Involvement of interleukin-18 in the infl ammatory response against oropharyngeal candidiasis

François Tardif1BDE, Jean-Paul Goulet1

BCDFG, Andrew Zakrzewski1BCDF,

Peter Chauvin2BCDF, Mahmoud Rouabhia1

ACDEFG

1 Faculté de médicine dentaire et Groupe de recherche en écologie buccale, Pavillon de médecine dentaire, Université Laval, Québec City, Canada

2 Faculty of Dentistry, McGill University, Montréal, Québec, Canada

Source of support: This work was supported by grants from the Natural Sciences and Engineering Research Council (Contract grant number: 227212-99), the Canadian Institutes of Health Research (Contract grant number: MOP-12591) and the Fonds de la Recherche en Santé du Québec (Contract grant number: 015006.01). Mahmoud Rouabhia is a senior research scholar of the FRSQ program.

Summary

Background: Oral candidiasis is a collective name for a group of disorders caused by the dimorphic fungus Candida albicans (C. albicans). Host defenses against C. albicans essentially fall into two categories: specifi c immune mechanisms and local oral mucosal epithelial cell defenses. The rationale of this study was to investigate the involvement of IL-18 in the infl ammatory response against oral candidiasis.

Material/Methods: We fi rst used human oral mucosa tissue and saliva to assess the production of Il-18. Second, we en-gineered human oral mucosa using only normal human oral epithelial cells and fi broblasts. Tissues were infected with C. albicans at different time points.

Results: Tissue and saliva analyses demonstrated that constitutively produced and secreted IL-18 was up-re-gulated following Candida-infection. With our engineered model, we showed that C. albicans signifi -cantly increased the secretion of active IL-18 by infected epithelial cells. Interestingly, a signifi cant secretion of IFNg functionally supported the up-regulation of active IL-18 in C. albicans-infected tissues. We also showed that rhIL-18 increased the expression and production of endogenous IL-18 and ICE in C. albicans-infected tissues, which was paralleled by a signifi cant increase in IFNg se-cretion.

Conclusions: These data suggest that (i) oral epithelial cells are involved in local host defenses against C. albicans infections, via IFNg induced-IL-18, and (ii) that IL-18 and IFNg secretions may be related to epi-thelial cells. Given that our experimental model closely mimics the natural interface between the

oral mucosa and C. albicans, it appears that IL-18 meets the requirements of being a cytokine that epithelial cells use to control C. albicans infections.

key words: oral candidiasis • Candida albicans • oral mucosa • IL-18 • IFNg • tissue engineering

Full-text PDF: http://www.MedSciMonit.com/pub/vol_10/no_8/4477.pdf

Word count: 4975 Tables: 2 Figures: 9 References: 43

Author’s address: Dr. Mahmoud Rouabhia, Faculté de médecine dentaire, Pavillon de médecine dentaire, Local 1728, Université Laval, Québec, G1K 7P4, Canada. e-mail: Mahmoud.rouabhia@fmd.ulaval.ca

Authors’ Contribution: A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection

Received: 2003.12.23Accepted: 2004.02.11Published: 2004.08.01

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PMID: 15277983BR

BACKGROUND

Candida albicans (C. albicans) colonizes the oral mucosa of approximately 80% of normal individuals as a commen-sal organism, causing no apparent damage and inducing no apparent infl ammation in the surrounding tissue [1,2]. However, under predisposing conditions, C. albicans multi-plies and penetrates the host tissue, causing infl ammation and tissue destruction [3,4]. Clinical assessments revealed that symptomatic oropharyngeal candidiasis (OPC) is often a manifestation of corticosteroid therapy [5], immunosup-pression after transplantation, and AIDS [6–8]. In contrast, OPC in healthy individuals is relatively rare.

Cell-mediated immunity (CMI) plays a critical role in the protection against mucosal C. albicans infections, as shown by the high incidence of OPC in individuals with reduced CMI [5,8]. This presumably is related to reduced numbers of Th1-type CD4+ T cells, which help protect against infec-tions, and/or a shift to Th2-type responses, which are asso-ciated with susceptibility to infection [6,8]. CMI is not the only defense against oral candidiasis. Recent attention has focused on innate immunity, and it has been reported that primary epithelial cells from murine, nonhuman primate, and human vaginal mucosa [9,10] as well as human oral mu-cosa [11,12] signifi cantly inhibit the growth of C. albicans in vitro. Epithelial cells also play active roles in mucosal immu-ne responses, including antigen presentation [13], antimi-crobial activity [14], and the production of multiple cyto-kines such as IL-1b and the recently described IL-18 [15]. IL-18 has been identifi ed as interferon-g (IFN-g) inducing factor and is a potent inducer of IFN-g production by activa-ted T cells [16,17]. Since its discovery, IL-18 has been shown to contribute to protective immunity against a variety of pa-thogens, including Cryptococcus, Leishmania, Salmonella, and Mycobacterium tuberculosis [17].

IL-18 is related to the IL-1 family both structurally and func-tionally [18]. Like IL-1, IL-18 is synthesized as a biological-ly inactive 24 kDa precursor protein. Cleavage to the acti-ve form is mediated by the IL-1 converting enzyme (ICE), also called caspase-1 [19]. Cleavage of IL-18 by ICE is essen-tial for IL-18 to become biologically active. IL-18 is produ-ced by activated macrophages, dermal keratinocytes, oste-oblasts, adrenal cortex cells, and intestinal epithelial cells [20,21], implying that it plays other physiological roles in addition to immune regulation.

Oral epithelial cells are involved in the pro-infl ammatory process through the production of cytokines either consti-tutively or after a variety of stimuli [22], implying that they may participate in controlling oral infections through an infl ammatory [23,24] process involving interleukins such as IL-1b and IL-18. In a previous study using monolayer cultures, we demonstrated that oral epithelial cell modula-te C. albicans growth through IL-18 expression and secre-tion and that IL-18 modulates IFNg secretion [12]. This oral monolayer culture model suggested the involvement of epithelial cells in the local defense against C. albicans in-fection. However, as oral mucosa is a complete, multi-cel-lular tissue, the use of a monolayer culture system to study C. albicans infection may not be the appropriate model to investigate oral mucosa involvement in host defenses aga-inst oral infections. To overcome this limitation we used

our engineered human oral mucosa (EHOM) consisting of a multi-layered epithelium and well-organized lamina propria interacting through a basement membrane. It is a stable, reproducible source of oral mucosal tissue for com-parative studies [24] and has the advantage of being more versatile and allowing the studies to be performed in a step-by step fashion for each type of immune and non-immune cells in the oral mucosa.

Considering the oral mucosa structure and based on the fact that epithelial cells are the cell type most often asso-ciated with C. albicans at mucosal surfaces and produce a variety of cytokines in response to microorganisms [15], and because IL-18 has a pro-infl ammatory role, we hypo-thesized that the oral mucosa either maintains an equili-brium status with commensal C. albicans or contributes to host defenses against fungal infections through epithelial cells that secrete IL-18. We thus sought to determine whe-ther human oral epithelial cells contribute to local immu-ne defenses through the production of IL-18 and IFNg in response to C. albicans.

MATERIALS AND METHODS

Tissue collection. Normal tissues were collected from pa-tients who attended our dental clinic for free gingival gra-fts to treat gingival recession, while pathological (candidia-sis) tissues were collected from patients with clinical signs of oral candidiasis according to the Newton classifi cation [25]. Tissue were collected before any medication was given. They were immediately fi xed for 15 min in 4% paraformaldehy-de, then embedded in paraffi n and used for immunohisto-chemical analyses. The refl ux saliva was also collected from both healthy patients and patients with candidiasis (the day of the clinical intervention). All patients provided informed consent, and the Laval University Ethics Committee appro-ved the procedure. Pathological specimens were reddish in color and presented a discreet form of palatal papillary hy-perplasia, two conditions that frequently coexist [26,27]. Saliva and tissue specimens from patients in which the pre-sence of the C. albicans was clinically suspected were incuba-ted in Sabouraud dextrose broth (Difco, Becton Dickinson, Franklin Lakes, NJ, USA) overnight at 37°C for confi rma-tion. Normal tissue and saliva from control patients were also incubated in Sabouraud to confi rm the absence of C. albicans. Normal C. albicans-free tissues were divided into two sections. One section was used to isolate epithelial cells and fi broblasts for subsequent studies and the second was used for immunohistochemistry staining. Both normal and pa-thologic tissue biopsies were cut into 1×1 mm pieces, fi xed in paraformaldehyde, embedded in optimal cutting tempe-rature medium (O.C.T, Sukura Finetek U.S.A, Inc, Torrance, CA, USA), and frozen at –80°C until used for IL-18 staining. Saliva samples were collected in tubes containing protease inhibitors (Sigma-Aldrich Canada Ltd, Oakville, ON), cen-trifuged (1,500×g, 10 min), aliquoted, and stored at –80°C until used for IL-18 quantifi cation.

Immunohistochemistry

Sections (5 µm) were cut from normal and infected tissu-es and immunostained with rabbit anti-human IL-18 (di-lution 1/25) (Cell Sciences Inc, Norwood, MA, USA) or rabbit serum (1:100). Before staining, the slides were tre-

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ated with citrate buffer (0.01M, pH 6) at 96°C for 40 min. Immunostaining was performed using the avidin-biotin pe-roxidase complex method [28]. Endogenous peroxidase background activity was blocked by incubating the tissues with 0.6% H2O2 in methanol for 10 min and then in nor-mal serum for 30 min. After washing with TBS, the samples were incubated overnight with the primary antibody at 4°C. The following day, the samples were washed with TBS (2×3 min) and incubated with a biotinylated secondary antibo-dy (dilution 1/100) for 60 min at room temperature fol-lowed by ABC-HRP complex (dilution 1/100) for 40 min. The slides were washed three times with TBS between each step. To detect bound antibodies, the samples were incuba-ted with amino-ethylcarbazole (AEC) substrate for 5 min. The reaction was stopped by rinsing with water. The sam-ples were then counterstained with Mayer Hematoxylin (di-lution 1/3). The slides were mounted with Crystal Mount (Biomeda, Foster City, CA, USA), examined using an opti-cal microscope, and photographed.

Oral fi broblast isolation and culturing

Small pieces of normal palatal mucosa were incubated in thermolysin (500 µg/ml; Sigma Chemical Co, St. Louis, MO, USA) for 24 h at 4°C to separate the lamina propria from the epithelium [24]. As previously reported, thermolysin sepa-rates the mucosa at the dermo-epidermal junction, yielding a dermis-free epithelium. The lamina propria was separated from the epithelium with forceps and placed in a solution of collagenase P (0.125 U/ml; Boehringer Mannheim, Laval, QC, Canada) for 45 minutes at 37°C to extract fi broblasts. Isolated cells (2×106) were seeded in 75cm2 fl asks (Falcon, Becton Dickinson) and grown in Dulbecco’s Modifi ed Eagle Medium (DMEM) (Life Technologies Inc, Gaithersburg, MD, USA) containing 10% fetal calf serum (Gibco, Burlington, ON, Canada), 100 IU/ml penicillin G, 25 µg/ml strepto-mycin, and 0.5 µg/ml fungizone. Following their characte-rization, the fi broblasts were used to produce EHOMs once the cultures had reached 100% confl uence.

Oral epithelial cell isolation and culturing

Normal human gingival epithelial cells were obtained by treating epithelial tissue with 0.05% trypsin and 0.1% EDTA. The epithelial cells (1×106) were then cultivated in Dulbecco’s modifi ed Eagle–Ham’s F12 medium supple-mented with 5% fetal calf serum (HyClone, Logan, UT, USA), 5 µg/ml insulin (Sigma), 0.4 µg/ml hydrocortiso-ne (Calbiochem, La Jolla, CA, USA), 10–10 M cholera toxin (ICN Biomedicals Inc, Aurora, OH, USA), 10 ng/ml EGF (Austral Biologicals, San Ramon, CA, USA), 100 IU/ml pe-nicillin G, 25 µg/ml streptomycin, 0.5 µg/ml fungizone, and 2×109 M 5’-triiodo-L-thyronine. The cultures were mainta-ined in an 8% CO2, 92% humidity atmosphere at 37°C. The cells were characterized using specifi c antibodies [24] and used at passage three. Once the cultures had reached 70% confl uency, the epithelial cells were detached and used to engineer human oral mucosa tissue (EHOM).

EHOM production

EHOM tissue was produced as described previously [24]. Briefl y, oral fi broblasts were mixed with bovine skin colla-gen to produce the lamina propria. The tissues were grown

in DMEM supplemented with 5% fetal calf serum for four days. The oral epithelial cells were then seeded on the lami-na propria. Once the epithelial cells had reached confl uen-ce, the EHOM was raised to an air-liquid interface for fi ve more days to allow the epithelium to stratify [24].

Infection conditions

On day three of the air-liquid interface incubation, the cul-ture medium was replaced with fungizone-free medium. On day four, a C. albicans LAM-1 (serotype A) culture was ini-tiated by growing the fungus in Sabouraud dextrose broth (Difco, Becton Dickinson) for 18 h at 30°C with rotary agi-tation (240 rpm). The cells were collected by centrifuga-tion (1,500×g, 10 min) and suspended at 108 cells per ml in Sabouraud and used for tissue infection. Before infection, the EHOMs were washed twice and fed serum-free, antibio-tic-free, fungizone-free medium. At this point, C. albicans (1.5×106/cm2) was seeded onto the EHOMs using sterile ray-on swabs (Spectrum Laboratories Inc, Houston, TX, USA). Half the C. albicans-infected EHOMs received 250 pg/ml of purifi ed recombinant mature human rhIL-18 (Chemicon International, Inc, Temecula, CA, USA). The remaining half served as controls. Supernatants, protein extracts, and total mRNA extracts were collected after 0, 2, 4, 6, 12, 24, and 48 hours of incubation.

Protein extract preparation

The epithelial structures were separated from the lamina propria using forceps and the thermolysin procedure descri-bed above. There was no lamina propria contamination of the epithelium at this step. Epithelial tissue was then used for total protein extraction. The tissues were lysed using a solution containing 2.3% SDS, 64 mM Tris-HCl (pH 6.8), and 10% glycerol. The lysates were boiled for 5 min and sonifi ed (SonicatorÒ, Heat Systems Ultrasonics, Plainview, NY, USA) for 10 seconds. A sample was taken for spectro-photometric analysis at 280 nm to determine the amount of protein in each sample, then b-mercaptoethanol at a fi -nal concentration of 5% (v/v) was added to each lysate. The protein lysates were stored at –80°C until the Western blot analyses were performed.

RNA extraction and RT-PCR analysis

The epithelial structures were separated from the lamina propria using forceps and the thermolysin procedure de-scribed above. Total cellular RNA was prepared from unin-fected and C. albicans-infected epithelium using the Qiagen RNeasy Total RNA kit (Qiagen Inc, Valencia, CA, USA). Total RNA was reverse-transcribed into cDNA using the Maloney-murine leukemia virus (M-MLV)-reverse transcriptase (Life Technologies) and random hexamers (Amersham Pharmacia Biotech Inc, Baie d’Urfé, QC, Canada). One microliter of each cDNA product was added to a 49 µL PCR mixture con-taining Taq polymerase (Qiagen Inc.) and forward and rever-se specifi c primers (BioSource International Inc, Camarillo, CA, USA) (see Table 1).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was measured in each sample as a positive control to confi rm the integrity of the RT-PCR. The conditions for the ICE RT-PCR were: 95°C for 5 minutes, 35 cycles at 94°C

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for 1 min, 60°C for 2 min, and 72°C for 2 min, with a fi nal extension at 72°C for 10 minutes. The conditions for the IL-18 PCR were: 95°C for 2 min, 35 cycles at 95°C for 1 min, 52°C for 30 sec, and 72°C for 1 min, with a fi nal extension at 72°C for 10 min. After the PCR procedures, a 4 µL volume of each product was separated on a 1.5% agarose gel conta-ining ethidium bromide. The gels were UV-illuminated, pho-tographed, and the relative band intensities were measured on digitized images using the Scion image program.

Western blotting analysis

Equal amounts (200 µg) of total protein were deposited on a 10% SDS-PAGE gel. After electrophoretic separation, the proteins were transferred to a nylon PVDF membra-ne (Millipore, MA, USA), blocked overnight with TBS-T (100 mM Tris pH 7.5, 0.9% NaCl, and 0.1% Tween 20), and complemented with 4% non-fat skim milk (blotto). The membrane was incubated for 1 h at room temperatu-re with goat anti-human IL-18 monoclonal antibody (MBL, Naka-ku Nagoya, Japan) diluted 1/1000 in blotto or with af-fi nity-purifi ed anti-human ICE rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1/1000 in blotto. Anti-human IL-18 monoclonal antibody reacts with pro-IL-18 (24 kDa). Anti-human ICE polyclo-nal antibody reacts with the 45 kDa precursor ICE prote-in. The membrane was washed (1×10 min and 1×5 min) in TBS-T and then incubated for 30 min at room temperature with horseradish peroxidase-labeled sheep anti-mouse IgG (Cedarlane Laboratories Ltd, Hornby, ON, Canada). After washing with TBS-Tween, the blots were developed using an ECL kit solution (Amersham International, UK) and expo-sed on Kodak biomax fi lm at room temperature for 1 min. As reported previously [29,30], unstimulated and LPS-sti-mulated gingival fi broblasts did not express or produce IL-18. Similar analyses performed on C. albicans-infected gingi-val fi broblasts showed that they did not express or produce IL-18 (data not shown).

Measurement of IL-18 secreted by EHOM tissues

The supernatants from normal tissue cultures (controls), C. albicans-infected tissues, rhIL-18-stimulated tissues, and rhIL-18 plus C. albicans-infected tissues were collected in tu-bes containing 1 µl of protease inhibitor cocktail for specifi c use with mammalian cell and tissue extracts (Sigma-Aldrich Canada Ltd,) and used to determine the levels of secreted IL-18 and IFNg. Immediately after the collection of the su-pernatants, cytokine measurements were performed in tripli-cate samples using an enzyme-linked immunosorbant assay

(ELISA) for IL-18 (Medical & Biological Laboratories Co. Ltd, Nagoya, Japan) and IFN-g (Pharmingen, BD Biosciences, Mississauga, ON, Canada). Supernatants were fi ltered thro-ugh 0.22 µm fi lters and used to quantify IL-18 and IFNg ac-cording to the manufacturer’s instructions. A 100 µl volume of undiluted supernatant was used to perform the quan-titative analysis. Plates were read at 450 nm and analyzed using an EAR-400 Easy Reader (SLT-Lab Instrumentation, Grödig, Austria). The sensitivity of the IL-18 ELISA kit was better than 13 pg/ml and the sensitivity of the IFNg ELISA kit was better than 1 pg/ml, according to the manufactu-rer. We also used the IL-18 ELISA kit to quantify IL-18 in saliva from normal patients and patients with candidiasis. The experiments were repeated six times and the means ±SD were calculated.

Statistical analysis

All experiments in this study were performed at least four times. The pictures shown are representative results. Experimental values are given as means ±SD. The statisti-cal signifi cance of differences between the control values and the test values was evaluated using a one-way ANOVA. Posteriori comparisons were done using Tukey’s adjustment technique. Normality and variance assumptions were veri-fi ed using the Shaprio-Wilk test and the Brown and Forsythe test respectively. All the assumptions were fulfi lled. P va-lues were declared signifi cant at the 0.05 level. Data were analyzed using the SAS version 8.2 statistical package (SAS Institute Inc, Cary, NC).

RESULTS

In situ evaluation of IL-18 production by oral mucosa cells

To assess the production of endogenous IL-18 by oral epithe-lial cells, immunostaining for human IL-18 was done using oral mucosa biopsies retrieved from healthy patients and patients with candidiasis. Normal tissue showed low consti-tutive pro-IL-18 production and deposition (Figure 1A, pa-nel A), and pathological tissue showed signifi cant staining of epithelial cells, mainly in the basal layer (Figure 1B, pa-nel A). These results were confi rmed by the ELISA using saliva. As indicated in Figure 1, panel B, saliva from heal-thy patients contained low basal levels of secreted active IL-18 secretion. Saliva from patients with candidiasis contained fi ve-fold more active IL-18 than the control saliva. This sug-gested that (i) oral epithelial cells are involved in local host defenses against C. albicans infections, that (ii) the defen-

ICESense 5’-ATCCGTTCCATGGGTGAAGGTACA-3’

620 bpAntisense 5’-CAAATGCCTCCAGCTCTGTAATCA-3’

IL-18Sense 5’-GCTTGAATCTAAATTATCAGTC-3’

320 bpAntisense 5’-GAAGATTCAAATTGCATCTTAT-3’

GAPDHSense 5’-ATGCAACGGATTTGGTCGTAT-3’

220 bpAntisense 5’-TCTCGCTCCTGGAAGATGGTG-3’

Table 1. Specifi c primers for reverse transcriptase polymerase chain reaction and predicted size of PCR product (bp).

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se involves IL-18, and that (iii) like other cells (e.g. macro-phages) epithelial cells secrete a signifi cant amount of IL-18. Based on these results, we conducted experiments to confi rm the link between oral epithelial cells, C. albicans, and IL-18 expression and secretion.

Expression of ICE and IL-18 by oral epithelial cells following infection with C. albicans

We fi rst measured ICE and IL-18 mRNA expression by epithe-lial cells in uninfected and C. albicans-infected EHOMs. Prior to infection, the oral epithelial cells constitutively expressed endogenous levels of ICE (Figure 2). ICE mRNA levels rose signifi cantly after infection by C. albicans. The increase was observable after 4 to 12 hours of contact with C. albicans. The expression of ICE mRNA remained at twice the pre-control levels after 24 and 48 hours of contact. Unlike ICE mRNA, IL-18 mRNA expression did not change. Uninfected cells expressed high IL-18 mRNA levels, which rose slightly but not signifi cantly when they were infected with C. albicans.

Production of ICE and IL-18 by oral epithelial cells following infection with C. albicans

Since only ICE mRNA levels rose on contact with C. albicans, we wanted to determine whether ICE and IL-18 proteins fol-lowed the same trend as the mRNAs. We performed Western Blot analyses using specifi c antibodies for ICE (45 kDa) and IL-18 (24 kDa). As indicated in Figure 3, the concentration

of inactive (p45) and active (p10) ICE proteins decreased with the onset of C. albicans infection. Indeed, the relative amount of p45 protein dropped from 0.9 (2 h) to less than 0.5 (6 h). On the other hand, only pro IL-18 (p24) could be detected in the cell lysates (Figure 3). While present at basal levels, the amount of pro IL-18 rose slightly following C. albicans infection. The main increase was noticed at 6 h post-infection. But, no active IL-18 (p18) was detected in the cell lysates by Western Blot analysis. This suggests that the C. albicans infection was modulating the secretion of the active form of IL-18. We thus performed an ELISA assay using culture supernatants from uninfected and C. albicans-infected tissues.

Quantifi cation of active IL-18, and IFNg in culture supernatants of human oral epithelial cells infected with C. albicans

As indicated in Figure 4, basal production (2.6±0.1 ng/ml) of active IL-18 protein was detected only in 24 h oral epi-thelial cell culture supernatants. The basal level increased following C. albicans infection. The concentration of IL-18 protein varied from 2±0.1 ng/ml 2 h post-infection to

Figure 1. Production and deposition of pro-IL-18 in oral mucosa tissues. Frozen cross-sections of normal (A) and Candida-infected (B) tissues stained with rabbit anti-human IL-18 (a,b) or rabbit serum (c). Note the low basal level (arrows) of IL-18 in the normal tissue (A) and the signifi cant amount of protein in the basal layer (arrows) of the infected tissue (B). Scale bar=50 μm. In panel B. IL-18 in saliva was evaluated using ELISA kit. Data are the means ±SD (n=7). ** P≤0.01 when the control value (healthy status) was compared to the test values (candidiasis).

A

B

Figure 2. C. albicans altered ICE but not IL-18 mRNA expression in oral epithelial cells. (A) Total cellular mRNA was obtained from the epithelia of uninfected and C. albicans-infected EHOMs (blastospore form; 1.5×106 C. albicans/cm2) and was analyzed by RT-PCR using specifi c primers for ICE, IL-18, and GADPH. Control (Ctrl) refers to the uninfected EHOM. (B) Changes in ICE and IL-18 mRNA levels were measured by the public domain NIH Image software package. Expression of GAPDH mRNA was used as a control. (A) is a representative gel of six diff erent gels. (B) is the mean ±SD of six diff erent experiments. ** P≤0.01, * P≤0.05. The results were obtained by comparing the control value to the test values.

A

B

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5.7±0.3 ng/ml 24 h post-infection. This clearly suggests that C. albicans stimulated the cells to secrete mature IL-18 and that the secretion was time dependant. To determine the biological signifi cance of this over-secretion of IL-18, a spe-cifi c IFN-g–ELISA was performed using oral epithelial cell culture supernatants. Uninfected tissues did not secrete IFN-g at levels that were detectable with our assay. When infec-ted with C. albicans, epithelial cells secreted reduced but si-gnifi cant amounts of IFN-g soon after contact (between 6 and 12 h). These data suggest that epithelial cells may pro-mote host defenses against C. albicans infections via IL-18-induced IFNg production. Although obvious, this study has to be confi rmed using IFNg blocking conditions.

Exogenous rhIL-18 enhances the expression of IL-18 and ICE mRNA

Since IL-1b can promote its own regulation [31,32] and sin-ce IL-18 is an IL-1-like cytokine, we decided to investigate the effect of exogenous recombinant mature human IL-18 (rhIL-18) on uninfected and C. albicans-infected EHOMs by adding to 250 pg/ml of purifi ed rhIL-18 to the culture me-dium. After various contact periods, total mRNA and prote-ins were extracted and used for ICE and IL-18 analyses. As

shown in Figure 5, exogenous rhIL-18 signifi cantly (P<0.01) increased the expression of IL-18 mRNA by normal uninfec-ted epithelial cells compared to untreated tissues. The ef-fect of exogenous rhIL-18 was time-dependent early in the stimulation period. Interestingly, when rhIL-18 was added to C. albicans-infected tissues, IL-18 mRNA rose to steady sta-te levels within 2 h of exposure. The effect was signifi cantly (P<0.01) higher than in uninfected, rhIL-18-stimulated tissu-es. The effect of exogenous rhIL-18 on IL-18 mRNA expres-sion by C. albicans-infected tissue was also time-dependent. It may be possible that exogenous rhIL-18 modulates ICE expression in both C. albicans-infected and uninfected tissu-es. Unexpectedly, exogenous rhIL-18 signifi cantly increased ICE mRNA expression in uninfected tissues (Figure 6). The higher level of ICE mRNA expression was obtained from 4 h to 12 h post-stimulation. When rhIL-18 was added to C. albicans-infected tissue cultures, the cells expressed high le-vels of ICE mRNA. However, maximum ICE mRNA expres-sion was reached 2 h after cell contact with both C. albicans and exogenous rhIL-18. Comparative analyses between rhIL-18-stimulated and rhIL-18 plus C. albicans-stimulated tissues revealed no differences except for the 2 h contact period when there was a signifi cant (p<0.05) difference. Since rhIL-18 enhances the expression of IL-18 and ICE mRNA, we in-vestigated the effect of exogenous rhIL-18 on the endoge-nous production of IL-18 and ICE proteins.

Figure 3. C. albicans transiently altered ICE and IL-18 protein production by oral epithelial cells. A: Total protein (200 μg) from lysates of unstimulated (Ctrl) and C. albicans-stimulated cells (blastospore form; 1.5×106 C. albicans/cm2) were separated on a SDS-PAGE gel and revealed by Western blot. Control (A) refers to 100 μg of monocyte lysate or 10 ng of purifi ed recombinant mature IL-18 for ICE and IL-18 respectively. Control (B) refers to uninfected EHOM. Changes in the levels of B, ICE, and IL-18 proteins were measured using the public domain NIH Image software package. (A) is a representative gel of six diff erent gels. (B) is the mean ±SD of six diff erent experiments. ** P≤0.01, * P≤0.05. These results were obtained by comparing the control value to the test values.

A

B

Figure 4. C. albicans increased the production of mature IL-18 by oral epithelial cells. EHOMs were infected with C. albicans (blastospore form; 1.5×106 C. albicans/cm2) for diff erent periods of time as indicated at the bottom of the fi gure. An uninfected EHOM was used as a control. The culture medium was collected from each experiment and used to determine the amount of the active form of IL-18 by ELISA. Data are the means ±SD of three separate experiments. Note the basal level of IL-18 secretion by oral epithelial cells and the time-dependent increase during the C. albicans infection. Ctrl is the supernatant from unstimulated oral epithelial cells cultured for 0 h (C-1) and 24 h (C-2). Plotted results are the means ± SD of six diff erent experiments. ** P≤0.01, * P≤0.05 when comparing the control values (C-1 or C-2) to the test values.

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The effect of exogenous rhIL-18 on IL-18 and ICE protein production

Adding rhIL-18 to the culture medium increased the pro-duction of intracellular pro-IL-18 protein over time (Figure 7). When tissues were primed with both C. albicans and rhIL-18, pro-IL-18 levels rose quickly and signifi cantly (2 h) and remained fairly stable for the remainder of the contact pe-riod. ICE protein secretion displayed a totally different pat-tern than ICE mRNA expression when the tissues were sti-mulated with rhIL-18 alone and with rhIL-18 plus C. albicans. rhIL-18 slightly increased inactive ICE secretion (Figure 8) in normal tissues. ICE secretion by C. albicans-infected tissu-es was also up-regulated by exogenous rhIL-18 (Figure 8). This suggests that ICE may be produced and used to co-nvert pro-IL-18 to active mature IL-18 protein. To test this hypothesis, we measured the levels of mature IL-18 in tis-sue culture supernatants.

Endogenous secretion of IL-18 by epithelial cells primed with C. albicans and rhIL-18

Since the levels of IL-18 mRNA and protein differed, de-pending on the type of stimulation (C. albicans or exoge-nous rhIL-18), we wanted to determine whether epithelial cells also processed the protein and released it differently.

Supernatants collected from uninfected and C. albicans-in-fected EHOMs that were stimulated or not with rhIL-18 (250 pg/ml) were analyzed by specifi c ELISA. As shown in Figure 9, exogenous rhIL-18 signifi cantly (P<0.001 or P<0.05) pro-moted the secretion of endogenous IL-18 by normal tissues beginning 2 h post-contact, with maximum release at 48 h. Interestingly, IL-18 production was observed only 2 and 24 h post-stimulation with both rhIL-18 and C. albicans, and was slightly lower at 4 and 6 h. This effect was initiated at an ear-ly stage (2 h) and was maintained, suggesting that epithelial cells are still able to mount a defense to control C. albicans infections even following IL-18 stimulation. This suggests that the endogenous secretion of IL-18 may be of biologi-cal signifi cance. The results reported in Table 2 show that C. albicans and exogenous rhIL-18 promoted the secretion of IFNg by oral epithelial cells. The highest level was obtained at 4 h (222.72±35 pg/ml), followed by a signifi cant decre-ase at later contact periods. Interestingly, IFNg production occurred 2 h later than IL-18 secretion, suggesting that epi-thelial cells need time to mount a defense against C. albicans infections via IL-18-induced-IFNg secretion.

DISCUSSION

Recent studies have reported that epithelial cells assist the pro-infl ammatory process by producing different cytokines,

Figure 5. Exogenous rhIL-18 increased IL-18 mRNA expression in uninfected and C. albicans-infected oral epithelial cells. Total cellular mRNA was obtained from the epithelia of uninfected and C. albicans-infected EHOMs (blastospore form; 1.5×106 C. albicans/cm2) that were treated with rhIL-18 (250 pg/ml) or left untreated. RNA was analyzed by RT-PCR using specifi c primers for IL-18 and GAPDH. Control (Ctrl) refers to the uninfected EHOM. Expression of GAPDH mRNA is shown as a control. B: IL-18 mRNA levels were measured using the public domain NIH Image software package. (A) is a representative gel of six diff erent gels. (B) is the mean ±SD of six diff erent experiments. ** P≤0.01, * P≤0.05. These results were obtained by comparing the control value to the test values. T. S, tissue stimulated

A

B

Figure 6. Exogenous rhIL-18 altered ICE mRNA expression in uninfected and C. albicans-infected oral epithelial cells. Total cellular mRNA was obtained from the epithelia of uninfected and C. albicans-infected EHOMs (blastospore form; 1.5×106 C. albicans/cm2) treated with rhIL-18 (250 pg/ml) or left untreated. RNA was analyzed by RT-PCR using specifi c primers for ICE and GAPDH. Control (Ctrl) refers to the uninfected EHOM. The expression of GAPDH mRNA is shown as a control. B: changes in ICE mRNA levels were measured using the public domain NIH Image software package (B). (A) is a representative gel of six diff erent gels. (B) is the mean ±SD of six diff erent experiments. * P≤0.05. These results were obtained by comparing the control value to the test values.

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B

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such as IL-1 or IL-18, either constitutively or in response to a variety of stimuli [32]. IL-18, a cytokine with pleiotrophic immunomodulatory functions, is secreted in greater amo-unts in a variety of human infl ammatory conditions, inclu-ding Crohn’s disease [33], rheumatoid arthritis [34], and tuberculoid leprosy [35]. The purpose of this study was to establish a link between oral candidiasis and IL-18 secretion. We demonstrated for the fi st time, using human oral muco-sa, that IL-18 is constitutively secreted by oral epithelial cells and is up-regulated following Candida-infection. We also de-monstrated that IL-18 is secreted as an active molecule in the saliva. The level of active IL-18 was 5-fold higher in sa-liva from patients with candidiasis compared to saliva from normal patients. IL-18 is thus involved in the infl ammatory defense mounted by different cells including epithelial cells against infections. Based on this interesting fi nding, we exa-mined the close relationship between epithelial cells and IL-18, and the defense against C. albicans. We demonstrated that oral epithelial cells express a signifi cant basal level of IL-18 mRNA that is not greatly changed by a C. albicans in-fection. Compared to our previous results [12] generated with oral epithelial cell monolayer cultures, IL-18 expres-sion was slightly upregulated in the EHOM model, sugge-sting that the presence of fi broblasts enhanced the defense mechanisms against C. albicans infection of epithelial cells. These results were consistent with reports that there are no changes in IL-18 mRNA transcript levels in Balb/c mo-use cornea infected with Pseudomonas aeruginosa [12,36]. It may be that C. albicans does not affect the level of IL-18 mRNA expression because IL-18 transcripts are present at saturating levels in both infected and uninfected epithe-

Figure 7. Exogenous rhIL-18 signifi cantly altered IL-18 protein production by uninfected and C. albicans-infected oral epithelial cells. A: Total proteins (200 μg) from each culture were separated on a SDS-PAGE gel and revealed by Western blot. Control (A) is 10 ng of purifi ed recombinant mature IL-18. Control (B) is uninfected EHOM. B: Changes in IL-18 levels were measured using the public domain NIH Image software package. (A) is a representative gel of six diff erent gels. (B) is the mean ±SD of six diff erent experiments. ** P≤0.01, * P≤0.05. These results were obtained by comparing the control values to the test values.

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B

Figure 8. rhIL-18 signifi cantly altered ICE protein production by uninfected and C. albicans-infected oral epithelial cells. A: Total proteins (200 μg) from each culture were separated on a SDS-PAGE gel and revealed by Western Blot. Control (A) is 100 μg of monocyte lysate for ICE. Control (B) is uninfected EHOM. B: Changes in ICE levels were measured using the public domain NIH Image software package. (A) is a representative gel of six diff erent gels. (B) is the mean ±SD of six diff erent experiments. * P≤0.01. These results were obtained by comparing the control values to the test values.

A

B

Figure 9. Exogenous rhIL-18 increased production of active IL-18 protein by uninfected and C. albicans-infected oral epithelial cells. Uninfected and C. albicans-infected EHOMs (blastospore form; 1.5×106 C. albicans/cm2) incubated in the presence or absence 250 pg/ml of rhIL-18 for diff erent periods of time as indicated at the bottom of the fi gure. The culture medium was collected from each experiment and used to determine the amount of the active form of IL-18 by ELISA. Data are the means ±SD (n=6). * P≤ 0.05, ** P≤ 0.01. Ctrl is the supernatant from unstimulated oral epithelial cells cultured for 0 h (C-1) and 24 h (C-2). T. S, tissue stimulated.

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lial cells, thus overwhelming any effect (up- or down-regu-lation) by C. albicans.

As previously reported [37], the high levels of IL-18 transcripts in both C. albicans-infected and uninfected tissues suggest that the biological activity of this cytokine is probably controlled post-translationally. This raises the question as to whether IL-18 transcripts from oral epithelial cells are translated into IL-18 proteins in both the presence and absence of infection. Western blot analyses of epithelial cell lysates showed that the basal level of pro-IL-18 protein (24 kD) produced by epithe-lial cells is slightly increased by yeast infections, suggesting IL-18 involvement in cell defenses against C. albicans. This is in disagreement with previous results from our group with cultured epithelial cells which had a signifi cant reduction of IL-18 production under the same conditions. This differen-ce may be explained by the interaction between epithelial cells and fi broblasts in the EHOM model compared to the monolayer epithelial cell culture model. Indeed, the EHOM model may better refl ect the real in vivo situation in the oral cavity insofar as there are cell (epithelial cells and fi brobla-sts) interactions to elaborate an appropriate defense against infection. Our data are in accordance with previously repor-ted data with mice [38,39] and human [35]. To be biologi-cally effi cient, IL-18 must thus be in its active form. Given its absence from epithelial cell lysates, as indicated by Western blot analysis, the active form may instead be secreted and re-leased into the culture supernatant. We have, in fact, found mature IL-18 in the culture supernatants of uninfected tissu-es. Interestingly, the basal levels of IL-18 signifi cantly incre-ased in C. albicans-infected tissues. This result is in agreement with other reports in the literature. Menacci et al. demonstra-ted that C. albicans-infected mice produce more IL-18 than uninfected mice [40]. Rouabhia et al. [12], reported that C. albicans upregulated the secretion of active IL-18 by oral epi-thelial cells following in vitro infections. The increased secre-tion of mature IL-18 by epithelial cells, which constitute the primary host-pathogen interface, suggests that IL-18 may play an important role in fi ghting C. albicans infections. Several studies have shown that IL-18 plays a key role in antimicrobial defenses against Cryptococcal, Leishmania, and Salmonella infec-tions [17,41]. Our fi nding may confi rm the hypothesis that epithelial cells contribute to host defenses against C. albicans

infection via IL-18. This interleukin requires post-translational enzymatic processing to be biologically activated [18,19]. RT-PCR and Western blot analyses of converting enzyme (ICE), using C. albicans-infected and uninfected oral mucosa engi-neered tissues, revealed higher mRNA expression and lower ICE production (p45) after infection. This down-regulation may be associated with the activation of ICE for the proces-sing and release of mature IL-18 by epithelial cells. These results are similar to those already reported in the literatu-re. Oral epithelial cell cultures had the same pattern of ICE mRNA expression and protein production as that obtained with the EHOM model [12]. Studies on ICE-defi cient mice showed that IL-18 production is impaired when the mice are infected with C. albicans, suggesting that ICE needs to be acti-vated during such infections [42]. On the other hand, infec-ted and uninfected EHOMs only produce inactive ICE (p45). It may be that active ICE is produced and then immediately released or is degraded in the culture medium, thereby be-coming undetectable by Western blot analysis of cell lysates. Further investigation is thus needed.

It has been reported that exogenous IL-18 restores Th1-mediated resistance to C. albicans infections [42]. In the same vein, we found that exogenous rhIL-18 (250 pg/ml), when added to the medium of uninfected and infected tis-sues, promoted the expression and production of endoge-nous IL-18. This fi nding points to a possible paracrine or autocrine activation of oral epithelial cells via their own mature IL-18.

The increase in endogenous IL-18 expression and produc-tion in response to exogenous rhIL-18 suggests that a co-nverting enzyme was activated. Using uninfected and C. albicans-infected rhIL-18-stimulated cultures, we showed that inactive ICE protein (p45) was slightly up-regulated in EHOMs treated with exogenous rhIL-18. Interestingly, the presence of both rhIL-18 and C. albicans decreased the level of ICE during the early stages of the infection, indi-cating that the enzyme was called on to convert the pro-IL-18 produced by the epithelial cells in response to the in-sult. Studies on IL-18-defi cient mice have shown that IL-18 plays an essential role in the infl ammatory processes of dif-ferent forms of infection. Indeed, in the absence of IL-18,

IFNg (pg/ml) production**

Contact time (h) 1.5×106 Candida/cm2 rhIL-18 (250 pg/ml) 1.5×106 Candida/cm2 plus rhIL-18 (250 pg/ml)

Control*** 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00

2 0.5 ± 0.81 17.53 ± 2.70* 7.56 ± 0.44*

4 0.35 ± 0.63 35.8 ± 10* 222.72 ± 35.14*

6 3.35 ± 2.30* 54.27 ± 4.63* 23.76 ± 3.42*

12 8.5 ± 2.92* 1.38 ± 0.94 0.07 ± 0.00

24 1.63 ± 0.54* 0.04 ± 0.00 0.08 ± 0.00

48 0.09 ± 0.02 2.0 ± 1.92* 7.9± 3.92*

Table 2. IFNγ produced by epithelial cells following engineered oral mucosa tissue contact with C. albicans alone or C. albicans and rhIL-18.

* P≤0.01 when comparing the control values to the test values;** mean ± SD (n = 3);*** cultured supernatant form C. albicans non-infected tissue

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IL-12 alone cannot promote Th1 cell expansion in vivo [38]. Furthermore, IL-18 is required for the sustained production of IFN-g and IL-12 during C. albicans infections [42]. IL-18 thus requires IFN-g to play its vital biological role [21]. We demonstrated that epithelial cells infected with C. albicans produce signifi cant amounts of IFN-g mainly at the mid-stage of the infection. IFN-g production was initiated follo-wing IL-18 stimulation, as confi rmed by the levels of IFN-g secreted by epithelial cells when the tissue was stimulated by rhIL-18 alone (Table 2). Interestingly we also demonstra-ted that the combination of rhIL-18 and C. albicans signifi -cantly enhanced the secretion of IFN-g at the early stages (4 and 6 h) of the infection. One possibility is that epithe-lial cells mount their defense against C. albicans at an early stage of the infection (4 to 6 h). The pathogen then domi-nates the cells via a complete inhibition of IFN-g secretion by infected oral epithelial cells even if the cells are stimula-ted by exogenous IL-18 and continue to produce endoge-nous active IL-18. A second possibility is that oral epithelial cells produce IFN-g via an IL-18-dependent mechanism in response to early events in an attempt to control C. albicans infections but block IFN production later on to escape the toxic effect of IFN-g. Both possibilities may explain the high levels of active IL-18 produced by C. albicans-infected epithe-lial cells 6 h and more post-infection as well as the absence of IFN-g production by these cells in response to both exo-genous and endogenous IL-18. Our data suggest that the-re is an independent relationship between the capacity of oral epithelial cell to secrete active IL-18 and IFN-g and the control of Candida infections, which is in agreement with previous reports pointing to an important role for endoge-nous IFN-g in resistance to both gastrointestinal and syste-mic candidiases [42,43].

CONCLUSIONS

In summary, given the involvement of IL-18 in infl amma-tory [33,34] and non-infl ammatory [42] diseases and the growing awareness of the importance of IL-18 in fi ne-tu-ning host immune defenses against pathogens [36], the present study provides important insights into IL-18 invo-lvement in controlling C. albicans pathogenesis by oral epi-thelial cells. Since our experimental model closely mimics the natural interface between the structured buccal muco-sa and C. albicans, it appears that IL-18 meets the require-ments of being a cytokine that epithelial cells use to con-trol C. albicans infections locally and, possibly, systematically. Efforts are presently underway to design an EHOM model using epithelial cells and fi broblasts isolated form patholo-gical tissues in order to perform mechanistic studies rela-ted to the pathogenicity of C. albicans and the local defen-se initiated by the oral mucosa.

Acknowledgments

The authors would like to thank Ms Yakout Mostefaoui for her technical support.

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