1. Introduction
Denture stomatitis (DS) is an inflammatory reaction of the oral mucosa underlying removable dental prostheses, mainly upper dentures [
1,
2]. Individuals in the 50–80 year age group most frequently suffer from DS, with greater involvement of the palatal mucosa [
3]. Although it is a disease of multifactorial etiology, DS is strongly related to the fungus
Candida albicans, which is commonly found in the biofilm that forms on the inner surface of acrylic dentures [
1,
2]. Systemic factors are also involved in the pathogenesis of DS, such as immune response depression [
4,
5], which is common in elderly people [
6,
7].
C. albicans is a commensal fungus constituent of the normal mucosal microbiota in humans [
8]. However, under suitably predisposing conditions, it is able to cause several mucosal diseases, including DS. The morphological transition from yeasts to filamentous hyphae and the virulence that is associated with this transition are crucial for the pathogenicity of
C. albicans [
9]. The filamentous hyphae grow by stretching and have the ability to penetrate oral tissues. This morphology is thought to be responsible for the initial inflammatory response in DS [
10].
Despite the damage that penetration of hyphae into cells causes, the epithelium attempts to combat fungal cell growth and invasion of tissue [
8]. The oral epithelium functions as a passive mechanical barrier that resists microbial infection. In addition to passive resistance, the epithelial cells actively respond by initiating an inflammation process, secreting cytokines and chemokines to alert various cell types for the activation of innate and adaptive immune responses [
4,
5,
11].
Focusing on the innate immune response, epithelial cells also produce antimicrobial molecules against pathogens to contribute to combating micro-organisms before the infection is established [
4,
5,
10]. The antimicrobial peptide β-defensins (hBD) is secreted by keratinocytes of oral mucosa [
12] and has been shown to present antifungal activity against
C. albicans [
13,
14]. Furthermore, these peptides participate in the modulation of innate and adaptive immune responses against a range of oral pathogens [
15]. Nitric oxide (NO), which is synthesized by a variety of cells, including keratinocytes, is also induced in response to microbial infection [
16], and plays an important role in a host’s defense against
C. albicans via direct cell destruction or anion formation [
17]. Moreover, NO was shown to be a potent antimicrobial agent against oral infections [
6,
18].
There is a lack of information on the role of epithelial cells in oral candidiasis, including in DS. Previous studies used an oral epithelial cell line derived from well-differentiated oral carcinomas (SCC15, ATCC; TR146; FaDu) [
10,
19,
20,
21], the immortalized oral keratinocyte line OKF6/TERT2 [
22,
23] and immortalized keratinocytes from human skin (HaCaTs) [
24,
25]. We did not find any studies involving a primary culture of human palate epithelial cells (HPECs), or studies on the relationship between the epithelial aggravation that
C. albicans causes and the epithelial defense responses against this micro-organism. The present study examines the direct and indirect effects of
C. albicans on HPEC defense over time. To complement the primary HPEC analyses, we performed experiments with the immortalized human gingival epithelial cell line OBA-9 in those cases where no or a low response was detected in the primary cells. The results show that aggressive events, such as the fungus’s invasion of HPECs and the induction of apoptosis in epithelial cells, were correlated with epithelial defense responses by NO and β-defensin 2 (hBD-2) production. In addition, the responses of the HPEC primary culture were found to be different from those of the immortalized keratinocyte cell line OBA-9, for example in terms of NO release.
2. Materials and Methods
2.1. Ethics Statement
Palatal mucosa samples (5 mm2) were biopsied from gingival graft patients after they had given written informed consent in compliance with the National Council of Health and Ethics Committee guidelines. All experimental procedures were approved by the Committee for Ethics in Research on Human Beings of the Bauru School of Dentistry, University of São Paulo (number 001/2012).
2.2. Isolation and Co-Culture of Human Palate Epithelial Cells
Volunteers were considered for inclusion in our study if they presented with normal health and did not suffer from any of the following conditions: diabetes mellitus, alcoholism, tobacco usage, periodontal disease or other oral pathology, gingival bleeding, and immune/endocrine/hematological alterations and the use of xerostomia, antifungal, or antibiotic medications, respectively. After disinfection of the samples in 70% alcoholic solution and the mechanical separation of epithelium from connective tissue, HPECs were obtained using either the direct explant method or the enzymatic method [
26].
2.2.1. Direct Explant Method
The separate samples for the explant method were cut into small fragments (1 mm2) and placed in culture dishes with the epithelial surface facing the plate. Then, the fragments were slightly humidified in a culture medium of Dulbecco’s Modified Eagle Medium (DMEM; Gibco® Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco® Invitrogen, Grand Island, NY, USA) and penicillin/streptomycin (100 IU/mL/100 μg/mL; Gibco® Invitrogen, Grand Island, NY, USA), which was dropped between the fragments. The plates were incubated in a humidified atmosphere at 37 °C with 5% CO2 for approximately 24 h. After this period, the fragments were flooded with an appropriate keratinocyte culture medium constituted of DMEM and F12 Nutrient Mixture (Ham; Gibco® Invitrogen, Grand Island, NY, USA) in a 2:1 ratio, with 10% Bovine Serum Product Fetal Clone III (Hyclone, Logan, UT, USA), penicillin/streptomycin (100 IU/mL/100 μg/mL), glutamine (4 mM; Gibco® Invitrogen, Grand Island, NY, USA), adenine (0.18 mM; Sigma Chemical Co., St. Louis, MO, USA), insulin (5 μg/mL; Sigma Chemical Co., St. Louis, MO, USA), hydrocortisone (0.4 μg/mL; Sigma Chemical Co., St. Louis, MO, USA), cholera toxin (0.1 nM; Sigma Chemical Co., St. Louis, MO, USA), and triiodothyronine (20 pM; Sigma Chemical Co., St. Louis, MO, USA). The culture plates were examined daily using an inverted microscope. When the migrated cells reached a diameter of 2–5 mm, Epidermal Growth Factor (EGF, 10 ng/mL; R&D Systems, Minneapolis, MN, USA) was also added to the DMEM/HamF12 medium. When the cells around the sample fragment started to change their morphology and increase their volume, they were removed by trypsinization (0.05% trypsin/0.02% EDTA; Gibco® Invitrogen, Grand Island, NY, USA) and their further propagation in culture was started.
2.2.2. Enzymatic Method
The initial fragments were cut into smaller pieces (0.5 × 0.5 mm) and immersed in a 0.05% trypsin/0.02% EDTA solution under low agitation at 37 °C. At intervals of 30 min, the supernatant cells were collected, the trypsin was neutralized with DMEM medium containing 10% FBS and penicillin/streptomycin (100 UI/mL/100 μg/mL), and this mixture was centrifuged at 1500 rpm for 5 min. The cells were resuspended in a keratinocyte culture medium (DMEM/HamF12, 2:1) supplemented as described in
Section 2.2.1. The cells (6 × 10
3 cells/cm
2) were seeded on a mitotically inactivated human gingival fibroblasts feeder-layer. The integrated keratinocytes/feeder-layer culture was kept in a humid incubator (5% CO
2/95% air, 37 °C) with a DMEM/HamF12 culture medium supplement. EGF (10 ng/mL) was added to the DMEM/HamF12 culture medium after it was first changed.
2.2.3. Mitomycin C Treatment of Human Fibroblasts
Human gingival fibroblasts were isolated from connective tissue, mechanically separated from the donated epithelial fragments, and cultured in DMEM culture medium supplemented with 10% FBS and penicillin/streptomycin (100 IU/mL/100 μg/mL). Fibroblasts used as feeder-layers (1.5 × 10
4 cells/cm
2) were mitotically inactivated with mitomycin C (8 μg/mL; Sigma Chemical Co., St. Louis, MO, USA) diluted in 10% SBF DMEM growth medium after incubation for 2 h at 37 °C. Then, the cells were washed, resuspended in culture medium, and seeded into cell culture bottles [
27].
2.2.4. Propagation of Keratinocytes on the Feeder-Layer
After a keratinocyte primary culture was developed using both methods, the propagation of these cells in subsequent cultures was carried out. The passages were fulfilled each time the culture reached the subconfluence stage. At that moment, trypsinization and centrifugation were performed. The integrated keratinocytes/feeder-layer culture was first trypsinized for 5 min to remove the fibroblasts that had been discarded. The keratinocytes were then removed by trypsinization for 15 min, counted, and seeded (6 × 10
3 cells/cm
2) on a new feeder-layer. The medium was changed every 2–3 days [
26].
2.3. OBA-9 Cell Culture
Cells from the immortalized human gingival epithelial cell line OBA-9 were obtained from the Institute of Biomedical Sciences (ICB-USP). The cells were subcultured in T25 Cell coat (Greiner Bio-One, Americana, SP, Brazil) containing keratinocyte culture medium (DKSFM, ThermoFisher, Waltham, MA, USA) supplemented with penicillin/streptomycin (100 IU/mL/100 μg/mL) and were kept in a humid incubator (5% CO2/95% air, 37 °C). Cells were trypsinized with Tryple Express (Thermo Fisher), plated at 104 cell/well in 96-well plates for the quantitative lactate dehydrogenase (LDH) assay, and plated at 105 cells/well in 24-well plates for the release of reactive oxygen species assay.
2.4. Fungal Growth Conditions and the Generation of Supernatants from C. albicans
C. albicans ATCC 90,028 was used for this study. Yeast cells were grown in trypticase soy broth medium (TSB, Himedia, Mumbai, Maharashtra, India) supplemented with 0.5% chloramphenicol (0.05 mg/mL; Sigma Chemical Co., St. Louis, MO, USA) for 24 h at 37 °C under gentle agitation. Then, the yeast cells were washed with phosphate-buffered saline (PBS), counted using a hemocytometer, resuspended in the appropriate keratinocyte culture medium (DMEM/HamF12, 2:1), and supplemented as described in
Section 2.2.1, but without an antibiotic. Concentrations of 10
3, 2.5 × 10
3, 4 × 10
3, 10
4, and 4 × 10
4 yeast cells/mL were used to test HPEC (10
5 cells/mL) interaction with
C. albicans in the fungal proportions 1/100, 1/40, 1/25, 1/10, and 1/2.5
Candida/HPEC, respectively [
28]. The fungal proportions 1/100, 1/40, and 1/10
Candida/cells were used to challenge cells of the OBA-9 cell line.
To analyze the effects of supernatant from
C. albicans on HPECs, yeast cells were cultured with DMEM/HamF12 (2:1) medium supplemented as described in
Section 2.2.1 but without antibiotics, to obtain the hyphal form, for 24 h at 37 °C in the different
Candida/HPEC ratios. These conditioned media were obtained by centrifugation (10,000 rpm for 10 min) and used in additional experiments [
28]. We did not evaluate the effects of hyphae supernatant on the OBA-9 cells.
2.5. HPEC Interaction with C. albicans via Either Direct (D.C.) or Indirect (I.C.) Cell–Cell Contact by Means of the Fungal Supernatants
For the experiments, 10
5 HPEC cells/mL were seeded on culture plates and incubated (5% CO
2/95% air, 37 °C) in complete DMEM/HamF12 (2:1) medium supplemented as described in
Section 2.2.1 for 24 h. After this period, the keratinocyte culture medium was discarded, and the cells were either kept together with
Candida supernatants, as a form of indirect contact (IC), or in direct contact with
C. albicans cells (DC) for an intercalated experimental time starting from 3 h to a maximum of 24 h. HPECs maintained in DMEM/HamF12 (2:1) medium supplemented as described in
Section 2.2.1 but without an antibiotic were used as an experimental control (CRTL/Medium, negative control). For the DC experiment,
Candida cell cultures were added to HPECs [
28,
29]. For the IC experiment, keratinocytes were incubated with the hyphae supernatant that was obtained through the fungal culture [
28].
2.6. HPEC Viability Assays
To evaluate the effect of
C. albicans on HPEC viability, a LIVE/DEAD cell viability assay was performed (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. HPECs were seeded onto a six-well culture plate and stimulated directly (DC) or indirectly (IC) with
C. albicans (1/100, 1/40, 1/25, 1/10, and 1/2.5
Candida/HPEC), under the conditions described in
Section 2.5, for 3, 6, 8, 10, 12, and 24 h. After the epithelial cells were stained (1 μL/mL of dye in PBS) for 20 min at 37 °C under light protection, a quantitative analysis was performed using an inverted fluorescence microscope (Leica DM IRBE, Mannheim, Baden-Württemberg, Germany) through 10 randomly captured fields per well at 200× magnification. The number of living cells expressed as a percentage of the total cell number was used to establish the quantitative cell viability index.
2.7. HPEC Invasion by C. albicans Post Direct Contact
HPECs were kept in DC with
Candida fungus (1/100, 1/40, 1/25, 1/10, and 1/2.5
Candida/HPEC) in 24-well plates containing sterile spherical glass coverslips (13 mm in diameter) for 3, 6, 8, 10, 12, and 24 h. The wells were washed with PBS to remove the extracellular fungus. The coverslips to which cells were attached were fixed with 2% paraformaldehyde in PBS for 15 min and washed again with PBS. Then, they were stained with 0.05 mg/mL acridine orange (Merck, Darmstadt, Germany) for 15 min in a dark room. After this, the coverslips were carefully removed and placed on glass slides with mounting medium. The slides were qualitatively evaluated by confocal laser-scanning microscopy (TCS-SPE, Leica, Wetzlar, Germany) at 630× magnification, which allowed us to differentiate the fungus inside the keratinocytes from those that were only bounded to the HPEC surface [
30]. The experiments were also performed in the absence of epithelial cells to compare the fungal biofilm that formed with those that developed in the presence of HPECs.
2.8. HPEC Apoptosis Assay
The cells to be analyzed for apoptosis were stained with Hoechst 33,258 [
31]. Initially, HPECs were kept in DC or IC in 24-well plates at 1/100, 1/40, and 1/10
Candida/HPEC for 3, 6, and 10 h. After washing, cells were incubated with Hoechst 33,258 (10 μg/mL in PBS; Invitrogen, Carlsbad, CA, USA) for 20 min at 37 °C to stain the DNA in each cell. Then, individual nuclei were visualized at 400× magnification to distinguish the normal uniform nuclear pattern from the characteristic condensed coalesced chromatin pattern of apoptotic cells [
20]. For this, 10 random microscopic fields per well were analyzed in an inverted fluorescence microscope. HPEC apoptosis is expressed as a percentage of overall cell numbers.
2.9. OBA-9 Cell Line D.C. Interaction and Lactate Dehydrogenase (LDH) Release Assay
To verify our previously obtained results, which revealed that the
C. albicans fungus has a slight cytotoxic effect on epithelial cells in vitro, a highly sensitive method that can measure low numbers of cells undergoing apoptosis [
32] was used to analyze OBA-9 cells. For this purpose, DC-induced cytotoxicity was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega Corporation, Madison, WI, USA). The standard protocol for the assays reported here was performed according to the manufacturer’s instructions. This colorimetric assay was used to quantitatively measure the LDH that was released into the media from damaged cells as a biomarker of cellular cytotoxicity and cytolysis. OBA-9 cells were plated in a 96-well plate (10
4 cells per well) in culture medium supplemented with 10% fetal bovine serum and incubated overnight at 37 °C in 5% CO
2. The cells were then challenged with different concentrations of
Candida (1/100, 1/40, and 1/10
Candida/OBA) for 3, 6, and 10 h before being subjected to the indicated assays. OBA-9 cells maintained in DKSFM medium supplemented as described in
Section 2.2.1 but without an antibiotic were used as the negative control (CRTL/Medium). The kit provided the LDH positive control. LDH activity was determined by spectrophotometric absorbance using a standard plate reader with a reference wavelength of 490 nm.
2.10. NO Production by HPEC and OBA-9 Cells
For NO detection in the experimental samples, nitrite (NO
−2) production was measured in the supernatants using the Griess method [
33]. The DC and IC assays were performed at ratios of 1/100, 1/40, and 1/10
Candida/HPEC for 3, 6, and 10 h. Briefly, 100 μL of supernatant sample was incubated with an equal volume of the Griess reagent (Sigma Chemical Co., St. Louis, MO, USA) at room temperature for 30 min. The absorbance was measured using a microplate reader (Spectra Max 250, Molecular Devices, Sunnywale, CA, USA) at 540 nm. The NO
-2 concentration was determined using a standard curve for NaNO
2 at a concentration range from 0.2 to 1.5 μM. The NO
-2 production by OBA-9 cells in the DC assay was also determined using the Griess method but using lipopolysaccharide (LPS) endotoxin as a positive control (CTRL/LPS).
2.11. Reactive Oxygen Species (ROS) Production by OBA-9 Cells
The amount of endogenous ROS in OBA-9 cells was determined using the fluorescent probe 2’,7-dichlorofluorescin diacetate (Cell Rox Deep Red Reagent; Life Technologies, Grand Island, NY, USA). OAB-9 cells (105 cells/well in a 24-well plate) were DC incubated with C. albicans (1/100, 1/40, and 1/10 Candida/OBA) for 3, 6, and 10 h (5% CO2/95% air, 37 °C). Cells in medium and Phorbol 12-myristate 13-acetate (PMA) were the negative (CTRL/Medium) and positive (CTRL/PMA) controls, respectively. Afterward, they were incubated with 5 μM of Cell Rox (5% CO2/95% air, 37 °C) for 30 min. The fluorescence intensities (FIs) of the cells were measured using the multiplate reader at 640/665 nm at 37 °C.
2.12. HPEC hBD-2 Gene Expression (RT-qPCR)
For the hBD-2 gene expression evaluation, HPECs were seeded in 96-well plates at a density of 104 cells per well. The DC and IC experiments were performed at 1/100, 1/40, and 1/10 Candida/HPEC for 3, 6, and 10 h. LPS from Escherichia coli (1 μg/mL) was used as a control (CTRL/LPS) under the same conditions. After the experimental period, RNA was extracted for reverse transcription (RT) followed by a quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA and complementary DNA (cDNA) were directly obtained from epithelial cells using a Cells-to-CTTM Kit (Ambion Inc., Life Technologies, Austin, TX, USA) according to the manufacturer’s instructions. In brief, the cells were washed two times with 1× PBS and incubated for 5 min with the 49.5 μL of lysis solution and 0.5 μL of DNAse that are provided with the kit. Following this incubation, 5 μL of stop solution was added for 2 min, and the RT was performed by adding the 2× RT buffer and the 20× enzyme mix to the lysate followed by incubation at 37 °C for 60 min and a subsequent 95 °C step for 5 min to stop the reaction. The qPCR was performed using TaqMan Gene Expression PCR Master Mix (Applied Biosystems, Life Technologies, Warrington, UK) and a proprietary primer (Applied Biosystems, Life Technologies, Warrington, UK) targeting mRNA of hBD-2 (Hs00175474_m1, forward 5’ CCAGCCATCAGCCATGAGGGT 3’ and reverse 5’ GGAGCCCTTTCTGAATCCGCA 3’). All experiments were performed in an aViiA 7 Real-Time PCR system (Applied Biosystems, Life Technologies, Warrington, UK) using the comparative cycle threshold (Ct) method (ΔΔCt). The human β-globulin gene was used as the reference gene for all of the reactions because it was the most stable reference gene in our experiments compared with β-actin (data not shown).
2.13. HPEC hBD-2 Production (ELISA)
The hBD-2 concentration was determined in the cell-free supernatants that were obtained after 3, 6, and 10 h of HPEC culture in DC and IC with C. albicans at 1/100, 1/40, and 1/10 Candida/HPEC. The utilized method was an enzyme-linked immunosorbent assay (ELISA) using a Human BD-2 ELISA Construction Kit (Antigenix America, Melville, NY, USA). The evaluations were performed according to the manufacturer’s instructions. The absorbance was read at 450 nm using a multiwell scanning spectrophotometer (ELISA reader; Amersham, Pharmacia Biotech, Cambridge, UK).
4. Discussion
In this study, we reported the survivability and immune responses of HPECs against in vitro direct contact with C. albicans cells (DC assay) or hyphae supernatant from C. albicans (IC assay) over time. Infected HPECs showed significant hBD-2 peptide production, which is an important antimicrobial defense mechanism, as well as an increase in NO production in one of the evaluated time periods. On the other hand, HPECs showed a decrease in cell viability at higher Candida/HPEC proportions, as well as under conditions of prolonged contact with the fungus or its hyphae supernatant. Furthermore, the contact between epithelial cells and fungal cells allowed the fungal cells to transition to their filamentous pathogenic form, proliferate, and penetrate into the epithelium. Moreover, the HPECs showed slight apoptosis and reduced NO production over time, particularly in the 1/40 Candida/HPEC ratio. As expected, the observed changes occurred mainly after the DC assay.
Although many studies that have evaluated the effect of
C. albicans on oral epithelium can be found in the literature [
10,
19,
20,
21,
22,
23], no previous study, to our knowledge, involved a HPECs primary culture. This seems to be the first report on the influence of
C. albicans on the epithelial biological response of HPECs primary. In most cases, the polymerized resin of maxillary dentures is contaminated by the
Candida fungus, resulting in an inflammatory reaction of the palatal mucosa known as DS, prosthetic stomatitis, or
Candida-related DS [
1,
2]. Our infection model was able to mimic the palatal epithelium response in maxillary denture wearers. It should be possible to extend the current studies to obtain more comprehensive results on
C. albicans pathogenicity mechanisms and host immune factors for the control of DS development.
Studies on
Candida-related DS have used epithelial cell lines derived from well-differentiated oral carcinomas (SCC15, ATCC; TR146; FaDu) [
10,
19,
20,
21] and immortalized human oral keratinocyte cell lines (OKF6/TERT2) created from telomerase 2 forced expression [
22,
23]. Other studies of human oral diseases have been performed with normal oral epithelial cells immortalized by transduction with simian virus 40 (SV40), such as OBA-9 cells [
34,
35,
36], which were used in this study. However, the use of SV40 results in effects that disrupt DNA repair in virally immortalized cell lines [
35,
36], affect apoptosis signaling, and induce the accumulation of mutations in some cells. This implies that they have additional effects other than immortalization [
36,
37], and highlights the importance of the results obtained in this study with an HPEC primary cell culture.
Firstly, in this work, preliminary HPEC viability and penetration tests after contact with
C. albicans allowed for the selection of
Candida/HPEC proportions and experimental times for the apoptosis, NO production, and hBD-2 production analyses. The results led to the selection of proportions of 1/100, 1/40, and 1/10
Candida/HPEC, and 3, 6, and 10 h experimental periods, to ensure that cells remained viable for further analysis. Direct interaction between
C. albicans cells HPECs occurred in phases of adhesion, invasion, and cell damage, similar to other studies [
8,
9]. On the other hand, in spite of the increase in cell damage with epithelial penetration by the fungal cells in their filamentous form, the qualitative assessment of the HPECs suggested that an antimicrobial defense response occurred against the fungus. Initially, the fungal filaments were observed only on the periphery of the epithelial colonies, indicating an initial colonization phase with fungal cell adhesion to the host cell, but suggesting that the HPECs repulsed the fungus. Also, in the absence of HPECs (CTRL/
Candida for 3 h of incubation), the fungal biofilm was observed to have a more intense presence and occupy the entire area of the culture plate, which was not observed in the biofilm developed in the presence of epithelial cells. In agreement with this finding, researchers have reported that epithelial cell lines are able to control fungal cell growth and invasion of tissue [
8] through their fungistatic activity [
14], and are even able to stop the progression of apoptosis [
22]. An intraepithelial invasion phase (active epithelial penetration) associated with the yeast-to-fungal filamentous transition was observed at 6 h, in particular in the 1/40
Candida/HPEC proportion, which intensified over time as the number of prolific filamentous forms increased. Finally, in a later phase (10 h), the fungus in its higher proportions resulted in significant epithelial cell death, coinciding with intracellular filamentous fungi and epithelial disintegration, and similar to an in vitro model using the oral epithelia TR146 line [
10,
38].
Therefore, in our experimental model, contact between
C. albicans and HPECs in the ratio 1/40
Candida/HPECs for 6 h caught our attention. Under these conditions, we observed an initial active epithelial penetration by the fungus simultaneous with significant epithelial apoptosis. However, although the epithelial cell death percentage did not exceed 2%, the apoptotic test demonstrates that, at an early stage, keratinocytes from the primary palate culture suffered from
C. albicans invasion. Studies have shown that apoptosis of oral epithelial cells by
C. albicans depends on its ability to physically interact with and invade the host cell [
19,
20]. On the other hand, the results of studies with keratinocytes from human skin (the HaCaT line) [
24] suggest that apoptosis induction depends on factors that the viable pathogen releases. Both DC and IC resulted in a similar amount of apoptosis based on our findings with primary HPECs. The morphological transformation into invasive hyphae cells is known to secrete soluble hydrolytic enzymes, such as secreted aspartyl proteinases (SAPs) [
2,
39] and phospholipases [
24] that can promote the disruption of physical integrity followed by oral epithelial cell death [
2,
22,
24]. Since our supernatant was obtained in the hyphae form, it is possible that it contained these soluble enzymes, which may explain the similar HPEC apoptosis observed in the DC and IC assays.
In localized oral candidiasis cases, epithelial tissue alterations occur and, consequently, host defense mechanisms activate [
4]. Our group has detected high salivary NO concentrations in individuals with DS, alerting us to the importance of this radical for disease control and the prevention of
C. albicans dissemination [
40]. In response to infectious agents, keratinocyte lines release NO [
6,
16,
41,
42], suggesting active control of oral infections [
11,
24,
43,
44]. In our study, HPEC cells at a 1/40
Candida/HPEC ratio after 6 h of incubation in the DC assay showed a significant increase in NO release, coinciding with the initial active epithelial penetration by the fungus. However, this NO level was not maintained over time. The presence of
C. albicans [
45] and its soluble factors [
24] have been shown to be capable of blocking NO production by macrophages after 24 h of incubation. In this study, it is possible that the fungus mediated the same effect on HPECs in the DC assay at later periods (10 h). Nevertheless, the IC assay did not induce NO levels above the baseline. It is possible that the aggression by the hyphae supernatant with the fungal secreted factors toward the HPECs was insufficient to stimulate a larger increase in NO levels at early periods.
To complement these analyses with primary HPECs, experiments with cell from the immortalized human gingival epithelial cell line OBA-9 were conducted, mainly for those experiments where a low or no response was detected in the primary cells. Despite the low cytotoxicity, DC induced a significant LDH release by OBA-9 cells at 6 h of incubation, similar to the HPEC apoptosis results. On the other hand, unlike the NO production by HPECs, the OBA-9 cells under DC with C. albicans did not produce this radical. The OBA-9 cells that were activated by C. albicans showed an ROS release peak at 6 h of incubation compared to basal levels. These results show that the immortalized OBA-9 cells may present distinct responses from primary culture HPECs with respect to the release of free radicals associated with the defense response.
Epithelial cells also release the antimicrobial peptides β-defensins to participate in the host defense mechanism’s activation [
10,
12,
15] and these peptides are considered to be one of the active mediators in the control of
C. albicans infection [
13,
14]. Since the hBD-2 peptide has been shown to be effective in
Candida cell death, being the main defensin involved in the epithelium–oral environment interface [
29,
46], we evaluated the hBD-2 production by HPECs. The results show that the peak of
hBD-2 gene expression by HPECs occurred at 6 h of incubation under baseline conditions and was maintained after the DC assay at the ratios of 1/100 and 1/40
Candida/HPEC. Furthermore, this expression was found to be higher than the base level expression. Analysis of hBD-2 levels in the HPEC supernatants confirmed this observation; significantly higher hBD-2 levels were detected in all cell supernatants exposed to fungal DC at 6 h of incubation compared to CTRL/Medium. Although the
hBD-2 gene expression at 10 h returned to baseline, these data suggest an important immune defense response since the hBD-2 levels were maintained in the HPEC supernatants at the ratios of 1/40 and 1/10
Candida/HPEC over time. Researchers have shown an increase in
hBD-2 mRNA expression and hBD-2 secretion during double the time (12 h) of our study and concomitantly with the presence of
C. albicans hyphae [
10]; however, this expression occurred in the RHOE epithelial cell line [
10], differently from our HPEC primary culture.
It is worth noting that, in the present work, this important defense response was not observed after the IC assay. This is because, after stimulation, there was a significant hBD-2 reduction compared to basal expression, especially at 6 h. Accordingly, a significant hBD-2 level in the HPEC supernatants exposed to fungal IC was not observed compared to unchallenged cells. On the other hand, the results showed an increase in hBD-2 level over time that kept up with the baseline level. This indicates that the hyphae supernatant, which possibly contained fungal soluble secreted factors, could not increase the amount of hBD-2 expression and secretion. This implies that direct fungus–epithelium interaction is required for HPECs to activate this defense response.
Regarding the HPEC innate immune response, the experimental conditions of a 1/40
Candida/HPEC proportion and 6 h of incubation produced the best HPEC response, which was characterized by concomitant peaks of epithelial hBD-2 and NO production. This strengthens the previously reported idea that these in vitro conditions seem to represent the best viable fungus/cell interaction to activate immune defense mechanisms in the epithelial lining of the human palate. Thus, HPECs showed a maximal defense response against
C. albicans that coincided with the initial active epithelial penetration by the fungus, as well as initial epithelial apoptosis signals. When the intraepithelial invasion was intensified and amplified over time by increasing the
C. albicans/epithelium proportion, the fungus inside the epithelial cells at a later time was able to subvert cellular defense mechanisms, as highlighted by other researchers [
19,
21,
22,
47].
Although there was a good immune response after 6 h in our experimental model, the HPECs had no potential to reverse the cell invasion. This may have occurred because of the pathogenicity of the fungus for these cells, leading to a decrease in
hBD-2 expression and NO production, or due to the reduction in antimicrobial action by the HPECs, which would have allowed the proliferation of the fungus to concentrations that are lethal for epithelium. Our results with HPECs did not support the hypothesis presented by other studies that the hBD-2 antimicrobial peptide expression by the epithelium prevents
C. albicans hyphal progression, allowing for recovery of the epithelium’s synthesis capacity and control of
Candida spp. growth and pathogenicity [
5,
10].
In summary, the results of this study show that C. albicans can damage human palate epithelium even in the absence of direct contact between the fungus and the epithelium. On the other hand, HPECs were able to provide an antimicrobial defense response against C. albicans that possibly interfered with the proliferation of fungal cells. However, simultaneously to these responses, the HPECs showed the first signs of active penetration by the fungus, for which there was the need for direct fungus–epithelium interaction. Moreover, according to our findings, the activation of the palatal epithelium apoptosis pathway, induced by the fungus or its hyphae supernatant, was observed before the onset of these epithelial defense mechanisms, especially NO and hBD-2 production. It is worth highlighting the importance of this defense response by human palate epithelial cells against C. albicans in the first hours of contact. However, this epithelial defensive capacity was not maintained for the entire experimental period; there was discrete reduction of NO release and return of hBD-2 gene expression to basal levels in addition to an increased epithelial invasion associated with slight apoptosis maintenance.