Entamoeba histolytica kills more humans than any other Category B protozoa by far. This R01 proposes work by a new investigator to identify mechanisms of innate resistance to intestinal amoebiasis in a new mouse model. We developed this model in 2002 and have found susceptibility to infection is mouse strain dependent, whereby C3H and CBA mouse strains are susceptible to amoebic colonization and colitis while C57BL/6 and other tested strains are resistant. Preliminary data with bone marrow chimeric mice indicate that these mouse-strain phenotypes are governed by non-hemopoietic cells, suggestive of the epithelium. Surprisingly, C57BL/6 IL10-/- mice lose resistance to infection, and this susceptibility is transferable with IL10-/- hemopoietic cells, suggesting that epithelial resistance can be modified. We therefore envision pathogenesis as a two-stage event where amoeba must first breach the epithelial barrier to colonize. We hypothesize this occurs in CBA mice because of a predisposition towards amoeba-induced epithelial apoptosis and in C57BL/6 IL10-/- mice because of their well-established epithelial barrier defects. Second, a neutrophil-mediated inflammatory response ensues that we have shown is phenotypically protective. We hypothesize this response is TLR-mediated. This grant application will therefore utilize experts in epithelial biology (Steven Cohn) and TLR signaling (Michael Smith).
Aim 1 will characterize the epithelium by laser capture microdissection and test the extent to which epithelial apoptosis predisposes to amoebic colonization using caspase 3-/- mice. The role of IL10 and IL10-regulated proinflammatory cytokines on epithelial health will be examined in a T84 epithelial cell monolayer model and these results then audited in the IL10-/- mouse model.
Aim 2 will characterize the anatomy of TLR expression in the mouse cecum and the pattern of amoeba-induced TLR activation in vitro. MyD88-/- mice will be generated on the susceptible CBA background and, if phenotypically important, the cellular anatomy of MyD88-dependent TLR activation ascertained via bone marrow chimeras. We acknowledge there are many potential directions to pursue in an early and complex model such as this. Some work must be descriptive. We have chosen these two aims because they try to synthesize background and preliminary data, can be tested in this newly available system, and may shed broad insight into how pathogens hijack the innate host response for their survival.
