in vivo. Second to adhesion, receptor-mediated contact with the host cell generally induces an immune response. Psoriasin binds to glucans, which are the key fungal pathogen- associated molecular patterns (reviewed in [30]). Binding of β-glucan to the C-type lectin dectin-1 was shown to mediate induction of pro-inflammatory cytokines in intestinal epithe- lial cells [31] and keratinocytes [32]. Therefore, we expected reduced immune induction in response to the β-glucan- depleted cell wall. Immune response to glucans, however, depends on the size and physical properties of their molecules. Large β-glucan particles induce cytokine production, produc- tion of reactive oxygen species, and phagocytosis by neutro- phils and macrophages, while soluble β-glucans act as antag- onists of these processes [33–35]. In line with these findings, we speculate that treatment of C. albicans induces cell wall changes with loss of outer material and increased exposure of β-glucan at the cell surface. The increased IL-8 observed sug- gests binding to large β-glucan and decreased availability of the soluble ones. In our in vitro experiments, the IL-8 production by vaginal Fig. 3 Psoriasin reduces Candida adhesion. a Adhesion of C. albicans in vaginal lavage simulant with different concentrations of psoriasin was epithelial cells was increased after pre-treatment of quantified by yeast metabolic activity using the XTT assay. b The same C. albicans with psoriasin, possibly by reducing the availabil- assay was performed with psoriasin pre-incubated with polysaccharides. ity of soluble β-glucan. Thus, inhibition of adhesion and in- Data are expressed in relation to corresponding samples without psoriasin crease of cytokine production, although relatively low, may (control, Co = 100) and shown as mean and standard deviation from three to six independent experiments; results were analyzed by one-way ANOVA together promote the mucosal immune protection against with Dunnett’s multiple comparison test versus control; *P < 0.05, C. albicans. **P < 0.01. c In a similar assay, Candida adhesion was measured in Interestingly, although psoriasin was clearly up-regulated vaginal lavage samples from healthy women and from patients with during the acute phase of VVC, it could not protect the host recurrent vulvovaginal candidiasis, and in the presence of glucan (n=18). Individual values with medians are shown, and results were analyzed by from infection. However, it can, however, not be ruled out that Kruskal-Wallis with Dunn’s multiple comparison test; *P < 0.05, patients with a single episode of VVC mount a higher and ***P<0.001 more effective psoriasin response compared to patients with reduced psoriasin is based on a selective cellular uptake, pos- recurrent infections. Further, gene polymorphism of DEFB1 sibly mediated by hydrophobin-like proteins, followed by in- has been suggested to be involved in susceptibility to human 2+ tracellular Zn -binding and subsequent induction of papillomavirus infection [36]. We therefore speculate that 544 J Mol Med (2018) 96:537–545 Fig. 4 Psoriasin promotes a pro-inflammatory response to Candida. a way ANOVA with Bonferroni’s multiple comparison test. b Relative Vaginal epithelial cells were infected with C. albicans pre-treated with mRNA expression of IL-8 and psoriasin was measured in vaginal tissue psoriasin or left untreated. After 6 h, induction of cytokine mRNA was from women during an acute infection with C. albicans. Individual values determined by RT-PCR. Data from four independent experiments are (n = 14) are shown, correlation between psoriasin mRNA (S100A7)and shown with mean and standard deviation; results were analyzed by two- IL8 was assessed by Spearman’stest, r =0.55, P <0.05 Open Access This article is distributed under the terms of the Creative polymorphisms in the psoriasin gene may be associated with Commons Attribution 4.0 International License (http:// recurrent C. albicans infections. creativecommons.org/licenses/by/4.0/), which permits unrestricted use, A strength of our study is the translational approach includ- distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link ing clinical as well as basic science aspects. A limitation is the to the Creative Commons license, and indicate if changes were made. rather small number of clinical samples from patients with recurrent vulvovaginitis. Although many women were recruit- ed for the first sample, not all fulfilled the strict inclusion References criteria. Moreover, clinical samples from women with sporad- ic VVC would have been an important comparison group. 1. Kullberg BJ, Arendrup MC (2015) Invasive candidiasis. N Engl J However, patients with sporadic VVC generally use over- Med 373:1445–1456 the-counter medications and rarely come for gynecology con- 2. Nobile CJ, Johnson AD (2015) Candida albicans biofilms and sultations. Furthermore, the observed effect of psoriasin was human disease. Annu Rev Microbiol 69:71–92 relatively limited, indicating that other factors may also be 3. Pivarcsi A, Nagy I, Koreck A, Kis K, Kenderessy-Szabo A, Szell M, Dobozy A, Kemeny L (2005) Microbial compounds induce the expres- involved. sion of pro-inflammatory cytokines, chemokines and human beta- defensin-2 in vaginal epithelial cells. Microbes Infect 7:1117–1127 4. Steele C, Fidel PL Jr (2002) Cytokine and chemokine production by human oral and vaginal epithelial cells in response to Candida Conclusion albicans. Infect Immun 70:577–583 5. Chromek M, Brauner A (2008) Antimicrobial mechanisms of the In conclusion, we describe a novel mechanism of action of urinary tract. J Mol Med 86:37–47 psoriasin expressed in the mucosal immunity against 6. Zasloff M (2002) Antimicrobial peptides of multicellular organ- C. albicans. Our results add to the understanding of the isms. Nature 415:389–395 7. Chromek M, Slamová Z, Bergman P, Kovács L, Podracká LU, host-pathogen interaction during Candida infections and Ehrén I, Hökfelt T, Gudmundsson GH, Gallo RL, Agerberth B may reveal new targets for antifungal treatment strategies. et al (2006) The antimicrobial peptide cathelicidin protects the uri- nary tract against invasive bacterial infection. Nat Med 12:636–641 Acknowledgements We thank Petra Lüthje for skillful laboratory work, 8. Teixeira V, Feio MJ, Bastos M (2012) Role of lipids in the interac- analyses, and valuable discussions of the results and Hilde Larson for tion of antimicrobial peptides with membranes. Prog Lipid Res 51: assisting with sampling of patient material. This research was funded by 149–177 the Swedish Research Council, Stockholm County Council (ALF pro- 9. Madsen P, Rasmussen HH, Leffers H, Honoré B, Dejgaard K, ject), and grants from the Karolinska Institutet (A.B.); K.H.S. held an MD Olsen E, Kiil J, Walbum E, Andersen AH, Basse B et al (1991) Fellowship of the Boehringer Ingelheim Fonds, Mainz, Germany. The Molecular cloning, occurrence, and expression of a novel partially funders had no role in study design, data collection and interpretation, secreted protein “Psoriasin” that is highly up-regulated in psoriatic or decision to submit the work for publication. skin. J Invest Dermatol 97:701–712 10. Glaser R, Harder J, Lange H, Bartels J, Christophers E, Schroder Compliance with ethical standards JM (2005) Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol 6:57–64 Conflict of interest The authors declare that they have no conflicts 11. Sohnle PG, Collins-Lech C, Wiessner JH (1991) The zinc- of interest reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J Infect Dis 164:137–142 J Mol Med (2018) 96:537–545 545 12. Hein K, Takahashi H, Tsumori T, Yasui Y, Nanjoh Y, Toga T, Wu Z, 26. Li X, de Leeuw E, Lu W (2005) Total chemical synthesis of human psoriasin by native chemical ligation. Biochemistry 44: Grötzinger J, Jung S, Wehkamp J et al (2015) Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide. 14688–14694 Proc Natl Acad Sci 112:13039–13044 27. Michalek M, Gelhaus C, Hecht O, Podschun R, Schröder JM, 13. Fidel PL Jr, Sobel JD (1996) Immunopathogenesis of recurrent Leippe M, Grötzinger J (2009) The human antimicrobial protein vulvovaginal candidiasis. Clin Microbiol Rev 9:335–348 psoriasin acts by permeabilization of bacterial membranes. Dev 14. Valore EV, Wiley DJ, Ganz T (2006) Reversible deficiency of an- Com Immunol 33:740–746 timicrobial polypeptides in bacterial vaginosis. Infect Immun 74: 28. Polke M, Hube B, Jacobsen ID (2015) Candida survival strategies. 5693–5702 Adv Appl Microbiol 91:139–235 15. Köten B, Simanski M, Gläser R, Podschun R, Schröder JM, Harder 29. Kalyoussef S, Nieves E, Dinerman E, Carpenter C, Shankar V, Oh J (2009) RNase 7 contributes to the cutaneous defense against J, Burd B, Angeletti RH, Buckheit KW, Fredricks DN, Madan RP, Enterococcus faecium. PLoS One 4:e6424. https://doi.org/10. Keller MJ, Herold BC (2012) Lactobacillus proteins are associated 1371/journal.pone.0006424 with the bactericidal activity against E. coli of female genital tract 16. Lüthje P, Brauner H, Ramos NL, Övregaard A, Gläser R, secretions. PLoS One 7:e49506. https://doi.org/10.1371/journal. Hirschberg AL, Aspenström P, Brauner A (2013) Estrogen sup- pone.0049506 ports urothelial defense mechanisms. Sci Transl Med 5:190ra180. 30. Barreto-Bergter E, Figueiredo RT (2014) Fungal glycans and the https://doi.org/10.1126/scitranslmed.3005574 innate immune recognition. Front Cell Infect Microbiol 4:145 17. Schröder JM (2010) Purification of antimicrobial peptides from 31. Cohen-Kedar S, Baram L, Elad H, Brazowski E, Guzner-Gur H, human skin. Methods Mol Biol 618:15–30 Dotan I (2014) Human intestinal epithelial cells respond to beta- 18. Owen DH, Katz DF (1999) A vaginal fluid simulant. Contraception glucans via Dectin-1 and Syk. Eur J Ímmunol 44:3729–3740 59:91–95 32. Hau CS, Tada Y, Shibata S, Uratsuji H, Asano Y, Sugaya M, 19. Tsai PW, Yang CY, Chang HT, Lan CY (2011) Human antimicro- Kadono T, Kanda N, Watanabe S, Tamaki K, Sato S (2011) High bial peptide LL-37 inhibits adhesion of Candida albicans by calcium, ATP, and poly(I:C) augment the immune response to beta- interacting with yeast cell-wall carbohydrates. PLoS One 6: glucan in normal human epidermal keratinocytes. J Invest Dermatol e17755. https://doi.org/10.1371/journal.pone.0017755 131:2255–2262 20. Sobel JD (2007) Vulvovaginal candidosis. Lancet 369:1961–1971 33. Brown GD, Herre J, Williams DL, Willment JA, Marshall AS, 21. Foxman B, Muraglia R, Dietz JP, Sobel JD, Wagner J (2013) Gordon S (2003) Dectin-1 mediates the biological effects of beta- Prevalence of recurrent vulvovaginal candidiasis in 5 European glucans. J Exp Med 197:1119–1124 countries and the United States: results from an internet panel sur- 34. Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM vey. J Low Gen Tract Dis 17:340–345 (2003) Collaborative induction of inflammatory responses by 22. Sobel JD (2016) Recurrent vulvovaginal candidiasis. Am J Obstet dectin-1 and toll-like receptor 2. J Exp Med 197:1107–1117 Gynecol 214:15–21 35. Goodridge HS, Reyes CN, Becker CA, Katsumoto TR, Ma J, Wolf 23. Cauchie M, Desmet S, Lagrou K (2017) Candida and its dual life- AJ, Bose N, Chan AS, Magee AS, Danielson ME et al (2011) style as a commensal and a pathogen. Res Microbiol 168:1–9 Activation of the innate immune receptor Dectin-1 upon formation 24. ErhartW,AlkasiO,BrunkeG,Wegener F,Maass N,ArnoldN, Arlt of a ‘phagocytic synapse’. Nature 472:471–475 A, Meinhold-Heerlein I (2011) Induction of human beta-defensins and psoriasin in vulvovaginal human papillomavirus-associated le- 36. Segat L, Zupin L, Moura RR, Coelho AVC, Chagas BS, de Freitas sions. J Infect Dis 204:391–399 AC, Crovella S (2014) DEFB1 polymorphisms are involved in 25. Lee KC, Eckert RL (2007) S100A7 (Psoriasin)–mechanism of an- susceptibility to human papillomavirus infection in Brazilian tibacterial action in wounds. J Invest Dermatol 127:945–957 gynaecological patients. Mem Inst Oswaldo Cruz 109:918–922 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Medicine Springer Journals
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Springer Berlin Heidelberg
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Copyright © 2018 by The Author(s)
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Biomedicine; Molecular Medicine; Human Genetics; Internal Medicine
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10.1007/s00109-018-1637-6
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Abstract

Candida albicans belongs to the normal microbial flora on epithelial surfaces of humans. However, under certain, still not fully understood conditions, it can become pathogenic and cause a spectrum of diseases, from local infections to life-threatening septicemia. We investigated a panel of antimicrobial proteins and peptides (AMPs), potentially involved in mucosal immunity against this pathogen. Out of six studied AMPs, psoriasin was most up-regulated during a mucosal infection, an acute episode of recurrent Candida vulvovaginitis, although candidacidal activity has not been demonstrated. We here show that psoriasin binds to β-glucan, a basic component of the C. albicans cell wall, and thereby inhibits adhesion of the pathogen to surfaces and increases IL-8 production by mucosal epithelial cells. In conclusion, we show a novel mechanism of action of psoriasin. By inhibiting C. albicans adhesion and by enhancing cytokine production, psoriasin contributes to the immune response against C. albicans. Key messages & The antimicrobial peptide psoriasin is highly up-regulated during a local mucosal infection, Candida albicans vulvovaginitis. & Psoriasin binds to β-glucan in the Candida albicans cell wall and thereby inhibits adhesion of the pathogen. & Binding of psoriasin to Candida albicans induces an im- mune response by mucosal epithelial cells. . . . . Keywords Candida albicans Psoriasin β-Glucan Adhesion Immune response Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00109-018-1637-6) contains supplementary material, which is available to authorized users. Candida albicans is a commensal fungus, colonizing mucosal and cutaneous surfaces of healthy individuals. As an opportunis- * Annelie Brauner tic pathogen, C. albicans can cause a variety of diseases, ranging Annelie.Brauner@ki.se from trivial local to life-threatening systemic infections. Immunocompromised patients are mostly at risk for severe fun- Department of Microbiology, Tumor and Cell Biology, Division of gal infections [1]. Indwelling catheters constitute another risk Clinical Microbiology, Karolinska Institutet and Karolinska factor, due to the ability of the fungus to adhere and form biofilm University Hospital, Stockholm, Sweden on foreign surfaces [2]. Yet, factors of the host-pathogen interac- Department of Clinical Sciences, Division of Obstetrics and tions that lead to asymptomatic carriage, to local infection, or to Gynecology, Danderyd Hospital and Karolinska Institutet, invasive systemic infection are still not fully understood. Stockholm, Sweden The mucosal epithelium with its normal microbial flora con- Department of Clinical Science, Intervention and Technology, stitutes an important dynamic barrier for invading microorgan- Division of Pediatrics, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden isms. The epithelial cells produce mucus, anti-adhesins, antimi- crobial proteins and peptides (AMPs), chemokines, and cyto- Department of Dermatology, Venerology and Allergology, University Hospital Schleswig-Holstein, Kiel, Germany kines [3–7]. AMPs are mostly cationic, amphipatic, and typically 538 J Mol Med (2018) 96:537–545 with between 12 and 50 amino acid residues in length [8]. The contraceptives did not differ between patients and control par- antimicrobial protein psoriasin (S100A7), however, is larger, not ticipants (Supplementary Table 1). cationic (MW 11,457; pI 6.77) [9] and has highly potent E. coli-cidal activity [10]. Unlike most AMPs, it is not acting Isolation and identification of Candida albicans via membrane effects, but similar to calprotectin [11], in a zinc- dependent fashion [10]. In an approach to explain the resistance For identification of C. albicans, vaginal swabs were cultured of lesional psoriatic skin towards dermatophyte infection, we on CHROMagar Candida and typical colonies were further recently identified the cysteine-reduced form of psoriasin as a analyzed by latex surface antigen agglutination (Bichro- fungicidal AMP, which acts via intracellular zinc-depletion and Dubli) at the Department of Clinical Microbiology, induction of apoptosis-like fungal cell death [12]. Cysteine- Karolinska University Hospital, Stockholm, Sweden. reduced psoriasin, but not its oxidized form, is a powerful Absence of fungal and bacterial growth in the samples from broad-spectrum fungicide for many dermatophytes and control participants was confirmed according to the same pro- Aspergillus fumigatus. Interestingly, however, both the oxidized cedure. Isolates were kept frozen at − 80 °C. For experiments, and the cysteine-reduced forms of psoriasin lack activity towards if not stated otherwise, isolates were cultured on Sabouraud C. albicans [12]. agar for 1–2 days at 37 °C and then in yeast peptone dextrose In this study, we sought to investigate the interaction be- (YPD) medium overnight at 30 °C. This culture was diluted tween C. albicans and epithelial defense mechanisms, in par- 1:100 in fresh medium and grown for another 3 h at 30 °C to ticular AMPs, during a typical mucosal C. albicans infection, the logarithmic growth phase. Under these conditions, vulvovaginal candidiasis. Although lacking a direct antifungal C. albicans grew as yeast cells. effect, psoriasin was strongly up-regulated during the acute C. albicans infection. This AMP supported the mucosal im- Vaginal lavage munity by alternative mechanisms, namely by inhibition of C. albicans adhesion and by affecting the immune response. Vaginal lavage was obtained according to a modified method by Valore et al. [14]. Briefly, 5 ml of distilled water was ad- ministered intravaginally, and lavage samples were collected Materials and methods 5 min later. The fluid was centrifuged at 300×g for 10 min, and the cell pellet and the sterile-filtered supernatant were stored at Participants and samples − 80 °C. The clinical part of the study was approved by the Regional Biopsy samples Ethical Review Board in Stockholm. All participants were recruited at the gynecology outpatient clinic at Danderyd Two biopsies were taken from the vaginal wall of each patient Hospital, Stockholm, Sweden, and informed consent was ob- with 3-mm forceps at the 3 and 9 o’clock positions approxi- tained. Recurrent vulvovaginal candidiasis (RVVC) was de- mately 3–4 cm from the vaginal introitus. One biopsy was fined according to Fidel [13], and only cases of primary fixed in 4% paraformaldehyde at 4 °C overnight, transferred RVVC were included, i.e., no known predisposing underlying to 70% ethanol and processed for immunohistochemical anal- conditions including pregnancy, antibiotic treatment, diabetes, ysis; a second biopsy was placed in RNAlater RNA and immunosuppression. Infection with C. albicans was con- Stabilization Reagent (Qiagen) and subjected to RNA isola- firmed microbiologically; patients infected with other tion and gene expression analysis. Candida spp. or bacterial vaginosis were excluded from the study. None of the patients had received any antibacterial or Immunofluorescence staining of sections antifungal treatment at the time of the first sampling. At the first visit, all patients showed typical symptoms of an acute Biopsies collected for immunohistochemical analysis were vulvovaginal C. albicans infection, and oral antifungal treat- paraffin-embedded and cut at 4 μm. Sections were ment was initiated, consisting of 50 mg/day fluconazole for deparaffinized in xylene or Neo-Clear, rehydrated in an etha- 1 week and 150 mg/week for 5 weeks. Samples were collected nol gradient and washed in water and phosphate-buffered sa- as described below, before and 2–3 weeks after antifungal line (PBS). Heat-mediated antigen retrieval was performed in treatment was completed. A total of 16 women, aged 20– citrate buffer (10 mM, pH 6). Sections were blocked for 39 years (median 32 years), were included in the study. All 30 min with Image-iT FX Signal Enhancer (Life patients were culture-negative for C. albicans and other uro- Technologies) and for 60 min with 10% serum from the spe- genital infections at the time of the second visit. Control sam- cies that the secondary antibody was raised in. Overnight in- ples were collected once from 27 healthy female volunteers, cubation with primary antibodies was carried out at 4 °C. aged 19–40 years (median, 25 years). The usage of Primary antibodies used were rabbit anti-HBD1 (1:100, J Mol Med (2018) 96:537–545 539 Santa Cruz Biotechnology), goat anti-HBD2 (1:50, R&D Enzyme-linked immunosorbent assays (ELISA) Systems), rabbit anti-HBD3 (1:100, Santa Cruz Biotechnology), rabbit anti-psoriasin (1:800, Abcam), and Vaginal lavage samples were analyzed using ELISA kits for rabbit anti-RNase 7 (1:500, Novus Biologicals). Sections were human β-defensins (HBD) 1, 2, and 3 (Alpha Diagnostic then incubated with Alexa Fluor-conjugated secondary anti- International); the human cathelicidin LL-37 (Hycult bodies (Life Technologies) for 30–60 min at room temperature Biotech); human ribonuclease (RNase) 7 (Icosagen); and IL- and mounted in ProlongGold Antifade mounting medium 8 (R&D Systems) according to the manufacturer’s instruc- with DAPI (Life Technologies). Images were acquired on a tions. Samples were tested in appropriate dilutions to fall into Leica SP5 confocal microscope using a ×40 objective. the concentration range covered by the internal standards. For HBD3, samples with signals above the limit of the detection system (2000 pg/ml) were assigned a value of 3000 pg/ml to Total RNA extraction and RT real-time PCR allow statistical evaluation. Total RNA extraction from vaginal biopsies and cultured cells Chemicals and reagents was performed using the RNeasy Mini kit (Qiagen). Tissue pieces were lysed in RLT lysis buffer and homogenized with Psoriasin was purified from human skin [17], dissolved in mortar and pestle and then passed through a QIAshredder. 0.01% acetic acid to a concentration of 1 mg/ml, and stored Cells were lysed in lysis buffer; samples were further processed at − 20 °C. A vaginal fluid simulant (VFS) was used as medi- according to the manufacturer’s protocol. Reverse transcription um for all reactions to mimic the vaginal milieu. This previ- with up to 1 μg of RNA was carried out using the DyNAmo ously described formulation resembles the vaginal material cDNA Synthesis Kit (Finnzymes) or the High Capacity cDNA with respect to pH, osmolarity, and chemical composition Reverse Transcription Kit (Applied Biosystems) according to the [18]; organic acids are represented by lactic and acetic acid manufacturers’ instructions. Gene expression was analyzed with and proteins by albumin. All mono- and polysaccharides were TaqMan Gene Expression Assays for HBD1 (DEFB1, purchased from Sigma; working dilutions were prepared Hs00608345_m1), HBD2 (DEFB4A, Hs00175474_m1) and freshly in VFS. HBD3 (DEFB103A, Hs00218678_m1), psoriasin (S100A7, Hs00161488_m1), human cathelicidin LL-37/hCAP-18 Candida albicans adhesion assays (CAMP, Hs00189038_m1), IL-1β (IL1B, Hs01555410_m1), IL-6 (IL6, Hs00985639_m1), and IL-8 (IL8, Hs00174103_m1). The adhesive capacity of C. albicans isolates was determined Expression of RNase 7 was examined using a SYBR Green- in a microtiter plate assay, using VFS as medium or vaginal based assay (Qiagen) with previously described primers [15] lavage. C. albicans adhesion was measured according to a and conditions [16]. GAPDH and 18S rRNA were used as inter- previously described protocol with modifications [19]. nal controls to calculate relative gene expression. C. albicans yeast cells from the logarithmic growth phase were collected by centrifugation, washed twice in PBS, the pellet was suspended in VFS, and the density was adjusted Western blot detection of psoriasin spectrophotometrically. A 100-μl volume containing approx- imately 1 × 10 colony-forming units (CFU) was added to For detection of psoriasin in cell pellets, cells were lysed in an wells on 96-well sterile, non-treated, flat-bottomed microtiter equal volume of Triton X-100 (1% in PBS, with protease plates (Corning) and incubated for 30 min at 37 °C with low inhibitors). The lysate was cleared by centrifugation and the agitation (100 rpm). Thereafter, wells were washed three times protein concentration was determined by BCA assay (Thermo with PBS to remove non-adherent cells, and the number of Scientific). Equal amounts of protein were heated in twofold adherent cells was quantified by metabolic activity using an tricine sample buffer (Bio-Rad) at 95 °C for 5 min and sub- XTT assay. To evaluate the influence of psoriasin on jected to polyacrylamide-gel-electrophoresis using 10–20% C. albicans adhesion, psoriasin (0.001–1 μM or vector) was tris-tricine gels (Bio-Rad), and the separated samples were added to duplicate wells. In selected experiments, psoriasin transferred to PVDF membranes (Invitrogen). After transfer, (1 μM or vector) was pre-incubated with polysaccharides membranes were blocked with milk-TBSTand incubated with β-(1,3)-D-glucan from baker’syeast, D-mannan from baker’s a polyclonal rabbit anti-psoriasin (1:800 in milk-TBST) anti- yeast, and chitin from crab shells (1 mg/ml in VFS, all from body for 1 h at room temperature at 4 °C. Incubation with an Sigma) for 30 min at 4 °C prior to the addition of C. albicans. anti-rabbit HRP-conjugated secondary antibody was carried To evaluate the effect of vaginal lavage on adhesion, VFS was out at room temperature for 60 min. Signals were detected replaced by vaginal lavage fluid from patients and control using SuperSignal West Pico Chemiluminescent reagents participants. The lavage contributed to 40% of the final reac- (Thermo Scientific). tion volume. In some experiments, lavage samples were 540 J Mol Med (2018) 96:537–545 treated with polysaccharides in the same manner as described Statistical analysis for psoriasin. All statistical analyses were performed using GraphPad Prism, Version 6. Data obtained from clinical material were analyzed Psoriasin-Candida binding assays with non-parametric tests; results are presented as individual values with median. For matched comparisons, the Wilcoxon A pull-down assay was adapted from a previously described matched-pairs signed rank test or Friedman test was used; protocol [19]. To demonstrate binding between C. albicans 7 comparison between groups was performed by Mann- cells and psoriasin, approximately 1.5 × 10 cells were incu- Whitney U test or Kruskal-Wallis test as appropriate. bated with 10 μg psoriasin in VFS (final volume 250 μl) and Variations in the number of participants included were due incubated for 30 min at 4 °C with agitation; alternatively, to limited material, and biological outliers were not excluded. 250 μl of vaginal lavage was used. Cells were collected by Data from other in vitro experiments are presented with mean centrifugation and washed twice with VFS. Binding of and standard deviation from at least three independent exper- psoriasin to polysaccharides was tested in a similar manner; iments (n ≥ 3). Results were analyzed by unpaired t test, one- because of the solubility of mannan, mannan-agarose was way ANOVA with Dunnett’s multiple comparison tests, or used. β-(1,3)-D-Glucan from baker’s yeast, chitin from crab two-way ANOVAwith Bonferroni’s multiple comparison cor- shells (0.25 mg/ml), or mannan-agarose (30 μl) were mixed rection, as appropriate. Technical outliers were detected by with 10 μg psoriasin in VFS (final volume 750 μl) and incu- Grubb’s test and excluded. All tests were performed two- bated overnight at 4 °C with agitation; samples without any sided, and differences with P values of < 0.05 were considered polysaccharide or psoriasin were kept alongside for control statistically significant. purposes. Polysaccharides were then pelleted by centrifuga- tion and washed twice with VFS. The final pellets were heated in tricine sample buffer (Bio-Rad) at 95 °C for 5 min and Results Western blotting was performed as described above. Candida albicans induces production of psoriasin Cell culture during mucosal infection The vaginal epithelial cell line AO was kindly provided by To describe the expression of epithelial AMPs at baseline and David J. Klumpp, Northwestern University, Chicago, IL, and in response to C. albicans, mRNA and protein levels of six was used as model of the vaginal epithelium. Cells were cul- AMPs were analyzed in vaginal biopsies from healthy women tured in EpiLife Medium with 60 μM calcium and supple- (n =27, Fig. 1a), from patients during acute C. albicans vul- mented with Human Keratinocyte Growth Supplement vovaginitis and from the same patients after successful anti- (Gibco) in a humidified incubator at 37 °C with 5% CO . fungal treatment (n =16; Fig. 1b and Supplementary Fig. S1). Out of six analyzed AMPs, psoriasin (encoded by the S1007A Cell infection gene) and RNase 7 showed the highest mRNA and protein levels (Fig. 1a). RNase 7 expression was not influenced by C. albicans from logarithmic growth phase were collected by infection with C. albicans (Supplementary Fig. S1). Psoriasin, centrifugation, washed in PBS. Aliquots of 3 × 10 cells were on the other hand, was strongly up-regulated during the acute transferred in low-binding 1.5-ml tubes. Cells were collect- infection episode, both on the mRNA (Fig. 1b) and on the ed by centrifugation, and the pellet was suspended in 98 μl protein levels as shown by immunohistochemistry (Fig. 1c) VFS with 2 μl psoriasin (1 mg/ml) or vector. The mixture in vaginal biopsies and by Western blot in the cell pellet from was incubated 30 min on ice, with occasional mixing. Cells the vaginal lavage samples (Fig. 1d–e). Increased production were collected by centrifugation at 4 °C, the supernatant of psoriasin was primarily detected in the pellet of the vaginal was discarded, and the pellet suspended in 200 μl0.2% lavage containing desquamated mucosal cells and yeast, and paraformaldehyde in PBS. Cells were fixed for 10 min at the levels of psoriasin in the lavage supernatant were not sig- room temperature, washed once, and suspended in cell cul- nificantly affected by infection (Supplementary Fig. S2). ture medium to a final concentration of 10 CFU/ml. From this suspension, 100 μl was added to a well of confluent Psoriasin interacts with β-glucan in the Candida AO cells containing 900 μl of fresh cell culture medium albicans cell wall and incubated for 6 h at 37 °C with 5% CO in a humidi- fied incubator. The medium was removed, and cells were Since psoriasin produced during acute mucosal infection was collected for RNA extraction and gene expression analysis. associated with the cell pellet and not increased in the super- All conditions were analyzed in duplicate. natant (Fig. 1d and Supplementary Fig. S2), we analyzed a J Mol Med (2018) 96:537–545 541 Fig. 1 Psoriasin dominates the response to vulvovaginal infection with green; cell nuclei, blue; ×40 objective). d Western blot of lavage Candida albicans. Vaginal biopsies and lavage samples were collected samples. e Psoriasin-specific bands on Western blots were quantified from healthy women (controls, Co) (a–e) and patients with recurrent using ImageJ, and signal intensity is expressed in arbitrary units (AU); vulvovaginal candidiasis (patients, P) (b–e). a Expression of one representative blot with patients’ samples during infection (inf) and antimicrobial proteins and peptides in vaginal tissue was determined on after treatment (after), and samples from controls are shown. Individual the mRNA level by RT-PCR and is expressed in relation to 18S rRNA or values with median are shown (a, b, e); analyses were performed with GAPDH levels (open diamonds, left axis); secreted proteins and peptides Mann-Whitney test to compared data from controls and patients, and were detected by ELISA (filled diamonds, right axis). b S100 mRNA in Wilcoxon matched-pairs signed rank test was used to evaluate data controls (open circles) and patients during infection and after treatment from patients during acute infection and after treatment; **P < 0.01, (filled circles). c Immunohistochemistry of tissue sections (psoriasin, ***P < 0.001, ****P <0.0001 possible interaction between psoriasin and C. albicans.We under conditions mimicking the vaginal milieu [18]. performed a pull-down assay using whole C. albicans cells Adherence of C. albicans cells to polystyrene was reduced and the polysaccharides β-glucan, chitin and mannan, the by psoriasin in a concentration-dependent manner (Fig. 3a). most important components of the fungal cell wall. Both pu- β-Glucan had the ability to completely neutralize the anti- rified psoriasin and the psoriasin present in vaginal lavage adhesive properties of psoriasin, which was not the case for strongly bound to whole C. albicans cells and to β-glucan, chitin or mannan (Fig. 3b). while chitin and mannan displayed only weak binding activity To confirm the effect also in vaginal lavage, samples from to psoriasin (Fig. 2a). healthy volunteers and from patients with acute C. albicans Electron microscopic investigation demonstrated a signifi- vulvovaginitis were analyzed in a similar adhesion assay. The cant thinning of the cell wall in psoriasin-exposed C. albicans presence of anti-adhesive activity was confirmed in vaginal (Fig. 2b, c). Moreover, immunogold-labelling revealed dimin- lavage fluid and could partially be blocked by β-glucan ished content of β-glucan in the affected cells (Fig. 2b). (Fig. 3c). Psoriasin inhibits Candida albicans adhesion Epithelial immune response to Candida albicans is to surfaces enhanced by psoriasin To evaluate the effect of psoriasin-mediated changes in the To further analyze the consequences of binding of psoriasin to candidal cell wall, we investigated adhesion of C. albicans C. albicans, vaginal epithelial cells were infected with 542 J Mol Med (2018) 96:537–545 Fig. 2 Psoriasin binds to Candida β-glucan and reduces its content in the cell wall. a C. albicans cells or cell wall polysaccharides were incubated with purified psoriasin or vaginal lavage (VL), the pellet was subjected to SDS-PAGE, and membranes were probed for psoriasin. b Due to the solubility of mannan, a mannan-agarose conjugate was used. C. albicans was incubated with psoriasin or vector (control) for 30 min at 4 °C and investigated by transmission electron microscopy, scale bars 1 μm(upperpanel); β-glucan was marked with immunogold-labeled anti-β-glucan antibodies (lower panel, enlarged). c The cell wall thickness from non-treated (Co) and psoriasin-exposed cells (Psoriasin) in five to eight images per experiment was measured as exemplified in the close-up; mean results from three independent experiments are presented as mean and standard deviation and compared by unpaired t test; *P <0.05 psoriasin-treated and untreated fungal cells. Overall, psoriasin women suffering from sporadic or recurrent VVC are other- treatment of C. albicans had an impact on the induction of wise healthy without known underlying conditions [23]. pro-inflammatory cytokines, with the most pronounced effect Epithelial cells lining the body surfaces are the first to come on IL-8 (Fig. 4a). Accordingly, IL8 mRNA levels in patients into contact with invading pathogens and are equipped with during acute infection correlated positively with expression AMPs to fend off potential intruders [6]. We analyzed a panel levels of psoriasin mRNA (Fig. 4b). However, psoriasin alone of AMPs potentially involved in mucosal immunity against did not influence IL-8 expression in epithelial cells in vitro C. albicans. Overall, the pattern resembled a previously re- (Supplementary Fig. S3). ported expression profile [24]. To our surprise, the most abundantly expressed and up- regulated AMP during acute C. albicans infection was Discussion psoriasin. This S100 protein is a powerful antimicrobial for E. coli and to a less degree also some other bacteria [10]. In its We here describe the expression of the antimicrobial protein cysteine-reduced form, psoriasin is a potent fungicidal for fil- psoriasin during mucosal infection with C. albicans and a amentous fungi. However, unexpectedly and for unknown novel mechanism of action of this protein. Psoriasin contrib- reasons, it is not killing C. albicans [10, 12]. utes to the inhibition of C. albicans adhesion and to the in- In search for alternative actions of psoriasin, we performed creased immune response to C. albicans by mucosal epithelial a pull-down assay using whole C. albicans cells or the major cells. components of the fungal cell wall. Psoriasin strongly bound C. albicans is part of the normal flora on skin and mucosal to whole yeast cells and to β-glucan. Accordingly, C. albicans membranes of humans, but as an opportunistic pathogen can cells were also able to precipitate psoriasin in vaginal lavage cause both local and systemic infections. Vulvovaginal candi- samples. diasis (VVC) is a common local infection in women of repro- The mode of antibacterial action by psoriasin is still not ductive age [20], and some will experience recurrent VVC, completely understood. Its activity appears to depend on its with several episodes of acute infections per year [21, 22]. The zinc-binding motifs [10, 25, 26], which also mediate binding majority of mucosal as well as skin infections caused by to E. coli but not Staphylococcus aureus [25]. Membrane in- C. albicans can be linked to impaired immunity or the pres- tegrity, however, was only affected in a Gram-positive species and at low pH [27]. The fungicidal activity of cysteine- ence of foreign bodies like indwelling catheters. In contrast, J Mol Med (2018) 96:537–545 543 apoptosis-like cell death in filamentous fungi [12]. It is possi- ble that the absence of hydrophobin-like molecules in C. albicans contributes to psoriasin’s inability to kill C. albicans. To study potential consequences of psoriasin-mediated changes of the C. albicans cell wall, two major steps in the pathogenesis of Candida infection were investigated, adhesion and induction of a cytokine response [28]. First, our experi- ments with vaginal lavage from healthy women and from patients with recurrent VVC confirmed the presence of anti- adhesive activity in vivo. This activity could partly be linked to psoriasin as demonstrated by an in vitro assay. Both the anti-adhesive activity present in vivo and the activity demon- strated when using purified psoriasin could partially be blocked by β-glucan. Vaginal lavage is known to have several inhibitory factors, such as proteins from commensal lactobacilli [29]. In the current study, the blocking effect of β-glucan in the lavage samples was not complete, suggesting the presence of additional anti-adhesins in vivo. Second to adhesion, receptor-mediated contact with the host cell generally induces an immune response. Psoriasin binds to glucans, which are the key fungal pathogen- associated molecular patterns (reviewed in [30]). Binding of β-glucan to the C-type lectin dectin-1 was shown to mediate induction of pro-inflammatory cytokines in intestinal epithe- lial cells [31] and keratinocytes [32]. Therefore, we expected reduced immune induction in response to the β-glucan- depleted cell wall. Immune response to glucans, however, depends on the size and physical properties of their molecules. Large β-glucan particles induce cytokine production, produc- tion of reactive oxygen species, and phagocytosis by neutro- phils and macrophages, while soluble β-glucans act as antag- onists of these processes [33–35]. In line with these findings, we speculate that treatment of C. albicans induces cell wall changes with loss of outer material and increased exposure of β-glucan at the cell surface. The increased IL-8 observed sug- gests binding to large β-glucan and decreased availability of the soluble ones. In our in vitro experiments, the IL-8 production by vaginal Fig. 3 Psoriasin reduces Candida adhesion. a Adhesion of C. albicans in vaginal lavage simulant with different concentrations of psoriasin was epithelial cells was increased after pre-treatment of quantified by yeast metabolic activity using the XTT assay. b The same C. albicans with psoriasin, possibly by reducing the availabil- assay was performed with psoriasin pre-incubated with polysaccharides. ity of soluble β-glucan. Thus, inhibition of adhesion and in- Data are expressed in relation to corresponding samples without psoriasin crease of cytokine production, although relatively low, may (control, Co = 100) and shown as mean and standard deviation from three to six independent experiments; results were analyzed by one-way ANOVA together promote the mucosal immune protection against with Dunnett’s multiple comparison test versus control; *P < 0.05, C. albicans. **P < 0.01. c In a similar assay, Candida adhesion was measured in Interestingly, although psoriasin was clearly up-regulated vaginal lavage samples from healthy women and from patients with during the acute phase of VVC, it could not protect the host recurrent vulvovaginal candidiasis, and in the presence of glucan (n=18). Individual values with medians are shown, and results were analyzed by from infection. However, it can, however, not be ruled out that Kruskal-Wallis with Dunn’s multiple comparison test; *P < 0.05, patients with a single episode of VVC mount a higher and ***P<0.001 more effective psoriasin response compared to patients with reduced psoriasin is based on a selective cellular uptake, pos- recurrent infections. Further, gene polymorphism of DEFB1 sibly mediated by hydrophobin-like proteins, followed by in- has been suggested to be involved in susceptibility to human 2+ tracellular Zn -binding and subsequent induction of papillomavirus infection [36]. We therefore speculate that 544 J Mol Med (2018) 96:537–545 Fig. 4 Psoriasin promotes a pro-inflammatory response to Candida. a way ANOVA with Bonferroni’s multiple comparison test. b Relative Vaginal epithelial cells were infected with C. albicans pre-treated with mRNA expression of IL-8 and psoriasin was measured in vaginal tissue psoriasin or left untreated. After 6 h, induction of cytokine mRNA was from women during an acute infection with C. albicans. Individual values determined by RT-PCR. Data from four independent experiments are (n = 14) are shown, correlation between psoriasin mRNA (S100A7)and shown with mean and standard deviation; results were analyzed by two- IL8 was assessed by Spearman’stest, r =0.55, P <0.05 Open Access This article is distributed under the terms of the Creative polymorphisms in the psoriasin gene may be associated with Commons Attribution 4.0 International License (http:// recurrent C. albicans infections. creativecommons.org/licenses/by/4.0/), which permits unrestricted use, A strength of our study is the translational approach includ- distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link ing clinical as well as basic science aspects. A limitation is the to the Creative Commons license, and indicate if changes were made. rather small number of clinical samples from patients with recurrent vulvovaginitis. Although many women were recruit- ed for the first sample, not all fulfilled the strict inclusion References criteria. Moreover, clinical samples from women with sporad- ic VVC would have been an important comparison group. 1. Kullberg BJ, Arendrup MC (2015) Invasive candidiasis. N Engl J However, patients with sporadic VVC generally use over- Med 373:1445–1456 the-counter medications and rarely come for gynecology con- 2. Nobile CJ, Johnson AD (2015) Candida albicans biofilms and sultations. Furthermore, the observed effect of psoriasin was human disease. Annu Rev Microbiol 69:71–92 relatively limited, indicating that other factors may also be 3. Pivarcsi A, Nagy I, Koreck A, Kis K, Kenderessy-Szabo A, Szell M, Dobozy A, Kemeny L (2005) Microbial compounds induce the expres- involved. sion of pro-inflammatory cytokines, chemokines and human beta- defensin-2 in vaginal epithelial cells. Microbes Infect 7:1117–1127 4. Steele C, Fidel PL Jr (2002) Cytokine and chemokine production by human oral and vaginal epithelial cells in response to Candida Conclusion albicans. Infect Immun 70:577–583 5. Chromek M, Brauner A (2008) Antimicrobial mechanisms of the In conclusion, we describe a novel mechanism of action of urinary tract. J Mol Med 86:37–47 psoriasin expressed in the mucosal immunity against 6. Zasloff M (2002) Antimicrobial peptides of multicellular organ- C. albicans. Our results add to the understanding of the isms. Nature 415:389–395 7. Chromek M, Slamová Z, Bergman P, Kovács L, Podracká LU, host-pathogen interaction during Candida infections and Ehrén I, Hökfelt T, Gudmundsson GH, Gallo RL, Agerberth B may reveal new targets for antifungal treatment strategies. et al (2006) The antimicrobial peptide cathelicidin protects the uri- nary tract against invasive bacterial infection. Nat Med 12:636–641 Acknowledgements We thank Petra Lüthje for skillful laboratory work, 8. Teixeira V, Feio MJ, Bastos M (2012) Role of lipids in the interac- analyses, and valuable discussions of the results and Hilde Larson for tion of antimicrobial peptides with membranes. Prog Lipid Res 51: assisting with sampling of patient material. This research was funded by 149–177 the Swedish Research Council, Stockholm County Council (ALF pro- 9. Madsen P, Rasmussen HH, Leffers H, Honoré B, Dejgaard K, ject), and grants from the Karolinska Institutet (A.B.); K.H.S. held an MD Olsen E, Kiil J, Walbum E, Andersen AH, Basse B et al (1991) Fellowship of the Boehringer Ingelheim Fonds, Mainz, Germany. The Molecular cloning, occurrence, and expression of a novel partially funders had no role in study design, data collection and interpretation, secreted protein “Psoriasin” that is highly up-regulated in psoriatic or decision to submit the work for publication. skin. J Invest Dermatol 97:701–712 10. Glaser R, Harder J, Lange H, Bartels J, Christophers E, Schroder Compliance with ethical standards JM (2005) Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol 6:57–64 Conflict of interest The authors declare that they have no conflicts 11. Sohnle PG, Collins-Lech C, Wiessner JH (1991) The zinc- of interest reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J Infect Dis 164:137–142 J Mol Med (2018) 96:537–545 545 12. Hein K, Takahashi H, Tsumori T, Yasui Y, Nanjoh Y, Toga T, Wu Z, 26. Li X, de Leeuw E, Lu W (2005) Total chemical synthesis of human psoriasin by native chemical ligation. Biochemistry 44: Grötzinger J, Jung S, Wehkamp J et al (2015) Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide. 14688–14694 Proc Natl Acad Sci 112:13039–13044 27. Michalek M, Gelhaus C, Hecht O, Podschun R, Schröder JM, 13. Fidel PL Jr, Sobel JD (1996) Immunopathogenesis of recurrent Leippe M, Grötzinger J (2009) The human antimicrobial protein vulvovaginal candidiasis. Clin Microbiol Rev 9:335–348 psoriasin acts by permeabilization of bacterial membranes. Dev 14. Valore EV, Wiley DJ, Ganz T (2006) Reversible deficiency of an- Com Immunol 33:740–746 timicrobial polypeptides in bacterial vaginosis. Infect Immun 74: 28. Polke M, Hube B, Jacobsen ID (2015) Candida survival strategies. 5693–5702 Adv Appl Microbiol 91:139–235 15. Köten B, Simanski M, Gläser R, Podschun R, Schröder JM, Harder 29. Kalyoussef S, Nieves E, Dinerman E, Carpenter C, Shankar V, Oh J (2009) RNase 7 contributes to the cutaneous defense against J, Burd B, Angeletti RH, Buckheit KW, Fredricks DN, Madan RP, Enterococcus faecium. PLoS One 4:e6424. https://doi.org/10. Keller MJ, Herold BC (2012) Lactobacillus proteins are associated 1371/journal.pone.0006424 with the bactericidal activity against E. coli of female genital tract 16. Lüthje P, Brauner H, Ramos NL, Övregaard A, Gläser R, secretions. PLoS One 7:e49506. https://doi.org/10.1371/journal. Hirschberg AL, Aspenström P, Brauner A (2013) Estrogen sup- pone.0049506 ports urothelial defense mechanisms. Sci Transl Med 5:190ra180. 30. Barreto-Bergter E, Figueiredo RT (2014) Fungal glycans and the https://doi.org/10.1126/scitranslmed.3005574 innate immune recognition. Front Cell Infect Microbiol 4:145 17. Schröder JM (2010) Purification of antimicrobial peptides from 31. Cohen-Kedar S, Baram L, Elad H, Brazowski E, Guzner-Gur H, human skin. Methods Mol Biol 618:15–30 Dotan I (2014) Human intestinal epithelial cells respond to beta- 18. Owen DH, Katz DF (1999) A vaginal fluid simulant. Contraception glucans via Dectin-1 and Syk. Eur J Ímmunol 44:3729–3740 59:91–95 32. Hau CS, Tada Y, Shibata S, Uratsuji H, Asano Y, Sugaya M, 19. Tsai PW, Yang CY, Chang HT, Lan CY (2011) Human antimicro- Kadono T, Kanda N, Watanabe S, Tamaki K, Sato S (2011) High bial peptide LL-37 inhibits adhesion of Candida albicans by calcium, ATP, and poly(I:C) augment the immune response to beta- interacting with yeast cell-wall carbohydrates. PLoS One 6: glucan in normal human epidermal keratinocytes. J Invest Dermatol e17755. https://doi.org/10.1371/journal.pone.0017755 131:2255–2262 20. Sobel JD (2007) Vulvovaginal candidosis. Lancet 369:1961–1971 33. Brown GD, Herre J, Williams DL, Willment JA, Marshall AS, 21. Foxman B, Muraglia R, Dietz JP, Sobel JD, Wagner J (2013) Gordon S (2003) Dectin-1 mediates the biological effects of beta- Prevalence of recurrent vulvovaginal candidiasis in 5 European glucans. J Exp Med 197:1119–1124 countries and the United States: results from an internet panel sur- 34. Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM vey. J Low Gen Tract Dis 17:340–345 (2003) Collaborative induction of inflammatory responses by 22. Sobel JD (2016) Recurrent vulvovaginal candidiasis. Am J Obstet dectin-1 and toll-like receptor 2. J Exp Med 197:1107–1117 Gynecol 214:15–21 35. Goodridge HS, Reyes CN, Becker CA, Katsumoto TR, Ma J, Wolf 23. Cauchie M, Desmet S, Lagrou K (2017) Candida and its dual life- AJ, Bose N, Chan AS, Magee AS, Danielson ME et al (2011) style as a commensal and a pathogen. Res Microbiol 168:1–9 Activation of the innate immune receptor Dectin-1 upon formation 24. ErhartW,AlkasiO,BrunkeG,Wegener F,Maass N,ArnoldN, Arlt of a ‘phagocytic synapse’. Nature 472:471–475 A, Meinhold-Heerlein I (2011) Induction of human beta-defensins and psoriasin in vulvovaginal human papillomavirus-associated le- 36. Segat L, Zupin L, Moura RR, Coelho AVC, Chagas BS, de Freitas sions. J Infect Dis 204:391–399 AC, Crovella S (2014) DEFB1 polymorphisms are involved in 25. Lee KC, Eckert RL (2007) S100A7 (Psoriasin)–mechanism of an- susceptibility to human papillomavirus infection in Brazilian tibacterial action in wounds. J Invest Dermatol 127:945–957 gynaecological patients. Mem Inst Oswaldo Cruz 109:918–922

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Journal of Molecular MedicineSpringer Journals

Published: May 7, 2018

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