Modelling endothelial cells to investigate lymphocyte proliferation under inflammatory conditions

Bordetella pertussis is a gram-negative bacteria and the causative agent for whooping cough, a toxin mediated respiratory disease, which causes extensive damage to epithelium in the trachea and bronchial mucosa. In the absence of a human model to study vascular leukocyte recruitment to the respiratory system, and tissue injury repair, we propose an in vitro model of endothelial cells (ECs) derived from embryonic stem cells (ESCs). We hypothesize that this model may be used to study the proliferation of Th17 and Treg cells during respiratory infection induced with B. pertussis antigens. Previous studies have shown that ECs constitutively express MHC II molecules allowing antigen presentation to recipient CD4 + T cells leading to a pro-inflammatory response. In addition, it has been demonstrated that human allogeneic EC induce Treg and Th17 proliferation. In this study, we differentiated mouse ESCs in vitro to endothelial cells in the presence of VEGF and bFGF. The cells were enriched for endothelial cell surface adhesion markers CD31 and CD144. Using flow cytometry, qPCR and immunofluorescence, we confirmed the expression of endothelial markers vWF and CD144 with 8% of our population double-positive for CD31 and CD144. The current model will be used to study pro-inflammatory response and T cell polarization will be assessed by activating endothelial cells with B. pertussis antigens or IFN-γ and then co-culturing with CFSE stained CD4 + T cells in polarizing conditions for 4 days. We anticipate that this study will contribute important information about endothelial regulatory response to injury and inflammation during whooping cough.