The effect of Demodex-associated Bacillus oleronius proteins and hyperosmolarity on a human conjunctival epithelial cell line

PURPOSE: Demodex mites act as a carrier vector for the bacterium Bacillus oleronius. Antigenic proteins from this bacterium are capable of producing an inflammatory response. This study investigated the potential effects of Demodex spp.-associated Bacillus oleronius proteins and dry eye-induced hyperosmolarity on an immortalised human conjunctival epithelial (Chang) cell line. METHODS: The Wong Kilbourne derivative of the Chang (WKD) HeLa-modified conjunctiva-derived epithelial cell line was cultured in a series of NaCl-hyperosmolar conditions with various concentrations ranging from 350mOsm/L to 550mOsm/L. Chang cells were then exposed to semi-purified B.oleronius proteins at 2 µg and 6 µg either alone or in conjunction with dry eye-induced hyperosmolar (HO) stress at 2 µg + HO500 mOsm/L and 6 µg + HO500 mOsm/L. The cell viability was assessed with the RealTime-Glo MT cell viability assay. RESULTS: Compared to controls, the cell viability appeared to decrease in a dose-dependent manner. B. oleronius concentration at 6 µg showed significantly lower cell viability than both controls (p < 0.001) and 2 µg (p < 0.001). When 500mOsm/L was added to the 2 µg and 6 µg concentrations, the lowest cell viability was observed (p < 0.001). At 24 hours, the B.oleronius 2 μg combined with HO500 resulted in 38% viable cells, compared to 81% viability in B.oleronius 2 μg alone (p < 0.001). Similarly, the B.oleronius 6 μg combined with HO500 showed only 23% metabolising cells compared to 60% viability cells in B.oleronius 6 μg alone (p < 0.001). CONCLUSION: The results suggest that the presence of bacterial proteins and hyperosmolarity have a dose and time-dependent effect on cellular viability. These experiments suggest that patients affected with the comorbidity of dry eye disease and Demodex blepharitis could experience more severe signs than those with Demodex-associated blepharitis alone.