Our results demonstrate that the number of neutrophils obtained from the saliva of elderly D.S. was significantly lower than that of the elderly control (0.2 ± 0.03 × 10⁵ and 1.3 ± 0.2 × 10⁵, respectively, Figure 1A). Similarly, young individuals with D.S. showed a lower number of salivary neutrophils in comparison to the young control (0.4 ± 0.06 × 10⁵ and 3.2 ± 0.4 × 10⁵, Figure 1A). Interestingly, young control subjects showed a significantly higher neutrophil counting than elderly controls (p = 0.0015).

Although the number of neutrophils isolated from the saliva of patients with D.S. was lower when compared to the respective control groups, the number of neutrophils obtained from the peripheral blood of subjects with denture stomatitis was higher than the respective controls (316.2 ± 51.9 × 10⁵ – elderly D.S. and 176.3 ± 35.4 × 10⁵ – elderly control; 305 ± 63.7 × 10⁵ – young D.S. and 136 ± 21.1 × 10⁵ – young control, Figure 1B).

In order to understand why a lower number of salivary neutrophils was found in elderly and young individuals carrying the disease, as well as in aged controls, we studied the expression of molecules involved in the chemotaxis and diapedesis of neutrophils from the blood of impaired patients and healthy volunteers. Our results showed a significantly lower expression of CXCR1 and CD62L on neutrophils from patients with Candida-related denture stomatitis than from young controls. Curiously, blood neutrophils of aged subjects not affected by the disease also expressed lesser amounts of CXCR1 and CD62L than matched young subjects (Figure 2B and 2C). Also, young controls presented a significant higher expression of CD11b on blood neutrophils than all other groups (Figure 2D).

Before performing the assays, the viability of salivary and blood neutrophils was analyzed. Our data showed a significantly higher rate of salivary apoptotic neutrophils in elderly groups (26.3 ± 2.4% – elderly D.S. and 24.4 ± 4.3%-elderly control, Figure 3A) than those from the respective young groups. While elderly groups showed the same rate of apoptotic neutrophils in the saliva, young D.S. had a higher rate of apoptotic neutrophils in the saliva than the matched controls (14.2 ± 2.2% – young D.S. and 8.5 ± 1.6% – young control, p = 0.04, Figure 3A). Although salivary neutrophils were apoptotic, blood neutrophils from aged subjects were as viable as those from the young ones, regardless of the disease (from 2 ± 0.5% to 3.4 ± 1.5%, Figure 3B).

Due to the different rate of viability of salivary neutrophils from aged people, the levels of cytokines known to affect neutrophil apoptosis, CXCL8, GM-CSF, and TNF-α were evaluated in the whole saliva from the subjects. CXCL8 was significantly elevated in the saliva from elderly patients and controls (5521 ± 1110 pg/mg and 3058 ± 410.5 pg/mg) when compared to young individuals, presenting or not D.S. (2025 ± 499.8 pg/mg and 1523 ± 391.3 pg/mg respectively, Figure 4A). In addition, elderly D.S. had higher levels of CXCL8 than matched controls (p = 0.0002). However, GM-CSF concentrations were lower in the saliva from elderly individuals, regardless of D.S. occurrence (194.9 ± 44.4 pg/mg, and 145.2 ± 40.6 pg/mg in D.S. or Control groups, respectively, Figure 4B). Although elderly D.S. and control groups did not exhibit differences, young D.S. presented significantly elevated concentration of GM-CSF (402 ± 98.8 pg/mg, Figure 4B) in comparison with matched controls (211 ± 39.4 pg/mg, p = 0.03).

We detected an increase of the TNF-α level in the saliva from elderly D.S. group (1026 ± 273.1 pg/mg) in comparison with its matched control (229.5 ± 95.8 pg/mg, Figure 4C). Although elderly D.S. displayed higher TNF-α level than young D.S. (638.3 ± 110.8 pg/mg), elderly controls had lower levels of TNF-α than young ones (696.7 ± 122.1 pg/mg, p = 0.003, Figure 4C). No differences were detected in TNF-α level when we compared young groups.

The ingestion of viable C. albicans was determined in purified salivary and blood neutrophils. The phagocytic activity against C. albicans by salivary neutrophils obtained from elderly D.S. was significantly lower when compared to the elderly controls (21.7 ± 2.4% and 34.4 ± 3.8%, respectively, Figure 5A), and this low phagocytic activity remained up to 120 min (24.3 ± 2.3%). Nevertheless, significant differences were observed between the phagocytic activity of salivary neutrophils from elderly and young controls at 120 min of the assay (Figure 1A, p < 0.01). At 30 min, salivary neutrophils from young controls were able to ingest C. albicans more efficiently than those neutrophils from young patients (49.9 ± 4.7% and 17.9 ± 3.5% respectively, Figure 5A). Although at 120 min of the assay the phagocytic activity of salivary neutrophils from young patients raised to 26 ± 6.2% (Figure 5A), this value was significantly lower than that of young controls (59.7 ± 4.9%). Therefore, salivary neutrophils from young and aged patients, and elderly control individuals showed reduced phagocytic activity.

In order to provide additional information on the role of neutrophils in denture stomatitis, besides the influence of ageing in the establishment of this disease, the phagocytic activity of systemic neutrophils was analyzed. At both 30 and 120 min evaluation times, peripheral blood neutrophils from elderly D.S. presented a significantly lower phagocytic activity (43.4 ± 4.1% and 37.1 ± 5.1%, respectively) than young D.S (62 ± 4.4% and 59.5 ± 8.8%, Figure 5B). In the elderly controls, a reduction in Candida phagocytosis was observed only after 120 min (53.5 ± 7.5%, Figure 5B). Although at 120 min of the assay the phagocytic activity of blood neutrophils from elderly controls raised, this value was significantly lower than that of young controls (71.1 ± 5.9%, Figure 5B). Taken together, our results demonstrated that systemic neutrophils from elderly groups had an impaired phagocytic function for C. albicans in comparison with the neutrophils from matched younger groups.

After demonstrating impaired phagocytic function of the salivary neutrophils from D.S. patients, the death of blastospores that were ingested by salivary and blood neutrophils was assessed. When it comes to salivary neutrophils, young controls neutralized more ingested blastoconidia than all the evaluated groups because they had less viable internalized C. albicans (57.7 ± 3.9% at 30 min and 47.8 ± 3.2% at 120 min, Figure 6A). Elevated rates of salivary neutrophil from aged (74.5 ± 5.7% and 77.4 ± 4.6% at 30 and 120 min) or young patients with D.S. showed viable phagocytosed blastoconidia at both experimental periods (77.4 ± 5.3% and 65.5 ± 6.2%, at 30 and 120 min, respectively, Figure 6A). Neutrophils from aged controls, in opposite to the younger ones, presented significant higher percentages of viable ingested C. albicans at 30 and 120 min (79.8 ± 2.3% and 66 ± 4.7%, sequentially, Figure 6A). These results confirm a deficient phagocytosis of C. albicans by salivary neutrophils from patients with D.S. In addition, our data showed that elderly people presented neutrophils with impaired ability to neutralize phagocytosed C. albicans.

Interestingly, blood neutrophils killed more C. albicans than salivary cells considering all the analyzed groups. The percentage mean of blood neutrophils from aged D.S. individuals that kept internalized viable yeasts was higher, but not statistically significant, than that found in the aged control at 30 min (70.3 ± 3.8% and 57.9 ± 5.7%, respectively, Figure 6B). At 120 min, it was observed a reduced difference between aged groups (57.8 ± 2.8% – D.S. and 45.7 ± 5.3%-Control, Figure 6B). Neutralization of C. albicans by blood neutrophils from young D.S. individuals was lower in comparison to matched controls since decreased percentages of such cells from young controls did not kill C. albicans at 30 and 120 min (27 ± 4.3% and 21.6 ± 3.5% of neutrophils containing viable phagocytosed yeast cells, respectively, Figure 6B).

In summary, there was no statistically significant difference in candidacidal activity between aged groups at any time of the assay. Moreover, blood neutrophils from elderly presenting or not D.S. and from young subjects presenting D.S. neutralized significantly fewer ingested blastoconidia than those from healthy young individuals at both times of evaluation. Photomicrographs 6C and 6D show the viable and dead ingested C. albicans inside blood neutrophils by fluorescence assay.

After obtaining the informed consent of the individual in compliance with resolution 196/96 of the National Council of Health and of the University of São Paulo (Bauru, São Paulo, Brazil) committee guidelines, the patients were drawn from the patient pool at the Bauru Dental School Prosthodontics Clinic (USP). Their selection was based on the inclusion and exclusion criteria described below. The exclusion criteria comprised patients suffering from the following conditions: diabetes mellitus, alcoholism, tobacco use, periodontal diseases or other oral pathologies, gingival bleeding, cancer and symptoms that could indicate cancer such as sudden changes in weight and appetite, immune/endocrine/hematological alterations and use of xerostomic, antifungal or antibiotic medications. The inclusion criteria comprised aged subjects presenting Candida-related denture stomatitis, which was confirmed by microbiological diagnosis (elderly D.S., n = 14) or healthy subjects (elderly control, n = 14), ranging from 60 to 85 years old (mean age = 69.4 ± 3 years old in D.S. and 68.6 ± 0.9 years old in control groups). Additionally, individuals ranging from 20 to 50 years old presenting Candida-related denture stomatitis (young D.S., n = 14, mean age = 33.8 ± 2.3 years) or not (young control, n = 14, mean age = 38.1 ± 3.9 years), were assessed. Age limits were determined based on the study of Wenisch et al. 29. In the sampling, all patients with Candida-related denture stomatitis were wearers of complete maxillary dentures for at least a two-year period, without natural maxillary teeth. Complete medical and dental histories were recorded. Participants that presented infectious diseases up to one month before saliva sampling, as well as active dental abscesses and collagen vascular diseases, were excluded from the study. None of the Candida-related denture stomatitis lesions had been treated in any manner prior to sample collection. None of the control volunteers presented oral lesions.

Denture stomatitis was clinically diagnosed and classified according to Newton 55. One elderly patient presented type III denture stomatitis (granular surface or inflammatory papillary hyperplasia of the palate) and the others showed type I denture stomatitis (pinpoint hyperemia or localized simple inflammation). After such classification, the microbiologic diagnosis was made by collecting material from the hard palate (in a denture stomatitis-associated erythematous area) with a sterile swab and then inoculating it in Sabouraud dextrose broth (Difco, Becton Dickinson, France) with 1% chloramphenicol (0.1 mg/mL, Sigma-Aldrich, St. Louis, USA) at room temperature. After one week, 0.1 mL from broth + sample was spread over Sabouraud dextrose agar plates with 1% chloramphenicol (0.1 mg/mL) to observe colony forming units (CFU). The plates were incubated for 48 h at 37°C.

Participants were advised to avoid eating, drinking, using chewing gum, mint, and brushing their teeth for at least two hours before collection. The samples were obtained by requesting subjects to swallow first, tilt their head forward, and then expectorate all saliva into 50 mL centrifuge tubes for 5 consecutive min without swallowing it. Nonstimulated flowing whole saliva samples were collected between 7:00 and 9:00 a.m. in sterilized tubes and centrifuged at 10,000 × g/20 min/4°C. The supernatants of saliva samples were frozen at -70°C until analysis. Interleukin-8 (IL-8, CXCL8), Granulocyte macrophage-colony stimulating factor (GM-CSF) and Tumoral Necrosis Factor-α (TNF-α) were quantified in saliva samples by the quantitative sandwich Enzyme-Linked Immunosorbent Assay (ELISA) using commercial capture and biotinylated detection antibodies (BD Pharmingen Corp., San Diego, CA) and the respective human recombinant cytokines (diluted in PBS) as standards, according to the manufacturer's instructions (cat.#555244 – IL8, # 555126 – GM-CSF, # 555212 – TNF). The concentrations of each cytokine were expressed as pg/mg protein. Previous studies confirmed that cytokines are stable in saliva 56.

Saliva protein content was quantified with Quick Start™ Bradford Protein assay kit (Bio-Rad, CA, USA) and the results expressed in mg/ml. Saliva from elderly stomatitis-positive and stomatitis-negative groups presented protein concentrations of 107.4 and 99.1 mg/ml, respectively. Young groups presented mean values of 117.9 mg/mL (D.S.) and 129.3 mg/mL (controls).

For immunostaining of purified blood neutrophils, phycoerithrin (PE) and fluorescein isothiocyanate (FITC) conjugated antibodies against human CXCR1 (CD181, cat. # 555939), CD62L (L-selectin, cat. # 555544) and CD11b (Mac-1, cat. # 555388), and the respective mouse isotype controls were used (BD Biosciences, San Diego, CA). Briefly, 1 × 10⁷ neutrophils were incubated in 20 μL of each antibody solution for 30 min at 4°C. After washing, the cells in 50 mL of phosphate-buffered saline solution were fixed by 1% paraformaldehyde, and then the binding of each antibody was detected using a flow cytometer. Cell acquisition (counting) was performed on a FACSort flow cytometer using the CellQuest software (BD Biosciences). The mean of fluorescence intensity was determined for each molecule in 30,000 events.

Salivary neutrophils were collected by a modified method described by Lukäc et al. 21. Briefly, volunteers and patients swished 10 mL of sterile 1.5% NaCl solution without swallowing it for 4 min and expectorated into a sterile beaker. This procedure was further repeated four times. Denture wearers were oriented to take their prosthesis off before the collection. After that, the collected material was centrifuged at 450 × g/10 min. The supernatant was discharged and the pellet suspended in 2 mL of RPMI 1640 medium containing 10% of fetal calf serum (10% FCS RPMI) (Gibco, Invitrogen, UK) including 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 30 mM HEPES, 100 U/mL penicillin, 0.1 mg/mL streptomycin. Samples were gently vortexed in order to avoid aggregation of neutrophils and the cell suspensions were sequentially filtered through 20 and 11 μm nylon filters (NY2002500 and 1102500, MilliUni, Millipore Corporation). The filtered suspension was washed once at 250 × g/10 min, the supernatant was discharged and the pellet was suspended in 1 mL of 10% FCS RPMI medium.

Heparinized whole blood (10 mL) was obtained from volunteers and patients by venipuncture, and blood neutrophils were purified as follows. Briefly, 5 mL of Histopaque 1119 (Sigma-Aldrich Brazil Ltda., São Paulo, Brazil) were poured into a 15 mL round bottom tube and overlaid with 3 mL of Histopaque 1083 (Sigma-Aldrich Brazil Ltda., São Paulo, Brazil), and 6 mL of whole blood were layered over the gradients. Tubes containing gradients and blood were centrifuged (460 × g) for 28 min at 25°C. The second band, which contained the neutrophils, was aspirated and washed twice with cold RPMI 1640.

The salivary and blood (systemic) neutrophils were identified through staining with Türk's solution. The viability of salivary and blood neutrophils was analyzed by positivity for annexin V-FITC or propidium iodide (Aposcreen Annexin V-FITC, R&D Systems, Minneapolis, USA). The cells were analyzed by fluorescence microscopy using a Axiostar plus HBO 50/AC (Zeiss, Germany) and the percentage of apoptotic cells was calculated from the proportion of positive neutrophils (green and/or red cells) in relation to total neutrophils 46.

C. albicans ATCC 10231 strains was obtained from the stock culture collection of the Department of Immunology and Microbiology, Bauru School of Dentistry, University of São Paulo. Yeast cells were grown on Sabouraud dextrose Agar and maintained at 28°C. Before infection, the blastoconidia were grown in Sabouraud dextrose broth for 48 h at room temperature. At the moment of the opsonization assay, these yeast cells were harvested by centrifugation (500 × g/6 min), washed and suspended at the desired concentration in Phosphate Buffered Saline (PBS).

Blastospores were opsonized in 1 mL of human serum during 30 min/37°C/5% CO₂, washed twice with PBS, and the pellet was suspended in PBS and maintained at 4°C until the procedures were carried out 57.

Blood or salivary neutrophils were cultured with viable C. albicans (at a ratio of 1:1 – opsonized yeast: viable neutrophil) in 10% FCS RPMI at 37°C/5% CO₂. After 30 and 120 min, the cell collection was centrifuged (350 × g/10 min) and the pellet was suspended in PBS. Neutrophils phagocytosing opsonized yeast forms of C. albicans were distributed on coverslips and analyzed after staining with 0.05 mg/mL of acridine orange solution in PBS and visualized with UV light under the fluorescence microscope Axiostar plus HBO 50/AC (Zeiss, Germany) and photographed by confocal laser scanning microscopy (TCS model, SPE, Leica, Mannheim, Germany).

Neutrophils phagocytosing or not C. albicans (showing green or orange colors, which indicate viable and dead blastospores, respectively) were counted 29. The results of the phagocytic activity as well as of the internalized fungal cell viability were expressed as the mean percentage of viable neutrophils containing at least one yeast cell by random counting of 10 fields. All assays were prepared and analyzed in duplicate.

Statistical analysis was performed using GRAPHPAD INSTAT version 2.04a. All results were analyzed through nonparametric tests. One-way ANOVA followed by Tukey's test was employed and significant differences were considered for P < 0.05. GraphPad PRISM (version 4) was used for graphic representations. To compare differences between two groups unpaired t-test followed Mann-Whitney test was applied and significant differences were considered for P < 0.05.