In vitro model for assessing the effect of T helper cell cytokines on the barrier function of alveolar type II epithelial cells

Abstract

Abstract There are still diseases of global burden for which satisfactory vaccines are unavailable, e.g. tuberculosis. Vaccines against intracellular microbes has relied on the activation of the adaptive T cell response. Our laboratory is interested in investigating whether the innate epithelial host defense could be integrated in a novel approach. In response to airborne pathogens, airway epithelia release antimicrobial peptides like human beta defensin-2 and -3 (HBD2/3), antimicrobial lipids, and chemokines, like IL-8, to attract other immune cells including T helper cells that are integral to the adaptive immune response. We hypothesize that T cell derived cytokines augment the antimicrobial function of airway epithelia and we have developed an in vitro model to generate pilot data. A549 cells, alveolar type II epithelial cells, were grown in air-liquid interface and stimulated with TNF-α & IFN-γ and IL-17 & IL-22, at 5 ng/mL each, for 24 h. IL-8 and HBD2/3 gene expression was quantified by RT-PCR and secretion of antimicrobial peptides was assessed by AU-PAGE and Western blot probing for HBD2/3. Lipid production was assessed with Bodipy and the physical barrier integrity with Phalloidin. IL-8 gene expression was 9-fold elevated when cells were stimulated with TNF-α & IFN-γ. Secretions from stimulated cells appeared to contain more cationic peptides that were not reactive with HBD2/3 polyclonal antibodies. Cellular Bodipy fluorescence was pronounced after IL-17 & IL22 stimulation. Phalloidin staining was consistent with tight junction formation in all samples. This data provides evidence that T helper cells may directly augment epithelial barrier function supporting a novel vaccine design that integrates the epithelial cell response.