Modulation of C. albicans-Induced Immune Response in Vaginal Epithelial Cells by Garcinoic Acid

Abstract

Vulvovaginal candidiasis (VVC) is a prevalent women's infection characterized by excessive inflammation and damage of the vaginal epithelium that, in its recurrent form (RVVC), causes more than three symptomatic episodes per year, impacting nearly 8% of women globally. Current antifungal treatments alleviate symptoms but often fail to restore the inflammatory homeostasis of mucosal tissue and prevent recurrences. α-Tocopherol (α-TOH) and garcinoic acid (GA), a vitamin E metabolite, with immunomodulatory properties, were investigated for the first time in vaginal epithelial cells exposed to C. albicans infection to assess their effects on inflammatory signaling parameters important to restore cellular homeostasis. For this purpose, the protein kinases MKK3/6, p38 stress kinase (SAPK), and ERK1/2 were studied together with c-Fos transcription factor and IL-6, IL-1α, and IL-1β secretion in A-431 vaginal epithelial cells pre-treated with GA or with α-TOH and then infected with C. albicans. GA, differently from α-TOH, significantly reduced the C. albicans-induced activation of p38-SAPK while increasing pro-survival MAPK ERK1/2 activity. This resulted in a significant reduction in the secretion levels of the inflammatory cytokines IL-6 and IL-1α, as well as IL-1β. Overall, our data indicate that GA holds potential for restoring the immuno-metabolic properties of the vaginal epithelium exposed to C. albicans infection, which may help to treat inflammatory symptoms in VVC/RVVC.

Keywords: C. albicans; VVC-RVVC; garcinoic acid; vaginal cells.

Figure 1

Effects of GA and α-TOH treatments on vaginal cell viability. A-431 cells were exposed to increasing levels of GA (A) or α-TOH (B) between 1 and 100 µM for 48 h and then assessed for viability by the MTT test. Data are the means ± SDs of triplicate determinations. * p < 0.05 (Ctr vs. all treatments). Ctr = uninfected cells.

Figure 2

Microscopic analysis of VECs infected for 3 h with C. albicans SC5314. The representative images, observed by optical microscopy, show the fungal cells in the transition to the hyphal form. The pictures were taken at magnifications of 200× (A,B) and 400× (C,D).

Figure 3

Effects of GA and α-TOH pre-treatments on phospho (P)-p38 and c-Fos levels in VECs infected with C. albicans. A-431 cells were pre-treated with 10 and 25 µM GA (A,B) or with 25 and 50 µM α-TOH (C,D) for 48 h and then infected for 3 h with C. albicans. P-p38 and c-Fos activation were determined by using Western blot analysis. The immunoblot images are representative of three independent experiments with similar results. The densitometric data depicted in the histograms are the means ± SDs of three independent experiments. * p < 0.05; ** p < 0.001 (UCV vs. C. albicans-infected cells); ** p < 0.001 (C. albicans-infected cells vs. C. albicans-infected cells + GA). Ctr = uninfected cells; UCV = uninfected cells + vehicle.

Figure 4

Effects of GA pre-treatment on phospho (P)-MKK3/6 and P-ERK1/2 levels of VECs infected with C. albicans. A-431 cells were pre-treated with 10 and 25 µM GA for 48 h and then exposed for 3 h to C. albicans. P-MKK3/6 and P-ERK1/2 activation was determined by using Western blot analysis. The immunoblot images are representative of three independent experiments with similar results (A). The densitometric data in the histograms depict the means ± SDs of three independent experiments (B). ** p < 0.001 (UCV vs. C. albicans-infected cells); * p < 0.05; ** p < 0.001 (C. albicans-infected cells vs. C. albicans-infected cells + GA). Ctr = uninfected cells; UCV = uninfected cells + vehicle.

Figure 5

Proposed model of mechanistic aspects of GA immuno-modulatory effects in vaginal epithelial cells exposed to C. albicans infection. The MKK3/6-p38/SAPK pathway and its regulatory interplay with pro-survival MAPK ERK1/2 are molecular targets of GA, with modulating activity on IL-6, IL-1 α, and IL-1β levels secreted by the infected vaginal epithelium. The effect of GA on these cytokines may preserve the immune-metabolic properties of the vaginal epithelium and support post-infection recovery and the restoration of cellular homeostasis. (A) Mechanism of action of C. albicans on vaginal epithelial cells. (B) Effect of pre-treatment with GA on vaginal cells infected with C. albicans. The dotted line in the arrows indicates that there is a trend toward a non-significant reduction in the proposed event; the solid line indicates a significant reduction/inhibition; red arrows indicate increased production; blue arrows indicate decreased secretion. The image was produced by using BioRender software (https://www.biorender.com; license number: C81D6A59-0002).