Defining mechanisms of disease initiation through analysis of Helicobacter pylori interactions with gastric microbial communities and the host

Abstract

Helicobacter pylori is an exceptionally prevalent bacterial pathogen that colonizes the human stomach where it can cause inflammation, and in some cases lead to gastric ulcers and cancer. H. pylori infection also seems to protect against development of certain diseases such as esophageal adenocarcinoma. The prevalence of H. pylori has been rapidly declining in economically developed countries and the disappearance of infection tracks with the reciprocal rise in esophageal adenocarcinoma. H. pylori may be modulating esophageal adenocarcinoma risk through its interactions with members of the upper gastrointestinal microbial communities. Before that hypothesis can be tested, a comprehensive survey of which microbial community members are present in health and disease is needed. Barrett’s esophagus, a metaplastic transition of the normal squamous epithelial lining of the esophagus to mucus-producing columnar epithelium, is the only known precursor lesion to esophageal adenocarcinoma. Using high throughput sequencing, we characterized the microbial composition of the upper gastrointestinal tract in individuals with Barrett’s esophagus. We found that bacterial communities of the stomach and esophagus showed overlapping community membership. Despite closer proximity, the stomach antrum and corpus communities were less similar than the antrum and esophageal samples. In this Barrett’s esophagus cohort, Streptococcus and Prevotella species dominated the upper GI and the ratio of these two species was associated with waist-to-hip ratio and hiatal hernia length, two known esophageal adenocarcinoma risk factors. Genomic instability is a predictor of cancer development in Barrett’s esophagus. We found that H. pylori-positive individuals had a significantly decreased incidence of aneuploidy and a trend toward lower incidence of esophageal adenocarcinoma. Our analysis also revealed that H. pylori can be found in esophageal tissues of infected individuals. The protective effect of H. pylori carriage on risk of esophageal adenocarcinoma development may be explained by H. pylori’s influence on members of the esophageal microbiota and/or direct interaction with esophageal epithelial cells. In the stomach, H. pylori is recognized by both the innate and adaptive immune system, but this rarely results in bacterial clearance. H. pylori establishes chronic colonization, in part, by evading numerous host defense strategies. However, H. pylori also seems to benefit from a restricted inflammatory response. During H. pylori infection cytotoxin associated gene A (CagA) is delivered to gastric epithelial cells through a type 4 secretion system (cag-T4SS). Along with CagA, proinflammatory bacterial factors gain access to the host cell cytosol through the cag-T4SS. Detection of those bacterial factors leads to activation of global transcription factor NF-κB, which regulates production of proinflammatory cytokines key for recruitment of immune cells to the site of infection. Until recently, the entire cag-T4SS-dependent inflammatory response in gastric epithelial cells was attributed to detection of bacterial cell wall fragments by host pathogen recognition receptor nucleotide-binding oligomerization domain 1 (NOD1). When we CRISPR/Cas9 targeted NOD1 in gastric epithelial cells, we found that the NF-κB mediated inflammatory response was attenuated, but not eliminated, suggesting that other pathogen recognition pathways play a role in H. pylori detection. We determined that bacterially-derived heptose-1,7-bisphosphate (HBP), a metabolic precursor in lipopolysaccharide (LPS) biosynthesis, is delivered to the host cytosol through the cag-T4SS, where it activates the host tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA)-dependent cytosolic surveillance pathway. We also found that CagA toxin contributes to the late NF-κB-driven response. The sequential activation of TIFA, NOD1, and CagA delivery drives the initial inflammatory response in gastric epithelial cells. To determine the in vivo relevance of NOD1 signaling, we infected Nod1-/- mice with a mouse adapted strain of H. pylori. We found that Nod1-/- mice were more susceptible to H. pylori infection than wild-type mice. Despite the modest contribution of NOD1 signaling that we observed in the gastric epithelial cell line, Nod1 appears to be important in restricting bacterial growth in a mouse model of infection. The discrepancy between our in vitro and in vivo findings motivated us to develop a better gastric epithelial cell model to study innate immune signaling pathways involved in H. pylori detection. Using mouse and human gastric tissues, we selected, expanded and propagated Lgr5-expressing progenitor cells in the presence of stem cell growth factors Wnt, R-spondin and Noggin. We differentiated these gastric organoids into primary-like gastric epithelial cells by withdrawing stem cell growth factors from the culture media. When we seed gastric organoids on a permeable support, they form a polarized gastric epithelial monolayer that can then be used for infection studies where bacteria are added to the apical side of the transwell. This novel ex vivo system enables host-pathogen interaction studies in a physiologically more relevant setting. Myeloid-derived cells detect H. pylori via pathogen recognition pathways that are distinct from those activated in gastric epithelial cells. We found that monocytes initiate a type I interferon (IFN) response following H. pylori exposure and that the response is cag-T4SS dependent. It remains unclear which bacterial factor(s) is initiating the type I IFN response and whether the response is synergistic or antagonistic of the NF-κB-driven inflammatory response. We can begin addressing these questions by using the gastric organoid co-culture system where we infect polarized epithelial cells with H. pylori and add myeloid-derived cells to the basal compartment to investigate the cross talk between bacteria, gastric epithelial cells and recruited immune cells. The studies presented in this dissertation enhance our understanding of how H. pylori initiates a proinflammatory signaling cascade in the host and better defines pathways that can be manipulated for therapeutic purposes.