Investigating the Three-Way Interaction between Gastric Epithelial Cells, Human Neutrophils, and Helicobacter pylori

Abstract

Abstract Neutrophils are the most abundant white blood cells in humans and are the first line of defense against pathogens with respect to innate immunity. Helicobacter pylori are gram-negative bacteria that infect the gastric epithelium and play an important role in the development of ulcers and gastric cancer. Large numbers of neutrophils are recruited to the gastric epithelium where they become heavily infected. We demonstrated previously that when neutrophils are infected by H. pylori it extends their short lifespan. In addition, these cells display unique characteristic such as nuclear hypersegmentation that are indicative of N1-like subtype differentiation which may increase bacterial virulence. Studies by other groups have examined H. pylori-epithelial cell interactions but notably absent are three-way co-cultures that examine simultaneous interactions of neutrophils with bacteria and gastric epithelial cells. Addressing this knowledge gap is the focus of my project. Specifically, I am investigating the dynamics of infection in these co-cultures and determining whether the infected PMNs alter epithelial cell function or viability. Using immunofluorescence I am determining bacterial localization, analyzing PMN nuclear morphology, and determining the extent of changes in epithelial cell junctions and the actin cytoskeleton over 24 hours. Additionally, I am using flow cytometry, lactate dehydrogenase release assays, western blotting and microscopy to quantify cell death, including apoptosis, to test the hypothesis that infected PMNs are more toxic to the epithelium than the bacteria alone. Ultimately, our studies are expected to advance understanding of the role of neutrophils in H. pylori pathogenesis.