INHIBITION OF CD73 AS A MODULATOR OF SALMONELLA VIRULENCE AND POTENTIAL THERAPY FOR SALMONELLA-INDUCED CALF SCOURS

The long-term goal for this project is to elucidate the mechanisms by which adenosine generated by the intestinal epithelium modulates inflammatory diseases. Epithelial cells create a critical boundary between antigens in the intestinal lumen and the underlying submucosa. Pathogens such as Salmonella enterica initiate infection in intestinal epithelial cells (IECs) to cause inflammatory disease. Thus, IECs represent a key point of host defense against pathogens. ATP is released into the intestinal lumen by pathogens, IECs, and infiltrating immune cells and acts as a pro-inflammatory signal during infection. Once near the intestinal epithelial cell surface, extracellular ATP is quickly converted to AMP by ecto-apyrase. AMP must then be converted to the anti- inflammatory molecule adenosine by CD73 for the resolution of inflammation. However, there is a critical knowledge gap in understanding the impact of CD73-generated adenosine on mechanisms of host-microbe interactions. Work from our lab has shown, S. Typhimurium is unable to carryout normal pathogenesis in the absence of intestinal epithelial CD73 (59). Additionally, it is clear that a number of immune cells also express CD73 and the role of adenosine production by these cells in mediating bacterial pathogenesis is not well understood. Therefore, based on our previous work, we hypothesize that CD73 inhibition alters adenosine generation and signaling pathways, limiting the ability of S. Typhimurium to cause calf scours. Three specific aims are proposed to test this hypothesis:Specific Aim 1: Examine the role of CD73 in the modulation of Salmonella infection in bovine IECs. Work in our lab has shown that epithelial CD73 acts as a mediator of S. Typhimurium infection in human cell models and in murine models in vivo. As an extension of these studies, we will examine the dynamics of CD73 modulation of S. Typhimurium infection in vitro in bovine intestinal epithelial cells (BIECs) and an ex vivo bovine intestinal organoid model. Here, we will determine the role of epithelial CD73 in S. Typhimurium pathogenesis in bovine cell models using the CD73 inhibitor AB680 (80). Additionally, we will employ the use of CRISPR technology to abrogate CD73 expression to confirm the importance of CD73 in the bovine cell models.Specific Aim 2: Determine if inhibition of CD73 will protect against Salmonella-induced experimental scours. Our previous studies in human epithelial cells and mouse models indicate that loss of epithelial CD73 abrogates S. Typhimurium infection and virulence. Here we will investigate the impact of CD73 inhibition on salmonellosis using a S. Typhimurium-mediated calf scours model. This infection is of economic relevance and has been employed successfully by our collaborator, Dr. Jutila, in earlier studies of therapeutic adjuvants in bovine calves (81). In this objective we propose to examine the capacity of the CD73 small molecule inhibitor AB680 to protect from S. Typhimurium-induced calf scours in dairy calves.Specific Aim 3: Elucidate the role of immune cell CD73 expression in modulation of Salmonella infection in murine disease models. We have previously demonstrated that loss of CD73 expression in the intestinal epithelium abrogates virulence in a mouse model of Salmonella infection (59). However, as described above, CD73 is broadly expressed in a number of cell types including, including immune cells, which impacts the function of these cells. As we have the CD73 floxed mouse (CD73fl/fl) in our colony, we are in position to examine the role of CD73 in specific cell lineages. CD73 has been shown influence T cell and myeloid cell responses. Here we will examine the impact of loss of CD73 in each of these cell lineages, utilizing specific cre recombinase mice, in our established Salmonella infection model.Successful completion of the work in this proposal will provide proof of principle that CD73 inhibition represents a novel and viable therapy for the treatment of Salmonella-induced calf scours. The results have a high potential of resulting in USDA funding and allow us to expand these studies to additional infectious pathogens that contribute to calf scours.