The role of complement in epithelial cell interactions with the gut microbiome

The intestinal epithelium is a highly dynamic organ with ongoing cell division, maturation into different cell types, and replacement, all in an environment heavily polluted with microbes. Our understanding of how epithelial cells sense these microorganisms and discharge anti-microbial defenses into the lumen is far from complete. In fact, the contribution of our single-greatest anti-microbial system, complement, has been almost totally neglected in this relationship. Complement is a network of proteins that upon activation become cleaved in a cascade fashion with the fragments assembling into molecules with new properties. The new properties include tagging the organisms for our white blood cells to destroy, the direct destruction of the microbes and dying cells, and inflammation. Inflammation is triggered by C3a and C5a. With NSERC funding I have emerged as a leader in the science of complement and the gut, including by discovering that epithelial cells possess receptors for and respond to C3a and C5a. Despite this progress it is unclear whether the intestine is fully armed with all the components of complement. This renewal will address this gap in our knowledge through 2 approaches; 1) measure the complement molecules made by cells growing as "organoids", a new way to grow intestinal epithelial cells devoid of all other cell types, and 2) determine how complement becomes activated in mice using a bacterial infection of their colons. Organoids are 3-dimensional tubular networks of fully mature small or large intestinal epithelium derived from intestinal stem cells, growing in petri dishes. I will grow organoids from mice and measure levels of complement molecules and receptors to determine which are present. When I know which complement molecules are present I will grow organoids from mice lacking these molecules and measure the impact on cell development. When I know which receptors for split complement molecules are present I will grow organoids and add the molecule that binds the receptor and measure the types and numbers of cells and their anti-microbial secretions. Importantly, using organoids I will study Paneth cells, a cell in the epithelium that protects us from bacteria but that is otherwise very difficult to study. Then, I will use mice deficient in certain complement molecules and infect them with a bacterial pathogen of the colon. I already know that the infection triggers complement activation, measured as increased C3a and C5a, but I do not know how or what complement molecules are in the cascade leading to these increases. I will use selected complement deficient mice to identify which molecules are activated by the infection. My future plan is to use this information to conduct experiments to discover how complement affects the bacteria living in the intestine (our microbiome).***********