An In Vitro Model to Assess Early Immune Markers following Co-Exposure of Epithelial Cells to Carbon Black (nano)Particles in the Presence of S. Aureus: Differential Induction of Cytokines

Human exposure to carbon black (CB) is inevitable due to its widespread applications in the medical, industrial and consumer sectors. With an ageing population, it is imperative that the effects of (nano)particle exposure in individuals with compromised immunity or infection are considered. Since barrier immunity provides the first line of defence against CB and the human skin and lung pathogen, Staphylococcus aureus, this work focuses on studying the impact of CB exposure upon compromised immunity during infection on human skin and lung epithelial cells in vitro. The principal aim of the work was to develop an epithelial cell model to characterise (co-)exposure to CB and S. aureus. The work used two human epithelial cell lines, HaCaT (skin) and A549 (lung), ELISA technology to assess the (pro-)inflammatory response, aseptic microbiology techniques to grow S. aureus and a Zetasizer, EDX spectroscopy, and both scanning and transmission electron microscopy (SEM and TEM) to characterise the CB under the conditions used in the study. Physicochemical characterisation of CB confirmed its shape, dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth, but in a biological media dependent manner. CB did not induce cytokines or antimicrobial peptides from skin and lung epithelial cells, when given alone. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both skin and lung epithelial cells. IL-6 and human beta defensin (hβD)-2 could only be detected when cells were stimulated with S. aureus. However, co-exposure to CB (100µg/ml) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus only induced levels). Furthermore, the same co-exposure induced significantly more hβD-2 (compared to S. aureus alone). The ability to detect pathogen responses to particle, in addition to epithelial responses to particle and pathogen is an advance on assessing cell responses under ‘healthy’ conditions and supports the need for developing exposure models under stressed or immunosuppressed conditions. This model will be useful for studying mechanisms of exposure in at-risk groups, including factory workers, the elderly and immunocompromised. Advanced models, that better represent human pathophysiology are essential for understanding cellular mechanisms of toxicity in the 21st Century.