Aim & Objectives: Development of an in-vitro model system to assess the effect of upper respiratory tract multispecies microbial communities comprising of commensals and potential pathogens on human respiratory epithelium.
Methods: Microbial communities for “Healthy” and “Diseased” state were represented by commensal bacteria Staphylococcus epidermidis & Corynebacterium amycolatum and potential pathogens Staphylococcus aureus & Haemophilus influenzea respectively. The microbial communities (single bacteria and combinations) were co-cultured with differentiated human respiratory epithelial cells (Calu-3) and this co-culture model was then used to assess differences in host response on exposure to either “healthy” / “disease” associated microbiota or their mixture.
Results: Experiments done so far consisted of optimising conditions for multispecies bacterial cultures and their co-culture with host respiratory epithelial cells. These mixed species co-cultures were also observed over time using confocal microscopy.
Preliminary results have shown successful co-culture of bacteria (single and combination) with Calu-3 cells for up to 72 hrs without any cytotoxic effect (LDH assay). Up to 80% of bacteria added to calu-3 cells attached to the mammalian differentiated cell layer and further increased in number over time. Barrier integrity of differentiated Calu-3 cells grown at air liquid interface (ALI) with bacteria was maintained throughout the treatment time. Inflammatory response from Calu-3 cells was assessed by measuring secreted cytokines (IL-6, IL-8 and IL-10). IL-6 levels were higher for co-cultures with S. epidermidis, H. influenzea and S.aureus + C. amycolatum at 24hrs while co-culture with C. amycolatum reduced IL-6 levels at 72hrs compared to Calu-3 cells alone. IL-8 secretion was increased by S. aureus, S. epidermidis, H. influenzea and S.aureus +C. amycolatum at 24 and 72 hrs compared to Calu-3 cells alone.
Conclusion: We have shown that mixed bacterial communities can be co-cultured with respiratory epithelial cells without causing cytotoxic effect or impacting barrier integrity of Calu-3 cells while being able to observe differential cytokine response by human host respiratory epithelial cells.