Oral and vaginal epithelial cell anti-Candida activity is acid labile and does not require live epithelial cells

Abstract

Background: Candida albicans is the causative agent of oral and vaginal candidiasis. Innate host defenses against C. albicans are important against each infection. Among these are oral and vaginal epithelial cells that have anti-Candida activity. The mechanism of action includes a requirement for cell contact with no role for soluble factors, and a putative role for carbohydrates based on the sensitivity of the activity to periodic acid.

Methods: Periodic acid treatment of epithelial cells as well as the property of partial resistance of antifungal activity to fixation was used to further dissect the mechanism of action.

Results: The results herein effectively now challenge a role for carbohydrates alone. Firstly, the putative carbohydrate(s) released into supernatants of periodic acid-treated epithelial cells could not compete with fresh epithelial cells for activity, and equivalent abrogation of activity was observed by periodic acid-treated cells irrespective of the amount of carbohydrate released. Instead, the similar abrogation of activity following treatment with other acids or when cocultured under acidic conditions suggests that the activity is acid-labile. Finally, while activity requires intact epithelial cells, it does not require live cells; activity was minimally affected by fixing epithelial cells prior to coculture where the majority of cells remained impermeable to Trypan blue but were defined as non-viable by positive nuclear staining with propidium iodide.

Conclusion: These results suggest that antifungal activity is dependent on contact by intact, but not necessarily live, epithelial cells through an acid-labile mechanism.

Fig. 1

The putative carbohydrate cannot compete with fresh epithelial cells for anti-Candida activity. Whole unstimulated saliva was collected from healthy volunteers (n = 3), and epithelial-enriched populations were isolated by nylon membrane retention. Candida was either pretreated with epithelial cell supernatants containing putative carbohydrates (PA sup) prior to the coculture with fresh epithelial cells (pretreatment), or treated throughout the coculture period (direct treatment). Controls included similar treatment with supernatants from PBS-treated cells (PBS sup) and periodic acid alone (PA). Figure shows cumulative data from three separate experiments. EC, epithelial cells. SEM, standard error of the mean.

Fig. 2

Abrogation of growth inhibition is independent of the amount of carbohydrate release. A retrospective study was conducted from experiments using whole unstimulated saliva collected from healthy volunteers, and the resulting oral epithelial-enriched populations were treated with periodic acid (PA) and examined for in vitro growth inhibition. Carbohydrate release was measured by the Dubois assay. A) Percent growth inhibition for high (> 10 μg/ml) and low (< 10 μg/ml) carbohydrate release. B) Scatter plot of carbohydrate release vs. percent abrogation of growth inhibition. Figure shows cumulative data from 39 separate experiments. SEM, standard error of the mean; CHO, carbohydrate.

Fig. 3

Effect of several acids on epithelial cell anti-Candida activity. Whole unstimulated saliva was collected from healthy volunteers (n = 4), and epithelial-enriched populations were treated with periodic acid (PA), trifluoromethane sulfonic acid (TFMS) or hydrochloric acid (HCl) and examined for in vitro growth inhibition of C. albicans by quantitative plate count. Figure shows cumulative data from four separate experiments. SEM, standard error of the mean.

Fig. 4

Viability requirement of epithelial cell antifungal activity. Whole unstimulated saliva was collected from healthy volunteers (n = 10), and epithelial-enriched populations were fixed with 1% paraformaldehyde, and stained with fluorescein diacetate (FDA, 50 μg/ml) and propidium=iodide (PI, 1 μg/ml) or Trypan blue. Controls included staining of unfixed cells. A) fixed cells stained with Trypan blue; B) fixed cells stained with FDA and PI; C) unfixed cells stained with Trypan blue; D) unfixed cells stained with FDA and PI. Arrows point to representative live cells. Magnification, 100×.

Fig. 5

Effects of fixation on cell viability evaluation and antifungal activity. Whole unstimulated saliva was collected from healthy volunteers (n = 10), and epithelial-enriched populations were fixed with 1% paraformaldehyde and examined for in vitro growth inhibition of C. albicans by [³H]-glucose uptake immediately or after 4 days in culture medium at 37°C. Cell viability was determined by Trypan blue (TB) dye exclusion and vital staining with FDA and PI. SEM, standard error of the mean.