An in Vitro 3D Oral Mucosa Model (3DOMM) for Infection Studies

Objectives: To assess the efficacy of primary oral keratinocytes (P3D oral mucosa model, P3DOMM) and immortalised HaCaT (3D oral mucosa model 3DOMM) cells to model single and dual infections of C. albicans and S. aureus for studying tissue colonisation and invasion, and subsequent pro-inflammatory cytokine production.
Methods: Models were infected with clinically relevant strains of S. aureus (IRAS ref. 208291) and/or the C. albicans type strain (SC5314) and subsequently evaluated for tissue invasion, cell pathology and pro-inflammatory cytokine production by histology, SEM, LDH production and ELISA. Different combinations of patient samples, and type strains, were added to HaCaT cells to compare isolates obtained from patients with single and dual colonisation of S. aureus and C. albicans for 24h prior to ELISA analyses for IL-6 and IL-8. Statistical analyses confirmed parametric vs. non-parametric data; p-values < 0.05 indicated statistical significance.
Results: HaCaT cells alone and in 3D mucosal models produced pro-inflammatory cytokines in response to infection. For HaCaT cells, 3DOMMs, and P3DOMMs, a significant increase in pro-inflammatory cytokine production (IL-8; IL-6 and IL-8; and IL-6, respectively) was observed between the unstimulated control and models stimulated with C. albicans or S. aureus, and dual infection. 3DOMMs only were capable of increasing both IL-6 and IL-8 in response to live infective stimuli. This means that despite the high levels of constitutive pro-inflammatory cytokine production identified, it is still possible to measure upregulation in response to live infection.
Conclusions: 3DOMM produced IL-6 and IL-8 pro-inflammatory cytokines in response to single and dual infections of C. albicans and S. aureus, as well as indicating its suitability to model microbial colonisation in a robust, reproducible, manner.