Each day the stomach is exposed to outside antigens, and the immune system must discriminate between innocuous environmental substances and harmful pathogens such as Helicobacter pylori. This is achieved through antigen sampling by antigen presenting cells (APCs) such as dendritic cells (DCs). In this study, we investigated apoptotic cell death as a potential mechanism for gastric antigen sampling. We hypothesized that apoptotic epithelial turnover exposes antigen to APCs through cell shedding and transient gaps in the epithelium. To test this hypothesis, we used H. pylori-infected human gastric organoids (HGOs), human DCs, and co-cultures of HGOs and DCs within our microphysiological system, the gastrointestinal organoid flow chip (GOFlowChip). Apoptotic cell death was measured using the NucView fluorescent biosensor. Infection of HGOs with H. pylori strain 60190 led to a significant increase of apoptotic cells after 72 hours. Using HGO infections with H. pylori CagA and VacA knockouts, we observed that the absence of the virulence factor CagA increased HGO apoptotic cell death, while the absence of VacA decreased apoptotic cell death when compared to wild type, confirming previous studies that have shown a pro-apoptotic role of VacA. We next determined the effect of H. pylori on HGO epithelial dynamics using confocal live imaging of mock or H. pylori infected organoids. Infected HGOs had increased epithelial turnover, and importantly, shed more cells to the basolateral side, where they can be phagocytosed by DCs in the gastric lamina propria. Additionally, infected HGOs had weakened epithelial barrier function, rupturing more frequently and releasing their luminal contents, which we showed using FITC dextran-injected organoids. We next determined whether DCs preferentially were recruited towards apoptotic epithelial cells. Using chemotaxis assays, we demonstrated that supernatants collected from HGOs treated with an apoptotic cell death inducer were a DC chemoattractant. Particle tracking of DCs, fluorescent H. pylori, and a fluorescent apoptotic cell death reporter within the GOFlowChip co-culture revealed recruitment of DCs to apoptotic organoids over 20 hours. Notably, while we detected DC phagocytosis of apoptotic and bacterial antigen, we did not observe significant dendrite-mediated antigen uptake by DCs. Taken together, we propose the following mechanism: H. pylori infection increases gastric epithelial cell death, which recruits DCs to sites of epithelial instability, allowing the passage of antigen to the basolateral side for sampling.

Mark Jutila

Appearances