Lactic acid in the vaginal milieu modulates the Candida-host interaction

Abstract

Vulvovaginal candidiasis (VVC) is one of the most common infections caused by Candida albicans. VVC is characterized by an inadequate hyperinflammatory response and clinical symptoms associated with Candida colonization of the vaginal mucosa. Compared to other host niches in which C. albicans can cause infection, the vaginal environment is extremely rich in lactic acid that is produced by the vaginal microbiota. We examined how lactic acid abundance in the vaginal niche impacts the interaction between C. albicans and the human immune system using an in vitro culture in vaginal simulative medium (VSM). The presence of lactic acid in VSM (VSM+LA) increased C. albicans proliferation, hyphal length, and its ability to cause damage during subsequent infection of vaginal epithelial cells. The cell wall of C. albicans cells grown in VSM+LA displayed a robust mannan fibrillar structure, β-glucan exposure, and low chitin content. These cell wall changes were associated with altered immune responses and an increased ability of the fungus to induce trained immunity. Neutrophils were compromised in clearing C. albicans grown in VSM+LA conditions, despite mounting stronger oxidative responses. Collectively, we found that fungal adaptation to lactic acid in a vaginal simulative context increases its immunogenicity favouring a pro-inflammatory state. This potentially contributes to the immune response dysregulation and neutrophil recruitment observed during recurrent VVC.

Keywords: Vulvovaginal candidiasis; candida albicans; host response; lactic acid; vaginal simulative medium.

Figure 1.

Growth in lactic acid-containing vaginal simulative medium (VSM) affects Candida albicans virulence traits. The virulence traits of C. albicans that were grown overnight in VSM with and without lactic acid, respectively, were evaluated in RPMI medium. a) Growth of C. albicans was measured by determining the optical density (OD) at 600 nm at 37°C for 24 h. b) C. albicans colony forming units (CFUs) associated with vaginal epithelial cells (VECs) after 24 h of infection at 37°C and 5% CO₂. c) C. albicans hyphal length after 3 h of incubation at 37°C. Statistical analysis was performed using a two-way ANOVA. d) Growth of C. albicans pre-cultured in VSM at pH 4.5 and pH 6 was compared by monitoring hyphal length (mm/mm²) over time at 37°C and 5% CO₂ for 10 h. Statistical analysis was performed using a two-way ANOVA with a Holm-Šídák’s multiple comparisons test. e) Adhesion of C. albicans cells to VECs after 1 h of infection at 37°C and 5% CO₂ (shown as % of adherent cells compared to the initial inoculum). Data are the mean of n = 3, pooled from 3 independent experiments. f) Invasion of VECs by C. albicans after 3 h of infection at 37°C and 5% CO₂ (shown as % of C. albicans hyphae that invaded VECs of total hyphae present). Data are the mean of n = 3, pooled from 3 independent experiments. g) Quantification of VEC damage as released lactate dehydrogenase (LDH) after 24 h of infection at 37°C and 5% CO₂. H) IL-8 release was measured in the cell culture supernatant of C. albicans infected VECs after 24 h. Statistical analysis was performed using a two-tailed Wilcoxon matched pairs signed-rank test. Results are shown as the mean ± SEM of at least 2 independent experiments. Statistical significance is shown as ** = p < 0.01 and **** = p < 0.0001.

Figure 2.

Lactic acid affects Candida cell architecture.

Figure 3.

Innate and adaptive cytokine profile in human peripheral blood mononuclear cells (PBMCs) upon stimulation with Candida albicans cells grown in lactic acid-depleted vaginal simulative medium (VSM). a) PBMCs from healthy volunteers were stimulated with live C. albicans grown either in VSM with or without lactic acid for 24 h at 37°C and 5% CO₂. Levels of IL-6, TNF, IL-1β, IL-1Ra, and IL-8 in cell culture supernatants were measure by ELISA. b) IFN-γ, IL-10, IL-17, and IL-22 levels were measured in supernatant of PBMCs stimulated for 7 days with thimerosal-killed C. albicans cells grown in VSM with or without lactic acid. Statistical analysis was performed using a two-tailed Wilcoxon matched pairs signed-rank test. Results are shown as the mean ± SEM pooled from at least two independent experiments. Statistical significance is shown as * = p < 0.05.

Figure 4.

Macrophage responses upon stimulation with Candida albicans cells grown in lactic acid-rich vaginal simulative medium (VSM). a) Inflammasome activation was assessed by measuring IL-1β levels in the cell culture supernatant from lipopolysaccharide (LPS)-primed human monocyte-derived macrophages (hMDMs) that were stimulated for 5 h at 37°C and 5% CO₂with either Nigericin (positive control) or C. albicans cells grown in VSM with or without lactic acid (LA). Results are presented as fold change of C. albicans grown in yeast extract-peptone-dextrose (YPD) medium. b) Macrophage death was assessed at 37°C and 5% CO₂by quantifying SYTOX green release by hMDMs stimulated with C. albicans cells grown overnight in VSM±LA. Data are shown as the mean of 5 different donors. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparisons test. c) hMDMs killing capacity is shown as C. albicans colony forming units (CFUs) after 5 h of stimulation at 37°C and 5% CO₂. Statistical analysis was performed using a test two-tailed Wilcoxon matched pairs signed-rank test. Results are shown as the mean ± SEM of at least 2 independent experiments.

Figure 5.

Induction of trained immunity by Candida albicans grown in the presence of lactic acid. a-e) Following the in vitro trained immunity protocol, monocytes isolated from healthy buffy coats were stimulated with either heat-killed (HK) C. albicans (positive control, grown in YPD) or C. albicans cells grown overnight in vaginal simulative medium (VSM) in the presence or absence of lactic acid. After 24 h, the training stimulus was washed, and cell were let to rest for 5 days. On day 6, cells were restimulated for 24 h with either lipopolysaccharide (LPS) or HK Escherichia coli. Levels of TNF (A), IL-6 (B), IL-1Ra (c), MCP-1 (d), and IL-8 (e) were measured in cell culture supernatants by ELISA. Statistical analysis was performed using a two-tailed Wilcoxon matched pairs signed-rank test. Results are shown as the mean ± SEM pooled from at least 3 independent experiments. Statistical significance is shown as * = p < 0.05 and ** = p < 0.01.

Figure 6.

Neutrophils show dysfunctional phenotype upon interaction with Candida albicans cells grown in lactic acid-rich vaginal simulative medium (VSM). Neutrophils isolated from healthy volunteers were stimulated with live and thimerosal-killed C.albicans cells grown in VSM either with or without lactic acid (LA) to assess their cellular response. a) Neutrophil phagocytosis capacity is expressed as % of FITC-positive cells after 2 h stimulation with thimerosal-killed FITC-labelled C. albicans. b) Neutrophils were stimulated with live C. albicans grown either in VSM±LA for 4 h at 37°C. Levels of IL-8 in cell culture supernatants were measured by ELISA. c) Killing capacity is shown as colony forming units (CFUs) after 3 h of stimulation at 37°C CO₂ with live C. albicans cells. d) Reactive oxygen species (ROS) production in response to live C. albicans during a 1 h timeframe at 37°C is reported both as time course [relative luminescence units (RLU)/sec, left panel] and area under curve (AUC, right panel). Time course was assessed for statistical differences between the two tested conditions by a two-way ANOVA and AUC means (right panel) were compared using the two-sided Wilcoxon signed rank test. e) The C. albicans CTA1p-GFP reporter strain was grown overnight in VSM with or without lactic acid. After washing, cells were exposed either to H₂0 or 2 mM H₂0₂ for 2 h at 37°C and fluorescence was quantified by flow cytometry. The mean fluorescence intensities (MFIs) were normalized to the corresponding VSM with lactic acid control and results are displayed as mean ± SEM with dots representing single cultures. Statistical analysis was performed using a two-way ANOVA with Holm-Šídák’s multiple comparisons test. f) Growth of C. albicans cells grown overnight in the tested VSM conditions were monitored in RPMI by determining the optical density (OD) at 600 nm at 37°C for 24 h in the presence or absence of 10 mm H₂0₂. Statistical analysis was performed using a two-way ANOVA. Statistical analysis was performed using a test two-tailed Wilcoxon matched pairs signed-rank test. g) Growth of C. albicans pre-cultured in VSM at pH 4.5 and pH 6 were monitored in RPMI by determining the optical density (OD) at 600 nm at 37°C for 24 h in the presence or absence of 1 mM H₂0₂. Statistical analysis was performed using a two-way ANOVA with a Holm-Šídák’s multiple comparisons test.Results are shown as the mean ± SEM of at least 2 independent experiments. Statistical significance is shown as * = p < 0.05, ** = p < 0.01, and **** = p < 0.0001.