Airway colonisation with Pseudomonas aeruginosa (PA) represents a hallmark of cystic fibrosis (CF). Mechanisms by which PA establishes pulmonary infection are undoubtedly complex and, in part, will reflect the capacity to interfere with host defense mechanisms. Dendritic cells (DCs) represent the most potent antigen-presenting cells in the lungs, with the unique ability to prime naïve T cells. PA-induced apoptosis has been demonstrated in epithelial cells and macrophages. We examined the capacity of PA to induce cell death in human dendritic cells, the cytotoxicity of clinical PA isolates, and the impact on antigen-presenting capacity.Methods
CD14+ monocytes were isolated from peripheral blood of healthy controls (n=11) and individuals with CF (n=5). Monocyte-derived DCs were generated by culture in the presence of IL-4 and GM-CSF. DCs were infected with live PA including isogenic laboratory strains of PA103 and PA isolates derived from the sputum of patients with CF. Heat-inactivated PA were utilised to evaluate the role of bacterial membrane components. Presence of early apoptosis and established cell death was analysed via annexin-V and 7-AAD incorporation, respectively. Cytotoxicity was further demonstrated via LDH release into the supernatant. Co-stimulatory molecules, CD40 and CD86, were measured via flow cytometry.Results
PA readily induced apoptosis and cell death in human DCs, with cytotoxicity seen within 3 h of infection. Induction of apoptosis by PA was an active process requiring live organisms, but was not dependent on a functional type III secretion system. A significant decrease in viable DCs was seen in response to infection with clinical PA strains at 3 h and 20 h compared with laboratory PA103 strains (p<0.05 and p<0.001, respectively). Due to increased cytotoxicity of clinical PA isolates, post-infection DCs demonstrated no increase in co-stimulatory molecule expression compared with uninfected DCs (p>0.05).Conclusions
These data demonstrate that human dendritic cells are susceptible to apoptosis induced by P aeruginosa, with clinical isolates of PA demonstrating high levels of cytotoxicity, and a subsequent reduction in DC antigen-presenting capacity. Elimination of these important antigen-presenting cells could lead to impairment of immune responses and thus a factor in the establishment of chronic PA colonisation in the CF lung.