The role of endothelial cells during infection 

There are more than 10 trillion EC in the human body, that cover 4000 m2 and these are an important replicative niche for a subset of micro-organisms such as Dengue virus, West Nile Virus, Listeria, TB, CMV, S. aureus and T. gondii. For many micro-organisms, the ability to infect EC is a key part of their pathogenesis. Indeed, EC express TLR and influence coagulation and neutrophil recruitment and their activation can lead to vascular damage and expression of adhesion molecules that promotes extravasation of inflammatory cells. Numerous in vitro studies have demonstrated that EC can be activated by cytokines to limit viral, bacterial and parasite replication. Furthermore, evidence that EC interact with the adaptive response is implicit in the ability of EC to present MHC class I & II restricted antigens. However, whether EC antigen presentation via MHC-I or MHC-II has a tolerogenic or inflammatory role during infection remains unclear. Therefore, there is a major knowledge gap in our understanding of the involvement of EC in orchestrating an immune response or how the immune system reacts to infected EC in vivo. 

EC constitutively express the chemokine fractalkine (CX3CL1) which is upregulated in response to inflammation and seminal intravital studies using mice which express GFP, driven by the CX3CR1 promoter, showed that at homeostasis CX3CR1+ monocytes patrol the luminal side of the entire microvasculature and in CX3CR1-KO mice this is 6 fold reduced. Because monocytes, microglia and resident macrophages are considered the major population of CX3CR1+ cells the majority of studies using CX3CR1-KO mice have focused on the role of CX3CR1 in the regulation of these cells in various models of inflammation. However, recent reports have shown that in several murine models of infections (Listeria, LCMV, TB, VSV, all of which can infect EC in vivo) there is a sizeable subset of pathogen specific T cells that express CX3CR1 and these cells are also present in humans. It has been proposed that CX3CR1 expression identifies a subset of effector memory T cells but there are no studies that have established a role for CX3CR1+ T cells in resistance to infection. These cells are also generated during infection with T. gondii and our preliminary data show that the loss of CX3CR1 results in an increased parasite burden in the vascular compartment. These observations form the foundation for the hypothesis that CX3CR1+ T cells are specialized to patrol the vascular compartment in order to eliminate infected EC.

Aim 1. Does the loss of antigen presentation via EC affect the outcome of infection? A number of genetic approaches have been used in attempts to specifically target EC and these include Tie2 and VE-cadherin (Cdh5) promoters to drive Cre expression. While these do target EC, these approaches result in significant off target effects because these promoters are widely active during development. While we have used bone marrow chimeras to target EC there remain significant well appreciated caveats with this approach. Therefore, we use an inducible ER-Cre (to limit off target effects) to provide a more reliable approach to target EC.

Aim 2. What is the role of CX3CR1+ CD8+ T cells in the recognition of infected EC? CD8+ T cells are important for resistance to T. gondii and the appearance of parasite specific CX3CR1+ CD8+ T cells in the blood of infected mice correlates with parasite control in this compartment. We hypothesize that these CX3CR1+ CD8+ T cells participate in surveillance for infected EC.