Candida albicans as a Trailblazer for Herpes Simplex Virus-2 Infection Against an In Vitro Reconstituted Human Vaginal Epithelium

Abstract

Little is known about the complex events driving host-pathogen and pathogen-pathogen interplay in polymicrobial infections. Using an in vitro model of a reconstituted vaginal epithelium (RVE) employing the A-431 cell line supplemented with synthetic vaginal fluid (SVF), we studied the consequences of single versus dual infections with Candida albicans and/or Herpes Simplex Virus-2 (HSV-2). Our data show (a) a relevant, SVF-enhanced expression of the differentiation marker cytokeratin 5/6 in the RVE; (b) the ability of Candida albicans to enhance HSV-2 in the dual infection model, with the virus titer almost doubling in the presence of SVF; (c) RVE damage (>20%), mostly attributable to Candida albicans and related to oxidative stress whether SVF is present; (d) the dysregulation of mucin-1, the production of which is enhanced (from 13 to 21 ng/mL) or impaired (from 21 to 10 ng/mL) in response to either SVF or infection, respectively; and (e) a partial-to-negligible cytokine response from the RVE, depending upon SVF presence. In conclusion, using an in vitro RVE model upgraded through the addition of synthetic vaginal fluid, we provide details on epithelial cell-pathogen-pathogen interaction, contributing to a better comprehension of the pathogenesis of polymicrobial infections at a mucosal level.

Keywords: A-431 cells; dual infections; pathogens interactions; synthetic vaginal fluid; vaginal infections.

Figure 1

Cytokeratin 5/6 staining in A-431 cells, cultured for 1 or 5 days, in the absence or presence of SVF. Cells were immunohistochemically stained, with an anti-cytokeratin 5/6 mouse monoclonal primary antibody, using BenchMark IHC/ISH automated instruments according to standard antigen retrieval protocol. Representative images from two experiments are shown (magnification 40×). Immunohistochemical staining for CK5/6 shows diffused expression in the RVE, with stronger intensity observed in SVF.

Figure 2

Quantification of fungal and viral loads in the RVE cultured with or without SVF. Five-day RVE cultures were infected with C. albicans (fungus:epithelial cell ratio = 0.5:1), and, after 3 h, HSV-2 (virus:cell ratio = 0.1:1) was added. Fungal and virus quantification was carried out after 24 h incubation using the CFU method on Sabouraud agar for C. albicans (A) and both RT-PCR (B) and the plaque reduction assay (C) for HSV-2. * p < 0.05.

Figure 3

Levels of A-431 cell damage after single or dual infection in the presence or absence of SVF. Five-day RVE cultures were infected with C. albicans (fungus:epithelial cell ratio = 0.5:1), and, after 3 h, HSV-2 (virus:cell ratio = 0.1:1) was added. At 24 h, the LDH assay was performed to quantify the percentage of LDH release in the RVE exposed to one or two pathogens, in the presence or in the absence of SVF. * p < 0.05.

Figure 4

Mitochondrial ROS production by A-431 cells, exposed to single or dual infection in the presence or absence of SVF. Five-day RVE cultures were infected with C. albicans (fungus:epithelial cell ratio = 0.5:1), and, after 3 h, HSV-2 was added (virus:cell ratio = 0.1:1). Then, MitoSOX™ Red was added to each well and the plates were evaluated using Fluoroskan at 37 °C, for 24 h. A representative heatmap of the quantification of mtROS production at 24 h is shown. The mock line refers to A-431 cells alone; an inoculum of C. albicans without cells was included as a negative control (abiotic support). The values corresponding to the different colors represent the Area Under the Curve calculated to summarize the curve of the 24 h determination into a single value.

Figure 5

Secretory profile of the RVE infected with C. albicans and/or HSV-2, in the presence or absence of SVF. Five-day RVE cultures were infected with C. albicans (fungus:epithelial cell ratio = 0.5:1), and, after 3 h, HSV-2 was added (virus:cell ratio = 0.1:1). Following 24 h incubation, quantification of the secretion product was performed on the supernatants using commercial ELISA. * p < 0.05; ** p < 0.01.